Oncogene database

1956

EGFR

epidermal growth factor receptor;EGFR;epidermal growth factor receptor

avian erythroblastic leukemia viral (v-erb-b) oncogene homolog|cell growth inhibiting protein 40|cell proliferation-inducing protein 61|erb-b2 receptor tyrosine kinase 1|proto-oncogene c-ErbB-1|receptor tyrosine-protein kinase erbB-1

7p12

protein-coding

Hematopoietic cell kinase (Hck) is a member of the Src-family of protein tyrosine kinases. We have found that upon enzymatic activation of Hck by the heavy metal mercuric chloride, there was a rapid increase in the levels of tyrosine phosphorylation of several proteins including the proto-oncogene p120(Cbl). Fibroblasts that are transformed with an activated allele of Hck exhibit constitutive Cbl phosphorylation. Upon Fcgamma receptor activation, a more physiologically relevant extracellular signal, Cbl is tyrosine phosphorylated and the Src-family selective inhibitor, PP1, can prevent this phosphorylation on Cbl. Hck phosphorylates Cbl in vitro and the interaction between Cbl and Hck is direct, requiring Hck s unique, SH3 and SH2 domains for optimal binding. Using a novel estrogen-regulated chimera of Hck we have shown a hormone-dependent association between Hck and Cbl in murine fibroblasts. This work suggests that Cbl serves as a key mediator of Hck induced signalling in hematopoietic cells.

The involvement of human papillomavirus (HPV) in the development of carcinomas of the uterine cervix has been firmly established. However, other genetic alterations also play an important role in the pathogenesis of cervical cancer. Therefore, we have investigated the role of several (onco)genes in cervical carcinoma. In tumors from 136 patients with stage I and II cancer of the uterine cervix, the expression of epidermal growth factor receptor (EGFR), c-erbB-2/neu, p53, and murine double minute 2 (MDM-2) was studied using immunohistochemistry. In 32 cases, amplification of EGFR, c-erbB-2/neu, MDM-2, and c-myc was studied by Southern blot hybridization. The expression levels of these proteins were correlated with HPV positivity, International Federation of Gynecologists and Obstetricians stage, lymph node metastases, tumor diameter, vessel invasion, and disease-free and overall survival. Moderate/strong expression of EGFR was observed in 54% of tumors. c-erbB-2/neu was focally positive in 12 cases. p53 showed moderate/strong expression in 32% of the tumors. Thirteen % of tumors showed a moderate/strong expression of MDM-2, and this expression was correlated to p53 expression (P<0.001). Only moderate/strong expression of EGFR was associated with reduced disease-free (P = 0.002) and overall survival (P = 0.003). In multivariate analysis, the association of EGFR overexpression with poor prognosis was independent from lymph node status. Gene amplification was found for EGFR (four cases), c-erbB-2/ neu (two cases), and c-myc (six cases). In two tumors, rearrangement of c-myc was found, probably due to the integration of HPV. In conclusion, overexpression of the EGFR is an independent predictor for prognosis in earlier stages (stage I and II) of cervical cancer. p53 and MDM-2 expression are correlated to each other and may play a role in the interaction with HPV. The importance of c-erbB-2/neu and c-myc amplification is relatively small in stage I and II cervical cancer.

Cytotoxic strategies which are directed to tumor-associated antigens might be most beneficial for cancer patients with minimal tumor load such as in an adjuvant setting after initial therapy. We have recently described a highly potent single chain antibody-toxin, scFv(14E1)-ETA, which consists of the variable domains of the antibody 14E1 genetically fused to a truncated form of Pseudomonas exotoxin A. ScFv(14E1)-ETA specifically recognizes the human epidermal growth factor receptor (EGFR) and the oncogenically activated receptor variant EGFRvIII, which have been implicated in the development of various human malignancies. Here we have investigated the antimetastatic activity of bacterially expressed scFv(14E1)-ETA and its disulfide-stabilized derivative ds-scFv(14E1)-ETA in a novel model for disseminated disease which is based on murine renal carcinoma cells subsequently transfected with the E. coli beta-galactosidase gene, and human full-length or variant EGFR cDNAs. Intravenous injection of these Renca-lacZ/EGFR and Renca-lacZ/EGFRvIII cells in syngenic Balb/c mice led to the formation of pulmonary metastases which were readily detectable upon excision of the lungs and X-gal staining. Systemic treatment of mice with scFv(14E1)-ETA resulted in the complete suppression of Renca-lacZ/EGFRvIII metastasis formation and drastically reduced the number of pulmonary Renca-lacZ/EGFR tumor nodules. The ds-scFv(14E1)-ETA derivative where the antibody variable regions are connected by an artificial disulfide bond displayed improved thermal stability at physiological temperature but due to reduced cytotoxic activity was less potent than the original scFv(14E1)-ETA in metastasis suppression.

Neoadjuvant chemotherapy prior to definitive radical surgery or radiotherapy may be effective in reducing tumor volume or clinical stage and may even enhance pelvic control and survival. However, there are significant limitations to the use of neoadjuvant therapy in the non-responder group. They include delayed total treatment course, the presence of drug resistant clones which result in accelerated tumor growth, and limited bone marrow reserve for subsequent definitive therapy. Thus, there is a need to identify parameters providing a more precise indication of the response to neoadjuvant chemotherapy in patients with invasive cervical cancer. From Jan. 1995 to Jan. 1996, neoadjuvant chemotherapy with 3 courses of cisplatin and vincristine was used in 32 patients with invasive cervical cancer (FIGO stage Ib to IIIb; tumor size greater than 2 cm). Prior to chemotherapy, quantitative tissue levels of epidermal growth factor receptor (EGFR) and c-erbB-2 oncogene protein were measured by using an enzyme-linked immunosorbent assay (ELISA). tumor size was estimated before and after chemotherapy. Relations between oncoproteins and reductions of tumor size were evaluated. tumor size prior to neoadjuvant chemotherapy did not show any correlation with either the concentrations of EGFR or c-erbB-2 oncoprotein. As well, the tumor reduction index did not manifest any correlation with EGFR, it did had an inverse linear correlation with the c-erbB-2 oncoprotein levels (Rs = -0.71, P < 0.05). The results of this study suggest that c-erbB-2 oncoprotein is associated with a reduced response to neoadjuvant chemotherapy in primary treatment of invasive cervical cancer and may be useful in directing therapeutic approaches.

Human glioma cells frequently overexpress epidermal growth factor receptor (EGFR). We found that the CrkII proto-oncogene product was associated with the EGFR in human glioma cells in the absence of epidermal growth factor (EGF). EGF stimulation of glioma cells induced the phosphorylation of tyrosine 221 of the CrkII protein, which correlates with its dissociation from the EGFR. By contrast, Shc and Grb2 were inducibly associated with the EGFR in response to EGF stimulation of glioma cells. In A431 cells, epidermoid carcinoma cells which overexpress EGFR, CrkII was tyrosine-phosphorylated and associated with the EGFR in an EGF-dependent manner. Therefore, the dissociation of CrkII from the EGFR upon stimulation with EGF appears to be specific to glioma cells. The Cbl oncogene product was also tyrosine-phosphorylated in U87MG glioma cells upon EGF stimulation. However, unlike in other cell lines, CrkII was not inducibly bound to Cbl in U87MG glioma cells. Thus, EGF-dependent binding of CrkII to phosphotyrosine-containing proteins appears to be suppressed in glioma cells. To evaluate the physiological role of dissociation of CrkII from EGFR, we expressed the CrkII-23 mutant in glioma cells. CrkII-23 mutant, which was isolated as a suppressor gene of the EGF-dependent transformation of NRK cells, binds constitutively to EGFR. We found that expression of CrkII-23 inhibited the anchorage-independent growth of the glioma cells in the presence of EGF. Taken together, these data implicate EGF-dependent dissociation of CrkII from EGFR in the oncogenicity of human glioma cells.

OBJECTIVE: Gastric carcinoma can be divided into two main histological and clinical types: diffuse and intestinal. The aim of this study was to investigate the expression of neu/c-erbB-2 oncoprotein, epidermal growth factor receptor (EGFR), cathepsin D (catD), progesterone receptor (PR) and tumor-associated glycoprotein-72 (TAG-72) in gastric carcinoma of these histological types. METHOD: In this randomized, prospective study we analyzed 85 biopsy samples from patients with gastric adenocarcinoma. The control group consisted of 40 specimens from normal gastric mucosa. Neu oncoprotein and PR were determined by ELISA. CatD and TAG-72 were quantified with immunoradiometric (IRMA) methods, and EGFR were studied by radioimmunoassay (RIA). RESULTS: Neu, EGFR, catD and TAG-72 concentrations were higher in the tumoral group (p = 0.02, p = 0.00001, p = 0.002 and p = 0.007, respectively). In diffuse adenocarcinomas, catD and PR expression was increased (p = 0.01 and p = 0.04 respectively), whereas TAG-72 concentration, which correlated with neu (r = 0.57), was higher in the intestinal type (p = 0.04). No significant differences in EGFR and neu concentrations were seen between the two histological types. CONCLUSIONS: The higher PR and catD concentrations in diffuse adenocarcinomas, and the overexpression of TAG-72 in the intestinal type, support the existence of two modes of gastric carcinogenesis.

A number of epidermal growth factor receptor (EGFR) deletion mutants have been identified in gliomas, in which the EGFR gene is frequently amplified and rearranged. We have previously characterized the structure of a gene in A-172 human glioma cells that encodes a 190-kDa EGFR mutant with tandem duplication of the tyrosine kinase (TK) and calcium-mediated internalization (CAIN) domains. Here we describe a 185-kDa tandem duplication mutant (TDM) that is expressed in KE and A-1235 glioma cells, along with certain functional characteristics of the mutants. The corresponding transcripts in KE and A-1235 cells contain 1053 additional nucleotides representing an in-frame duplication of exons 18 through 25 which encode the entire TK region and a portion of the CAIN domain. As with duplication of the entire TK/CAIN region (exons 18-26) in A-172 cells, duplication of exons 18-25 is associated with a specific genomic rearrangement between flanking introns. Involved introns contain homology to recombination signal sequence (RSS) heptamers present in the V(D)J region of the T lymphocyte receptor gene. In defined medium, both oncogenic TDM are constitutively autophosphorylated and inefficiently downregulated. High-affinity binding is reduced in EGFR.TDM/18-26, although the t1/2 of receptor internalization is not prolonged.

Oncogenic transformation confers resistance to chemotherapy through a variety of mechanisms, including suppression of apoptosis, increased drug metabolism, and modification of target proteins. Oncogenic epidermal growth factor receptor family members, including EGFRvIII and HER2, are expressed in a broad spectrum of human malignancies. Cell lines transfected with EGFRvIII and HER2 are more resistant to paclitaxel-mediated cytotoxicity, and tubulin polymerization induced by paclitaxel is suppressed compared with cells expressing wild type epidermal growth factor receptor. Because differential expression of beta-tubulin isotypes has been proposed to modulate paclitaxel resistance, we analyzed beta-tubulin isotypes expressed in cell lines transfected with different oncogenes. EGFRvIII- and HER2-expressing cells demonstrated equivalent total beta-tubulin protein compared with cells transfected with wild type receptor or untransfected controls. EGFRvIII-expressing cells demonstrated increases in class IVa (2.5-fold) and IVb (3.1-fold) mRNA, and HER2-expressing cells showed increases in class IVa (2. 95-fold) mRNA. expression of oncogenic Ha-Ras did not change class IV RNA levels significantly. Inhibition of EGFRvIII kinase activity using a mutant allele with an inactivating mutation in the kinase domain decreased expression of class IVa by 50% and partially reversed resistance to paclitaxel. expression of oncogenic epidermal growth factor receptor family members is associated with modulation of both beta-tubulin isotype expression and paclitaxel resistance in cells transformed by expression of the receptor. This effect on tubulin expression may modulate drug resistance in human malignancies that express these oncogenes.

Lymphatic metastases are an important indicator of the malignancy of epithelial cancers. Empirical clinical observations associating specific genetic abnormalities with tumour progression, allied with basic laboratory investigations, are providing not only improved prognostic and diagnostic opportunities, but also a detailed understanding of the molecular machinery of metastasis. One such association--between the c-erbB oncogene family and metastasis--has proved particularly instructive. Functional links between over-expression (and occasionally mutational activation) of c-erbB-1 (EGFR) and c-erbB-2 and specific phenotypes of metastatic cells have been elucidated. Activated c-erbB oncogenes potentiate tumour cell adhesion to endothelial cells and upregulate VEGF, potentially facilitating angiogenesis and vascular invasion. In addition, cells over-expressing these oncogenes frequently show aberrant cell-cell and cell-matrix interactions, mediated by changes in integrin and cadherin function. Thirdly, both EGFR and c-erbB-2 signalling can significantly upregulate specific matrix metalloproteinases, key enzymes involved in angiogenesis and invasion. Finally, c-erbB receptors linked to the actin cytoskeleton and highly expressed on invadopodia, are thought to assist cell migration. Taken together, these observations suggest that such receptors can act as "master switches" in metastasis, whose activation co-ordinately controls events normally utilised in development, now subverted by the metastatic cell. As such, they represent ideal targets for therapeutic intervention.

Paraffin-embedded tissue slides from 88 infiltrating ductal breast carcinoma were examined by immunohistochemistry technique with the use of monoclonal antibody against human p65 antigen and polyclonal antibody against p65-like protein present in fetal bovine serum. Immunohistochemical analysis of expression of growth factor receptors (EGFR), protein product of oncogene c-erb B2 as well as protein product of mutated anti-oncogene p53 was also done. It was established that there is no correlation between p65 and c-erbB2, EGFR or p53 expression. In low differentiated tumors (grade III) high p53 index and high EGFR and c-erbB2 expression was connected with low p65 expression. The lack of c-erbB2 and EGFR and low p53 expression was combined usually with high p65 oncoprotein levels.

Many ligand-independent receptor tyrosine kinases are tumorigenic. The biochemical signals that mediate ligand-independent transformation of cells by these transmembrane receptors are poorly defined. In this report, we demonstrate that a constitutively activated mutant epidermal growth factor receptor (v-ErbB) induces the formation of a transformation-specific signaling module that complexes with myosin II. The components of this signaling complex include the signal adapter proteins Shc, Grb2, and Nck, and tyrosine-phosphorylated forms of p21-activated kinase (Pak), caldesmon, and myosin light chain kinase. Transformation-specific, tyrosine phosphorylation of Pak enhances the catalytic activity of this serine/threonine kinase. Furthermore, the tyrosine phosphorylation of Pak is Rho-, but not Ras-, Rac-, or Cdc42-dependent. These results demonstrate that a ligand-independent epidermal growth factor receptor mutant can transduce oncogenic signals that are distinct from ligand-dependent, mitogenic signals. In addition, these data provide evidence for the coupling of oncogenic receptor tyrosine kinases with the actomyosin molecular motor. This myosin-associated signaling module may mediate some of the biochemical changes of myosin found in v-ErbB- transformed fibroblasts, thereby contributing to the regulation of the mechanical forces governing cellular adhesion, cytoskeletal tension, and, hence, anchorage-independent cell growth.

mutations in the epidermal growth factor receptor have been identified in several human tumor types, including gliomas. These receptor mutants have deletions in their extracellular ligand-binding domains and are, therefore, no longer regulated by ligand, resulting in constitutive activation of the receptor kinase. These mutants have been proposed to transduce oncogenic signals via ligand-independent signaling pathways. Avian viral homologues of these oncogenic epidermal growth factor receptors exhibit structurally homologous deletions and form tumors in chickens. One such mutant, S3v-ErbB, transforms fibroblasts in vitro, and transformation has been correlated with the formation of a novel tyrosine phosphoprotein complex. V-ErbB-mediated complex formation and transformation have been shown to occur independently of Ras activation. The major aims of this study are to further characterize this ligand-independent v-ErbB oncogenic signaling pathway. Here we show that both v-ErbB-mediated phosphoprotein complex formation and transformation are inhibited by a dominant negative mutant of Rho. This inhibition is specific for dominant negative Rho; dominant negative mutants of Rac and Cdc42 have no effect on transformation or on tyrosine phosphorylation of the phosphoprotein complex. Based on these observations, we propose that S3v-ErbB stimulates a Rho-dependent tyrosine kinase, resulting in complex formation and ultimately oncogenic transformation.

We established a new cancer cell line, NOS-1, which was derived from a human oral primary squamous cell carcinoma. Geneticin treatment was adopted to eliminate contaminating fibroblasts and to enrich tumor cells in the early stage of the culture. The NOS-1 cells showed epithelial morphological features with light and electron microscopy, and immunohistochemical analysis confirmed their epithelial origin. Overexpression of mutant p53 protein, a p53 point mutation at codon 248 with transition from CGG to TGG, and amplification of the erbB-1 oncogene/epidermal growth factor receptor gene were also observed in NOS-1 cells. The NOS-1 cells formed tumors in nude mice when transplanted subcutaneously into their backs. Further, they were transplantable orthotopically in the tongues of nude mice, and the transplanted tumors in the tongue showed diffuse invasion without forming capsules. The NOS-1 cells were useful for elucidating the mechanism involving p53 inactivation and erbB-1 oncogene amplification, as well as treatment of oral cancer.

Ten human head and neck squamous carcinoma (HNSCC) cell lines were established in order to study the role of c-erbB signaling pathways in HNSCC progression. Five cell lines were derived from primary tumors at four different sites, and five were from lymph node metastases in the neck. Two pairs of lines were derived from the primary tumor and metastatic lymph node in the same patient. Basic characteristics including morphology, doubling time, phenotypes, cytogenetic profiles and tumorigenicity in nude mice were described. We examined the expression of c-erbB receptors and ligands in early passage new HNSCC lines and compared with five long-term established lines, normal keratinocytes and fibroblasts. Amplification of c-erbB-1 (EGFR) gene was observed in only one cell line whereas no amplification of other c-erbB genes was found. Overexpression of EGFR, c-erbB-2, c-erbB-3 and c-erbB-4 mRNAs was observed in 10, 14, 10 and 8 out of 15 head and neck cell lines respectively. Overexpression of c-erbB-3 and c-erbB-4 was more frequently observed in newly derived HNSCC lines than in long-established cell lines. The majority of tumor cells also expressed multiple c-erbB ligands. One selected cell line, SIHN-006, was shown to exhibit tyrosine phosphorylation via ALL four receptors. These new cell lines could provide a useful experimental model to study the co-operative signaling of type I tyrosine kinase receptors in HNSCC progression.

Approximately two decades ago, the epidermal growth factor receptor (EGFR) was discovered to be the proto-oncogene of the mutant, constitutively active oncogenic v-erbB tyrosine kinase, which induces avian erythroblastosis. Unlike the mutant oncogene, the EGFR requires activation by binding of ligand to its extracellular (EC) domain, whereas its cellular effects depend on activation of its cytoplasmic tyrosine kinase. The overexpression of EGFR and its ligands in several human carcinomas and their association with accelerated tumor progression provided a rationale for targeting this network with tumor-selective strategies. Two of those antireceptor approaches, both solidly based on the known structure and function of the EGFR, are discussed. The first strategy involves the development of humanized monoclonal antibodies against the nonconserved receptor s EC domain. These antibodies block ligand binding and can induce receptor endocytosis and downregulation. The second approach is the generation of ATP mimetics that compete with ATP for binding to the receptor s kinase pocket, thus disabling signal transduction. Preclinical and early clinical studies already suggest that both of these approaches, either alone or in combination with standard anticancer therapies, will be able to alter the natural history of EGFR-expressing cancers with little to no toxicity to the tumor-bearing host.

We developed a multiparameter flow cytometric technique for the simultaneous measurement of cellular DNA content and c-erbB-2 or epidermal growth factor receptor (EGFR) expression. The method provides a high resolution of DNA content and well preserved c-erbB-2 and EGFR immunostaining under saturated antibody conditions, allowing good control for background fluorescence and satisfactory cell morphology. Four different protocols for the short-term preservation of cells used for multiparameter flow cytometric assay of EGFR and c-erbB-2 were assessed in cell suspensions prepared by mechanical disaggregation in 10 gynecologic tumors. The protocols at 4 degrees C were: storage in 50% methanol, and storage in buffer after formalin fixation. Tissues were also cryopreserved as cell suspensions and tissue blocks. When the oncoprotein expression and DNA histograms were compared with those in fresh suspensions, cryopreservation was found to be the best method: oncoprotein expression was well preserved and there was a good correlation between oncoprotein expression and the quality of the DNA histograms. The currently developed methods for cell preservation make the technique generally available for clinical cancer studies.

OBJECTIVE: To explore the effect of expression of epidermal growth factor receptor (EGFR), transforming growth factor beta (TGF beta), c-erbB-2 oncogene in myoepithelioma and malignant myoepithelioma. METHODS: 20 cases of myoepithelioma and 20 cases of malignant myoepithelioma were examined by immunohistochemical methods. RESULTS: ALL cases showed overexpression of EGFR and TGF beta, the intensity of expression didn t associate with the benignity and malignity of tumors. The overexpression of c-erbB-2 oncoprotein was seen in 5 cases of myoepithelioma and 13 cases of malignant myoepithelioma, and the cases of malignant myoepithelioma were stained more intense than that of myoepithelioma. CONCLUSION: The synergy of EGFR and TGFR beta had significant relation to the proliferative and differentiation processes of myoepithelial tumor. The activated c-erbB-2 oncogene, that is similar with EGFR in structure, plays an important promoting role in unrestricted progression of malignant myoepithelioma cells.

Asbestos is a ubiquitous naturally occurring fiber causing multiple cancers and fibroproliferativedisease. The mechanisms of epithelial cell hyperplasia, a hallmark of the initiation of lung cancers by asbestos, have been unclear. We demonstrate here that mice expressing a dominant-negative mutant epidermal growth factor receptor (EGFR) under the control of the human lung surfactant protein-C promoter exhibit decreased pulmonary epithelial cell proliferation without alterations in asbestos-induced inflammation. In contrast to transgene-negative littermates, inhalation of asbestos by mice expressing the mutant EGFR does not result in early and elevated expression of early response proto-oncogenes (fos/jun or activator protein 1 family members). Additionally, quantitative reverse transcriptase-PCR analysis for levels of c-jun and c-fos in bronchiolar epithelium isolated by laser capture microdissection demonstrates increases in expression of these genes in asbestos-exposed epithelial cells. Results show that the EGFR mediates both asbestos-induced proto-oncogene expression and epithelial cell proliferation, providing a rationale for modification of its phosphorylation in preventive and therapeutic approaches to lung cancers and mesothelioma.

Beta-catenin functions as a cytoskeletal linker protein in cadherin-mediated adhesion and as a signal mediator in wnt-signal transduction pathways. We use a novel integrative approach, combining evolutionary, genomic, and three-dimensional structural data to analyze and trace the structural and functional evolution of beta-catenin genes. This approach also enabled us to examine the effects of gene duplication on the structure and function of beta-catenin genes in Drosophila, C. elegans, and vertebrates. By sampling a large number of different taxa, we identified both ancestral and derived motifs and residues within the different regions of the beta-catenin proteins. Projecting amino acid substitutions onto the three- dimensional structure established for mouse beta-catenin, we identified specific domains that exhibit loss and gain of selective constraints during beta catenin evolution. Structural changes, changes in the amino acid substitution rate, and the appearance of novel functional domains in beta-catenin can be mapped to specific branches on the metazoan tree. Together, our analyses suggest that a single, beta-catenin gene fulfilled both adhesion and signaling functions in the last common ancestor of metazoans some 700 million years ago. In addition, gene duplications facilitated the evolution of beta-catenins with novel functions and allowed the evolution of multiple, single-function proteins (cell adhesion or wnt-signaling) from the ancestral, dual-function protein. Integrative methods such as those we have applied here, utilizing the natural experiments present in animal diversity, can be employed to identify novel and shared functional motifs and residues in virtually any protein among the proteomes of model systems and humans.

Relay of information from the extracellular environment into the cell often results from a peptide growth factor binding to its cognate cell surface receptor; this event is an integral mechanism by which many cellular functions occur, including cell growth, motility, and survival. In recent years, however, this requirement for ligand binding has been shown to be surpassed by several distinct mechanisms, including cell surface receptor cross-talk (e.g., between epidermal growth factor receptor [EGFR] and G-coupled receptors), receptor-extracellular matrix interactions (e.g., EGFR: integrin complexes), and finally by structural mutations within the receptor itself. While ALL of these pathways result in so-called ligand-independent signaling by the EGF receptor, to date, only structural mutations in the receptor have been shown to result in qualitative changes in downstream targets of the receptor, which specifically result in oncogenic signaling, transformation, and tumorigenicity. In this review, we describe aspects of the known signaling properties of the retroviral oncogene v-ErbB as a model of ligand-independent oncogenic signaling, and compare these properties to results emerging from ongoing studies on structurally related EGF receptor mutants originally identified in human tumors. A better understanding of the signaling pathways used by these uniquely oncogenic receptor tyrosine kinase mutants may ultimately reveal new targets for the development of novel therapeutics selective for the inhibition of tumor cell growth.

A comparative study was undertaken between cancer of the uterine cervix (n = 50) and female breast cancer (n = 50) with reference to the expression of c-erbB-2 oncoprotein (HER-2/neu) and that of epidermal growth factor receptor (EGF-R), both being highly homologous structurally. expressions of EGF-R and c-erbB-2 oncoprotein were viewed in breast and cervical cancer tissues by immunochemical staining. Cervical cancer cases showed much higher expression of EGF-R which also revealed significant association with the expression of c-erbB-2 oncoprotein and tumour grading. Among breast cancer cases, over-expression of EGF-R correlated significantly with metastasis of lymph node; and expression of c-erbB-2 oncoprotein showed a significant relationship with histological grading of the tumour. Moreover, an association was noticed between the tumour grade and the concomitant immuno positive expression of EGF-R and c-erbB-2. Our study revealed an existence of a conflicting pattern in the expression of EGF-R and c-erbB-2 oncoprotein between carcinomas of the breast and uterine cervix.

Both epidermal growth factor receptor (EGFr) and the oncogene product of c-erbB2 have been shown to be expressed by human malignancies, and in some cases to relate to clinical outcomes. This study investigates the correlation between the presence of these receptor proteins and known prognostic indicators of colorectal cancer. Indirect immunoperoxidase staining of 32 freshly frozen surgical specimens revealed an overall expression of EGFr and c-erbB2 of 43% and 38%, respectively. A significantly higher rate of EGFr expression was found in tumours of more advanced stage (Dukes C and D), poor differentiation and those exhibiting vascular and lymphatic invasion. The presence of the c-erbB2 protein did not correlate with any of these variables. expression of these molecules appeared to be independent and positive staining for both receptors occurred in only 19% of cases. EGFr may play a future role as a prognostic tool in colorectal cancer.

BACKGROUND: This study prospectively examines the prognostic role of p53 oncoprotein (p53), Ki67-antigen (Ki67), tumor angiogenesis (MVD), epidermal growth factor receptor (EGFR), and HER2 receptor protein (HER2) expression in Chinese with undifferentiated nasopharyngeal carcinoma (NPC). METHODS: Seventy-eight Chinese were recruited from October 1995 to July 1997 at the Prince of Wales Hospital, Hong Kong. Pretreatment immunohistochemical preparations of the primary tumor were made, and clinical data were collected prospectively until October 30, 2000. The markers were correlated with overall survival (OS), disease-free survival (DFS), time to progression (TTP), and UICC stage. RESULTS: On univariate analysis, EGFR expression correlated with poorer OS (p =.0001), DFS (p =.01), shorter TTP (p =.0001), and advanced T stage (p =.036). Strong EGFR expression, when compared with weak or moderate, was associated with poorer OS (p =.04) and shorter TTP in a subgroup of patients with UICC stage III-IV disease. HER2 expression was associated with advanced UICC stage (p =.006). The presence of p53 expression correlated with poorer DFS (p =.01) and a trend toward shorter TTP (p =.06). No correlation was found with Ki67-antigen or MVD. On multivariate analysis, only EGFR expression was significantly linked to shorter OS and TTP. CONCLUSIONS: EGFR expression in undifferentiated NPC is associated with a poor clinical outcome. A prognostic role of p53 and HER2 expression is suggestive but not consistently defined in this study. The relatively high prevalence of positive staining for EGFR supports the use of molecular targeted therapy in this disease.

We have compared the protein tyrosine kinase activities of the chicken epidermal growth factor receptor (chEGFR) and three ErbB proteins to learn whether cancer-activating mutations affect the kinetics of kinase activity. In immune complex assays performed in the presence of 15 mM Mn2+, ErbB proteins and the chEGFR exhibited highly reproducible tyrosine kinase activity. Under these conditions, the ErbB and chEGFR proteins had similar apparent Km [Km(app)] values for ATP. The ErbB proteins appeared to be activated, as they had at least 3-fold-higher relative Vmax(app) for autophosphorylation and approximately 2-fold higher relative Vmax(app) for the phosphorylation of the exogenous substrate TK6 (a bacterially expressed fusion protein containing the C-terminal domain of the human EGFR). The ErbB kinases had both higher Km(app) and higher Vmax(app) for the phosphorylation of the exogenous substrate TK6 than did the chEGFR. The ratios of the Vmax(app) to the Km(app) for TK6 phosphorylation suggested that the ErbB proteins had lower catalytic efficiencies for the exogenous substrate than did the chEGFR. The three tested ErbB proteins had cytoplasmic domain mutations that conferred distinctive disease potentials. These mutations did not affect the kinetics for the phosphorylation of the exogenous substrate TK6. Two of the ErbB proteins contained ALL of the sites used for autophosphorylation. In these, a mutation that broadened oncogenic potential to endothelial cells caused an additional increase in Vmax(app) for autophosphorylation. Thus, mutations that change the EGFR into an ErbB oncogene cause multiple changes in the kinetics of protein tyrosine kinase activity.

erb-B gene is an oncogene from avian leukosis virus. The carcinogenic capability of erb-B is due to the close similarity of expressional proteins to the epidermal growth factor receptor (EGFr). In this study the amplification and the over-expression of erb-B were found in human oral squamous (cell carcinomas by DNA and RNA dot blot hybridization). Results showed that erb-B was correlated with the oral epidermoid tumors but not with group of non-epidermoid tumors in the head and neck region. Southern blotting analysis showed that there were rearrangement of erb-B gene in the tumor genosomes, which is perhaps one of the ways to activate the proto-oncogene of erb-B.

Overexpression and autocrine activation of the epidermal growth factor receptor (EGF-R) cause transformation of cultured cells and correlate with tumor progression in cancer patients. Dimerization and transphosphorylation are crucial events in the process by which receptors with tyrosine kinase activity generate normal and transforming cellular signals. Interruption of this process by inactive receptor mutants offers the potential to inhibit ligand-induced cellular responses. Using recombinant retroviruses, we have examined the effects of signalling-incompetent EGF-R mutants on the growth-promoting and transforming potential of ligand-activated, overexpressed wild-type EGF-R and the v-erbB oncogene product. expression of a soluble extracellular EGF-R domain had little if any effect on the growth and transformation of NIH 3T3 cells by either tyrosine kinase. However, both a kinase-negative EGF-R point mutant (HERK721A) and an EGF-R lacking 533 C-terminal amino acids efficiently inhibited wild-type EGF-R-mediated, de novo DNA synthesis and cell transformation in a dose-dependent manner. Furthermore, coexpression with the v-erbBES4 oncogene product in NIH 3T3 cells resulted in transphosphorylation of the HERK721A mutant receptor and reduced soft-agar colony growth but had no effect in a focus formation assay. These results demonstrate that signalling-defective receptor tyrosine kinase mutants differentially interfere with oncogenic signals generated by either overexpressed EGF-R or the retroviral v-erbBES4 oncogene product.

The expressions of epidermal growth factors (EGF), epidermal growth factor receptors (EGFR), and the c-erbB-2 oncoprotein were immunohistochemically examined in 25 cases of human pancreatic carcinoma and epineoplastic pancreatitis and in 10 non-cancerous/non-inflammatory pancreatic tissues. The positive rates of EGF, EGFR, and the c-erbB-2 oncoprotein in cancer tissues were 72%, 36%, and 28%, respectively. EGF was stained mainly in the cytoplasm and partly on the surfaces of the cancer cells. EGFR and the c-erbB-2 oncoprotein were stained mainly on the surfaces of the cancer cells and partly in the cytoplasm. The expressions of these 3 products correlated significantly with tumor invasion into the anterior and posterior areas surrounding the pancreas. In the EGF, EGFR, and c-erbB-2 positive cancer tissues, some stromal cells, that is fibroblasts and endothelial cells, were also positive. In the adjacent pancreatic tissues with inflammation, these products were noted in some ductal cells, acinar cells, fibroblasts and endothelial cells. No distinct staining was detected in non-cancerous/non-inflammatory tissues. The survival period for patients who tested positive for these three proteins was statistically shorter than for those who tested negative. These results suggest that the coexpression of EGF and EGFR and the expression of the c-erbB-2 oncoprotein are related to the existence of the invasion of human pancreatic cancer. Furthermore, an immunohistochemical examination of these three products is useful in forming a prognosis for pancreatic cancer patients.

PURPOSE: A study was undertaken to define the prognostic value of the expression of the c-erbB-2 oncoprotein in a series of breast cancer patients when compared by multivariate analysis with expression of the epidermal growth factor receptor (EGFR), DNA ploidy, and conventional clinicopathologic features. PATIENTS AND METHODS: Prognostic indicators were analyzed in 165 primary breast cancers. The c-erbB-2 oncoprotein was recognized by the polyclonal antibody 21N using an immunocytochemical method. expression of the EGFR was stated immunocytochemically using the monoclonal antibody EGFR1. DNA ploidy was assessed in paraffin-embedded sections using a standard flow-cytometric method. RESULTS: Overall, 27% of carcinomas had membrane 21N-staining and were classified as c-erbB-2-positive. Overexpression of the c-erbB-2 oncoprotein was poorly associated with EGFR expression and the conventional pathologic features, and it was weakly associated with DNA ploidy and nodal status. Univariate analysis showed that c-erbB-2 expression, nodal status, DNA ploidy, and EGFR provided significant prognostic information concerning 4-year relapse-free survival (RFS) with the odds ratios (ORs) of not relapsing of 2.94, 2.83, 2.34, and 2.20, respectively. Regarding overall survival (OS) at 4 years, only nodal status and DNA ploidy had prognostic significance, with the ORs of not dying of 2.68 and 2.80, respectively. Applying multivariate analysis to RFS, 21N when adjusted for nodal status, EGFR, and DNA ploidy (full model) failed to retain prognostic value (P = .202), whereas nodal status was the most significant indicator of relapse (P = .027) followed by DNA ploidy (P = .056) and EGFR (P = .093). CONCLUSIONS: This study suggests that overexpression of the c-erbB-2 oncoprotein appears to be an important indicator of relapse in stage I-II breast cancer when singly evaluated. Multivariate analysis shows that the determination both of nodal status and DNA ploidy improves our ability to identify subsets of patients with different prognoses, and allows for a better selection of patients for systemic adjuvant treatments.

The expression of oncogene products related to cell growth (c-erbB-2, c-myc, ras p21, EGFR) was investigated in benign (15 cases) and malignant breast lesions (20 cases) by means of immunohistochemistry using the avidin-biotin-peroxidase technique with polyclonal and monoclonal antibodies. The aim of this study was to evaluate the relationship between the staining positivity and various morphological and biological features, such as tumour type, grading, hormone receptor status and cell kinetic parameters. In benign breast lesions, as expected, the kinetic parameters were low, both for Ki-67 and LI. ALL the specimens showed a diploid condition (the DI being equal to 1) and we found a limited degree of immunoreactivity for ALL the growth factors and oncogene products. In breast cancer we studied the distribution of immunohistochemical positivity for EGFR, c-erbB-2, c-myc, ras p21 and Ki-67, which was related to age, nodal status, ER and PgR receptor status, LI, DI and histopathological grading. A significant positive correlation was found both between ras p21 expression and nodal status and ER-ICA positivity. We observed a strong correlation between LI and Ki-67 and an inverse relation between Ki-67 and ER expression. These findings suggest the importance of studying the relationship between prognostic factors which may provide preoperative prediction in the biological behaviour of breast cancer, not only on biopsy specimens, but also on fine needle aspirates.

The HER-2/neu oncogene (a member of the Erb-like oncogene family) is distinct from but closely related to the c-erb B gene which encodes the epidermal growth factor receptor (EGFr). HER-2/neu gene amplification was found in a large number of mammary carcinomas and there was a strong correlation between this phenomenon and poor prognosis. In our study HER-2/neu oncogene expression was determined in 16 malignant ovarian tumors, 2 ovarian lymphomas and 5 normal ovaries. The HER-2/neu gene was found both in normal ovaries and malignant tumors, without any apparent difference among the various histological types. In ALL the specimens examined, HER-2/neu expression did not seem to be related to EGF binding capacity.

The amplification grade of oncogene c-erbB-2 was examined by the polymerase-chain-reaction-method in DNA s of 56 primary mammary carcinomas. 26 (46.4%) of these showed the amplified oncogene c-erbB-2. In the strongly amplified cases, the expression of the c-erbB-2 oncoprotein was verifiable immunohistochemically. Between the progesterone receptor status (PR) and the amplified c-erbB-2 oncogene there was a statistically proven dependency. No correlation was observed between the amplified c-erbB-2 oncogene and the epidermal growth-factor receptor (EGFR).

An animal model for malignant mastocytosis is described in mice reconstituted with bone marrow cells expressing the v-erbB oncogene. The lethal mast cell disease is characterized by massive infiltration of bone marrow, spleen, and several other visceral organs by connective tissue mast cells, which normally reside in the skin and the peritoneal cavity. As is frequently found in malignant mastocytosis, the v-erbB-induced mast cell disease was accompanied in some primary recipients by an acute myelogenous leukemia (AML) that killed ALL secondary recipients regardless of whether the AML was already evident in the primary host. The infiltrating mast cells stained strongly positive with berberine sulfate, suggesting that they were terminally differentiated and in vitro they showed only a weak proliferative capacity. The leukemias were clonal but apparently of different origin than the malignant mast cells, implying the transformation of two independent cell populations. Leukemic cells expressed various myeloid-specific markers as well as the B220 antigen, normally associated with the B-cell lineage. However, the Ig heavy chain genes were still in germ line configuration. In culture, these cells proliferated in the absence of exogenous growth factors and had the capacity to differentiate into mature myeloid cells. Preliminary experiments suggest that v-erbB may use parts of a signal transduction pathway normally coupled to the c-kit receptor. The v-erbB-induced malignant mast cell disease should provide a useful animal model for elucidating the cause for malignant mastocytosis in humans and to explore possible therapeutic strategies.

The effect of the v-erbA and/or v-erbB oncogenes on cellular gene expression was investigated after separation by two-dimensional polyacrylamide gel electrophoresis of [35S]methionine-labelled proteins from chicken embryo fibroblasts (CEF), infected by either the avian erythroblastosis virus (AEV) carrying both oncogenes, or by viruses carrying only one of them. We observed significant changes in the synthesis of 34 proteins in AEV-transformed CEF as compared with control cells. The synthesis of 24 of them was increased while the synthesis of the other 10 proteins was decreased. The expression of v-erbB alone is necessary and sufficient to induce changes in the synthesis of 27 proteins while the 7 remaining modifications are observed only in cells expressing v-erbB together with v-erbA. Moreover, the deregulation of protein synthesis by v-erbB-expressing viruses was correlated with the morphological transformation state of cells.

Abnormalities of epidermal growth factor receptor (EGFR) and c-erbB-2 have been demonstrated to be correlated with aggressive biologic behavior in a variety of human cancers. To analyze the possible roles of these oncogenes in ovarian neoplasms, immunolocalization of EGFR and c-erbB-2 oncogene product was performed in 45 cases of human ovarian mucinous and serous cystadenomas, carcinomas of low malignant potential (LMP), and invasive carcinomas by employing antibodies against these oncogene products. EGFR immunoreactivity was present in 15 of 35 LMP and invasive carcinomas and 1 of 10 cystadenomas. On the contrary, immunoreactivity of p185, which is an oncogene product of c-erbB-2, was detected only in five cases of carcinoma and in no benign cystadenoma. These results indicate that EGFR may be involved in the neoplastic process in epithelial ovarian adenocarcinoma, especially mucinous carcinoma, but involvement of c-erbB-2 is probably not as prevalent as considered previously. Four of the five cases immunohistochemically positive for p185 were also positive for EGFR, which suggests that expression of EGFR and p185 is to some extent correlated in human ovarian carcinoma.

The expression of c-erbB-2 oncoprotein and epidermal growth factor receptor (EGFR) was examined by immunocytochemical and radioreceptor assays in 115 patients with primary breast cancer. In 48 of 115 patients (42%), the assays were found to be positive for the expression of c-erbB-2 oncoprotein, and, in 44 of 115 (35%) patients, the assays were positive for the expression of EGFR. There was no correlation between the expression of c-erbB-2 oncoprotein and EGFR. Clinical survey demonstrated that both c-erbB-2 oncoprotein expression and EGFR expression have independent prognostic values. Furthermore, when patients were divided into three groups on the basis of the expression of both c-erbB-2 oncoprotein and EGFR, those who were found to be positive for the expression of both c-erbB-2 oncoprotein and EGFR showed a worse prognosis than other groups. These results suggest that the combination of the expression of both c-erbB-2 oncoprotein and EGFR may be important in selecting patients who have a poor prognosis.

expression of epidermal growth factor receptor (EGFR) and ERBB2 oncoprotein were studied in paraffin-embedded normal (n = 2), hyperplastic (n = 17), and malignant (n = 147) prostatic tissues by immunohistochemistry. Strong immunoreactivity was detected in the epithelial cells of ALL normal and hyperplastic prostates with a new EGFR antibody (Mab31G7). In prostatic carcinomas, the EGFR immunoreactivity was variable with 47% showing uniform, 39% partial, and 14% no staining. tumors with partial or uniform EGFR immunoreactivity were locally more advanced and of higher histological grade than the EGFR-negative tumors. EGFR-positive tumors also had two to three times higher S-phase fraction, suggesting that EGFR expression conferred proliferative advantage. Patients who had either partially or uniformly EGFR-positive carcinomas had a worse 10-yr progression-free (p = 0.05), overall (p = 0.03), and prostatic carcinoma-specific (p = 0.007) survival than those with EGFR-negative carcinomas. However, according to a multivariate analysis, EGFR did not have independent prognostic value. None of the normal, hyperplastic, or malignant prostate tissues showed clearly positive ERBB2 immunoreactivity with MAb1 antibody.

The erbB oncogenes of two transducing viruses that arose in chicken 5005 have been molecularly characterized. One of these viruses, AEV-5005, caused erythroblastosis. The other, AAV-5005, caused angiosarcoma. Both viruses had identical 5 junctions of viral and host sequences, indicating that both arose from the same proviral insertion. The erbB oncogenes of both viruses encoded transmembrane, kinase and C-terminal domains of the chicken epidermal growth factor receptor. The C-terminal domain of the AEV-5005 erbB was complete, whereas that of AAV-5005 contained a 59 amino acid internal deletion (amino acids 993-1051 of the chicken epidermal growth factor receptor). Oncogenicity tests using retroviral constructs containing the erbB sequences from AEV-5005 and AAV-5005 demonstrated that both erbB genes caused erythroblastosis and that the 59 amino acid deletion conferred the ability to induce angiosarcoma. The 59 amino acid deletion also caused increased levels of erbB autophosphorylation in cells grown in the presence of sodium orthovanadate.

OBJECTIVE: To detect the relations of c-erbB-2 oncogene protein, epidermal growth factor receptor (EGFR) and transforming growth factor-beta1 (TGF-beta1) to the progression or metastasis of pancreatic carcinoma. METHODS: Using streptavidinbiotin complex (SABC) method, c-erbB-2 oncongene protein, we examined immunohistochemically EGFR and TGF-beta1 expressions in wax-tissue sections from 10 individuals with normal pancreas (NP), 13 patients with chronic pancreatitis (CP) and 36 patients with pancreatic ductal adenocarcinoma (PC). RESULTS: The positive expression rates of c-cerbB-2 oncogene protein, EGFR and TGF-beta1 in the NP, CP and PC groups were 0, 0, 10%; 7.7%, 7.7%, 7.7%; and 41.7%, 50.0%, 44.4%, respectively. The positive expression rates of the three specific proteins increased more significantly in the PC group than in the NP and CP groups (P<0.05). The individual expression of c-erbB-2, EGFR and TGF-beta1 was not related to the age and sex of the patients as well as the site, size and histopathological grade of tumors (P>0.05), but to the clinical stage of tumors (P<0.01). The coexpression rate of the three proteins was 27.8% (10/36). This coexpression in the PC group was correlated with the histopathological grades and clinical stages of tumors (P<0.01). CONCLUSION: Detection of c-erbB-2 oncogene protein, EGFR, and TGF-beta1 expressions in pancreatic tissue is helpful to judge the malignancy, progression, and metastasis of PC.

Oncogenic forms of the small GTPase Ras increase the resistance of cells to killing by ionizing radiation (IR). Although not ALL of the signaling pathways for radioresistance are well defined, it is now clear that Ras-dependent signaling pathways involved in radioresistance include those mediated by phosphatidylinositol 3 -kinase (PI3-K) and Raf. Nevertheless, PI3-K and Raf together are not sufficient to reconstitute ALL of the resistance conferred by Ras, indicating that other effectors must also contribute. We show here that Ras-driven autocrine signaling through the epidermal growth factor receptor (EGFR) also contributes to radioresistance in Ras-transformed cells. Conditioned media (CM) collected from RIE-1 rat intestinal epithelial cells expressing oncogenic Ras increased the survival of irradiated cells. Ras-CM contains elevated levels of the EGFR ligand transforming growth factor alpha (TGF-alpha). Both Ras-CM and TGF-alpha stimulated EGFR phosphorylation, and exogenous TGF-alpha mimicked the effects of Ras-CM to increase radioresistance. Blocking EGFR signaling with the EGFR/HER-2 kinase inhibitor (KI) GW572016 decreased the postradiation survival of irradiated Ras-transformed cells and normal cells but had no effect on the survival of unirradiated cells. Ras-CM and TGF-alpha also increase PI3-K activity downstream of the EGFR and increase postradiation survival, both of which are abrogated by GW572016. Thus, Ras utilizes autocrine signaling through EGFR to increase radioresistance, and the EGFR KI GW572016 acts as a radiosensitizer. The observation that Ras-transformed cells can be sensitized to killing by ionizing radiation with GW572016 demonstrates that EGFR KIs could potentially be used to radiosensitize tumors in which radioresistance is dependent on Ras-driven autocrine signaling through EGFR.

While some low molecular weight GTPases such as Ras and RhoA contribute to malignant transformation, a closely related family member, RhoB, has tumor-suppressive activity, but little is known about its regulation by oncogenes. In this study, we show that H-Ras, N-Ras, K-Ras, EGFR and ErbB2 but not v-Src suppress RhoB promoter transcriptional activity in NIH3T3 cells and human cancer cell lines derived from lung (A-549), pancreatic (Panc-1) and cervical (C33A) tumors. The EGFR and ErbB2 suppression of RhoB promoter activity is mediated by Ras. Furthermore, Ras suppresses basal as well as 5-fluorouracil (5-FU)-induced RhoB promoter activity and RhoB protein levels. Ectopic expression of RhoB, but not the closely related family member RhoA, antagonizes the ability of EGFR, ErbB2, H-Ras, N-Ras and K-Ras but not v-Src to transform NIH3T3 cells. Furthermore, RhoB, but not RhoA, inhibits colony formation and proliferation and induces anoikis in A-549 cells and Ras-transformed NIH3T3 cells. Finally, Ras-mediated resistance to 5-FU-induced apoptosis is reversed by RhoB. These results demonstrate that RhoB expression is negatively regulated by oncogenes that are prevalent in human cancers, and that ectopic expression of RhoB antagonizes the ability of these oncogenes to induce transformation. Taken together the data suggest that certain oncogenes suppress RhoB as one of the critical steps leading to malignant transformation.

OBJECTIVE: The purpose of this study was to investigate the expression of epidermal growth factor receptor and c-erbB-2 oncoprotein in trophoblast populations of placenta accreta. STUDY DESIGN: Paraffin sections of 43 placental specimens with (cases) and 43 without (controls) placenta accreta were studied immunohistochemically for epidermal growth factor receptor and c-erbB-2 expression in the syncytiotrophoblast, villous cytotrophoblast, and extravillous cytotrophoblast. Epidermal growth factor receptor and c-erbB-2 protein levels were also performed by Western blot analysis. Controls were matched for gestational age in this case-control study. RESULTS: The percentage of strong/intermediate immunoreactivity of epidermal growth factor receptor in the syncytiotrophoblast was significantly higher in cases (98%) than controls (79%; odds ratio,11.1; 95% CI, 1.3-92.0; P = .03), although strong/intermediate immunoreactivity of c-erbB-2 in the syncytiotrophoblast was significantly lower in cases (35%) than controls (81%; odds ratio, 0.1; 95% CI, 0.1-0.3; P < .001). Epidermal growth factor receptor and c-erbB-2 expression in the villous and extravillous cytotrophoblastic cells were not significantly different between controls and cases ( P > .05). Most immunoreactive bands on Western blots were consistent with the trends of immunohistochemical findings. CONCLUSION: The development of placenta accreta is related closely to the differential expression of epidermal growth factor receptor and c-erbB-2 in the syncytiotrophoblast.

PURPOSE: To evaluate EGFR and HER2 copy number changes and to assess their significance to tumor progression in a large group of patients with larynx cancer through the construction of a tissue microarray (TMA) consisting of 1,385 biopsies. METHODS: Fluorescent in situ hybridization (FISH) was applied to analyze the tumors. FISH was successful for EGFR in 1,080 (77.98%) and for HER2 in 683 (49.31%). RESULTS: HER2 was amplified in 1.02% of cases. Amplification did not correlate with the tumor phenotype-clinical stage, and grade. The low frequency of amplification of HER2 oncogene in larynx tumors showed that the mechanism responsible for the high level of receptor overexpression still remains unclear in the majority of cases. Amplification of EGFR was found in 10.37% of cases. The analysis revealed a lack of correlation between amplification of the oncogenes and the tumor phenotype. We observed a lack of difference between the samples of primary tumors and advanced disease carcinomas--tumors with regional/distant metastases and recurrent tumors regarding oncogene amplification. CONCLUSION: These results suggest that EGFR amplification is a relatively rare event in larynx carcinogenesis that obviously does not predispose to tumor progression.

Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) is considered to be the major oncogenic protein of EBV-encoded proteins and has always been the core of the oncogenic mechanism of EBV. Advanced studies on nuclear translocation of the epidermal growth factor receptor (EGFR) family have greatly improved our knowledge of the biological function of cell surface receptors. In this study, we used the Tet-on LMP1 HNE2 cell line as a cell model, which is a dual-stable LMP1-integrated nasopharyngeal carcinoma (NPC) cell line and the expression of LMP1 which could be regulated by the Tet system. We found that LMP1 could regulate the nuclear accumulation of EGFR in a dose-dependent manner quantitatively and qualitatively. We also demonstrated that the nuclear localization sequence of EGFR played some roles in the location of the protein within the nucleus under LMP1 regulation and EGFR in the nucleus could bind to the promoters of cyclinD1 and cyclinE, respectively. We further demonstrated that EGFR is involved in the acceleration of the G1/S phase transition by LMP1 through binding to cyclinD1 and cyclinE directly. These findings provided a novel view that the acceleration of LMP1 on the G1/S transition via the nuclear accumulation of EGFR was critical in the process of nasopharyngeal carcinoma.

Multicellular organisms rely on complex networks of signaling cascades for development, homeostasis, and responses to the environment. These networks involve diffusible signaling molecules, their receptors, and a variety of downstream effectors. Alterations in the expression or function of any one of these factors can contribute to disease, including cancer. Many viruses have been implicated in cancer, and some of these modulate cellular signal transduction cascades to carry out their life cycles. High-risk human papillomaviruses (HPVs), the causative agents of most cervical and anogenital cancers, encode three oncogenes. One of these, E5, has been postulated to transform cells in tissue culture by modulating growth factor receptors. In this study, we generate and characterize transgenic mice in which the E5 gene of the most common high-risk HPV, HPV16, is targeted to the basal layer of the stratified squamous epithelium. In these mice, E5 alters the growth and differentiation of stratified epithelia and induces epithelial tumors at a high frequency. Through the analysis of these mice, we show a requirement of the epidermal growth factor receptor for the hyperplastic properties of E5.

We have investigated the expression of the products of two well-characterized oncogenes, erbB and Ha-ras, in the primary cultures of human gynecologic tumor cells by indirect immunofluorescent analysis. Epidermal growth factor receptor (c-erbB gene product) and p21 (ras gene product) were highly expressed in 21 out of 26 cases (81%) and 14 out of 18 cases (78%), respectively. Furthermore, they were frequently co-localized in the same region of the cells. These results suggest that these two oncogene products may play a part in gynecologic tumorigenesis and that they can interact with each other.

The HER-2/neu oncogene (a member of the Erb-like oncogene family) is distinct from but closely related to the c-erb B gene which encodes the epidermal growth factor receptor (EGFr). HER-2/neu gene amplification was found in a large number of mammary carcinomas and there was a strong correlation between this phenomenon and poor prognosis. In our study HER-2/neu oncogene expression was determined in 16 malignant ovarian tumors, 2 ovarian lymphomas and 5 normal ovaries. The HER-2/neu gene was found both in normal ovaries and malignant tumors, without any apparent difference among the various histological types. In ALL the specimens examined, HER-2/neu expression did not seem to be related to EGF binding capacity.

One of the most prominent features of malignant melanoma is the fast generation of metastasizing cells, resulting in the poor prognosis of patients with this tumor type. For this process, cells must gain the ability to migrate. The oncogenic receptor Xmrk (Xiphophorus melanoma receptor kinase) from the Xiphophorus melanoma system is a mutationally activated version of the epidermal growth factor receptor that induces the malignant transformation of pigment cells. Here, we show that the activation of Xmrk leads to a clear increase of pigment cell motility in a fyn-dependent manner. Stimulation of Xmrk induces its interaction with the focal adhesion kinase (FAK) and the interaction of active, receptor-bound fyn with FAK. This results in changes in FAK activity and induces the modulation of stress fibers and focal adhesions. Overexpression of dominant-negative FAK shows that the activity of innate FAK and a receptor-induced focal adhesion turnover are a prerequisite for pigment cell migration. Our findings show that in our system, Xmrk is sufficient for the induction of pigment cell motility and underlines a role of the src family protein tyrosine kinase fyn in melanoma development and progression.

Somatic mutations in the epidermal growth factor receptor (EGFR) occur frequently in lung cancer and confer sensitivity to EGFR kinase inhibitors gefitinib and erlotinib. These mutations, which occur in the kinase domain of the protein, also render EGFR constitutively active and transforming. Signal transducers and activators of transcription 3 (STAT3) transduces signals from a number of oncogenic tyrosine kinases and contributes to a wide spectrum of human malignancies. Here, we show that STAT3 is activated by mutant EGFRs and is necessary for its downstream phenotypic effects. Inhibiting STAT3 function in fibroblasts abrogates transformation by mutant EGFR. In non-small-cell lung cancer cells, STAT3 activity is regulated by EGFR through modulation of STAT3 serine phosphorylation. Inhibiting STAT3 function increases apoptosis of these cells, suggesting that STAT3 is necessary for their survival. Finally, a group of genes constituting a STAT3 signature is enriched in lung tumors with EGFR mutations. Thus, STAT3 is a critical mediator of the oncogenic effects of somatic EGFR mutations and targeting STAT3 may be an effective strategy for treating tumors characterized by these mutations.

Her-2/neu protein product was immunocytochemically analyzed in 139 breast cancers. Epidermal growth factor receptors were similarly analyzed in 74 breast cancers from the same patient pool. These results were also separated on the basis of estrogen receptor proteins and of combined aneuploidy with elevated S-phase from flow cytometry. Invasive breast cancer yielded a positive label for Her-2/neu protein (26%) and for epidermal growth factor receptor (25%), with no significant difference. Correlations with estrogen receptor labeling yielded differences significant inversely for both Her-2/neu protein (p less than 0.02) and epidermal growth factor receptor (p less than 0.01). Positive Her-2/neu protein labels correlated with a positive combination of aneuploidy and elevated S-phase (37%) and a negative combination of aneuploidy and elevated S-phase (21%), with a statistically nonsignificant difference. Positive epidermal growth factor receptor cases with aneuploidy and an elevated S-phase (75%) and without aneuploidy and elevated S-phase (42%) did differ with significance at p less than 0.05. There were eight cases positive for both Her-2/neu protein and epidermal growth factor receptor, four of six cases with negative estrogen receptor, four of six cases with negative estrogen receptor, six of six cases aneuploid, and five of six cases with an elevated S-phase. ALL eight cases had threatening disease--either stage III or stage IV, with one case of extensive ductal carcinoma in situ (comedo). Correlation of negative Her-2/neu protein with negative epidermal growth factor receptor was significant (p less than 0.05) in 74 cases. However, positive Her-2/neu protein did not correlate with positive epidermal growth factor receptor; there was a trend toward inverse correlation. We conclude that epidermal growth factor receptor labeling results show similarities to Her-2/neu protein results, but epidermal growth factor receptor tended to correlate with unfavorable ploidy and S-phase. Epidermal growth factor receptor labeling might be useful in breast cancers with macrocysts reported to show high epidermal growth factor activity.

A novel multiparameter flow-cytometric method was used to quantify the expression of epidermal growth factor receptor (EGFR) and c-erbB-2 oncoprotein on 85 cryopreserved normal tissues (30 ovary, 29 endometrium, 16 cervix) and 67 carcinomas (31 ovarian, 18 cervical, 15 endometrial, 3 vulvar). Overexpression of the EGFR and c-erbB-2 oncoproteins was found in respectively 3/31 (9%) and 10/31 (32%) ovarian carcinomas, 13/18 (72%) and 7/18 (38%) cervical carcinomas, and 2/15 (13%) and 2/15 (13%) endometrial carcinomas. oncoprotein expression was significantly higher in the malignant tumors (for ALL tumor sites) than in the corresponding normal tissues (P less than 0.034 for ALL combinations). Aneuploid tumors expressed levels of EGFR and c-erbB-2 oncoprotein significantly higher than those of DNA diploid tumors (P = 0.042 and P = 0.048, respectively). oncoprotein could be detected in nearly ALL normal tissues: expression was higher in premenopausal than in postmenopausal patients (EGFR, P = 0.07; c-erbB-2, P less than 0.001). The present study supports the idea that EGFR and c-erbB-2 may play an important role in the autocrine, paracrine, and/or endocrine growth control and differentiation of normal tissues. Alteration in the expression of these oncoproteins is probably involved in malignant transformation and tumorigenesis.

OBJECTIVES: To investigate whether hyaluronan (HA) and CD44 (hereinafter HA-CD44) promotes head and neck squamous cell carcinoma (HNSCC) chemotherapy resistance and whether HA-CD44 promotes epidermal growth factor receptor (EGFR)-mediated oncogenic signaling to alter chemotherapy sensitivity in HNSCC. Hyaluronan, a glycosaminoglycan component of the extracellular matrix, is a ligand for the transmembrane receptor CD44, which acts through multiple signaling pathways to influence cellular behavior. We recently determined that HA-CD44 promotes phospholipase C-mediated calcium signaling and cisplatin resistance in HNSCC. DESIGN: Cell line study. MAIN OUTCOME MEASURES: tumor cell growth with various chemotherapeutic drugs (methotrexate, doxorubicin hydrochloride, adriamycin, and cisplatin) was measured in the presence or absence of HA and other inhibitors of the EGFR-mediated signaling pathway. Immunoblotting was used to study EGFR signaling. Migration assays provided one measure of tumor progression. RESULTS: The addition of HA, but not HA plus anti-CD44 antibody, resulted in a 2-fold reduced ability of methotrexate and an 8-fold reduced ability of adriamycin to cause HNSCC cell death. Immunoblotting studies demonstrated that HA can promote an association between CD44 and EGFR as well as CD44-dependent activation of EGFR-mediated signaling. Migration assays demonstrated that HA-CD44 can promote tumor migration with EGFR signaling. The presence of AG1478, an EGFR inhibitor, and U0126, an extracellular signal-regulated kinase inhibitor, inhibited HA-mediated tumor growth, migration, and chemotherapy resistance. CONCLUSIONS: Our results indicate that HA promotes CD44/EGFR interaction, EGFR-mediated oncogenic signaling, and chemotherapy resistance in HNSCC. Perturbation of HA-CD44-mediated signaling may be a promising and novel strategy to treat HNSCC.

The proto-oncogenes c-erbB-2 and epidermal growth factor (EGF) receptor which encode 2 closely homologous transmembrane glycoproteins have been found amplified and/or overexpressed in a range of epithelial malignancies. In a series of 46 head and neck squamous cell cancers (SCCs), immunohistochemical reactivity for the EGF receptor was detected in ALL cases, particularly at the invading edge of cellular islands of SCC and in the basal cells of normal adjacent squamous epithelium. Southern blot analysis demonstrated EGF receptor gene amplification in 3 cases. In contrast, strong membrane staining for the c-erbB-2 oncoprotein was not detected in any sample, and there were no cases of c-erbB-2 gene amplification. Despite a close structural and (presumed) functional homology between these 2 receptor-oncoproteins in the development of malignancy, we report that their expression in SCCs is markedly different. Furthermore, unlike the situation for breast cancer, quantitation of the c-erbB-2 or EGF receptor oncoproteins is unlikely to yield important prognostic information in this group of patients.

AIM: Recent studies had suggested substantial molecular differences between tumours from different ethnic groups. In this study, the molecular differences between the incidences of colorectal carcinoma in Saudi and Swiss populations are investigated. METHOD: 518 cases of colon cancer tumours (114 from Saudi Arabia and 404 from Switzerland) were analysed in a tissue microarray format. Fluorescence in situ hybridisation (FISH) was used to estimate frequencies of copy number changes of known oncogenes, including HER2, TOPO2A, CCND1, EGFR and C-MYC. RESULTS: Using FISH, amplifications were mostly low level (gene-to-centromere ratio 2 to 4), which is in contrast with other tumour types with more frequent gene amplifications. The amplifications were particularly frequent for MYC (Saudi 9% and Swiss 14.2%) but unrelated to clinical outcome and pathological information. Remarkably, there were four tumours exhibiting classic high-level gene amplification for HER2 (Swiss 1.3%), a pattern often accompanied by response to trastuzumab (Herceptin) in breast cancer. Occasional high-level amplifications were also observed for CCND1 (Saudi 1/106, 0.9%; Swiss 2/373, 0.5%) and EGFR (Swiss 2/355; 0.6%). CONCLUSIONS: Rare high-level amplifications of therapeutic target genes were found in patients with colon cancer. Although no molecular differences were found between incidences of colon cancer cases in Swiss and Saudi populations, these observations emphasise the urgent need for clinical studies investigating the effect of targeted therapies.

The surgical material obtained from 57 patients was immunohistochemically studied. Immunohistochemistry with quantitative and semiquantitative analyses of the results of test was used to define the expression of oncomarkers p53, EGRF, ret-oncogene, and thyreoglobulin. The expression of thyreoglobulin, EGRF, and ret-oncogene reflects the malignant potential of papillary carcinoma of the thyroid and and may be recommended as a predictive marker. The low expression of thyreoglobulin and the high expression of p53 and ret-oncogene are markers of a poor prognosis and tumor recurrence. EGFR expression is not of predictive value. MB3 proposed by the authors may be recommended for the determination of the malignant potential of papillary carcinoma of the thyroid.

Because functionally significant substrates for the tyrosyl protein kinase activity of pp60v-src are likely to include membrane-associated proteins involved in normal growth control, we have tested the hypothesis that pp60v-src could phosphorylate and alter the signaling activity of transmembrane growth factor receptors. We have found that the epidermal growth factor (EGF) receptor becomes constitutively phosphorylated on tyrosine in cells transformed by the src oncogene and in addition displays elevated levels of phosphoserine and phosphothreonine. High-performance liquid chromatography phosphopeptide mapping revealed two predominant sites of tyrosine phosphorylation, both of which differed from the major sites of receptor autophosphorylation; thus, the src-induced phosphorylation is unlikely to occur via an autocrine mechanism. To determine whether pp60v-src altered the signaling activity of the EGF receptor, we analyzed the tyrosine phosphorylation of phospholipase C-gamma, since phosphorylation of this enzyme occurs in response to activation of the EGF receptor but not in response to pp60v-src alone. We found that in cells coexpressing pp60v-src and the EGF receptor, phospholipase C-gamma was constitutively phosphorylated, a result we interpret as indicating that the signaling activity of the EGF receptor was altered in the src-transformed cells. These findings suggest that pp60v-src-induced alterations in phosphorylation and function of growth regulatory receptors could play an important role in generating the phenotypic changes associated with malignant transformation.

mutations in the ERBB2 gene were recently found in approximately 2% of primary non-small cell lung cancer (NSCLC) specimens; however, little is known about the functional consequences and the relevance to responsiveness to targeted drugs for most of these mutations. Here, we show that the major lung cancer-derived ERBB2 mutants, including the most frequent mutation, A775insYVMA, lead to oncogenic transformation in a cellular assay. Murine cells transformed with these mutants were relatively resistant to the reversible epidermal growth factor receptor (EGFR) inhibitor erlotinib, resembling the resistant phenotype found in cells carrying the homologous mutations in exon 20 of EGFR. However, the same cells were highly sensitive to the irreversible dual-specificity EGFR/ERBB2 kinase inhibitor HKI-272, as were those overexpressing wild-type ERBB2. Finally, the NSCLC cell line, Calu-3, overexpressing wild-type ERBB2 owing to a high-level amplification of the ERBB2 gene were highly sensitive to HKI-272. These results provide a rationale for treatment of patients with ERBB2-mutant or ERBB2-amplified lung tumors with HKI-272.

Monoclonal antibodies (mAbs) against the extracellular domain of the epidermal growth factor receptor (EGFR) have been introduced for the treatment of metastatic colorectal cancer (mCRC). We have reported recently that increased copy number of the EGFR can predict response to anti-EGFR mAbs and that patients might be selected for treatment based on EGFR copy number. Here, we show that mutations activating the RAS/RAF signaling pathway are also predictive and prognostic indicators in mCRC patients, being inversely correlated with response to anti-EGFR mAbs. In cellular models of CRCs, activation of the RAS signaling pathway by introduction of an activated K-RAS allele (Gly(12)Val) impairs the therapeutic effect of anti-EGFR mAbs. In cancer cells carrying constitutively active RAS, the pharmacologic inhibition of the mitogen-activated protein kinase (MAPK) signaling cascade improves anti-EGFR treatment based on mAbs. These results have implications for the identification of patients who are likely to respond to anti-EGFR treatment. They also provide the rationale for combination therapies, targeted simultaneously to the EGFR and RAS/RAF/MAPK signaling pathways in CRC patients.

The epidermal growth factor receptor (EGFR) and the protein products of c-erbB-2 and c-met proto-oncogenes belong to a family of growth factor receptors with tyrosine kinase activity. In human colonic carcinomas, the expression of the EGFR and c-erbB-2 have been studied at the protein level only, while c-met expression has not been reported. We have examined the mRNA expression of these genes in human normal colorectal mucosa and primary carcinomas. The results demonstrate that the normal mucosa shows highly variable levels of EGFR and c-erbB-2 mRNAs, but expresses consistently low amounts of c-met mRNA. Colorectal carcinomas did not express significantly higher levels of the EGFR and c-erbB-2 mRNAs than the normal mucosa. In contrast, c-met was consistently and significantly overexpressed (mean sixfold) in carcinomas as compared with normal mucosa. Seventy percent of paired normal-tumor specimens showed a tumor to normal c-met mRNA ratio of greater than 4. The expression of c-met mRNA was also enhanced in the adenomas, suggesting that over-expression of this proto-oncogene may have mechanistic significance in the early stages of human colorectal carcinogenesis.

Pancreatic and colorectal carcinomas frequently express oncogenic/mutant K-Ras that contributes to both tumorigenesis and clinically observed resistance to radiation treatment. We have previously shown that farnesyltransferase inhibitors (FTI) radiosensitize many pancreatic and colorectal cancer cell lines that express oncogenic K-ras at doses that inhibit the prenylation and activation of H-Ras but not K-Ras. In the present study, we have examined the mechanism of FTI-mediated radiosensitization in cell lines that express oncogenic K-Ras and found that wild-type H-Ras is a contributor to radiation survival in tumor cells that express oncogenic K-Ras. In these experiments, inhibiting the expression of oncogenic K-Ras, wild-type H-Ras, or epidermal growth factor receptor (EGFR) led to similar levels of radiosensitization as treatment with the FTI tipifarnib. Treatment with the EGFR inhibitor gefitinib led to similar levels of radiosensitization, and the combinations of tipifarnib or gefitinib plus inhibition of K-Ras, H-Ras, or EGFR expression did not provide additional radiosensitization compared with tipifarnib or gefitinib alone. Finally, supplementing culture medium with the EGFR ligand transforming growth factor alpha was able to reverse the radiosensitizing effect of inhibiting K-ras expression. Taken together, these findings suggest that EGFR-activated H-Ras signaling is initiated by oncogenic K-Ras to promote radiation survival in pancreatic and colorectal cancers.

FSH regulates ovarian granulosa cell differentiation not only by activating adenylyl cyclase and protein kinase A (PKA) but also by other complex mechanisms. Using primary rat granulosa cell cultures, we provide novel evidence that FSH rapidly activates two small GTP-binding proteins RAP1 and RAS. FSH activation of RAP1 requires cAMP-mediated activation of exchange factor activated by cAMP/RAPGEF3 whereas FSH activation of RAS and downstream signaling cascades involves multiple factors. Specifically, FSH activation of RAS required Rous sarcoma oncogene (SRC) family tyrosine kinase (SFK) and epidermal growth factor receptor (EGFR) tyrosine kinase activities but not PKA. FSH-induced phosphorylation of ERK1/2 was blocked by dominant-negative RAS as well as by inhibitors of EGFR tyrosine kinase, metalloproteinases involved in growth factor shedding, and SFKs. In contrast, FSH-induced phosphorylation of protein kinase B (PKB/AKT) and the Forkhead transcription factor, FOXO1a occurred by SFK-dependent but RAS-independent mechanisms. The SFKs, c-SRC and FYN, and the SRC-related tyrosine kinase ABL were present and phosphorylated rapidly in response to FSH. Lastly, the EGF-like factor amphiregulin (AREG) activated RAS and ERK1/2 phosphorylation in granulosa cells by mechanisms that were selectively blocked by an EGFR antagonist but not by an SFK antagonist. However, AREG-mediated phosphorylation of PKB and FOXO1a required both EGFR and SFK activation. Moreover, we show that FSH induces AREG and that activation of the EGFR impacts granulosa cell differentiation and the expression of genes characteristic of the luteal cell phenotype. Thus, FSH orchestrates the coordinate activation of three diverse membrane-associated signaling cascades (adenylyl cyclase, RAS, and SFKs) that converge downstream to activate specific kinases (PKA, ERK1/2, and PKB/FOXO1a) that control granulosa cell function and differentiation.

Activating mutations in the epidermal growth factor receptor (EGFR) characterize a subset of non-small cell lung cancers (NSCLC) with extraordinary sensitivity to targeted tyrosine kinase inhibitors (TKI). A single secondary EGFR mutation, T790M, arising in cis with the primary activating mutation, confers acquired resistance to these drugs. However, the T790M mutation is also detected in the absence of drug selection, suggesting that it may provide a growth advantage. We show here that although T790M alone has only a modest effect on EGFR function, when combined with the characteristic activating mutations L858R or del746-750, it results in a dramatic enhancement of EGFR activity. The double mutants show potent ligand-independent receptor autophosphorylation associated with altered cellular phenotypes, soft agar colony formation, and tumorigenesis in nude mice. The significant gain-of-function properties of these double mutants may explain their initial presence before drug selection and their rapid selection as the single drug resistance mutation during therapy with gefitinib/erlotinib, and suggests that they may contribute to the adverse clinical course of TKI-resistant NSCLC.

The Epidermal growth factor receptor (EGFR) is a membrane spanning glycoprotein, which frequently has been implicated in various cancer types. The mechanisms by which EGFR becomes oncogenic are numerous and are often specific for each cancer type. In some tumors, EGFR is activated by autocrine/paracrine growth factor loops, whereas in others activating mutations promote EGFR signaling. Overexpression and/or amplification of the EGFR gene are prevalent in many cancer types leading to aberrant EGFR signaling. In addition, failure to attenuate receptor signaling by receptor downregulation can also lead to cellular transformation. Heterodimerization of EGFR with ErbB2 inhibits downregulation of EGFR and thereby prolongs growth factor signaling. This also indicates that cross-talk between EGFR and heterologous receptor systems serves as another mechanism for oncogenic activation of EGFR. Because of its role in tumor promotion, the EGFR has been intensely studied as a therapeutic target. There are currently two major mechanisms by which the EGFR is targeted: antibodies binding to the extracellular domain of EGFR and small-molecule tyrosine-kinase inhibitors. However, tumorigenesis is a multi-step process involving several mutations, which might explain why EGFR therapeutics has only been partially successful. This highlights the importance of pinpointing the mechanisms by which EGFR becomes oncogenic in a particular cancer. In this review, each of the above mentioned mechanisms will be discussed, as a detailed molecular and genetic understanding of how EGFR contributes to the malignant phenotype might offer new promise for the design, development and clinical evaluation of future tumor-specific anticancer approaches.

BACKGROUND: Epidermal growth factor receptor (EGFR) mutations are present in the majority of patients with non-small cell lung cancer (NSCLC) responsive to the EGFR tyrosine kinase inhibitors (TKIs) gefitinib or erlotinib. These EGFR-dependent tumors eventually become TKI resistant, and the common secondary T790M mutation accounts for half the tumors with acquired resistance to gefitinib. However, the key proapoptotic proteins involved in TKI-induced cell death and other secondary mutations involved in resistance remain unclear. The objective of this study was to identify the mechanism of EGFR TKI-induced apoptosis and secondary resistant mutations that affect this process. METHODS AND FINDINGS: To study TKI-induced cell death and mechanisms of resistance, we used lung cancer cell lines (with or without EGFR mutations), Ba/F3 cells stably transfected with EGFR mutation constructs, and tumor samples from a gefitinib-resistant patient. Here we show that up-regulation of the BH3-only polypeptide BIM (also known as BCL2-like 11) correlated with gefitinib-induced apoptosis in gefitinib-sensitive EGFR-mutant lung cancer cells. The T790M mutation blocked gefitinib-induced up-regulation of BIM and apoptosis. This blockade was overcome by the irreversible TKI CL-387,785. Knockdown of BIM by small interfering RNA was able to attenuate apoptosis induced by EGFR TKIs. Furthermore, from a gefitinib-resistant patient carrying the activating L858R mutation, we identified a novel secondary resistant mutation, L747S in cis to the activating mutation, which attenuated the up-regulation of BIM and reduced apoptosis. CONCLUSIONS: Our results provide evidence that BIM is involved in TKI-induced apoptosis in sensitive EGFR-mutant cells and that both attenuation of the up-regulation of BIM and resistance to gefitinib-induced apoptosis are seen in models that contain the common EGFR T790M and the novel L747S secondary resistance mutations. These findings also suggest that induction of BIM may have a role in the treatment of TKI-resistant tumors.

BACKGROUND: Epidermal growth factor receptor (EGFR) mutations are present in the majority of patients with non-small cell lung cancer (NSCLC) responsive to the EGFR tyrosine kinase inhibitors (TKIs) gefitinib or erlotinib. These EGFR-dependent tumors eventually become TKI resistant, and the common secondary T790M mutation accounts for half the tumors with acquired resistance to gefitinib. However, the key proapoptotic proteins involved in TKI-induced cell death and other secondary mutations involved in resistance remain unclear. The objective of this study was to identify the mechanism of EGFR TKI-induced apoptosis and secondary resistant mutations that affect this process. METHODS AND FINDINGS: To study TKI-induced cell death and mechanisms of resistance, we used lung cancer cell lines (with or without EGFR mutations), Ba/F3 cells stably transfected with EGFR mutation constructs, and tumor samples from a gefitinib-resistant patient. Here we show that up-regulation of the BH3-only polypeptide BIM (also known as BCL2-like 11) correlated with gefitinib-induced apoptosis in gefitinib-sensitive EGFR-mutant lung cancer cells. The T790M mutation blocked gefitinib-induced up-regulation of BIM and apoptosis. This blockade was overcome by the irreversible TKI CL-387,785. Knockdown of BIM by small interfering RNA was able to attenuate apoptosis induced by EGFR TKIs. Furthermore, from a gefitinib-resistant patient carrying the activating L858R mutation, we identified a novel secondary resistant mutation, L747S in cis to the activating mutation, which attenuated the up-regulation of BIM and reduced apoptosis. CONCLUSIONS: Our results provide evidence that BIM is involved in TKI-induced apoptosis in sensitive EGFR-mutant cells and that both attenuation of the up-regulation of BIM and resistance to gefitinib-induced apoptosis are seen in models that contain the common EGFR T790M and the novel L747S secondary resistance mutations. These findings also suggest that induction of BIM may have a role in the treatment of TKI-resistant tumors.

mutations of the p53 gene are uncommon in synovial sarcoma, a high-grade tumor genetically characterized by the chromosomal translocation t:(X;18), which results in the fusion of SS18 with members of SSX gene family. Although implicated in tumorigenesis, the mechanisms by which SS18-SSX promotes tumor growth and cell survival are poorly defined. Here, we show that SS18-SSX1 negatively regulates the stability of the tumor suppressor p53 under basal conditions. Overexpression of SS18-SSX1 enhanced p53 ubiquitination and degradation in a manner dependent on the ubiquitin ligase activity of HDM2. The negative effect of SS18-SSX1 expression on p53 was mediated by its ability to promote HDM2 stabilization through inhibition of HDM2 autoubiquitination. Furthermore, SS18-SSX1 expression altered the induction of p53-regulated genes in response to cellular stress by abrogating the transactivation of HDM2, PUMA, and NOXA but not p21. Our data uncover a novel mechanism whereby SS18-SSX1 can negatively regulate p53 tumor-suppressive function by increasing the stability of its negative regulator HDM2 and suggest that chemical compounds that target the p53-HDM2 regulatory axis may be of therapeutic benefit for the treatment of synovial sarcoma.

OBJECTIVES: Clear-cell renal cell carcinoma (RCC) is the most prevalent form of kidney cancer and is frequently associated with loss of von Hippel-Lindau (VHL) gene function, resulting in the aberrant transcriptional activation of genes that contribute to tumor growth and metastasis, including transforming growth factor-alpha (TGF-alpha), a ligand of the epidermal growth factor receptor (EGFR) tyrosine kinase. To determine the functional impact of EGFR activation on RCC, we suppressed critical components of this pathway: EGFR, Akt-1, and MEK-1. METHODS: Stable transfection of RCC cells with plasmids bearing shRNA directed against each of these genes was used to individually suppress their expression. Transfectants were characterized for growth and invasiveness in vitro and tumorigenesis in vivo. RESULTS: RCC cell transfectants displayed significantly reduced growth rate and matrix invasion in vitro and RCC tumor xenograft growth rate in vivo. Analysis of tumor cells that emerged after extended periods in each model showed that significant EGFR suppression was sustained, whereas Akt-1 and MEK-1 knock-down cells had escaped shRNA suppression. CONCLUSIONS: EGFR, Akt-1, and MEK-1 are individually critical for RCC cell invasiveness in vitro and tumorigenicity in vivo, and even partial suppression of each can have a significant impact on tumor progression. The emergence of transfectants that had escaped Akt-1 and MEK-1 suppression during tumorigenicity experiments suggests that these effectors may each be more critical than EGFR for RCC tumorigenesis, consistent with results from clinical trials of EGFR inhibitors for RCC, where durable clinical responses have not been seen.

The epidermal growth factor receptor (EGFR)-src-signal transducers and activators of transcription 3 (STAT3) oncogenic pathway plays a central role in tumorigenesis and is involved not only in cell transformation but also in tumor escape to genotoxic treatments. Despite its importance, the molecular mechanisms by which this signaling pathway induces resistance to DNA damage remain most of the time to be characterized. In this study, we show that the EGFR-src pathway is activated in response to topoisomerase I inhibition. After treatment, this signaling cascade induced the activation of STAT3 and the binding of the transcription factor to the promoter of the Eme1 gene. Eme1 is an endonuclease involved in the processing of DNA damage after topoisomerase I inhibition. These results suggest a model by which the STAT3-mediated activation of Eme1 prevents DNA damage and enhances cell survival in response to topoisomerase inhibition. This survival pathway was inhibited by a combined treatment with a src inhibitor, SKI, and with cetuximab, a monoclonal antibody directed against the EGFR that is widely used in the treatment of colorectal cancers. We therefore propose that the benefit of anti-EGFR therapy relies on an increase of DNA damage generated by topoisomerase I inhibition.

The epidermal growth factor receptor (EGFR) and other members of the EGFR/ErbB receptor family of receptor tyrosine kinases (RTKs) are important regulators of proliferation, angiogenesis, migration, tumorigenesis and metastasis. Overexpression, mutations, deletions and production of autocrine ligands contribute to aberrant activation of the ErbB proteins. The signalling output from EGFR is complicated given that other ErbB proteins are often additionally expressed and activated in the same cell, resulting in formation of homo-and/or heterodimers. In particular, association of EGFR with ErbB2 prevents its down-regulation, underscoring the importance of the cellular background for EGFR effects. Signalling from ErbB proteins can either be terminated by dissociation of ligand resulting in dephosphorylation, or blunted by degradation of the receptors. Although proteasomal targeting of ErbB proteins has been described, lysosomal degradation upon ligand-induced endocytosis seems to play the major role in EGFR down-regulation. Preclinical and clinical data have demonstrated that EGFR is a central player in cancer, especially in carcinomas, some brain tumours and in non-small cell lung cancer. Such studies have further validated EGFR as an important molecular target in cancer treatment. This review focuses on mechanisms involved in ligand-induced EGFR activation and endocytic down-regulation. A better understanding of EGFR biology should allow development of more tumour-selective therapeutic approaches targeting EGFR-induced signalling.

Hominoid- and human-specific genes may have evolved to modulate signaling pathways of a higher order of complexity. TBC1D3 is a hominoid-specific oncogene encoded by a cluster of eight paralogs on chromosome 17. Initial work indicates that TBC1D3 is widely expressed in human tissues ( Hodzic, D., Kong, C., Wainszelbaum, M. J., Charron, A. J., Su, X., and Stahl, P. D. (2006) Genomics 88, 731-736 ). In this study, we show that TBC1D3 expression has a powerful effect on cell proliferation that is further enhanced by epidermal growth factor (EGF) in both human and mouse cell lines. EGF activation of the Erk and protein kinase B/Akt pathways is enhanced, both in amplitude and duration, by TBC1D3 expression, whereas RNA interference silencing of TBC1D3 suppresses the activation. Light microscopy and Western blot experiments demonstrate that increased signaling in response to EGF is coupled with a significant delay in EGF receptor (EGFR) trafficking and degradation, which significantly extends the life span of EGFR. Moreover, TBC1D3 suppresses polyubiquitination of the EGFR and the recruitment of c-Cbl. Using the Ras binding domain of Raf1 to monitor GTP-Ras we show that TBC1D3 expression enhances Ras activation in quiescent cells, which is further increased by EGF treatment. We speculate that TBC1D3 may alter Ras GTP loading. We conclude that the expression of TBC1D3 generates a delay in EGFR degradation, a decrease in ubiquitination, and a failure to recruit adapter proteins that ultimately dysregulate EGFR signal transduction and enhance cell proliferation. Altered growth factor receptor trafficking and GTP-Ras turnover may be sites where recently evolved genes such as TBC1D3 selectively modulate signaling in hominoids and humans.

The epidermal growth factor receptor (EGFR) variant Xmrk of the Mexican fish Xiphophorus is one of the first oncogenes described. Its overexpression in pigment cells of the skin occurs after certain repeated cross-breedings between the platyfish Xiphophorus maculatus and the swordtail Xiphophorus hellerii. Xmrk overexpression results in melanoma with a high malignant potential. The Xiphophorus melanoma model provides a valuable tool for the understanding of melanoma development in general. The xmrk gene is the duplicated version of the proto-oncogene egfr-b. Compared to this, it contains 14 nonsynonymous mutations. Two of these mutations lead to a permanent protein dimerization which results in constitutive signaling. For a better understanding of the structural conditions responsible for this effect, the authors modeled the extracellular domain of Egfr-b, based on the crystal structure of the closely related human EGFR, and introduced the mutations G336R or C555S. The resulting protein models show that in either mutation the formation of an intramolecular disulfide bridge is very likely prevented, leading to an intermolecular disulfide bridge between released cysteine residues. In the G336R model, the distance between the membrane-proximal domains from both chains is smaller than in the C555S model, confirming the previous observation than Egfr-b-G336R is more tumorigenic than Egfr-b-C555S in vivo.

Epidermal growth factor receptor (EGFR) mutation is frequently observed in human cancer and contributes to the growth, survival and therapeutic resistance of tumors. EGFRvIII is an oncogenic EGFR mutant resulting from the deletion of exons 2-7 and is the most common EGFR mutant observed in glioblastoma multiforme, an aggressive brain tumor. EGFRvIII is constitutively active but poorly ubiquitinated, leading to inefficient receptor trafficking to lysosomes and unattenuated oncogenic signaling. The mechanism by which EGFRvIII evades downregulation is not fully understood although recent studies suggest that its interaction with the ubiquitin ligase Cbl may be compromised. In this study, we examine the regulation of EGFRvIII by the recently identified negative regulator, LRIG1, which targets EGFR through recognition of its extracellular domain. Here, we determine whether the extracellular domain deletion in EGFRvIII renders it refractory to LRIG1 regulation. We find that EGFRvIII retains interaction with LRIG1 and is in fact more sensitive to LRIG1 action than wild-type receptor. We demonstrate that LRIG1 regulation of EGFRvIII is distinct from the only other known mechanism of EGFR regulation, Cbl-mediated degradation. Ectopic expression of LRIG1 in EGFRvIII(+) glioblastoma cells opposes EGFRvIII-driven tumor cell proliferation, survival, motility and invasion. Finally, RNAi-mediated silencing of LRIG1 alters EGFRvIII intracellular trafficking and leads to enhanced EGFRvIII expression, suggesting that loss of LRIG1 in tumors may contribute to a permissive environment for EGFRvIII overexpression, contributing to EGFRvIII oncogenesis.

BACKGROUND: Lung cancer with EGFR mutation was shown to be a specific clinical entity. In order to better understand the biology behind this disease we used a genome wide characterization of loss of heterozygosity and amplification by Single Nucleotide Polymorphism (SNP) Array analysis to point out chromosome segments linked to EGFR mutations. To do so, we compared genetic profiles between EGFR mutated adenocarcinomas (ADC) and KRAS mutated ADC from 24 women with localized lung cancer. RESULTS: Patterns of alterations were different between EGFR and KRAS mutated tumors and specific chromosomes alterations were linked to the EGFR mutated group. Indeed chromosome regions 14q21.3 (p = 0.027), 7p21.3-p21.2 (p = 0.032), 7p21.3 (p = 0.042) and 7p21.2-7p15.3 (p = 0.043) were found significantly amplified in EGFR mutated tumors. Within those regions 3 genes are of special interest ITGB8, HDAC9 and TWIST1. Moreover, homozygous deletions at CDKN2A and LOH at RB1 were identified in EGFR mutated tumors. We therefore tested the existence of a link between EGFR mutation, CDKN2A homozygous deletion and cyclin amplification in a larger series of tumors. Indeed, in a series of non-small-cell lung carcinoma (n = 98) we showed that homozygous deletions at CDKN2A were linked to EGFR mutations and absence of smoking whereas cyclin amplifications (CCNE1 and CCND1) were associated to TP53 mutations and smoking habit. CONCLUSION: ALL together, our results show that genome wide patterns of alteration differ between EGFR and KRAS mutated lung ADC, describe two models of oncogenic cooperation involving either EGFR mutation and CDKN2A deletion or cyclin amplification and TP53 inactivating mutations and identified new chromosome regions at 7p and 14q associated to EGFR mutations in lung cancer.

The clinical efficacy of selective kinase inhibitors suggests that some cancer cells may become dependent on a single oncogene for survival. RNAi has been increasingly used to understand such "oncogene addiction" and validate new therapeutic targets. However, RNAi approaches suffer from significant off-target effects that limit their utility. Here, we combine carefully titrated lentiviral-mediated short hairpin RNA knockdown of the epidermal growth factor receptor (EGFR) with heterologous reconstitution by EGFR mutants to rigorously analyze the structural features and signaling activities that determine addiction to the mutationally activated EGFR in human lung cancer cells. EGFR dependence is differentially rescued by distinct EGFR variants and oncogenic mutants, is critically dependent on its heterodimerization partner ErbB-3, and surprisingly, does not require autophosphorylation sites in the cytoplasmic domain. Quantitative "oncogene rescue" analysis allows mechanistic dissection of oncogene addiction, and, when broadly applied, may provide functional validation for potential therapeutic targets identified through large-scale RNAi screens.

Persistent activation of the Hedgehog (HH)/GLI signaling pathway has been implicated in the development of a number of human cancers. The GLI zinc finger transcription factors act at the end of the HH signaling cascade to control gene expression, and recent studies have shown that the activity of GLI proteins can be additionally modified by integration of distinct signals, such as the MEK/extracellular signal-regulated kinase (ERK) and phosphinositide-3 kinase (PI3K)/AKT pathway. However, little is known about the identity of the upstream activators of these HH/GLI interacting signaling pathways in cancer. Here, we provide evidence that integration of the HH/GLI and epidermal growth factor receptor (EGFR) pathway synergistically induces oncogenic transformation, which depends on EGFR-mediated activation of the RAS/RAF/MEK/ERK but not of the PI3K/AKT pathway. EGFR/MEK/ERK signaling induces JUN/activator protein 1 activation, which is essential for oncogenic transformation, in combination with the GLI activator forms GLI1 and GLI2. Furthermore, pharmacologic inhibition of EGFR and HH/GLI efficiently reduces growth of basal cell carcinoma (BCC) cell lines derived from mice with activated HH/GLI signaling. The results identify the synergistic integration of GLI activator function and EGFR signaling as a critical step in oncogenic transformation and provide a molecular basis for therapeutic opportunities relying on combined inhibition of the HH/GLI and EGFR/MEK/ERK/JUN pathway in BCC.

Glioblastoma multiforme (GBM) is a highly lethal brain tumor for which little treatment is available. The epidermal growth factor receptor (EGFR) signaling pathway is thought to play a crucial role in GBM pathogenesis, initiating the early stages of tumor development, sustaining tumor growth, promoting infiltration, and mediating resistance to therapy. The importance of this pathway is highlighted in the fact that EGFR is mutationally activated in over 50% of GBM tumors. Consistent with this, we show here that concomitant activation of wild-type and/or mutant (vIII) EGFR and ablation of Ink4A/Arf and PTEN tumor suppressor gene function in the adult mouse central nervous system generates a fully penetrant, rapid-onset high-grade malignant glioma phenotype with prominent pathological and molecular resemblance to GBM in humans. Studies of the activation of signaling events in these GBM tumor cells revealed notable differences between wild-type and vIII EGFR-expressing cells. We show that wild-type EGF receptor signals through its canonical pathways, whereas tumors arising from expression of mutant EGFR(vIII) do not use these same pathways. Our findings provide critical insights into the role of mutant EGFR signaling function in GBM tumor biology and set the stage for testing of targeted therapeutic agents in the preclinical models described herein.

Activated EGF receptor (EGFR) plays an oncogenic role in several human malignancies. Although the intracellular effects of EGFR are well studied, its ability to induce and modulate tumor angiogenesis is less understood. We found previously that oncogenic EGFR can be shed from cancer cells as cargo of membrane microvesicles (MVs), which can interact with surfaces of other cells. Here we report that MVs produced by human cancer cells harboring activated EGFR (A431, A549, DLD-1) can be taken up by cultured endothelial cells, in which they elicit EGFR-dependent responses, including activation of MAPK and Akt pathways. These responses can be blocked by annexin V and its homodimer, Diannexin, both of which cloak phosphatidylserine residues on the surfaces of MVs. Interestingly, the intercellular EGFR transfer is also accompanied by the onset of VEGF expression in endothelial cells and by autocrine activation of its key signaling receptor (VEGF receptor-2). In A431 human tumor xenografts in mice, angiogenic endothelial cells stain positively for human EGFR and phospho-EGFR, while treatment with Diannexin leads to a reduction of tumor growth rate and microvascular density. Thus, we propose that oncogene-containing tumor cell-derived MVs could act as a unique form of angiogenesis-modulating stimuli and are capable of switching endothelial cells to act in an autocrine mode.

BACKGROUND: The incidence of multiple lung adenocarcinomas is rising, making it difficult to determine the stage and assign treatment in an increasing number of patients following surgery. Clinical guidelines have been developed to distinguish independent non-small cell lung cancers from metastases, that is, criteria developed by Martini and Melamed and the American College of Chest Physicians (ACCP). However, these guidelines can be difficult to apply and may give conflicting results. Here, we report on seven patients in whom epidermal growth factor receptor (EGFR) and Kirsten-rat sarcoma 2 viral oncogene homolog (KRAS) tumor mutation status was used to determine clonal relationships among multiple lung lesions. METHODS: We identified seven patients whose paired lung adenocarcinomas were found to harbor distinct EGFR or KRAS mutations. We assessed these patients disease status using established clinical guidelines. We also explored the use of comprehensive histologic subtyping (CHS) of tumor sections to distinguish multiple primaries. RESULTS: According to the Martini-Melamed criteria, six of the seven patients had multiple primary lung tumors. By ACCP criteria, three patients had multiple primaries, and three patients had metastases. Classification of the seventh patient by ACCP criteria was indeterminate. mutational testing suggested that ALL paired tumors were multiple primary adenocarcinomas, which was consistent with results from CHS. CONCLUSIONS: Assuming that independent tumor clones harbor distinct mutations, these seven cases highlight discrepancies between the existing clinical criteria used to distinguish independent tumor foci from metastases. EGFR/KRAS mutation testing of multiple lung adenocarcinomas can assist in differentiating multiple primary lung adenocarcinomas from metastatic lesions. Use of CHS in this setting should also be further explored.

Despite the importance of epidermal growth factor receptor (EGFR) in animal development and malignant transformation, surprisingly little is known about the regulation of its expression. Here, we report a novel zinc finger and G-patch domain-containing protein, ZIP. We demonstrated that ZIP acts as a transcription repressor through the recruitment of the nucleosome remodelling and deacetylase complex. Transcriptional target analysis revealed that ZIP regulates several cellular signalling pathways including EGFR pathways that are critically involved in cell proliferation, survival, and migration. We showed that ZIP inhibits cell proliferation and suppresses breast carcinogenesis, and that ZIP depletion leads to a drastic tumour growth in vivo. We found that ZIP is downregulated in breast carcinomas and that its level of expression is negatively correlated with that of EGFR. Our data indicate that ZIP is a novel transcription repressor and a potential tumour suppressor. These findings may shed new light on the EGFR-related breast carcinogenesis and might offer a potential new target for breast cancer therapy.

The C-erbB-2 gene was first found in human genomic DNA as a sequence which had homology in nucleotide sequence to the V-erbB by molecular hybridization under relaxed conditions. The product of this gene is a receptor type protein-tyrosine kinase which has a structure highly related to that of epidermal growth factor receptor (EGF-r: C-erbB-1). The proto-neu gene is a rat counterpart of the C-erb B-2 gene. The C-erbB-2 gene is also called as the HER-2 gene. The C-erbB-2 gene acquires the ability to transform NIH 3 T 3 cells by, 1) mutation which alters valine 659 in transmembrane region to glutamic acid as was found in neu gene activation, 2) deletion of c-terminal regulatory domain or 3) gene-amplification or overexpression. C-erbB-2 expresses in human embryos on mucous membranes and glands, but only faintly in adult tissues. High expression or gene amplification in human tumor appeared to be an indication for high risk of metastasis or high degree of malignancy.

Activating epidermal growth factor receptor (EGFR) mutations are common in many cancers including glioblastoma. However, clinical responses to EGFR inhibitors are infrequent and short-lived. We show that the Src family kinases (SFK) Fyn and Src are effectors of oncogenic EGFR signaling, enhancing invasion and tumor cell survival in vivo. expression of a constitutively active EGFR mutant, EGFRvIII, resulted in activating phosphorylation and physical association with Src and Fyn, promoting tumor growth and motility. Gene silencing of Fyn and Src limited EGFR- and EGFRvIII-dependent tumor cell motility. The SFK inhibitor dasatinib inhibited invasion, promoted tumor regression, and induced apoptosis in vivo, significantly prolonging survival of an orthotopic glioblastoma model expressing endogenous EGFRvIII. Dasatinib enhanced the efficacy of an anti-EGFR monoclonal antibody (mAb 806) in vivo, further limiting tumor growth and extending survival. Examination of a large cohort of clinical samples showed frequent coactivation of EGFR and SFKs in glioblastoma patients. These results establish a mechanism linking EGFR signaling with Fyn and Src activation to promote tumor progression and invasion in vivo and provide rationale for combined anti-EGFR and anti-SFK targeted therapies.

The neu oncogene protein, p185, and epidermal growth factor receptor (EGFR) were localized immunohistochemically in benign and malignant human breast tissues using monoclonal antibodies. Both benign and malignant epithelial cells were positive for these oncogene proteins in acetone-postfixed frozen sections. Stromal cells were negative for p185, but occasionally positive for EGFR. Myoepithelial cells were consistently positive for EGFR, and p185 was localized predominantly in duct-lining cells, where the basolateral plasma membrane was the normal expression site of both substances. Paraformaldehyde-prefixed frozen sections were less sensitive for antigen demonstration. Based on the intensity of immunoreactivity, 11 of 37 acetone-postfixed breast carcinomas (30%) were judged neu overexpressors, while none of 24 benign tissues overexpressed neu. Epidermal growth factor receptor was demonstrated in 18 of 36 acetone-postfixed cancer tissues (50%) and was overexpressed in three (8%). At the cellular level, heterogenous expression of p185 and EGFR was occasionally observed in both benign and malignant tissues, and a single case of cancer overexpressing both neu and EGFR showed reciprocal patterns of staining, indicating their independent expression. In some carcinomas, EGFR was localized only in stromal cells. Our findings confirmed mutually independent expression of the two closely related protooncogenes in benign and malignant breast tissues.

The epidermal growth factor receptor (EGFR) is a primary contributor to glioblastoma (GBM) initiation and progression. Here, we examine how EGFR and key downstream signaling networks contribute to the hallmark characteristics of GBM such as rapid cancer cell proliferation and diffused invasion. Additionally, we discuss current therapeutic options for GBM patients and elaborate on the mechanisms through which EGFR promotes chemoresistance. We conclude by offering a perspective on how the potential of integrative systems biology may be harnessed to develop safe and effective treatment strategies for this disease.

By immunohistochemical staining C-erbB-2 (neu) oncogene was found on the cell membrane in 19 out of 44 primary breast cancers from a pathology archive. No obvious relation was found between neu oncogene, age, and lymph node status and tumor size. There was a tendency towards smaller primary tumors and more estrogen receptor-negative tumors in the oncogene-positive group. No case of distant metastasis during follow-up was found among the oncogene-negative patients, while 6 oncogene-positive patients developed such metastases. This suggests that the neu oncogene is an independent prognostic factor, which might predict the development of distant metastasis. Further studies including more patients and long-term survival analysis are, however, needed in order to evaluate the prognostic significance of the neu oncogene.

BACKGROUND: Activating mutations in one allele of an oncogene (heterozygous mutations) are widely believed to be sufficient for tumorigenesis. However, mutant allele specific imbalance (MASI) has been observed in tumors and cell lines harboring mutations of oncogenes. METHODOLOGY/PRINCIPAL FINDINGS: We determined 1) mutational status, 2) copy number gains (CNGs) and 3) relative ratio between mutant and wild type alleles of KRAS, BRAF, PIK3CA and EGFR genes by direct sequencing and quantitative PCR assay in over 400 human tumors, cell lines, and xenografts of lung, colorectal, and pancreatic cancers. Examination of a public database indicated that homozygous mutations of five oncogenes were frequent (20%) in 833 cell lines of 12 tumor types. Our data indicated two major forms of MASI: 1) MASI with CNG, either complete or partial; and 2) MASI without CNG (uniparental disomy; UPD), due to complete loss of wild type allele. MASI was a frequent event in mutant EGFR (75%) and was due mainly to CNGs, while MASI, also frequent in mutant KRAS (58%), was mainly due to UPD. Mutant: wild type allelic ratios at the genomic level were precisely maintained after transcription. KRAS mutations or CNGs were significantly associated with increased ras GTPase activity, as measured by ELISA, and the two molecular changes were synergistic. Of 237 lung adenocarcinoma tumors, the small number with both KRAS mutation and CNG were associated with shortened survival. CONCLUSIONS: MASI is frequently present in mutant EGFR and KRAS tumor cells, and is associated with increased mutant allele transcription and gene activity. The frequent finding of mutations, CNGs and MASI occurring together in tumor cells indicates that these three genetic alterations, acting together, may have a greater role in the development or maintenance of the malignant phenotype than any individual alteration.

Little is known about the factors that control the initiation of growth of primordial follicles. The objective of the present study was to investigate the effect of c-erbB(2) on the onset of primordial follicle development, and whether c-erbB(2) mediates the effect of epidermal growth factor (EGF) in this process. We synthesized three pairs of siRNAs targeting the c-erbB(2) mRNA and transferred them into the newborn rat ovary cultured in vitro with Metafectene. After siRNAs transfection, the efficiency of siRNAs was tested by examining c-erbB(2) mRNA and protein levels. The level of c-erbB(2) mRNA was reduced by 49.6%, 46.7% and 82.6% respectively after transfecting siRNA1, siRNA2 and siRNA3, and the level of ErbB(2) protein also reduced remarkably after siRNA3 transfection. c-erbB(2) siRNA3 significantly inhibited the primordial follicle initiation and development; EGF augmented primordial follicles formation, but the effect was abolished by c-erbB(2) siRNA3. ALL of these results suggest that c-erbB(2) plays an important role in primordial follicle development and folliculogenesis initiation, and mediates the effect of EGF on primordial follicle development.

Overexpression of the epidermal growth factor (EGF) receptor (c-erbB) proto-oncogene is a frequent occurrence in human carcinoma and appears to accompany autocrine or paracrine transforming growth factor-alpha expression, which in model systems can result in activation of EGF receptor tyrosine kinase activity and phenotypic transformation. Here we have investigated the transcriptional regulation of the EGF receptor gene, by run-on transcription in isolated nuclei derived from epithelioid tumor lines. The level of transcription was measured at various points on the 100-kilobase pair EGF receptor gene locus, on either sense or antisense DNA strands. We find the level of sense strand transcription along exon 1 is 8-fold higher than transcription in exons 2-26. Primary EGF receptor transcripts appear to pause or terminate prematurely between exons 1 and 2. Termination was mapped to a sequenced region approximately 2 kilobase pairs 3 of exon 1, proximal to a previously reported DNase I hypersensitive site and an enhancer-like activity. Transcription in the CpG-rich region surrounding exon 1 is bidirectional, with antisense transcripts initiating in intron 1 and extending through the coding first exon. Activation of protein kinase C results in a 5-fold induction of EGF receptor transcription, accompanied by a slow release in the block RNA elongation between exon 2 and exon 26, showing that EGF receptor RNA synthesis may be altered by changes in de novo transcription and by a block to RNA elongation.

A mutation of the epidermal growth factor receptor (EGFR) that results in a tandem kinase domain duplication (TKD-EGFR) has been described in glioblastoma multiforme biopsies and cell lines. Although the TKD-EGFR confers tumorigenicity, little is known about the molecular underpinnings of receptor dysregulation. Therefore, we transfected B82L mouse fibroblast cells devoid of endogenous EGFR to determine the molecular mechanisms of receptor activation when expressed in cells as well as the contribution of each duplicated kinase domain to receptor phosphorylation. The TKD-EGFR displayed chronically elevated basal autophosphorylation at five known phosphotyrosine sites. The chronically phosphorylated TKD-EGFR was also resistant to competitive inhibition of ligand-binding compared with wild-type EGFR (WT-EGFR) and showed undetectable levels of basal dimerization, suggesting the TKD-EGFR escapes known mechanisms of receptor downregulation. Immunofluorescence analyses revealed a substantial portion of the TKD-EGFR resides in the cytosol in an activated state, although surface-localized subsets of the receptor retain ligand responsiveness. Kinase activity-deficient knockouts of the N-terminal or the C-terminal kinase domains generated TKD-EGFRs that recapitulate the autophosphorylation/localization patterns of a constitutively activated receptor versus a WT-like EGFR, respectively. Investigation of the molecular activity of the TKD-EGFR yields evidence for a unique mechanism of constitutive activity and dual kinase domain activation.

MYCN, a proto-oncogene normally expressed in the migrating neural crest, is in its amplified state a key factor in the genesis of human neuroblastoma (NB). However, the mechanisms underlying MYCN-mediated NB progression are poorly understood. Here, we present a MYCN-induced miRNA signature in human NB involving the activation and transrepression of several miRNA genes from paralogous clusters. Several family members derived from the miR-17 approximately 92 cluster, including miR-18a and miR-19a, were among the up-regulated miRNAs. expression analysis of these miRNAs in NB tumors confirmed increased levels in MYCN-amplified samples. Specifically, we show that miR-18a and miR-19a target and repress the expression of estrogen receptor-alpha (ESR1), a ligand-inducible transcription factor implicated in neuronal differentiation. Immunohistochemical staining demonstrated ESR1 expression in human fetal sympathetic ganglia, suggesting a role for ESR1 during sympathetic nervous system development. Concordantly, lentiviral restoration of ESR1 in NB cells resulted in growth arrest and neuronal differentiation. Moreover, lentiviral-mediated inhibition of miR-18a in NB cells led to severe growth retardation, outgrowth of varicosity-containing neurites, and induction of neuronal sympathetic differentiation markers. Bioinformatic analyses of microarray data from NB tumors revealed that high ESR1 expression correlates with increased event-free survival in NB patients and favorable disease outcome. Thus, MYCN amplification may disrupt estrogen signaling sensitivity in primitive sympathetic cells through deregulation of ESR1, thereby preventing the normal induction of neuroblast differentiation. Collectively, our findings demonstrate the molecular consequences of abnormal miRNA transcription in a MYCN-driven tumor and offer unique insights into the pathology underlying MYCN-amplified NB.

KRAS and epidermal growth factor receptor (EGFR) are the two most frequently mutated proto-oncogenes in adenocarcinoma of the lung. The occurrence of these two oncogenic mutations is mutually exclusive, and they exhibit many contrasting characteristics such as clinical background, pathological features of patients harboring each mutation, and prognostic or predictive implications. Lung cancers harboring the EGFR mutations are remarkably sensitive to EGFR tyrosine kinase inhibitors such as gefitinib or erlotinib. This discovery has dramatically changed the clinical treatment of lung cancer in that it almost doubled the duration of survival for lung cancer patients with an EGFR mutation. In this review, we describe the features of KRAS mutations in lung cancer and contrast these with the features of EGFR mutations. Recent strategies to combat lung cancer harboring KRAS mutations are also reviewed.

The MYCN oncogene encodes a transcription factor which is amplified in up to 40% of high risk neuroblastomas. MYCN amplification is a well-established poor prognostic marker in neuroblastoma, however the role of MYCN expression and the mechanisms by which it acts to promote an aggressive phenotype remain largely unknown. This review discusses the current evidence identifying the direct and indirect downstream transcriptional targets of MYCN from recent studies, with particular reference to how MYCN affects the cell cycle, DNA damage response, differentiation and apoptosis in neuroblastoma.

Activated Cdc42-associated Kinase, ACK1, is a non-receptor tyrosine kinase with numerous interacting partners, including Cdc42 and EGFR. Gene amplification and overexpression of ACK1 were found in many cancer types such as those of the lung and prostate. Previously, we identified both somatic- and germ line missense mutations in the ACK1 coding sequence, by surveying 261 cancer cell lines and 15 control tissues. Here, we verified and characterized the non-synonymous mutation, ACK-S985 N, located in the ubiquitin association domain of the protein. Both overexpression and silencing experiments in MCF7 and A498 cells, respectively, demonstrated a role of the ACK1 S985 N mutation in enhancing cell proliferation, migration and anchorage-independent growth as well as the epithelial-mesenchymal transition. Further, we showed that the ACK1 S985 N mutant is unable to bind ubiquitin, unlike the wild type kinase. This contributed to ACK1 protein stability and stabilized EGFR after EGF stimulation, thereby prolonging mitogenic signaling in cancer cells. In addition, the ACK1 S985 N-EGFR interaction is enhanced, but not the ubiquitination of the receptor. Intriguingly, silencing of ACK1 in A498 cells sensitized the renal carcinoma cells to gefitinib, against which they are otherwise resistant. The work demonstrates that other than gene amplification, a single somatic mutation in ACK1 can result in extended protein stability enabling the oncoprotein to exert its oncogenic function in tumor progression. It also provides a rationale to target ACK1 in combination with other chemotherapeutic drugs, such as EGFR inhibitors, to potentiate therapeutic action against resistant tumors.

PURPOSE: The purpose of this study was to investigate the prognostic significance of the expression of EGFR and C-erbB-2 gene products by immunohistochemical analysis for curatively resected gastric adenocarcinoma. MATERIALS AND METHODS: Between January 1996 and December 2001, 739 patients with curatively resected gastric cancer patients underwent immunohistochemical staining for EGFR and C-erbB-2 proteins, and we retrospectively analyzed their correlation with the clinical outcome. RESULTS: The over expressions of EGFR and C-erbB-2 were 25.4% and 26.2%, respectively. The over expressions of EGFR was associated with the more poorly differentiated tumor (p=0.000) and with neuronal invasion (p=0.03). Over expression of C-erbB-2 was associated with less vascular invasion (p=0.001). tumor depth or node metastasis was not related to the over expression of EGFR or C-erbB-2. The seven-year overall survival and relapse-free survival rates were 87.2% and 75.8%, respectively. Upon multivariate Cox regression analysis, the tumor stage, tumor size and patient age were important prognostic factors for overall survival, and tumor stage was the important factor for relapse-free survival. Over expressions of EGFR or c-erbB-2 were not significant prognostic factors. CONCLUSION: Immunohistochemical staining of EGFR and C-erbB-2 gene products were not independent prognostic factors for predicting the overall survival and the relapse-free survival in curatively resected gastric cancer.

The introduction of KRAS testing as a diagnostic tool to select patients for epidermal growth factor receptor (EGFR)-targeted cetuximab- or panitumumab-based therapies for metastatic colorectal cancer is widely regarded as a key advance in the field of personalized cancer medicine. Oncologists are now facing emerging issues in the treatment of metastatic colorectal cancer, including: (i) the identification of additional genetic determinants of primary resistance to EGFR-targeted therapy for further improving selection of patients; (ii) the explanation of rare cases of patients carrying KRAS-mutated tumors who have been reported to respond to either cetuximab or panitumumab and (iii) the discovery of mechanisms of secondary resistance to anti-EGFR antibody therapies. Here we discuss the potential role of comprehensive dissection of the key oncogenic nodes in the EGFR signaling cascade to predict resistance and sensitivity to EGFR monoclonal antibodies in metastatic colorectal cancer. Current data suggest that, together with KRAS mutations, the evaluation of BRAF and PIK3CA/PTEN alterations could also be useful for selecting patients with reduced chance to benefit from EGFR-targeted therapy. Furthermore, measuring EGFR gene copy number also appears relevant to positively identify responders. Up until now, each of these markers has been mainly assessed as a single event, often in retrospective analyses and patients series. As these molecular alterations display overlapping patterns of occurrence, this adds considerable complexity to the drawing of an algorithm suitable for clinical decision-making. We suggest that in the near future comprehensive molecular analysis of the entire oncogenic pathway triggered by the EGFR should be performed, thus enhancing the prediction ability of individual markers.

The EGF-receptor is frequently mutated in a large variety of tumors. Here we review the most frequent mutations and conclude that they commonly enhance the intrinsic tyrosine kinase activity, or they represent loss-of-function of suppressive regulatory domains. Interestingly, the constitutive activity of mutant receptors translates to downstream pathways, which are subtly different from those stimulated by the wild-type receptor. cancer drugs intercepting EGFR signaling have already entered clinical application. Both kinase inhibitors specific to EGFR, and monoclonal antibodies to the receptor are described, along with experimental approaches targeting the HSP90 chaperone. Deeper understanding of signaling pathways downstream to mutant receptors will likely improve the outcome of current EGFR-targeted therapies, as well as help develop new drugs and combinations.

cancer cells frequently overexpress tissue factor (TF) and become procoagulant. This conversion may be driven by genetic transformation, including through the expression of the oncogenic epidermal growth factor receptor (EGFR) and its mutant, EGFRvIII, present in glioblastoma multiforme (GBM). Here we show that the EGFRvIII-dependent GBM cell transformation is associated with the onset of the simultaneous overexpression of TF, protease-activated receptors 1 and 2 (PAR1 and PAR2), and ectopic synthesis of factor VII (FVII). Efficient generation of factor Xa by these cells still requires exogenous FVIIa. However, as a result of EGFRvIII-dependent transformation, GBM cells become hypersensitive to TF/PAR-mediated signaling and produce ample angiogenic factors (vascular endothelial growth factor and interleukin-8) on exposure to FVIIa and PAR1- or PAR2-activating peptides. Thus, oncogenes may cause complex changes in the ability of GBM cancer cells to interact with the coagulation system, thereby exacerbating its influence on angiogenesis and disease progression.

Activating mutation of K-ras and inactivation of DPC4 are two common genetic alterations that occur in the development and progression of pancreatic ductal adenocarcinomas (PDAC). A separate common event in PDAC progression is increased expression of phosphotyrosine kinase receptors (PTKRs). In our study, we examined whether activating mutations of K-ras and loss of Smad4 play a role in causing the aberrant expression of PTKRs. Immortalized human pancreas ductal cells (HPNE) were genetically modified by expressing oncogenic K-ras and/or by shRNA knockdown of Smad4. EGFR and erbB2 protein levels but not Ron or IGF-1R were substantially upregulated in HPNE cells that express K-ras((GD12)). The increased expression of EGFR in HPNE cells that expressed K-ras((GD12)) was mediated by both stabilizing EGFR protein and by increasing EGFR transcription. TGF-beta signaling partially suppressed K-ras((GD12)) induced EGFR transcription in Smad4 intact HPNE cells; whereas knockdown of Smad4 in cells expressing K-ras((GD12)) further enhanced expression of EGFR and erbB2. The upregulation of EGFR and erbB2 was associated with an increase of invasion, which was blocked by a kinase inhibitor of EGFR. Our study indicates for the first time, that oncogenic ras and loss of Smad signaling cooperate to upregulate EGFR and erbB2, which plays a role in promoting invasion.

Overexpression/amplification of human epidermal growth factor receptor (HER)2/neu (erbB-2) oncogene plays a causal role in carcinogenesis and correlates with a poor clinical prognosis. However, little is known about HER2 in gastric cancer. In this study, we explored the pharmacological activities of natural triterpenoid corosolic acid (CRA) in HER2 signaling and its role in gastric cancer development and progression. In this study, CRA dramatically inhibited HER2 expression in a dose- and time-dependent manner, effectively inhibited cell proliferation, and induced G(0)/G(1) arrest through the induction of p27(kip1) and cyclin D(1) down-regulation. CRA exposure enhanced apoptotic cell death, as confirmed by caspase-3 and poly (ADP-ribose) polymerase cleavage activities. CRA inhibited signaling pathways downstream of HER2, including phospho-proteins such as Akt and Erk. In addition, CRA combined with adriamycin and 5-fluorouracil enhanced this growth inhibition, but not with docetaxel and paclitaxel. These findings demonstrate that CRA suppresses HER2 expression, which in turn promotes cell cycle arrest and apoptotic cell death of gastric cancer cells, providing a rationale for future clinical trials of CRA in the treatment of HER2-positive gastric cancers.

Epidermal growth factor receptor (EGFR)-targeting therapeutics have shown efficacy in the treatment of colorectal cancer patients. Clinical studies have revealed that activating mutations in the KRAS protooncogene predict resistance to EGFR-targeted therapy. However, the causality between mutant KRAS and resistance to EGFR inhibition has so far not been demonstrated. Here, we show that deletion of the oncogenic KRAS allele from colorectal tumor cells resensitizes those cells to EGFR inhibitors. Resensitization was accompanied by an acquired dependency on the EGFR for maintaining basal extracellular signal-regulated kinase (ERK) activity. Deletion of oncogenic KRAS not only resensitized tumor cells to EGFR inhibition but also promoted EGF-induced NRAS activation, ERK and AKT phosphorylation, and c-FOS transcription. The poor responsiveness of mutant KRAS tumor cells to EGFR inhibition and activation was accompanied by a reduced capacity of these cells to bind and internalize EGF and by a failure to retain EGFR at the plasma membrane. Of 16 human colorectal tumors with activating mutations in KRAS, 15 displayed loss of basolateral EGFR localization. Plasma membrane localization of the EGFR could be restored in vitro by suppressing receptor endocytosis through Rho kinase inhibition. This caused an EGFR-dependent increase in basal and EGF-stimulated ERK phosphorylation but failed to restore tumor cell sensitivity to EGFR inhibition. Our results demonstrate a causal role for oncogenic KRAS in desensitizing tumor cells not only to EGFR inhibitors but also to EGF itself.

Selective enhancement of tumor response to radiation therapy is a highly attractive objective, but it has not been met clinically. Gain-of-function Ras (gf) signaling via hyperactivation of receptor tyrosine kinases, such as the epidermal growth factor receptor (EGFR), or via oncogenic mutation of Ras is shown to confer radioresistance and requires the engagement of the Raf/MEK/ERK pathway. However, upstream mediators of such interaction in cancer cells that could be targeted for radiosensitization have not been identified and characterized. Here, we provide original observations both in vitro and in vivo that kinase suppressor of Ras1 (KSR1) is a new target for reversing gf Ras-mediated radioresistance. We employed EGFR-dependent A431 squamous cell carcinoma (SCC) and genetically defined the molecular function of KSR1 in irradiation-induced Raf/MEK/ERK activation. In vitro KSR1 inactivation via genetic inhibition of its expression or kinase function abrogated ionizing radiation-induced activation of the Raf/MEK/ERK2 cascade, enhanced the cytotoxic effect of radiation, and achieved radiosensitization associated with inhibition of DNA damage repair and enhancement of clonogenic death. In vivo pharmacologic inactivation of KSR1 by KSR1 AS-ODN infusion leads to radiosensitization in EGFR-dependent A431 SCC and in oncogenic K-Ras-driven A549 human non-small cell lung carcinoma. These observations collectively establish KSR1 as a novel target for radiosensitization and show the feasibility of using KSR1 AS-ODN as a radiosensitizer for treating gf Ras-dependent human malignancies. Identification of such mediators of gf Ras signaling in response to irradiation holds promises for improving the therapeutic efficacy of radiation therapy and our ability to eradicate tumor.

PURPOSE: Inhibitors of antiapoptosis protein (IAP) have been implicated in the resistance to cisplatin. Therefore, verifying which pathway is involved in cIAP2 upregulation may be helpful in finding a feasible pathway inhibitor to increase the chemotherapeutic efficacy in human papillomavirus (HPV)-infected lung cancer. EXPERIMENTAL DESIGN: Specific inhibitors of different pathways were used to verify which pathway is involved in cIAP2 transcription. cIAP2 promoter fragments with various deletions and/or mutations were constructed by site-directed mutagenesis. cIAP2, epidermal growth factor receptor (EGFR), and phospho-AKT (p-AKT) expressions in 136 lung tumors were evaluated by immunohistochemistry. RESULTS: Our data show that two NF-kappaB (-209 to -200 and -146 to -137) and one CREB (cyclic AMP-responsive element binding protein; -52 to -42) binding sites in cIAP2 promoter region were responsible for cIAP2 upregulated by E6 in TL-1 cells. Moreover, CREB was phosphorylated by EGFR/phosphatidylinositol 3-kinase (PI3K) pathway. To test the involvement of cIAP2 on cisplatin resistance, IC(50) was lowered to 8.6 mumol/L in TL-1 cells with cIAP2 short hairpin RNA (shRNA) transfection and compared with 39.7 mumol/L in TL-1 cells with nonspecific shRNA. Pretreatment with EGFR or PI3K inhibitor in TL-1 cells diminished the resistance to cisplatin. Among the tumor groups, cIAP2 expression correlated significantly with HPV16/18 E6, EGFR, and p-AKT. We followed up 46 of 136 patients who had tumor recurrence and/or metastasis and underwent chemotherapy. tumors with cIAP2-positive immunostaining were associated with a poorer tumor response to chemotherapy compared with those with negative immunostaining. CONCLUSIONS: cIAP2 upregulated by E6 via EGFR/PI3K/AKT cascades may contribute to cisplatin resistance, revealing that the EGFR or PI3K inhibitor combined with cisplatin may improve the chemotherapeutic efficacy in HPV-infected lung cancer.

Malignant gliomas are the most common type of intrinsic central nervous system (CNS) tumors with high mortality and morbidity. beta-catenin is overexpressed in human glioblastoma and knockdown of beta-catenin inhibits glioblastoma cell proliferation and invasive ability, and induces apoptotic cell death. Furthermore, treating the nude mice carrying established subcutaneous LN229 gliomas with siRNA targeting beta-catenin intratumorally also delayed the tumor growth. However, the mechanisms of down-regulation of beta-catenin that represses glioblastoma malignancy behavior remain to be elucidated. We utilized text-mining of MEDLINE abstracts with natural language processing to establish the beta-catenin biologic association network, and identified several interactions of this network with the EGFR pathway. In both in vitro and in vivo studies, our results confirmed down-regulation of beta-catenin induced reduced expression of EGFR, STAT3 and AKT1 mRNA and protein, besides, the level of phosphorylated Akt also decreased. A similar reduction in expression of CyclinD1, MMP2 and MMP9, downstream genes of the EGFR pathway, was observed. These results suggest that the Wnt/beta-catenin pathway regulates glioma cell proliferation and invasion, in part via the EGFR pathway.

MicroRNAs (miRNA) mediate distinct gene regulatory pathways triggered by epidermal growth factor receptor (EGFR) activation, which occurs commonly in lung cancers with poor prognosis. In this study, we report the discovery and mechanistic characterization of the miRNA miR-7 as an oncogenic "oncomiR" and its role as a key mediator of EGFR signaling in lung cancer cells. EGFR activation or ectopic expression of Ras as well as c-Myc stimulated miR-7 expression in an extracellular signal-regulated kinase (ERK)-dependent manner, suggesting that EGFR induces miR-7 expression through a Ras/ERK/Myc pathway. In support of this likelihood, c-Myc bound to the miR-7 promoter and enhanced its activity. Ectopic miR-7 promoted cell growth and tumor formation in lung cancer cells, significantly increasing the mortality of nude mice hosts, which were orthotopically implanted with lung cancers. Quantitative proteomic analysis revealed that miR-7 decreased levels of the Ets2 transcriptional repression factor ERF, the coding sequence of which was found to contain a miR-7 complementary sequence. Indeed, ectopic miR-7 inhibited production of ERF messages with a wild-type but not a silently mutated coding sequence, and ectopic miR-7 rescued growth arrest produced by wild-type but not mutated ERF. Together, these results identified that ERF is a direct target of miR-7 in lung cancer. Our findings suggest that miR-7 may act as an important modulator of EGFR-mediated oncogenesis, with potential applications as a novel prognostic biomarker and therapeutic target in lung cancer.

We have previously shown that an intracellular mechanism down regulates epidermal growth factor (EGF) receptor levels in rodent fibroblasts transformed by the src oncogene (W. J. Wasilenko, L. K. Shawver, and M. J. Weber, J. Cell. Physiol. 131:450-457, 1987). We now report that this down regulation is due to an inhibition of EGF receptor biosynthesis. With Rat-1 (R1) cells infected with a temperature-sensitive src mutant, we found that 125I-labeled EGF binding to cells began to decrease soon after the activation of pp60v-src by shift down to the permissive temperature for transformation. This effect of src on EGF receptors was reversible. Pulse-chase studies with [35S]methionine-labeled cells revealed that the tyrosine protein kinase activity of pp60v-src had little if any effect on EGF receptor degradation rate. By contrast, the expression of pp60v-src caused a large reduction in the apparent rate of EGF receptor biosynthesis. Northern (RNA) blot analysis demonstrated that pp60v-src also caused marked reductions in the steady-state level of EGF receptor mRNA. These data indicate that one way the expression of the src oncogene can affect the machinery of growth control is by affecting the expression of specific genes for growth factor receptors.

The epidermal growth factor receptor (EGFR) plays an important role in cancer by activating downstream signals important in growth and survival. Inhibitors of EGFR are frequently selected as treatment for cancer including lung cancer. We performed an unbiased and comprehensive search for EGFR phosphorylation events related to somatic activating mutations and EGFR inhibitor (erlotinib) sensitivity. EGFR immunoprecipitation combined with high resolution liquid chromatography-mass spectrometry and label free quantitation characterized EGFR phosphorylation. Thirty (30) phosphorylation sites were identified including 12 tyrosine (pY), 12 serine (pS), and 6 threonine (pT). Site-specific phosphorylation was monitored by comparing ion signals from the corresponding unmodified peptide. Phosphorylation sites related to activating mutations in EGFR as well as sensitivity to erlotinib were identified using 31 lung cancer cell lines. We identified three sites (pY1092, pY1110, pY1172) correlated with activating mutations and three sites (pY1110, pY1172, pY1197) correlated with erlotinib sensitivity. Five sites (pT693, pY1092, pY1110, pY1172, and pY1197) were inhibited by erlotinib in concentration-dependent manner. Erlotinib sensitivity was confirmed using liquid chromatography coupled to multiple reaction monitoring (LC-MRM) and quantitative Western blotting. This LC-MS/MS strategy can quantitatively assess site-specific EGFR phosphorylation and can identify relationships between somatic mutations or drug sensitivity and protein phosphorylation.

PURPOSE: Emerging evidence suggests that aberrant expression of oncogenes contributes to development of lung malignancy. The thyroid transcription factor 1 (TITF-1) gene functions as a lineage survival gene abnormally expressed in a significant fraction of non-small cell lung cancers (NSCLC), in particular lung adenocarcinomas. EXPERIMENTAL DESIGN: To better characterize TITF-1 abnormality patterns in NSCLC, we studied TITF-1 s gene copy number using FISH and quantitative PCR, as well as its protein expression by immunohistochemistry analysis in a tissue microarray comprising surgically resected NSCLC (N = 321) including 204 adenocarcinomas and 117 squamous cell carcinomas (SCC). TITF-1 copy number and protein expression were correlated with patients clinicopathologic characteristics, and in a subset of adenocarcinomas with EGFR and KRAS mutation status. RESULTS: We found that increased TITF-1 protein expression was prevalent in lung adenocarcinomas only and was significantly associated with female gender (P < 0.001), never-smokers (P = 0.004), presence of EGFR mutations (P = 0.05), and better overall survival (all stages, P = 0.0478; stages I and II, P = 0.002). TITF-1 copy number gain(CNG) was detected by FISH analysis in both adenocarcinomas (18.9%; high CNG, 8.3%) and SCCs (20.1%; high CNG, 3.0%), and correlated significantly with the protein product (P = 0.004) and presence of KRAS mutations (P = 0.008) in lung adenocarcinomas. Moreover, multivariate analysis revealed that TITF-1 copy number gain was an independent predictor of poor survival of NSCLC (P = 0.039). CONCLUSIONS: Our integrative study demonstrates that the protein versus genomic patterns of TITF-1 have opposing roles in lung cancer prognosis and may occur preferentially in different subsets of NSCLC patients with distinct oncogene mutations.

Human lung adenocarcinomas with activating mutations in EGFR (epidermal growth factor receptor) often respond to treatment with EGFR tyrosine kinase inhibitors (TKIs), but the magnitude of tumour regression is variable and transient. This heterogeneity in treatment response could result from genetic modifiers that regulate the degree to which tumour cells are dependent on mutant EGFR. Through a pooled RNA interference screen, we show that knockdown of FAS and several components of the NF-kappaB pathway specifically enhanced cell death induced by the EGFR TKI erlotinib in EGFR-mutant lung cancer cells. Activation of NF-kappaB through overexpression of c-FLIP or IKK (also known as CFLAR and IKBKB, respectively), or silencing of IkappaB (also known as NFKBIA), rescued EGFR-mutant lung cancer cells from EGFR TKI treatment. Genetic or pharmacologic inhibition of NF-kappaB enhanced erlotinib-induced apoptosis in erlotinib-sensitive and erlotinib-resistant EGFR-mutant lung cancer models. Increased expression of the NF-kappaB inhibitor IkappaB predicted for improved response and survival in EGFR-mutant lung cancer patients treated with EGFR TKI. These data identify NF-kappaB as a potential companion drug target, together with EGFR, in EGFR-mutant lung cancers and provide insight into the mechanisms by which tumour cells escape from oncogene dependence.

Many microRNAs (miRNAs) play crucial roles in regulating expression of oncogenes or tumor suppressor genes. The epidermal growth factor receptor (EGFR) is frequently overexpressed in a wide range of solid tumors and is an important therapeutic target; however, the therapeutic outcome of currently available anti-EGFR agents is often limited due to diverse molecular resistance mechanisms. In this study, we evaluated the potential of targeting miRNA-7 for overcoming radio-resistance of cancer cells with activated EGFR-associated signaling. A panel of human cancer cell lines with increased EGFR-PI3K-Akt signaling was transfected with pre-miR-7 or control miRNA. Ectopic overexpression of miR-7 attenuated EGFR and Akt expression and radiosensitized SQ20B squamous cell carcinoma of the larynx, MDA-MB-468 breast cancer cells, A549 lung carcinoma cells, and U251 and U87 malignant glioma cells. In contrast, antisense-mediated inhibition of mature miR-7 expression led up-regulation of EGFR and its downstream effectors, and increased radio-resistance of U251 glioma cells. Overexpression of miR-7 prolonged radiation-induced gammaH2AX foci formation and downregulation of DNA-dependent protein kinases (DNA-PKcs). miR-7 may be a useful therapeutic target for overcoming the radio-resistance of human cancers with activated EGFR-PI3K-AKT signaling.

The role of the extracellular domain of the v-erbB oncogene in the transformation of chicken fibroblasts and erythroblasts has been examined. A virus was isolated in which the complete extracellular domain and the transmembrane domain of the v-erbB were replaced with sequences from the gag and env genes. The resulting virus, GEE35, was capable of transforming both fibroblasts and erythroblasts as efficiently as wild-type v-erbB. Biochemical and immunochemical analysis of the v-erbB protein encoded by GEE35 revealed two proteins of apparent molecular weights of approximately 130,000 daltons. One of these proteins was an N-linked glycosylated membrane protein, whereas the other was cytoplasmic. The possible roles of either of these two proteins in transformation is discussed.

expression of the product of the c-erbB-2 gene, a proto-oncogene related to, but distinct from c-erbB-1 encoding the epidermal growth factor receptor (EGF-R), was investigated in human urinary bladder carcinomas. In addition, levels of EGF-R and transferrin receptor were also analyzed using an immunohistochemical approach, and the results compared with histological pattern and grading, and tumor staging. Increased expression of c-erb B-2 product was found in 32% of cases (7/22), a positive reaction being observed in 60% of transitional cell carcinoma (TCC) Grade 3 lesions (3/5), 20% of Grade 2 TCCs (2/10) and 100% of adenocarcinomas (AC) (2/2), but in none of the cases of squamous cell carcinoma (SCC). Although no statistical correlation with staging was evident, TCCs or SCCs of high grade and stage often showed EGF-R-positive staining, whereas other well differentiated lesions and normal bladder epithelium were generally negative. Most cases of urinary bladder carcinoma were positive for the transferrin receptor, which was not detected in normal bladder. The results thus suggested that a positive reaction for c-erbB-2 product is correlated with TCC histological grading or AC morphology. A high intensity of EGF-R staining in human bladder carcinomas may be associated with poor differentiation and invasion, whereas transferrin receptor expression might reflect tumor growth.

Emergence of acquired resistance is virtually inevitable in patients with a mutation in the epidermal growth factor receptor gene (EGFR) treated with EGFR tyrosine kinase inhibitors (TKIs). Several novel TKIs that may prevent or overcome the resistance mechanisms are now under clinical development. However, it is unknown how tumor cells will respond to intensive treatment using these novel TKIs. We previously established HCC827EPR cells, which are T790M positive, through combined treatment with erlotinib and a MET-TKI from erlotinib-hypersensitive HCC827 cells. In this study, we treated HCC827EPR cells sequentially with an irreversible EGFR-TKI, CL-387,785, to establish resistant cells (HCC827CLR), and we analyzed the mechanisms responsible for resistance. In HCC827CLR cells, PTEN expression was downregulated and Akt phosphorylation persisted in the presence of CL-387,785. Akt inhibition restored CL-387,785 sensitivity. In addition, withdrawal of CL-387,785 reduced cell viability in HCC827CLR cells, indicating that these cells were "addicted" to CL-387,785. HCC827CLR cells overexpressed the EGFR, and inhibition of the EGFR or MEK-ERK was needed to maintain cell proliferation. Increased senescence was observed in HCC827CLR cells in the drug-free condition. Through long-term culture of HCC827CLR cells without CL-387,785, we established HCC827-CL-387,785-independent cells, which exhibited decreased EGFR expression and a mesenchymal phenotype. In conclusion, PTEN downregulation is a newly identified mechanism underlying the acquired resistance to irreversible EGFR-TKIs after acquisition of T790M against erlotinib. This series of experiments highlights the flexibility of cancer cells that have adapted to environmental stresses induced by intensive treatment with TKIs.

Phosphoinositide-3-kinase catalytic alpha polypeptide (PIK3CA) encodes the p110alpha subunit of the mitogenic signaling protein phosphoinositide 3-kinase (PI3K). PIK3CA mutations in the helical binding domain and the catalytic subunit of the protein have been associated with tumorigenesis and treatment resistance in various malignancies. Characteristics of patients with PIK3CA-mutant lung adenocarcinomas have not been reported. We examined epidermal growth factor receptor (EGFR), Kirsten rate sarcoma viral oncogene homolog (KRAS), v-Raf murine sarcoma viral oncogene homolog B1 (BRAF), human epidermal growth factor receptor 2 (HER2), PIK3CA, v-akt murine thymoma vial oncogene homolog 1 (AKT1), v-ras neuroblastoma viral oncogene homolog (NRAS), dual specificity mitogen-activated protein kinase kinase 1 (MEK1), and anaplastic lymphoma kinase (ALK) in patients with adenocarcinoma of the lung to identify driver mutations. Clinical data were obtained from the medical records of individuals with mutations in PIK3CA. Twenty-three of 1,125 (2%, 95% CI: 1-3) patients had a mutation in PIK3CA, 12 in exon 9 (10 E545K and 2 E542K), and 11 in exon 20 (3 H1047L and 8 H1047R). The patients (57% women) had a median age of 66 at diagnosis (range: 34-78). Eight patients (35%) were never smokers. Sixteen of 23 (70%, 95% CI: 49-86) had coexisting mutations in other oncogenes-10 KRAS, 1 MEK1, 1 BRAF, 1 ALK rearrangement, and 3 EGFR exon 19 deletions. We conclude that PIK3CA mutations occur in lung adenocarcinomas, usually concurrently with EGFR, KRAS, and ALK. The impact of PIK3CA mutations on the efficacy of targeted therapies such as erlotinib and crizotinib is unknown. Given the high frequency of overlapping mutations, comprehensive genotyping should be carried out on tumor specimens from patients enrolling in clinical trials of PI3K and other targeted therapies.

Although it is known that mTOR complex 2 (mTORC2) functions upstream of Akt, the role of this protein kinase complex in cancer is not well understood. Through an integrated analysis of cell lines, in vivo models, and clinical samples, we demonstrate that mTORC2 is frequently activated in glioblastoma (GBM), the most common malignant primary brain tumor of adults. We show that the common activating epidermal growth factor receptor (EGFR) mutation (EGFRvIII) stimulates mTORC2 kinase activity, which is partially suppressed by PTEN. mTORC2 signaling promotes GBM growth and survival and activates NF-kappaB. Importantly, this mTORC2-NF-kappaB pathway renders GBM cells and tumors resistant to chemotherapy in a manner independent of Akt. These results highlight the critical role of mTORC2 in the pathogenesis of GBM, including through the activation of NF-kappaB downstream of mutant EGFR, leading to a previously unrecognized function in cancer chemotherapy resistance. These findings suggest that therapeutic strategies targeting mTORC2, alone or in combination with chemotherapy, will be effective in the treatment of cancer. SIGNIFICANCE: This study demonstrates that EGFRvIII-activated mTORC2 signaling promotes GBM proliferation, survival, and chemotherapy resistance through Akt-independent activation of NF-kappaB. These results highlight the role of mTORC2 as an integrator of two canonical signaling networks that are commonly altered in cancer, EGFR/phosphoinositide-3 kinase (PI3K) and NF-kappaB. These results also validate the importance of mTORC2 as a cancer target and provide new insights into its role in mediating chemotherapy resistance, suggesting new treatment strategies.

Despite many evidences supporting the concept of "oncogene addiction" and many hypotheses rationalizing it, there is still a lack of detailed understanding to the precise molecular mechanism underlying oncogene addiction. In this account, we developed a mathematic model of epidermal growth factor receptor (EGFR) associated signaling network, which involves EGFR-driving proliferation/pro-survival signaling pathways Ras/extracellular-signal-regulated kinase (ERK) and phosphoinositol-3 kinase (PI3K)/AKT, and pro-apoptotic signaling pathway apoptosis signal-regulating kinase 1 (ASK1)/p38. In the setting of sustained EGFR activation, the simulation results show a persistent high level of proliferation/pro-survival effectors phospho-ERK and phospho-AKT, and a basal level of pro-apoptotic effector phospho-p38. The potential of p38 activation (apoptotic potential) due to the elevated level of reactive oxygen species (ROS) is largely suppressed by the negative crosstalk between PI3K/AKT and ASK1/p38 pathways. Upon acute EGFR inactivation, the survival signals decay rapidly, followed by a fast increase of the apoptotic signal due to the release of apoptotic potential. Overall, our systems biology modeling together with experimental validations reveals that inhibition of survival signals and concomitant release of apoptotic potential jointly contribute to the tumor cell death following the inhibition of addicted oncogene in EGFR addicted cancers.

Cheung and colleagues demonstrate that amplified CRKL can function as a driver oncogene in lung adenocarcinoma, activating both RAS and RAP1 to induce mitogen-activated protein kinase signaling. In addition, they show that CRKL amplification may be another mechanism for primary or acquired resistance to epidermal growth factor receptor kinase inhibitors.

OBJECTIVE: To study the expression of the epidermal growth factor receptor (EGFR) and the oncogene c-erbB2 on pulmonary fibrosis induced by bleomycin (BLM) in rats. METHODS: Fifty-four Wistar rats were randomly divided into three groups, the pulmonary fibrosis group (BLM), Iressa group and the control group. There were 18 rats in each group. Control group were injected with saline 0.2-0.3 ml in trachea. Iressa group and BLM group were injected with BLM intratracheal. After the fibrosis models were build, Iressa group were given orally Iressa (200 mg/kg)1 h before modeling in Iressa group, saline were fed 10 ml/kg in BLM group and control group. The three groups were fed 5 times per week; and were sacrificed after treatment on days 1, 14 and 28 respectively. The lungs were harvested for histological studies. RESULTS: The lung tissue in Iressa group showed fibrosis and inflammatory cell infiltration, the same as shown in the BLM group. The pulmonary fibrosis score was significantly lower than the BLM group on the 28 th day (2.17 +/- 0.41 vs 3.50 +/- 0.84, P < 0.01). Compared with the control group, c-erbB2 and EGFR were hyper expressed significantly both in BLM group and Iressa group at ALL time points (P < 0.01); c-erbB2 expression had no changes between the Iressa group and the BLM (P > 0.05), that were gradually decreased, and was significantly different at each time point (P < 0.01). EGFR expression was increased gradually on the 14th and 28th day (0.17 +/- 0.02 and 0.28 +/- 0.04) in Iressa group ,that was significantly lower than the BLM group (0.27 +/- 0.04 and 0.34 +/- 0.02) (P < 0.01). EGFR expression increased significantly on the 28th day than on the 14th day in the Iressa group (P < 0.01). CONCLUSION: The expression of C-erbB2 and EGFR are enhanced in different stages of alveolitis and pulmonary fibrosis, c-erbB2 and EGFR may be participated in different stages of pulmonary fibrosis.

BACKGROUND: The discovery of distinct subsets of nonsmall cell lung cancer (NSCLC) characterized by activation of driver oncogenes has greatly affected personalized therapy. It is hypothesized that the dominant oncogene in NSCLC would be associated with distinct patterns of metastatic spread in NSCLC at the time of diagnosis. METHODS: A total of 209 consecutive patients with stage IV nonsquamous NSCLC with an EGFR (epidermal growth factor receptor) mutation (N = 39), KRAS (v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog) mutation (N = 49), ALK (anaplastic lymphoma receptor tyrosine kinase) gene rearrangement (N = 41), or wild-type for ALL 3 (triple negative, N = 80) were included. The percentage of patients with metastatic disease at a given site was compared between each molecular cohort (EGFR, KRAS, or ALK) and the triple negative cohort. RESULTS: ALK gene rearrangement was significantly associated with pericardial disease (odds ratio [OR] = 4.61; 95% confidence interval [CI] = 1.30, 16.37; P = .02) and pleural disease (OR = 4.80; 95%

AIMS: The epidermal growth factor receptor (EGFR) is an available target of effective anti-EGFR therapy for human breast cancer. KRAS, the human homolog of the Kirsten rat sarcoma-2 virus oncogene, encodes a main downstream signaling molecule in the EGFR pathway. The aim of this study was to assess the presence of EGFR and KRAS gene mutations in breast cancer. MATERIALS AND METHODS: EGFR and KRAS gene mutations were investigated in formalin-fixed, paraffin-embedded tissues from 143 Chinese female patients with breast cancer by means of real-time quantitative polymerase chain reaction (RT-PCR). RESULTS: Based on RT-PCR, 2/143 (1.4%) samples and 1/143 (0.7%) had EGFR and KRAS gene mutations, respectively. Overall, none of the cases was identified with mutations of both of these two genes. CONCLUSIONS: In this study, both EGFR and KRAS mutations were present rarely in this cohort of samples with breast cancer. This suggested that mutation analyses for EGFR and KRAS are not useful as screening tests for sensitivity to anti-EGFR therapy for breast carcinomas.

Oncogenic KRAS mutations in colorectal cancer (CRC) are associated with lack of benefit from epidermal growth factor receptor (EGFR)-directed antibody (Ab) therapy. However, the mechanisms by which constitutively activated KRAS (KRAS(G12V)) impairs effector mechanisms of EGFR-Abs are incompletely understood. Here, we established isogenic cell line models to systematically investigate the impact of KRAS(G12V) on tumor growth in mouse A431 xenograft models as well as on various modes of action triggered by EGFR-Abs in vitro. KRAS(G12V) impaired EGFR-Ab-mediated growth inhibition by stimulating receptor-independent downstream signaling. KRAS(G12V) also rendered tumor cells less responsive to Fc-mediated effector mechanisms of EGFR-Abs-such as complement-dependent cytotoxicity (CDC) and Ab-dependent cell-mediated cytotoxicity (ADCC). Impaired CDC and ADCC activities could be linked to reduced EGFR expression in KRAS-mutated versus wild-type (wt) cells, which was restored by small interfering RNA (siRNA)-mediated knockdown of KRAS4b. Immunohistochemistry experiments also revealed lower EGFR expression in KRAS-mutated versus KRAS-wt harboring CRC samples. Analyses of potential mechanisms by which KRAS(G12V) downregulated EGFR expression demonstrated significantly decreased activity of six distinct transcription factors. Additional experiments suggested the CCAAT/enhancer-binding protein (C/EBP) family to be implicated in the regulation of EGFR promoter activity in KRAS-mutated tumor cells by suppressing EGFR transcription through up-regulation of the inhibitory family member C/EBPbeta-LIP. Thus, siRNA-mediated knockdown of C/EBPbeta led to enhanced EGFR expression and Ab-mediated cytotoxicity against KRAS-mutated cells. Together, these results demonstrate that KRAS(G12V) signaling induced C/EBPbeta-dependent suppression of EGFR expression, thereby impairing Fc-mediated effector mechanisms of EGFR-Abs and rendering KRAS-mutated tumor cells less sensitive to these therapeutic agents.

The epidermal growth factor receptor (EGFR), a ubiquitously expressed receptor tyrosine kinase, is an important factor in carcinogenesis. Transcriptional intermediary factor 2 (TIF2), a member of the p160 nuclear receptor co-activator gene family, is linked to the proliferation of cancer cells. However, the direct interplay between the EGFR and the nuclear receptors remains unclear. Our previous study demonstrated that nuclear EGFR could directly bind to the cyclin D1 promoter under the regulation of the oncoprotein latent membrane protein 1 (LMP1), but it also indicated that other factors are involved in the activation of target genes. In this study, we found that LMP1 upregulated the expression of TIF2 and promoted the interaction of EGFR with TIF2 in nasopharyngeal carcinoma. Furthermore, we demonstrated that the intact complex was linked with cyclin D1 promoter activity in an LMP1-dependent manner. The physiological functions of the intact complex were associated with cell proliferation and cell cycle progression. These findings suggest that TIF2 is a novel binding partner for nuclear EGFR and is involved in regulating its target gene expression.

Epidermal growth factor receptor (EGFR) is a member of the ErbB family of receptor tyrosine kinases (RTK). EGFR overexpression or mutation in many different forms of cancers has highlighted its role as an important therapeutic target. Gefitinib, the first small molecule inhibitor of EGFR kinase function to be approved for the treatment of nonsmall cell lung cancer (NSCLC) by the FDA, demonstrates clinical activity primarily in patients with tumors that harbor somatic kinase domain mutations in EGFR. Here, we compare wild-type EGFR autophosphorylation kinetics to the L834R (also called L858R) EGFR form, one of the most common mutations in lung cancer patients. Using rapid chemical quench, time-resolved electrospray mass spectrometry (ESI-MS), and Western blot analyses, we examined the order of autophosphorylation in wild-type (WT) and L834R EGFR and the effect of gefitinib (Iressa) on the phosphorylation of individual tyrosines. These studies establish that there is a temporal order of autophosphorylation of key tyrosines involved in downstream signaling for WT EGFR and a loss of order for the oncogenic L834R mutant. These studies also reveal unique signature patterns of drug sensitivity for inhibition of tyrosine autophosphorylation by gefitinib: distinct for WT and oncogenic L834R mutant forms of EGFR. Fluorescence studies show that for WT EGFR the binding affinity for gefitinib is weaker for the phosphorylated protein while for the oncogenic mutant, L834R EGFR, the binding affinity of gefitinib is substantially enhanced and likely contributes to the efficacy observed clinically. This mechanistic information is important in understanding the molecular details underpinning clinical observations as well as to aid in the design of more potent and selective EGFR inhibitors.

Glucose is an essential fuel for cell survival and its availability limits aberrant cellular proliferation. We have hypothesized that specific cancer mutations regulate metabolic response(s) to glucose deprivation (GD). By means of somatic knock-in cellular models, we have analyzed the response to glucose deprivation in cells carrying the frequent (delE746-A750)EGFR, (G13D)KRAS or (E545K)PIK3CA cancer alleles. We demonstrate that, in mammary epithelial cells, glucose has an essential antioxidant function and that these cells are very sensitive to GD. Conversely, isogenic cells carrying the (delE746-A750)EGFR or the (E545K)PIK3CA, but not the (G13D)KRAS allele, display high tolerance to GD by stimulating the expression of anti-oxidant genes (MnSOD and catalase). This adaptive transcriptional response is mediated by the activation of WNT/beta-catenin and FOXO4 signalling. Our data highlights a new functional synergism between oncogenic EGFR and PIK3CA with WNT/beta-catenin conferring high tolerance to oxidative stress generated by nutrient deprivation.

The epidermal growth factor (EGF) receptor (EGFR) has a key role in normal embryonic development, adult tissue homeostasis and many pathological processes, in particular tumour formation. Aberrant EGFR activation occurs in many cancer types, and inhibition of this receptor is a promising anti-tumour strategy. Besides overexpression of the wild-type receptor, mutated oncogenic EGFR variants are often associated with malignant transformation. In human non-small-cell lung cancers, kinase mutants of the EGFR are rather common. Human glioblastoma often express the truncated EGFRvIII version as well as other dimerized and permanently activated mutants of the receptor, which are considered as tumour drivers. Similarly, the mutated and dimerized EGFR variant Xiphophorus melanoma receptor kinase (Xmrk) is causative for the development of malignant pigment cell tumours in medaka and Xiphophorus melanoma models. It is generally believed that oncogenic receptors that are active due to dimerizing mutations are ligand independent. Here, we show that different EGFR variants from fish and human efficiently induce autocrine loops by inducing EGFR ligands such as amphiregulin and HB-EGF. Importantly, the pre-dimerized oncogenic EGFR versions Xmrk from Xiphophorus and human EGFR(C600F), though already active in absence of ligands, respond to ligand stimulation with enhanced oncogenic signalling. In summary, our data show that autocrine or paracrine loops are still acting on pre-dimerized oncogenic EGFRs and contribute to their pro-tumorigenic signalling.

Monoclonal antibodies against the epidermal growth factor receptor (EGFR) are effective cancer therapeutics, but tumors harboring RAS mutations are resistant. To functionally dissect RAS-mediated resistance, we have studied clinically approved anti-EGFR antibodies, cetuximab and panitumumab, in cancer models. Both antibodies were equally cytotoxic in vitro. However, cetuximab, which also triggers antibody-dependent cellular cytotoxicity (ADCC), was more effective than panitumumab in vivo. Oncogenic RAS neutralized the activity of both antibodies in vivo. Mechanistically, RAS upregulated BCL-XL in cancer cell lines and in primary colorectal cancers. Suppression of BCL-XL by short hairpin RNA or treatment with a BH3 mimetic overcame RAS-mediated antibody resistance. In conclusion, RAS-mutant tumors escape anti-EGFR antibody-mediated receptor blockade as well as ADCC in vivo. Pharmacological targeting of RAS effectors can restore sensitivity to antibody therapy.

MicroRNAs (miRNAs) are emerging as cooperating factors that promote the activity of oncogenes in tumor formation and disease progression. This poses the challenge of identifying the miRNA targets responsible for these interactions. In this study, we identify the growth regulatory miRNA bantam and its target, Socs36E, as cooperating factors in EGFR-driven tumorigenesis and metastasis in a Drosophila model of epithelial transformation. bantam promotes growth by limiting expression of Socs36E, which functions as a negative growth regulator. Socs36E has only a modest effect on growth on its own, but behaves as a tumor suppressor in combination with EGFR activation. The human ortholog of SOCS36E, SOCS5, behaves as a candidate tumor suppressor in cellular transformation in cooperation with EGFR/RAS pathway activation.

General properties of the family of tyrosine kinase ERBB receptors are considered in connection with their role in the generation of cascades of signal transduction in normal and tumor cells. Causes of acquisition of oncogene features by genes encoding these receptors and their role in tumorigenesis are analyzed. Anti-ERBB monoclonal antibodies approved for therapy are described in detail, and mechanisms of their antitumor activity and development of resistance to them are reviewed. The existing and the most promising strategies for creating and using monoclonal antibodies and their derivatives for therapy of cancer are discussed.

Increased inducible nitric oxide synthase (NOS2) expression in breast tumors is associated with decreased survival of estrogen receptor negative (ER-) breast cancer patients. We recently communicated the preliminary observation that nitric oxide (NO) signaling results in epidermal growth factor receptor (EGFR) tyrosine phosphorylation. To further define the role of NO in the pathogenesis of ER- breast cancer, we examined the mechanism of NO-induced EGFR activation in human ER- breast cancer. NO was found to activate EGFR and Src by a mechanism that includes S-nitrosylation. NO, at physiologically relevant concentrations, induced an EGFR/Src-mediated activation of oncogenic signal transduction pathways (including c-Myc, Akt, and beta-catenin) and the loss of PP2A tumor suppressor activity. In addition, NO signaling increased cellular EMT, expression and activity of COX-2, and chemoresistance to adriamycin and paclitaxel. When connected into a network, these concerted events link NO to the development of a stem cell-like phenotype, resulting in the upregulation of CD44 and STAT3 phosphorylation. Our observations are also consistent with the finding that NOS2 is associated with a basal-like transcription pattern in human breast tumors. These results indicate that the inhibition of NOS2 activity or NO signaling networks may have beneficial effects in treating basal-like breast cancer patients.

INTRODUCTION: Many patients with oncogene-driven non-small-cell lung cancer (NSCLC) treated with tyrosine kinase inhibitors experience limited sites of disease progression. This study investigated retrospectively the benefits of local ablative therapy (LAT) to central nervous system (CNS) and/or limited systemic disease progression and continuation of crizotinib or erlotinib in patients with metastatic ALK gene rearrangement (ALK+) or EGFR-mutant (EGFR-MT) NSCLC, respectively. METHODS: Patients with metastatic ALK+ NSCLC treated with crizotinib (n = 38) and EGFR-MT NSCLC treated with erlotinib (n = 27) were identified at a single institution. Initial response to the respective kinase inhibitors, median progression-free survival (PFS1), and site of first progression were recorded. A subset of patients with either nonleptomeningeal CNS and/or four sites or fewer of extra-CNS progression (oligoprogressive disease) suitable for LAT received either radiation or surgery to these sites and continued on the same tyrosine kinase inhibitors. The subsequent median progression-free survival from the time of first progression (PFS2) and pattern of progression were recorded. RESULTS: Median progression-free survival in ALK+ patients on crizotinib was 9.0 months, and 13.8 months for EGFR-MT patients on erlotinib. Twenty-five of 51 patients (49%) who progressed were deemed suitable for local therapy (15 ALK+, 10 EGFR-MT; 24 with radiotherapy, one with surgery) and continuation of the same targeted therapy. Post-LAT, 19 of 25 patients progressed again, with median PFS2 of 6.2 months. DISCUSSION: oncogene-addicted NSCLC with CNS and/or limited systemic disease progression (oligoprogressive disease) on relevant targeted therapies is often suitable for LAT and continuation of the targeted agent, and is associated with more than 6 months of additional disease control.

Our laboratory has previously shown that some gefitinib-insensitive head and neck squamous cell carcinoma (HNSCC) cell lines exhibit dominant autocrine fibroblast growth factor receptor (FGFR) signaling. Herein, we deployed a whole-genome loss-of-function screen to identify genes whose knockdown potentiated the inhibitory effect of the FGFR inhibitor, AZ8010, in HNSCC cell lines. Three HNSCC cell lines expressing a genome-wide small hairpin RNA (shRNA) library were treated with AZ8010 and the abundance of shRNA sequences was assessed by deep sequencing. Under-represented shRNAs in treated cells are expected to target genes important for survival with AZ8010 treatment. Synthetic lethal hits were validated with specific inhibitors and independent shRNAs. We found that multiple alternate receptors provided protection from FGFR inhibition, including receptor tyrosine kinases (RTKs), v-erb-b2 erythroblastic leukemia viral oncogene homolog 2 (ERBB2), and hepatocyte growth factor receptor (MET). We showed that specific knockdown of either ERBB2 or MET in combination with FGFR inhibition led to increased inhibition of growth relative to FGFR tyrosine kinase inhibitor (TKI) treatment alone. These results were confirmed using specific small molecule inhibitors of either ERBB family members or MET. Moreover, the triple combination of FGFR, MET, and ERBB family inhibitors showed the largest inhibition of growth and induction of apoptosis compared with the double combinations. These results reveal a role for alternate RTKs in maintaining progrowth and survival signaling in HNSCC cells in the setting of FGFR inhibition. Thus, improved therapies for HNSCC patients could involve rationally designed combinations of TKIs targeting FGFR, ERBB family members, and MET.

The two essential requirements for pathologic specimens in the era of personalized therapies for non-small cell lung carcinoma (NSCLC) are accurate subtyping as adenocarcinoma (ADC) versus squamous cell carcinoma (SqCC) and suitability for EGFR molecular testing, as well as for testing of other oncogenes such as EML4-ALK and KRAS. Actually, the value of EGFR expressed in patients with NSCLC in predicting a benefit in terms of survival from treatment with an epidermal growth factor receptor targeted therapy is still in debate, while there is a convincing evidence on the predictive role of the EGFR mutational status with regard to the response to tyrosine kinase inhibitors (TKIs).This is a literature overview on the state-of-the-art of EGFR oncogenic mutation in NSCLC. It is designed to highlight the preclinical rationale driving the molecular footprint assessment, the progressive development of a specific pharmacological treatment and the best method to identify those NSCLC who would most likely benefit from treatment with EGFR-targeted therapy. This is supported by the belief that a rationale for the prioritization of specific regimens based on patient-tailored therapy could be closer than commonly expected.

BACKGROUND: The prognostic and therapeutic implications of the spectrum of v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) oncogene substitutions in lung cancer remain poorly understood. The objective of this study was to determine whether KRAS oncogene substitutions differed with regard to prognosis or predictive value in lung adenocarcinoma. METHODS: KRAS oncogene substitutions and mutant allele-specific imbalance (MASI) were determined in patients with lung adenocarcinoma, and the associations with overall survival (OS), recurrence-free survival (RFS), and chemotherapy interactions were assessed. RESULTS: KRAS mutational analysis was performed on 988 lung adenocarcinomas, and 318 KRAS mutations were identified. In this predominantly early stage cohort (78.6% of patients had stage I-III disease), OS and RFS did not differ according to the type of KRAS substitution (OS, P = .612; RFS, P = .089). There was a trend toward better OS in the subset of patients with KRAS codon 13 mutations (P = .052), but that trend was not significant in multivariate analysis (P = .076). RFS did not differ according to codon type in univariate analysis (P = .322). There was a marked difference in RFS based on the presence of MASI in univariate analysis (P = .004) and multivariate analysis (P = .009). A test for interaction was performed to determine whether the effect of chemotherapy on OS and RFS differed based on KRAS substitution type, codon type, or the presence of MASI. That test indicated that there were no differences in the effects of chemotherapy for any of variables examined. CONCLUSIONS: KRAS codon 13 mutations and MASI were identified as candidate biomarkers for prognosis, and it may be useful to incorporate them into prospective studies evaluating novel therapies in KRAS-mutant lung adenocarcinoma.

MicroRNAs (miRNAs) are generated by two-step processing to yield small RNAs that negatively regulate target gene expression at the post-transcriptional level. Deregulation of miRNAs has been linked to diverse pathological processes, including cancer. Recent studies have also implicated miRNAs in the regulation of cellular response to a spectrum of stresses, such as hypoxia, which is frequently encountered in the poorly angiogenic core of a solid tumour. However, the upstream regulators of miRNA biogenesis machineries remain obscure, raising the question of how tumour cells efficiently coordinate and impose specificity on miRNA expression and function in response to stresses. Here we show that epidermal growth factor receptor (EGFR), which is the product of a well-characterized oncogene in human cancers, suppresses the maturation of specific tumour-suppressor-like miRNAs in response to hypoxic stress through phosphorylation of argonaute 2 (AGO2) at Tyr 393. The association between EGFR and AGO2 is enhanced by hypoxia, leading to elevated AGO2-Y393 phosphorylation, which in turn reduces the binding of Dicer to AGO2 and inhibits miRNA processing from precursor miRNAs to mature miRNAs. We also identify a long-loop structure in precursor miRNAs as a critical regulatory element in phospho-Y393-AGO2-mediated miRNA maturation. Furthermore, AGO2-Y393 phosphorylation mediates EGFR-enhanced cell survival and invasiveness under hypoxia, and correlates with poorer overall survival in breast cancer patients. Our study reveals a previously unrecognized function of EGFR in miRNA maturation and demonstrates how EGFR is likely to function as a regulator of AGO2 through novel post-translational modification. These findings suggest that modulation of miRNA biogenesis is important for stress response in tumour cells and has potential clinical implications.

In this study we selected three breast cancer cell lines (SKBR3, SUM149 and SUM190) with different oncogene expression levels involved in ERBB2 and EGFR signaling pathways as a model system for the evaluation of selective integration of subsets of transcriptomic and proteomic data. We assessed the oncogene status with reads per kilobase per million mapped reads (RPKM) values for ERBB2 (14.4, 400, and 300 for SUM149, SUM190, and SKBR3, respectively) and for EGFR (60.1, not detected, and 1.4 for the same 3 cell lines). We then used RNA-Seq data to identify those oncogenes with significant transcript levels in these cell lines (total 31) and interrogated the corresponding proteomics data sets for proteins with significant interaction values with these oncogenes. The number of observed interactors for each oncogene showed a significant range, e.g., 4.2% (JAK1) to 27.3% (MYC). The percentage is measured as a fraction of the total protein interactions in a given data set vs total interactors for that oncogene in STRING (Search Tool for the Retrieval of Interacting Genes/Proteins, version 9.0) and I2D (Interologous Interaction Database, version 1.95). This approach allowed us to focus on 4 main oncogenes, ERBB2, EGFR, MYC, and GRB2, for pathway analysis. We used bioinformatics sites GeneGo, pathwayCommons and NCI receptor signaling networks to identify pathways that contained the four main oncogenes and had good coverage in the transcriptomic and proteomic data sets as well as a significant number of oncogene interactors. The four pathways identified were ERBB signaling, EGFR1 signaling, integrin outside-in signaling, and validated targets of C-MYC transcriptional activation. The greater dynamic range of the RNA-Seq values allowed the use of transcript ratios to correlate observed protein values with the relative levels of the ERBB2 and EGFR transcripts in each of the four pathways. This provided us with potential proteomic signatures for the SUM149 and 190 cell lines, growth factor receptor-bound protein 7 (GRB7), Crk-like protein (CRKL) and Catenin delta-1 (CTNND1) for ERBB signaling; caveolin 1 (CAV1), plectin (PLEC) for EGFR signaling; filamin A (FLNA) and actinin alpha1 (ACTN1) (associated with high levels of EGFR transcript) for integrin signalings; branched chain amino-acid transaminase 1 (BCAT1), carbamoyl-phosphate synthetase (CAD), nucleolin (NCL) (high levels of EGFR transcript); transferrin receptor (TFRC), metadherin (MTDH) (high levels of ERBB2 transcript) for MYC signaling; S100-A2 protein (S100A2), caveolin 1 (CAV1), Serpin B5 (SERPINB5), stratifin (SFN), PYD and CARD domain containing (PYCARD), and EPH receptor A2 (EPHA2) for PI3K signaling, p53 subpathway. Future studies of inflammatory breast cancer (IBC), from which the cell lines were derived, will be used to explore the significance of these observations.

There is no available targeted therapy for triple-negative or its more aggressive subtype, basal-like breast cancer. Multiple therapeutic strategies based on translational knowledge have not improved the treatment options for triple negative patients. As understanding of molecular pathways that drive tumor development is rapidly increasing, it is imperative to adapt our treatment strategies to perturbations in molecular pathways driving the malignant process. Basal-like breast cancers over-express EGFR (without mutations or EGFR gene amplifications) and have p53 mutations. While EGFR drives the malignant behavior in triple negative breast cancer (TNBC), anti-EGFR therapies have fallen short of the expected results in clinical trials. Here we bring evidence that the less than optimal results of the anti-EGFR therapies may be explained in part by the increased potency of the EGFR signaling due to increased endosomal recycling. The functional connection between EGFR and endosomal trafficking in TNBC is mutant p53 found in the most aggressive forms of TNBC. Mutant p53 acquires oncogenic functions and binds p63 protein, a member of p53 family with tumor suppressor activities. In the absence of functional p63 there is an upregulation of endosomal recycling EGFR and integrin to the membrane with increased proinvasive abilities of cancer cells. Blocking endosomal trafficking combined with anti-EGFR treatments may result in better clinical outcomes in TNBC.

BACKGROUND: Lung cancers in women, in nonsmokers, and in patients with adenocarcinoma from Asia have more prevalent mutations in the epidermal growth factor receptor (EGFR) gene than their counterparts. However, the etiology of EGFR mutations in this population remains unclear. The authors hypothesized that the human papillomavirus (HPV) type 16/18 (HPV16/18) E6 oncoprotein may contribute to EGFR mutations in Taiwanese patients with lung cancer. METHODS: One hundred fifty-one tumors from patients with lung cancer were enrolled to determine HPV16/18 E6 and EGFR mutations using immunohistochemistry and direct sequencing, respectively. Levels of 8-oxo-7,8-dihydro-2 -deoxyguanosine (8-oxo-dG) in lung tumors and cells were evaluated using immunohistochemistry and liquid chromatography-mass spectrometry/mass spectrometry. An supF mutagenesis assay was used to determine H2 O2 -induced mutation rates of lung cancer cells with or without E6 expression. RESULTS: Patients with E6-positive tumors had a greater frequency of EGFR mutations than those with E6-negative tumors (41% vs 20%; P = .006). Levels of 8-oxo-dG were correlated with EGFR mutations (36% vs 16%; P = .012). Two stable clones of E6-overexpressing H157 and CL-3 cells were established for the supF mutagenesis assay. The data indicated that the cells with high E6 overexpression had higher H2 O2 -induced SupF gene mutation rates compared with the cells that expressed lower levels of E6 and compared with vector control cells. CONCLUSIONS: HPV16/18 E6 may contribute in part to EGFR mutations in lung cancer, at least in the Taiwanese population.

Deletion of 3p12-22 is frequent in multiple cancer types, indicating the presence of critical tumor-suppressor genes (TSGs) at this region. We studied a novel candidate TSG, CMTM7, located at the 3p22.3 CMTM-gene cluster, for its tumor-suppressive functions and related mechanisms. The three CMTM genes, CMTM6, 7 and 8, are broadly expressed in human normal adult tissues and normal epithelial cell lines. Only CMTM7 is frequently silenced or downregulated in esophageal and nasopharyngeal cell lines, but uncommon in other carcinoma cell lines. Immunostaining of tissue microarrays for CMTM7 protein showed its downregulation or absence in esophageal, gastric, pancreatic, liver, lung and cervix tumor tissues. Promoter CpG methylation and loss of heterozygosity were both found contributing to CMTM7 downregulation. Ectopic expression of CMTM7 in carcinoma cells inhibits cell proliferation, motility and tumor formation in nude mice, but not in immortalized normal cells, suggesting a tumor inhibitory role of CMTM7. The tumor-suppressive function of CMTM7 is associated with its role in G1/S cell cycle arrest, through upregulating p27 and downregulating cyclin-dependent kinase 2 (CDK2) and 6 (CDK6). Moreover, CMTM7 could promote epidermal growth factor receptor (EGFR) internalization, and further suppress AKT signaling pathway. Thus, our findings suggest that CMTM7 is a novel 3p22 tumor suppressor regulating G1/S transition and EGFR/AKT signaling during tumor pathogenesis.

Despite the progress made in targeted anticancer therapies in recent years, challenges remain. The identification of new potential targets will ensure that the arsenal of cancer therapies continues to expand. FAM83B was recently discovered in a forward genetic screen for novel oncogenes that drive human mammary epithelial cell (HMEC) transformation. We report here that elevated FAM83B expression increases Phospholipase D (PLD) activity, and that suppression of PLD1 activity prevents FAM83B-mediated transformation. The increased PLD activity is engaged by hyperactivation of epidermal growth factor receptor (EGFR), which is regulated by an interaction involving FAM83B and EGFR. Preventing the FAM83B/EGFR interaction by site-directed mutation of lysine 230 of FAM83B suppressed PLD activity and MAPK signaling. Furthermore, ablation of FAM83B expression from breast cancer cells inhibited EGFR phosphorylation and suppressed cell proliferation. We propose that understanding the mechanism of FAM83B-mediated transformation will provide a foundation for future therapies aimed at targeting its function as an intermediary in EGFR, MAPK and mTOR activation.

Anticancer agents that have minimal effects on normal cells and tissues are ideal cancer drugs. Here, we show specific inhibition of human cancer cells carrying oncogenic mutations in the epidermal growth factor receptor (EGFR) gene by means of oncogenic allele-specific RNA interference (RNAi), both in vivo and in vitro. The allele-specific RNAi (ASP-RNAi) treatment did not affect normal cells or tissues that had no target oncogenic allele, whereas the suppression of a normal EGFR allele by a conventional in vivo RNAi caused adverse effects, i.e., normal EGFR is vital. Taken together, our current findings suggest that specific inhibition of oncogenic EGFR alleles without affecting the normal EGFR allele may provide a safe treatment approach for cancer patients and that ASP-RNAi treatment may be capable of becoming a safe and effective, anticancer treatment method.

The success in lung cancer therapy with programmed death (PD)-1 blockade suggests that immune escape mechanisms contribute to lung tumor pathogenesis. We identified a correlation between EGF receptor (EGFR) pathway activation and a signature of immunosuppression manifested by upregulation of PD-1, PD-L1, CTL antigen-4 (CTLA-4), and multiple tumor-promoting inflammatory cytokines. We observed decreased CTLs and increased markers of T-cell exhaustion in mouse models of EGFR-driven lung cancer. PD-1 antibody blockade improved the survival of mice with EGFR-driven adenocarcinomas by enhancing effector T-cell function and lowering the levels of tumor-promoting cytokines. expression of mutant EGFR in bronchial epithelial cells induced PD-L1, and PD-L1 expression was reduced by EGFR inhibitors in non-small cell lung cancer cell lines with activated EGFR. These data suggest that oncogenic EGFR signaling remodels the tumor microenvironment to trigger immune escape and mechanistically link treatment response to PD-1 inhibition. SIGNIFICANCE: We show that autochthonous EGFR-driven lung tumors inhibit antitumor immunity by activating the PD-1/PD-L1 pathway to suppress T-cell function and increase levels of proinflammatory cytokines. These findings indicate that EGFR functions as an oncogene through non-cell-autonomous mechanisms and raise the possibility that other oncogenes may drive immune escape.

Genes functioning in epidermal growth factor receptor (EGFR) signaling pathways are among the most frequently activated oncogenes in human cancers. We have conducted a comparative analysis of functional footprints (that is, effect on signaling and transcriptional landscapes in cells) associated with oncogenic and tumor suppressor mutations in EGFR pathway genes in human cancers. We have found that mutations in the EGFR pathway differentially have an impact on signaling and metabolic pathways in cancer cells in a mutation- and tissue-selective manner. For example, although signaling and metabolic profiles of breast tumors with PIK3CA or AKT1 mutations are, as expected, highly similar, they display markedly different, sometimes even opposite, profiles to those with ERBB2 or EGFR amplifications. On the other hand, although low-grade gliomas and glioblastomas, both brain cancers, driven by EGFR amplifications are highly functionally similar, their functional footprints are significantly different from lung and breast tumors driven by EGFR or ERBB2. Overall, these observations argue that, contrary to expectations, the mechanisms of tumorigenicity associated with mutations in different genes along the same pathway, or in the same gene across different tissues, may be highly different. We present evidence that oncogenic functional footprints in cancer cell lines have significantly diverged from those in tumor tissues, which potentially explains the discrepancy of our findings with the current knowledge. Nevertheless, our analyses reveal a common inflammatory response signature in EGFR-driven human cancers of different tissue origins. Our results may have implications in the design of therapeutic strategies in cancers driven by these oncogenes.

Site-specific antibodies to the src-homologous domain (residues 373-383) of the erbB gene product neutralized the tyrosine kinase activity of the epidermal growth factor receptor, suggesting that the region against which the antibodies were directed may be functionally important for the kinase activity. In the immunofluorescence experiment, the site-specific antibodies detected the epidermal growth factor receptor and the erbB gene product only when the cells were permeabilized prior to staining, while monoclonal anti-epidermal growth factor receptor antibody, which recognizes the epidermal growth factor binding domain, gave a positive surface stain with viable nonpermeabilized A431 cells. This result supports the view that the epidermal growth factor binding domain and the src-homologous domain are located at the cell surface and inner face of the plasma membrane, respectively.

BACKGROUND: Anaplastic lymphoma kinase-positive non-small cell lung carcinoma patients are generally highly responsive to the dual anaplastic lymphoma kinase and MET tyrosine kinase inhibitor crizotinib. However, they eventually acquire resistance to this drug, preventing the anaplastic lymphoma kinase inhibitors from having a prolonged beneficial effect. The molecular mechanisms responsible for crizotinib resistance are beginning to emerge, e.g., in some anaplastic lymphoma kinase-positive non-small cell lung carcinomas the development of secondary mutations in this gene has been described. However, the events behind crizotinib-resistance currently remain largely uncharacterized. Thus, we report on an anaplastic lymphoma kinase-positive non-small cell lung carcinoma patient with concomitant occurrence of epidermal growth factor receptor and V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog mutations upon development of crizotinib-resistance. CASE PRESENTATION: A 61-year-old Caucasian never-smoking male was diagnosed with anaplastic lymphoma kinase -positive pulmonary adenocarcinoma, stage T4N3M1b. Treatment with crizotinib initially resulted in complete objective response in the thorax and partial response in the abdomen, but after 8 months of therapy the patient acquired resistance and progressed. Biopsies from new metastases revealed development of epidermal growth factor receptor and V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog mutations concomitant with the original anaplastic lymphoma kinase gene rearrangement and without signs of anaplastic lymphoma kinase fusion gene amplification or secondary anaplastic lymphoma kinase mutations. CONCLUSION: To our knowledge, this is the first report of an anaplastic lymphoma kinase-positive pulmonary adenocarcinoma, which upon emergence of crizotinib resistance acquired 2 new somatic mutations in the epidermal growth factor receptor and V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog genes, respectively, concomitant with the original anaplastic lymphoma kinase rearrangement. Thus, these 3 driver mutations, usually considered mutually exclusive, may coexist in advanced non-small cell lung carcinoma that becomes resistant to crizotinib, presumably because heterogeneous tumor clones utilize epidermal growth factor receptor and/or V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog signaling to circumvent the inhibition of anaplastic lymphoma kinase-mediated signaling by crizotinib. The identification of new targetable somatic mutations by tumor re-biopsy may help clarify the mechanism behind the development of the acquired crizotinib resistance and pave the way for combined strategies involving multiple targeted therapies.

Activating K-Ras mutations and inactivating mutations of Smad4 are two common genetic alterations that occur in the development and progression of pancreatic ductal adenocarcinomas (PDAC). To further study the individual and combinatorial roles of these two mutations in the pathogenesis of PDAC, immortalized human pancreas nestin postive cells (HPNE) were genetically modified by either expressing oncogenic K-Ras (HPNE/K-Ras), by shRNA knock down of Smad4 (HPNE/ShSmad4) or by creating both alterations in the same cell line (HPNE/K-Ras/ShSmad4). We previously found that expression of oncogenic K-Ras caused an increase in expression of EGFR and loss of Smad4 further enhanced the up regulation in expression of EGFR and that this increase in EGFR was sufficient to induce invasion. Here we further investigated the mechanism that links mutational alterations and EGFR expression with invasion. The increase in EGFR signaling was associated with up regulation of MMP9 and uPA protein and activity. Moreover, the increase in EGFR signaling promoted a nuclear translocation and binding of RelA (p65), a subunit of NF-kappaB, to the promoters of both MMP-9 and uPA. Treatment of HPNE/K-Ras/ShSmad4 cells with an inhibitor of EGFR reduced EGF-mediated NF-kappaB nuclear translocation and inhibitors of either EGFR or NF-kappaB reduced the increase in MMP-9 or uPA expression. In conclusion, this study provides the mechanism of how a combination of oncogenic K-Ras and loss of Smad4 causes invasion and provides the basis for new strategies to inhibit metastases.

BACKGROUND: The dismal outcome of malignant peripheral nerve sheath tumor (MPNST) highlights the necessity of finding new therapeutic methods to benefit patients with this aggressive sarcoma. Our purpose was to investigate epidermal growth factor receptor (EGFR) as a potential therapeutic target in MPNSTs. PATIENTS AND METHODS: We performed a microarray based-comparative genomic hybridization (aCGH) profiling of two cohorts of primary MPNST tissue samples including 25 patients treated at The University of Texas MD Anderson cancer Center (MD Anderson) and 26 patients from Tianjin Medical University cancer Institute & Hospital (TMUCIH). Fluorescence in situ hybridization (FISH) method was used to validate the gene amplification detected by aCGH analysis. Another independent cohort of 56 formalin fixed paraffin embedded (FFPE) MPNST samples was obtained to explore EGFR protein expression by immunohistochemical analysis. Cell biology detection and validation were performed on human MPNST cell lines ST88-14 and STS26T. RESULTS: aCGH and pathway analysis of the 51 MPNSTs identified significant gene amplification events in EGFR pathway, including frequent amplifications of EGFR gene itself, which was subsequently validated by FISH assay. High expression of EGFR protein was associated with poor disease-free and overall survival of human MPNST patients. In human MPNST cell lines ST88-14 and STS26T, inhibition of EGFR by siRNA or Gefitinib led to decreased cell proliferation, migration, and invasion accompanied by attenuation of PI3K/AKT and MAPK pathways. CONCLUSION: These results suggest that EGFR is a potential therapeutic target for MPNST.

The diagnostic testing, treatment and prognosis of non-small cell lung cancer (NSCLC) has undergone a paradigm shift since the discovery of sensitizing mutations in the epidermal growth factor receptor (EGFR) gene in a subset of NSCLC patients. Several additional oncogenic mutations, including gene fusions and amplifications, have since been discovered, with a number of drugs that target each specific oncogene. This review focuses on oncogenes in NSCLC other than EGFR and their companion "targeted therapies." Particular emphasis is placed on the role of ALK, ROS1, RET, MET, BRAF, and HER2 in NSCLC.

OBJECTIVE: The oncoprotein, gankyrin, is known to facilitate cell proliferation through phosphorylation and degradation of retinoblastoma protein. In the present study, we evaluated the expression of gankyrin and phosphorylated retinoblastoma protein in human testis and testicular germ cell tumors. METHODS: The effects of suppression of gankyrin by locked nucleic acid on phosphorylation status of retinoblastoma and cell proliferation were analyzed using western blot analysis and testicular tumor cell line NEC8. The expressions of gankyrin, retinoblastoma and retinoblastoma protein were analyzed in 93 testicular germ cell tumor samples and five normal human testis by immunohistochemistry. The retinoblastoma protein expression was determined using an antibody to retinoblastoma protein, Ser795. RESULTS: Gankyrin was expressed in NEC8 cells as well as a normal human testis and testicular tumors. Suppression of gankyrin by locked nucleic acid led to suppression of retinoblastoma protein and cell proliferation in NEC8 cells. Immunohistochemistry of normal testis showed that gankyrin is expressed dominantly in spermatocytes. In testicular germ cell tumors, high expressions of gankyrin and phosphorylated-retinoblastoma protein were observed in seminoma and embryonal carcinoma, whereas the expressions of both proteins were weak in histological subtypes of non-seminoma. Growing teratoma and testicular malignant transformation tissues expressed phosphorylated-retinoblastoma protein strongly, but gankyrin faintly. CONCLUSION: Gankyrin is dominantly expressed in normal spermatocytes and seminoma/embryonal carcinoma, and its expression correlates well with retinoblastoma protein expression except in the growing teratoma and testicular malignant transformation cases. These data provide new insights into the molecular mechanisms of normal spermatogenesis and pathogenesis of testicular germ cell tumors.

It is known that human papillomavirus (HPV) infection can cause squamous cell neoplasms at several sites, such as cervix uteri carcinoma and oral squamous carcinoma. There is little information on the expression of HPV and its predictive markers in tumours of the major and minor salivary glands of the head and neck. We therefore assessed oral salivary gland neoplasms to identify associations between HPV and infection-related epidermal growth factor receptor (EGFR), cyclin-dependent kinase inhibitor 2A (CDKN2A/p16) and tumour protein p53 (TP53). Formalin-fixed, paraffin-embedded tissue samples from oral salivary gland carcinomas (n=51) and benign tumours (n=26) were analysed by polymerase chain reaction (PCR) analysis for several HPV species, including high-risk types 16 and 18. Evaluation of EGFR, CDKN2A, TP53 and cytomegalovirus (CMV) was performed by immunohistochemistry. Epstein-Barr virus (EBV) was evaluated by EBV-encoded RNA in situ hybridisation. We demonstrated that salivary gland tumours are not associated with HPV infection. The expression of EGFR, CDKN2A and TP53 may be associated with tumour pathology but is not induced by HPV. CMV and EBV were not detectable. In contrast to oral squamous cell carcinomas, HPV, CMV and EBV infections are not associated with malignant or benign neoplastic lesions of the salivary glands.

Grade IV glioblastoma is characterized by increased kinase activity of epidermal growth factor receptor (EGFR); however, EGFR kinase inhibitors have failed to improve survival in individuals with this cancer because resistance to these drugs often develops. We showed that tumor necrosis factor-alpha (TNFalpha) produced in the glioblastoma microenvironment activated atypical protein kinase C (aPKC), thereby producing resistance to EGFR kinase inhibitors. Additionally, we identified that aPKC was required both for paracrine TNFalpha-dependent activation of the transcription factor nuclear factor kappaB (NF-kappaB) and for tumor cell-intrinsic receptor tyrosine kinase signaling. Targeting aPKC decreased tumor growth in mouse models of glioblastoma, including models of EGFR kinase inhibitor-resistant glioblastoma. Furthermore, aPKC abundance and activity were increased in human glioblastoma tumor cells, and high aPKC abundance correlated with poor prognosis. Thus, targeting aPKC might provide an improved molecular approach for glioblastoma therapy.

BACKGROUND: To understand the mechanism of frequent and early lymph node metastasis in high-risk human papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma (OPSCC), this study investigated whether beta-catenin is regulated by the HPV oncoprotein and contributes to OPSCC metastasis. METHODS: expression levels of p16, beta-catenin, and epidermal growth factor receptor (EGFR) were examined in OPSCC samples (n = 208) by immunohistochemistry. The expression and subcellular localization of beta-catenin and EGFR activation were also studied in HPV-positive and HPV-negative head and neck squamous cell carcinoma cell lines with western blot analysis. HPV16 E6 small interfering RNA was used to elucidate the effect of the HPV oncoprotein on beta-catenin translocation. The involvement of EGFR in beta-catenin translocation was confirmed by treatment with erlotinib. Moreover, the invasive capacity was evaluated after HPV16 E6/E7 repression. RESULTS: The results showed that the membrane weighted index of beta-catenin was inversely correlated with p16 positivity (P < .001) and lymph node metastasis (P = .026), whereas nuclear staining of beta-catenin was associated with p16-positive OPSCC (P < .001). A low level of membrane beta-catenin expression was significantly associated with disease-free and overall survival (P < .0001 in both cases). Furthermore, the membrane weighted index of EGFR was inversely correlated with p16 positivity (P < .001) and positively correlated with membrane beta-catenin (P < .001). The in vitro study showed that HPV16 E6 repression led to reductions of phospho-EGFR and nuclear beta-catenin, which were also observed after erlotinib treatment, and inhibition of invasion. CONCLUSIONS: The findings suggest that HPV16 E6 mediates the translocation of beta-catenin to the nucleus, which may be regulated by activated EGFR.

INTRODUCTION: The EGFR plays a central role in regulating cancer cell growth and survival, representing an attractive therapeutic target in NSCLC. AREAS COVERED: For the purpose of this review article, data from Phase II and III trials with anti-EGFR agents, including EGFR-tyrosine kinase inhibitors (TKIs) and mAbs, were collected and analysed. EXPERT OPINION: Eight large Phase III trials demonstrated that EGFR-TKIs are the best option we can offer today as front-line therapy exclusively in EGFR mutant NSCLC. In patients with EGFR wild type or unknown lung cancer, platinum-based chemotherapy remains the standard of care, with no consistent benefit produced by the addition of an anti-EGFR treatment. In pretreated NSCLC, EGFR-TKIs are considered more effective than standard monotherapy with cytotoxics in presence of classical EGFR mutations, whereas in the EGFR wild-type population, a similar efficacy with docetaxel or pemetrexed in terms of survival has been demonstrated. New agents targeting EGFR are under investigation, particularly in individuals with squamous cell histology and those with acquired resistance to EGFR-TKIs.

mutations in the epidermal growth factor receptor (EGFR) kinase domain occur in 10-30% of lung adenocarcinoma and are associated with tyrosine kinase inhibitor (TKI) sensitivity. We sought to identify the immediate direct and indirect phosphorylation targets of mutant EGFRs in lung adenocarcinoma. We undertook SILAC strategy, phosphopeptide enrichment, and quantitative MS to identify dynamic changes of phosphorylation downstream of mutant EGFRs in lung adenocarcinoma cells harboring EGFR(L858R) and EGFR(L858R/T790M) , the TKI-sensitive, and TKI-resistant mutations, respectively. Top canonical pathways that were inhibited upon erlotinib treatment in sensitive cells, but not in the resistant cells include EGFR, insulin receptor, hepatocyte growth factor, mitogen-activated protein kinase, mechanistic target of rapamycin, ribosomal protein S6 kinase beta 1, and Janus kinase/signal transducer and activator of transcription signaling. We identified phosphosites in proteins of the autophagy network, such as ULK1 (S623) that is constitutively phosphorylated in these lung adenocarcinoma cells; phosphorylation is inhibited upon erlotinib treatment in sensitive cells, but not in resistant cells. Finally, kinase-substrate prediction analysis from our data indicated that substrates of basophilic kinases from, AGC and Calcium and calmodulin-dependent kinase groups, as well as STE group kinases were significantly enriched and those of proline-directed kinases from, CMGC and Casein kinase groups were significantly depleted among substrates that exhibited increased phosphorylation upon EGF stimulation and reduced phosphorylation upon TKI inhibition. This is the first study to date to examine global phosphorylation changes upon erlotinib treatment of lung adenocarcinoma cells and results from this study provide new insights into signaling downstream of mutant EGFRs in lung adenocarcinoma. ALL MS data have been deposited in the ProteomeXchange with identifier PXD001101 (http://proteomecentral.proteomexchange.org/dataset/PXD001101).

expression of the oncogene hepatocyte growth factor receptor (MET) and phosphorylation of the MET protein have been associated with both primary and acquired resistance to tyrosine kinase inhibitors (TKIs) used in therapy targeting the epidermal growth factor receptor (EGFR) in patients with non-small cell lung cancers (NSCLCs). Therefore, simultaneous inhibition of both of these receptor tyrosine kinases (RTKs) should improve disease treatment. Our previous study of microRNA (miRNA) expression signatures of lung squamous cell carcinoma (lung-SCC) revealed that microRNA-206 (miR206) was significantly reduced in lung-SCC tissues, suggesting that miR206 functions as a tumor suppressor in the disease. Furthermore, putative miR206 binding sites were annotated in the 3 -UTRs of MET and EGFR RTKs in miRNA databases. The aim of the study was to investigate the functional significance of miR206 in lung-SCC and to confirm the inhibition of both MET and EGFR oncogenic signaling by expression of miR206 in cancer cells. We found that restoration of mature miR206 inhibited cancer cell proliferation, migration, and invasion in EBC-1 cells through downregulation of both mRNA and protein levels of MET and EGFR. Interestingly, phosphorylation of ERK1/2 and AKT signaling were inhibited by restoration of miR206 in cancer cells. Overexpression of MET and EGFR were observed in clinical specimens of lung-SCC. tumor-suppressive miR206 inhibited dual signaling networks activated by MET and EGFR, and these findings will provide new insights into the novel molecular mechanisms of lung-SCC oncogenesis and new therapeutic approaches for the treatment of this disease.

We studied the effects of herpes simplex virus type 1 (HSV-1) inoculation and topical 7,12-dimethylbenz[a]anthracene (DMBA) application, alone or in combination, on the carcinogenesis and on the amplification and expression of various cellular proto-oncogenes in hamster buccal pouch tissue. Topical DMBA treatment produced tumor formation in pouches, but HSV-1 inoculation, alone caused no neoplastic changes. In pouch tissues receiving both DMBA application and HSV-1 inoculation, the development of initial leukoplakia and tumor has hastened and enhanced in comparison with those receiving DMBA alone. Topical DMBA application to pouch tissue induced an amplification and an increase in the expression of cellular erb-B-1 (c-erb-B-1) proto-oncogene in the epithelial tissue, whereas repeated infection with HSV-1 alone did not. Topical DMBA combined with HSV-1 inoculation, however, resulted in greater amplification and expression of c-erb-B-1 proto-oncogene in the pouch epithelial tissue compared to the DMBA alone. These data indicate that HSV-1 inoculation significantly increases the carcinogenic activity of DMBA, in part, by probably enhancing DMBA-induced amplification and expression of c-erb-B-1 proto-oncogene in hamster buccal pouch tissue.

Constitutive activation of EGFR due to overexpression or mutation in tumor cells leads to dysregulated downstream cellular signaling pathways. Therefore, EGFR as well as its downstream effectors have been identified as important therapeutic targets. The FDA-approved small-molecule inhibitors of EGFR, gefitinib (Iressa) and erlotinib (Tarceva), are clinically effective in a subset of patients with non-small cell lung cancer (NSCLC) whose tumors harbor activating mutations within the kinase domain of EGFR. The current study examined effects of these drugs in 32D cells expressing native (WT) or oncogenic (L858R) EGFR as well as in cancer cell lines A431 and H3255. Distinct patterns for gefitinib and erlotinib inhibition of EGFR autophosphorylation at individual tyrosines were revealed for wild-type (WT) and L858R EGFR. Phosphorylation of Y845 has been shown to be important in cancer cells and Y1045 phosphorylation is linked to Cbl-mediated ubiquitination and degradation. Dramatic differences were observed by greater potency of these drugs for inhibiting downstream effectors for L858R EGFR including Cbl and STAT5. Selective targeting of Cbl may play a role in oncogene addiction and effects on STAT5 identify features of signaling circuitry for L858R EGFR that contribute to drug sensitivity and clinical efficacy. These data provide new understanding of the EGFR signaling environment and suggest useful paradigms for predicting patient response to EGFR-targeted therapy as well as combination treatments. IMPLICATIONS: This study offers fundamental insights for understanding molecular mechanisms of drug sensitivity on oncogenic forms of EGFR and downstream signaling components as well as considerations for further drug optimization and design of combination therapy.

It is a matter of debate whether the amplification of c-erbB-2 oncogene or production of the oncoprotein in breast cancers correlate with the presence of lymph node metastasis and with a poor prognosis. This study was aimed at elucidating the immunohistochemical localization of oncogene products which are related to cell growth, c-erbB-2 product, epidermal growth factor receptor (EGFR), c-myc protein and estrogen receptor (ER), in benign and malignant lesions of the breast. Fresh frozen sections of 25 breast cancers and 11 fibroadenomas from Japanese women were studied by indirect immunoperoxidase method with proper fixation. C-erbB-2 product and EGFR were localized on the cell membrane whereas c-myc protein and ER were observed in the nuclei. Immunohistochemical expression of oncogene products and ER were not only observed in the mammary carcinomas but also in the fibroadenomas. However immunoreactivities of EGFR and ER were more frequently seen in the fibroadenomas (p less than 0.05). In breast cancers, the incidence of immunoreactivity for c-erbB-2 was higher in the cases with lymph node metastasis than cases without nodal metastasis (p less than 0.05) and there was reciprocal correlation between the expressions of EGFR and ER (p less than 0.05). Regarding the size of the primary tumour, there was no statistically significant correlation with the expressions of c-erbB-2, EGFR, c-myc or ER. Histological grade correlated only with the expression of ER (p less than 0.05).

BRAF and NRAS are the two most frequent oncogenic driver mutations in melanoma and are pivotal components of both the EGF and FGF signaling network. Accordingly, we investigated the effect of BRAF and NRAS oncogenic mutation on the response to the stimulation and inhibition of epidermal and fibroblast growth factor receptors in melanoma cells. In the three BRAF mutant, two NRAS mutant and two double wild-type cell lines growth factor receptor expression had been verified by qRT-PCR. Cell proliferation and migration were determined by the analysis of 3-days-long time-lapse videomicroscopic recordings. Of note, a more profound response was found in motility as compared to proliferation and double wild-type cells displayed a higher sensitivity to EGF and FGF2 treatment when compared to mutant cells. Both baseline and induced activation of the growth factor signaling was assessed by immunoblot analysis of the phosphorylation of the downstream effectors Erk1/2. Low baseline and higher inducibility of the signaling pathway was characteristic in double wild-type cells. In contrast, oncogenic BRAF or NRAS mutation did not influence the response to EGF or FGF receptor inhibitors in vitro. Our findings demonstrate that the oncogenic mutations in melanoma have a profound impact on the motogenic effect of the activation of growth factor receptor signaling. Since emerging molecularly targeted therapies aim at the growth factor receptor signaling, the appropriate mutational analysis of individual melanoma cases is essential in both preclinical studies and in the clinical trials and practice.

The aim of this study was to determine the distribution of known oncogenic driver mutations in female never-smoker Asian patients with lung adenocarcinoma. We analyzed 214 mutations across 26 lung cancer-associated genes and three fusion genes using the MassARRAY LungCarta Panel and the ALK, ROS1, and RET fusion assays in 198 consecutively resected lung adenocarcinomas from never-smoker females at a single institution. EGFR mutation, which was the most frequent driver gene mutation, was detected in 124 (63%) cases. mutation of ALK, KRAS, PIK3CA, ERBB2, BRAF, ROS1, and RET genesoccurred in 7%, 4%, 2.5%, 1.5%, 1%, 1%, and 1% of cases, respectively. Thus, 79% of lung adenocarcinomas from never-smoker females harbored well-known oncogenic mutations. Mucinous adenocarcinomas tended to have a lower frequency of known driver gene mutations than other histologic subtypes. EGFR mutation was associated with older age and a predominantly acinar pattern, while ALK rearrangement was associated with younger age and a predominantly solid pattern. Lung cancer in never-smoker Asian females is a distinct entity, with the majority of these cancers developing from oncogenic mutations.

Organ shaping and patterning depends on the coordinated regulation of multiple processes. The Drosophila compound eye provides an excellent model to study the coordination of cell fate and cell positioning during morphogenesis. Here, we find that loss of vav oncogene function during eye development is associated with a disorganised retina characterised by the presence of additional cells of ALL types. We demonstrate that these defects result from two distinct roles of Vav. First, and in contrast to its well-established role as a positive effector of the EGF receptor (EGFR), we show that readouts of the EGFR pathway are upregulated in vav mutant larval eye disc and pupal retina, indicating that Vav antagonises EGFR signalling during eye development. Accordingly, decreasing EGFR signalling in vav mutant eyes restores retinal organisation and rescues most vav mutant phenotypes. Second, using live imaging in the pupal retina, we observe that vav mutant cells do not form stable adherens junctions, causing various defects, such as recruitment of extra primary pigment cells. In agreement with this role in junction dynamics, we observe that these phenotypes can be exacerbated by lowering DE-Cadherin or Cindr levels. Taken together, our findings establish that Vav acts at multiple times during eye development to prevent excessive cell recruitment by limiting EGFR signalling and by regulating junction dynamics to ensure the correct patterning and morphogenesis of the Drosophila eye.

Although oncogene-targeted therapy often elicits profound initial tumor responses in patients, responses are generally incomplete because some tumor cells survive initial therapy as residual disease that enables eventual acquired resistance. The mechanisms underlying tumor cell adaptation and survival during initial therapy are incompletely understood. Here, through the study of EGFR mutant lung adenocarcinoma, we show that NF-kappaB signaling is rapidly engaged upon initial EGFR inhibitor treatment to promote tumor cell survival and residual disease. EGFR oncogene inhibition induced an EGFR-TRAF2-RIP1-IKK complex that stimulated an NF-kappaB-mediated transcriptional survival program. The direct NF-kappaB inhibitor PBS-1086 suppressed this adaptive survival program and increased the magnitude and duration of initial EGFR inhibitor response in multiple NSCLC models, including a patient-derived xenograft. These findings unveil NF-kappaB activation as a critical adaptive survival mechanism engaged by EGFR oncogene inhibition and provide rationale for EGFR and NF-kappaB co-inhibition to eliminate residual disease and enhance patient responses.

Members of the EGFR family are potent mediators of normal cell growth and development. HER2 possesses an active tyrosine kinase domain, but no direct ligand has been identified. To investigate the differential effect of HER2 in breast cell lines, HER2 was overexpressed in MCF-10A, MCF7 and MDA-MB-231 cells. HER2 overexpression promoted proliferation, survival and migration in MCF-10A and MCF-7 cells. No significant differences were seen in proliferation, survival or migration between MDA-MB-231 vec and HER2 cells. The activity of downstream HER2 proteins increased in MCF-10A HER2 and MCF-7 HER2 cells but not in MDA-MB-231 HER2 cells. Exogenously expressed HER2 failed to associate with EGFR or HER3 in MDA-MB-231 cells, while overexpression of HER2 enhanced HER family dimerization in MCF-10A and MCF-7 cells.

Pleckstrin homology-like domain family A member 2 (PHLDA2) is located within the tumor suppressor region of 11p15, and its expression is suppressed in several malignant tumor types. We recently identified PHLDA2 as a robustly induced, novel downstream target of oncogenic EGFR/ErbB2 signaling. In an immunohistochemical study, we find that PHLDA2 protein expression correlates positively with AKT activation in human lung cancers corroborating our data that PHLDA2 is induced upon oncogenic activation and might serve as a biomarker for AKT pathway activation. We show that PHLDA2 overexpression inhibits AKT phosphorylation while decreased PHLDA2 expression increases AKT activity. We further find that PHLDA2 competes with the PH domain of AKT for binding of membrane lipids, thereby directly inhibiting AKT translocation to the cellular membrane and subsequent activation. Indeed, PHLDA2 overexpression suppresses anchorage-independent cell growth and decreased PHLDA2 expression results in increased cell proliferation and reduced sensitivity to targeted agents of EGFR/ErbB2-driven cancers demonstrating functional relevance for this interaction. In summary, our studies demonstrate that PHLDA2 is strongly regulated by EGFR/ErbB2 signaling and inhibits cell proliferation via repressing AKT activation in lung cancers in a negative feedback loop. We highlight a novel action for PHLDA2 as a potential biomarker for AKT pathway activation.

Somatic, activating mutations in EGFR identify a significant minority of patients with non-small cell lung cancer (NSCLC). Although these mutations are associated with an approximately 70% response rate to some EGFR tyrosine kinase inhibitors (gefitinib, erlotinib, and afatinib), patients develop resistance (i.e., "acquired resistance") after a median of 9 to 12 months. In patients with clinical acquired resistance, repeat biopsy of tumors has identified a number of relevant mechanisms of resistance, but by far the most frequent event is the acquisition of EGFR T790M, a mutation in the "gatekeeper" residue that confers resistance to gefitinib, erlotinib, and afatinib. This emphasizes the critical dependence upon EGFR signaling for some tumors, a property that has been exploited therapeutically. Dual EGFR blockade using afatinib and cetuximab led to a 29% radiographic response rate. More recently, drugs that target EGFR T790M (e.g., rociletinib, AZD9291, and others) have entered clinical trials, with impressive results observed in phase I clinical trials. The development of these newer drugs, with efficacy after resistance to first-line EGFR tyrosine kinase inhibitor, has led to exploration of these strategies in multiple disease settings: at resistance, in the first line, and in adjuvant treatment of those with completely resected early-stage disease who would otherwise die of recurrent/metastatic disease. This example of translational research that identifies mechanisms of resistance to first-generation drugs, and then targets those mechanisms yielding clinical benefit, is a paradigm for how targeted therapies can be developed.

Exosomes are approximately 100-nm vesicles that consist of a lipid bilayer of cellular membranes secreted in large quantities from various types of normal and disease-related cells. Endocytosis has been reported as a major pathway for the cellular uptake of exosomes; however, the detailed mechanisms of their cellular uptake are still unknown. Here, we demonstrate the active induction of macropinocytosis (accompanied by actin reorganisation, ruffling of plasma membrane, and engulfment of large volumes of extracellular fluid) by stimulation of cancer-related receptors and show that the epidermal growth factor (EGF) receptor significantly enhances the cellular uptake of exosomes. We also demonstrate that oncogenic K-Ras-expressing MIA PaCa-2 cells exhibit intensive macropinocytosis that actively transports extracellular exosomes into the cells compared with wild-type K-Ras-expressing BxPC-3 cells. Furthermore, encapsulation of the ribosome-inactivating protein saporin with EGF in exosomes using our simple electroporation method produces superior cytotoxicity via the enhanced cellular uptake of exosomes. Our findings contribute to the biological, pharmaceutical, and medical research fields in terms of understanding the macropinocytosis-mediated cellular uptake of exosomes with applications for exosomal delivery systems.

Human lung adenocarcinomas (LUAD) contain mutations in EGFR in approximately 15% of cases and in KRAS in approximately 30%, yet no individual adenocarcinoma appears to carry activating mutations in both genes, a finding we have confirmed by re-analysis of data from over 600 LUAD. Here we provide evidence that co-occurrence of mutations in these two genes is deleterious. In transgenic mice programmed to express both mutant oncogenes in the lung epithelium, the resulting tumors express only one oncogene. We also show that forced expression of a second oncogene in human cancer cell lines with an endogenous mutated oncogene is deleterious. The most prominent features accompanying loss of cell viability were vacuolization, other changes in cell morphology, and increased macropinocytosis. Activation of ERK, p38 and JNK in the dying cells suggests that an overly active MAPK signaling pathway may mediate the phenotype. Together, our findings indicate that mutual exclusivity of oncogenic mutations may reveal unexpected vulnerabilities and therapeutic possibilities.

BACKGROUND: The KIF5B-RET rearrangement is detected with the frequency of 1 ~ 2% in triple marker -negative lung adenocarcinomas, i.e., EGFR, KRAS and EML4-ALK wild type. These mutational changes are known to be mutually exclusive, but the co-existence of ALK rearrangement with activating mutations of EGFR is rarely found. METHODS: We examined the KIF5B-RET fusion gene in frozen tissues from 154 surgically resected lung tumors using RT-PCR with direct sequencing and the mutation status of EGFR and KRAS genes using PNA clamping. We tested KIF5B-RET translocation in Formalin Fixed Paraffin Embedded using fluorescence in situ hybridization. We also measured RET mRNA and protein expression by RT-PCR and immunohistochemistry, respectively. RESULTS: The existence of KIF5B-RET fusion gene was identified in 9 patients. The mean age was 67.2 and M: F ratio 4:5. Of 9 patients, 3 patients harbored wild type of EGFR and KRAS gene. However, KIF5B-RET fusion gene coincided with EGFR or KRAS mutation in 6 patients. These six pts were also positive for both RET break-apart probes (23.9%) and KIF5B-RET fusion (44.4%). However, there were no correlations between RET mRNA and protein expression in the KIF5B-RET-positive patients. The median disease free survival and overall survival were 23.9 months and 29.5 months, respectively. CONCLUSIONS: Taken together, our data suggest one-step screening platform for KIF5B-RET as well as EGFR, K-RAS, ALK oncogenic mutations be necessary for lung adenocarcinoma patients because EGFR or KRAS mutation are not infrequently found in KIF5B-RET-positive patients.

The effect of the N-ras oncogene on the propensity of transformed cells to disseminate from the tumor and to metastasize, using NIH 3T3 cells transformed either with human melanoma DNA containing the N-ras oncogene or with the cloned N-ras from human neuroblastoma, was investigated. The results show that NIH 3T3 expressing these genes readily formed tumors after subcutaneous injection in nude mice. Spontaneous lymph node metastasis was observed after a first cycle of transfection in one animal inoculated with cells containing human melanoma N-ras oncogene, and in 95 per cent of the animals after the second and third rounds of transfection, indicating that the metastatic capacity was transferred. In ALL cases human N-ras oncogene was found in both the metastases and the associated tumors. No control NIH 3T3 cells formed tumors or metastases in nude mice, and NIH 3T3 cells transfected with cloned N-ras activated oncogene formed tumors in 100 per cent of injected mice, but no spontaneous metastases. Thus human activated N-ras gene may not be sufficient to confer metastatic behavior in nude mice and the metastatic ability of human melanoma DNA transfected cells may be due to, among other possibilities, expression of other gene sequences from melanoma DNA co-transfected with the N-ras oncogene, or to specific activated murine sequences switched on during the initial process of transfection.

We have characterized the expression of transforming growth factor alpha (TGF alpha) and its receptor, the epidermal growth factor receptor (EGF-R), in normal and malignantly transformed human mammary epithelial cells. Human mammary epithelial cells were derived from a reduction mammoplasty (184), immortalized by benzo-a-pyrene (184A 1N4), and further transformed by the oncogenes simian virus 40 T (SV40 T), v-Ha-ras, and v-mos alone or in combination using retroviral vectors. 184 and 184A 1N4 cells require EGF for anchorage-dependent clonal growth. In mass culture, they secrete TGF alpha at high concentrations and exhibit an attenuated requirement for exogenous EGF/TGF alpha. SV40 T transformed cells have 4-fold increased EGF-R, have acquired the ability to clone in soft agar with EGF/TGF alpha supplementation, but are not tumorigenic. Cells transformed by v-mos or v-Ha-ras are weakly tumorigenic and capable of both anchorage dependent and independent growth in the absence of EGF/TGF alpha. Cells transformed by both SV40 T and v-Ha-ras are highly tumorigenic, are refractory to EGF/TGF alpha, and clone with high efficiency in soft agar. The expression of v-Ha-ras is associated with a loss of the high (but not low) affinity binding component of the EGF-R. Malignant transformation and loss of TGF alpha/EGF responsiveness did not correlate with an increase in TGF alpha production. Thus, TGF alpha production does not appear to be a tumor specific marker for human mammary epithelial cells. Differential growth responses to EGF/TGF alpha, rather than enhanced production of TGF alpha, may determine the transition from normal to malignant human breast epithelium.

Previous reports have indicated that the C termini of the membrane-associated tyrosine kinases encoded by c-src and c-fms proto-oncogenes have a negative effect on their biological activity and that this effect is mediated by their C-terminal tyrosine residue. To determine whether this was true for the human epidermal growth factor (EGF) receptor, which is also a membrane-associated tyrosine kinase proto-oncogene, we have constructed two premature termination mutants, dc19 and dc63, that delete the C-terminal 19 and 63 amino acids, respectively, from the human full-length receptor (hEGFR). The smaller deletion removes the C-terminal tyrosine residue, while the larger deletion removes the two most C-terminal tyrosines; similar deletions are found in v-erbB. As previously shown for the gene encoding the full-length EGF receptor, the two C-terminal mutants induced EGF-dependent focal transformation and anchorage-independent growth of NIH 3T3 cells. However, both dc19 and dc63 were quantitatively less efficient than the gene encoding the full-length receptor, with dc63 being less active than dc19. Although the C-terminal mutants displayed lower biological activity than the gene encoding the full-length receptor, the mutant receptors were found to be similar in several respects to the full-length receptor. These parameters included receptor localization, stability in the absence of EGF, receptor half-life in the presence of EGF, EGF binding, extent of EGF-dependent autophosphorylation in vitro, and EGF-dependent phosphorylation of an exogenous substrate in vitro. Therefore, the C-terminal 63 amino acids of the human receptor have no detectable influence on EGF-dependent early events. We conclude that in contrastFAU - Velu, T J

The hamster cheek pouch is an excellent model for the experimental induction of oral tumor by chemical carcinogens. We have previously shown that the cellular oncogene c-erb B1 is overexpressed in the cheek pouch of the Syrian hamster when transformed by the carcinogenic chemical dimethyl-benzanthracene (DMBA). Here we report that this cellular oncogene is amplified in one such chemically-induced oral carcinoma (HCP-T12) and in a cell line (HCPC-1) established from a similarly derived tumor. Furthermore we present preliminary evidence that the gene amplification is most likely through an extrachromosomal pathway. These data together suggest that the amplification of the c-erb B1 oncogene might be responsible for its overexpression in these chemically-induced oral carcinomas.

The epidermal growth factor (EGF) receptor is the functional target of the mitogen EGF and the cellular homolog of the avian erythroblastosis virus erbB oncogene product. Regulation of expression of the proto-oncogene encoding the EGF receptor can be elucidated by studying the structure and function of the gene promoter outside the confines of the cell. Previously, we reported the isolation of the human EGF receptor gene promoter. The promoter is highly GC rich, contains no TATA or CAAT box, and has multiple transcription start sites. An S1 nuclease-sensitive site has now been found 80 to 110 base pairs (bp) upstream from the major in vivo transcription initiation site. Two sets of direct repeat sequences were found in this area; both conform to the motif TCCTCCTCC. When deletion mutations were made in this region of the promoter by using either Bal 31 exonuclease or S1 nuclease, we found that in vivo activity dropped three- to fivefold, on the basis of transient-transfection analysis. Examination of nuclear protein binding to normal and mutated promoter DNAs by gel retardation analysis and DNase I footprinting revealed that two specific factors bind to the direct repeat region but cannot bind to the S1 nuclease-mutated promoter. One of the specific factors is the transcription factor Sp1. The results suggest that these nuclear trans-acting factors interact with the S1 nuclease-sensitive region of the EGF receptor gene promoter and either directly or indirectly stimulate transcription.

A common site of ecotropic murine leukemia virus integration designated Evi-2 (ecotropic viral integration site-2) has been identified in BXH-2 myeloid tumors. As part of experiments to determine whether Evi-2 identified a new proto-oncogene locus involved in myeloid disease, we determined its chromosomal location. We mapped Evi-2 to mouse Chromosome 11 using standard recombinant inbred strain and genetic backcross analysis. We then determined the location of Evi-2 relative to other proto-oncogene and growth factor loci located on Chromosome 11 by interspecific backcross analysis. The loci included in this study were the proto-oncogene loci, Erbb, Erba, and Rel, as well as, Il-3 (interleukin-3), Csfgm (granulocyte-macrophage colony stimulating factor), and Trp53-1 (transforming protein p53). ALL loci except Erbb had been previously mapped to Chromosome 11 with the use of somatic cell hybrids and consequently their positions on Chromosome 11 were not known. One proto-oncogene, Erbb-2 (analogous to the neu proto-oncogene), and one growth factor locus, Csfg (granulocyte colony-stimulating factor), which had not been mapped in the mouse were also localized on Chromosome 11 using the interspecific backcross mice. Recombination between Evi-2 and all proto-oncogene and growth factor loci was demonstrated, suggesting that Evi-2 may ultimately identify a new proto-oncogene involved in myeloid disease. This study revealed a number of interesting conserved linkage groups common to mouse and man.

Five human glioblastoma cell lines were analyzed for oncogene activation with a panel of probes. Abnormal expression of the epidermal growth factor receptor (EGFr) gene was detected in four of five lines; N-ras oncogene overexpression was found in ALL five cell lines. These results were subsequently confirmed with fresh brain tumor and nonneoplastic brain tissue biopsy samples; increased expression of the N-ras proto-oncogene was observed in five of five glioblastomas, ALL of which also showed EGFr gene overexpression, but not in well-differentiated gliomas or in nonneoplastic brain tissue specimens. No significant differences in Ha-ras and Ki-ras expression were observed. Preliminary histochemical observations showed that intracellular levels of transforming growth factor alpha, a putative biochemical link between these two oncogenes, were significantly higher in glioblastoma cells than in controls.

Two separate enhancers were identified both upstream and downstream of the promoter of the human epidermal growth factor receptor gene. Transcriptional enhancer activity was found to be associated with a region 1172 and 852 base pairs upstream of the most upstream RNA start site, and within a 530-base pair fragment located in the first intron about 2000 base pairs downstream of the most upstream RNA start site. These two separate enhancers stimulated promoter activity cooperatively in HeLa cells synthesizing the epidermal growth factor receptor. The enhancer downstream of the promoter functioned only in the presence of the other upstream enhancer. Several areas of sequences homology with viral and cellular enhancers were noted in both the upstream and downstream enhancers. The region essential for the downstream enhancer activity has 10 nuclear factor-binding sites.

The genome of avian erythroblastosis virus (AEV), a defective acute leukemia retrovirus, carries two distinct cell-derived oncogenes in the structure 5 delta gag-erbA-erbB-delta env3 . The nucleotide sequence of the v-erbA gene was recently reported. In order to determine the boundary between the two adjacent oncogenes, the sequence of the v-erbA/v-erbB junction of AEV was compared to that of a recombinant lambda phage containing a chicken cellular sequence representing the 5 part of c-erbB. The four C-terminal amino acids of v-erbA are in fact encoded by a c-erbB intron-derived sequence thus demonstrating that the virus acquired a truncated c-erbA gene. Furthermore the 7 to 10 amino acid residues upstream from the 4 C-terminal amino acids mentioned above appeared to be derived from env-related sequences. The splice acceptor site at the beginning of the only open reading frame for v-erbB is also present and functional in c-erbB when expressed to generate a truncated EGF (epidermal growth factor) receptor. Thus AEV joins a truncated erbA gene to a truncated erbB gene through env-derived sequences and intronic sequences from c-erbB.

Three glycoprotein-processing inhibitors were used to resolve whether correct glycosylation was required for the oncogenic activity of erb B. The two glucosidase-I inhibitors, 1-deoxynojirimycin and 2,5-dihydroxymethyl 3,4-dihydroxypyrrolidine, arrested processing of v-erb B at the immature 68-kd form whereas, in the presence of the alpha-mannosidase-II inhibitor (swainsonine), cells synthesised an abnormally processed 70-kd form of v-erb B. Transport of incorrectly processed v-erb B to the cell surface was, however, unaffected, suggesting that correct processing is not a prerequisite for intracellular routing of v-erb B. Two systems were used to assess whether incorrectly processed erb B could maintain the transformed state. The first asked whether inhibitor treatment would release temperature-sensitive avian erythroblastosis virus (AEV) transformed erythroblasts kept at the viral permissive temperature from the erb B-induced block in differentiation, as seen when cells are normally shifted to the non-permissive temperature. The second tested the ability of AEV-transformed fibroblasts to grow in soft agar. In both systems, ALL three processing inhibitors did not alter the transformed phenotype suggesting that correct carbohydrate processing is not required for the transforming activity of erb B. In addition, none of the three processing inhibitors were found to have any effect on the normal maturation of bone marrow CFU-E or induced differentiation of temperature-sensitive AEV-transformed erythroblasts.

Both interleukin 2 (IL-2) and epidermal growth factor (EGF) receptors exist in two forms that differ with respect to affinity for their ligand. Only the high-affinity receptors appear to be responsible for the proliferation signal delivered upon binding of the growth factor. Fibroblasts transfected with IL-2 receptor cDNA generate only low-affinity receptors for IL-2, but fusion of membranes from these fibroblasts with T-cell membranes converts some receptors to high affinity, indicating the involvement of a T cell-specific factor in the generation of high-affinity receptors. We have constructed a chimeric cDNA molecule containing the extracellular IL-2-binding domain of the IL-2 receptor cDNA and the transmembrane and intracellular tyrosine kinase domains of the EGF receptor cDNA. When transfected into fibroblasts, this IL-2-EGF receptor cDNA generated high-affinity receptors for IL-2. Moreover, fibroblasts transfected with the chimeric molecule were morphologically transformed and produced rapidly growing tumors in nude mice.

Repeated local applications of dimethylbenzanthracene (DMBA) induce epidermoid carcinomas in 14 weeks in the cheek pouch of Syrian hamsters. Characteristic histopathological changes similar to the ones observed in human oral cancers can be detected during the development of these tumors in the hamster cheek pouch. Results presented in this report demonstrate that the cellular proto-oncogene c-erbB is amplified and expressed in an epidermoid carcinoma cell line (HCPC-1), derived from one such cheek pouch tumor. The expression of the gene cannot be detected in the untreated cheek pouch tissue whereas the level of expression of the gene is significantly high in the tumor cell line. The expression of c-erbB gene can also be detected in cheek pouch tissue at an early stage (8-9 weeks) of tumor development and in ALL tumor-bearing tissue of subsequent stages. The stage of expression of c-erbB gene does not coincide with the stage in which extensive hyperplasia of the epithelial cells (5 weeks) is observed. Rather, this coincides with the onset of the early invasion of the connective tissue by the dysplastic epithelium. The expression of c-erbB gene increases in parallel with the increased tumor burden. These results implicate the possible role of c-erbB gene in the genesis of DMBA-induced oral epithelial carcinomas.

Recent studies have demonstrated that the amount of epidermal growth factor receptor (EGF-R) is increased in squamous cell carcinoma cells and that the amino acid sequence of EGF-R shows great homology with the v-erb B transforming protein. In this study, we examined the tissue localization of EGF-R and myc oncogene product in normal squamous epithelium, dysplasia, carcinoma in situ, invasive squamous cell carcinoma of the uterine cervix and in metastatic lymph nodes by means of the avidin/biotin immunoperoxidase technique. Normal squamous epithelium was negative for EGF-R and myc product. The lesions of dysplasia and carcinoma in situ had a positive staining for EGF-R, but were still negative for myc product. There were differences in the staining intensity of EGF-R and myc product among the types of invasive carcinoma. Staining for EGF-R and myc product was negative in small cell non-keratinizing carcinoma, whereas strong staining for both EGF-R and myc product was observed in large cell non-keratinizing and keratinizing carcinoma. The intensity of positive staining for EGF-R and myc product declined in the lesions of cancer pearl. Metastatic lymph nodes were remarkably stained for EGF-R and myc product, while non-metastatic lymph nodes were negative for EGF-R and myc product. Our observations suggest that the amplified expression of EGF-R and myc product may accompany the malignant transformation of squamous epithelium of the uterine cervix, together with the metastasis.

The expression of EGF receptor mRNAs in regenerating rat liver was measured using two nonoverlapping cDNA probes for the human gene from a highly conserved region. These probes (pE7 and pE62) both hybridized to RNA species of 10 and 6 kb. The 10 and 6 kb RNA species were shown to decrease in the first 12 hours after partial hepatectomy. However, significant increases above control levels were noted at 24h and 72h. The level of alpha-actin mRNA increased as has been previously reported. These results suggest that a transcriptional and/or a posttranscriptional regulatory mechanism exists in regenerating rat liver with respect to EGF receptor gene expression.

The epidermal growth factor (EGF) receptor gene EGFR has been placed in a retrovirus vector to examine the growth properties of cells that experimentally overproduce a full-length EGF receptor. NIH 3T3 cells transfected with the viral DNA or infected with the corresponding rescued retrovirus developed a fully transformed phenotype in vitro that required both functional EGFR expression and the presence of EGF in the growth medium. Cells expressing 4 x 10(5) EGF receptors formed tumors in nude mice, while control cells did not. Therefore, the EGFR retrovirus, which had a titer on NIH 3T3 cells that was greater than 10(7) focus-forming units per milliliter, can efficiently transfer and express this gene, and increased numbers of EGF receptors can contribute to the transformed phenotype.

Cultured NIH-3T3 cells devoid of endogenous epidermal growth factor (EGF) receptors were transfected with cDNA expression constructs encoding either normal human EGF receptor or a receptor mutated in vitro at Lys-721, a residue that is thought to function as part of the ATP-binding site of the kinase domain. Unlike the wild-type EGF-receptor expressed in these cells, which exhibited EGF-dependent protein tyrosine kinase activity, the mutant receptor lacked protein tyrosine kinase activity and was unable to undergo autophosphorylation and to phosphorylate exogenous substrates. Despite this deficiency, the mutant receptor was normally expressed on the cell surface, and it exhibited both high- and low-affinity binding sites. The addition of EGF to cells expressing wild-type receptors caused the stimulation of various responses, including enhanced expression of proto-oncogenes c-fos and c-myc, morphological changes, and stimulation of DNA synthesis. However, in cells expressing mutant receptors, EGF was unable to stimulate these responses, suggesting that the tyrosine kinase activity is essential for EGF receptor signal transduction.

Chromosome analysis in a series of human glioblastoma cell lines (HeRo, HeRo-SV1, A172, T406, T508, T705) has indicated characteristic changes in the karyotype, the most striking and consistent of which is a significant increase in the copy number of chromosome 7, with up to 8 copies per metaphase. As determined by Spurr et al., chromosome 7 represents the genomic locus for the oncogene erbB (7pter-q22). Therefore, we have compared the number of chromosomes 7 to the levels of expression of the erbB oncogene. Interestingly, in ALL of them erbB-specific mRNA was found to be increased at levels even higher than expected from the number of chromosomes 7 found. In contrast, in an astrocytoma of slightly lower grade of malignancy (cell line T567), neither polysomy 7 nor significant expression of the erbB oncogene was noted.

Although mutations in ras genes are thought to be important for the development of about 20% of human tumors, almost nothing is known about the way in which these mutations lead to cellular transformation. The known biochemical properties of the 21-kilodalton ras proteins suggest that they may behave as G proteins, regulating the proliferation of cells in response to growth factor stimulation of a receptor. Although the putative receptor(s) has not been identified, several lines of evidence, in particular the fact that rodent cell lines containing ras oncogenes produce transforming growth factor alpha, have suggested that the epidermal growth factor (EGF) receptor is involved in ras transformation. Here we show that murine fibroblasts with no EGF receptors can be transformed to a completely malignant phenotype with a mutated ras gene. It appears, therefore, that the EGF receptor is not required for ras-mediated transformation of these cells.

A three-dimensional model for the ATP-binding site of the oncogene product v-src is proposed by the use of interactive computer graphics. A similar model would apply to sequence related proteins such as other oncogene products, the epidermal growth factor receptor, cAMP-dependent protein kinase and the cell division control protein CDC28. The model was proposed on the basis of the conservation of certain key residues between the oncogene product family of proteins and several nucleotide binding proteins of known structures.

Each of six peptides derived from the human epidermal growth factor (EGF) receptor very closely matches a part of the deduced sequence of the v-erb-B transforming protein of avian erythroblastosis virus (AEV). In all, the peptides contain 83 amino acid residues, 74 of which are shared with v-erb-B. The AEV progenitor may have acquired the cellular gene sequences of a truncated EGF receptor (or closely related protein) lacking the external EGF-binding domain but retaining the transmembrane domain and a domain involved in stimulating cell proliferation. Transformation of cells by AEV may result, in part, from the inappropriate acquisition of a truncated EGF receptor from the c-erb-B gene.

The FBR murine osteosarcoma virus complex, isolated from a radiation-induced osteosarcoma of an X/Gf mouse causes the rapid appearance of osteosarcomas in newborn mice and transforms fibroblasts in vitro. The two components of the FBR-viral complex have been isolated separately in tissue culture: FBR-MuLV by end-point dilution and FBR-MuSV by the establishment of mouse [FBR-NP 117 (NIH 3T3)] and rat non-producer cell lines [FBR-NP415 (REF)]. The host range and RNase Tl fingerprint analysis of FBR-MuLV demonstrated a pattern closely related to, but distinguishable from, Akv-MuLV. Transformed cells from both mice and rats contain a rescuable FBR-MuSV genome. These pseudotypes produce foci in tissue culture and induce osteosarcomas in susceptible mouse strains. An FBR-MuSV (FBR-MuLV) cDNA probe detects a 5.2 kb HindIII and a 9.5 kb EcoRI FBR-MuSV-specific fragment in FBR-MuSV-transformed non-producer rat cells. The same fragments hybridized with a fos specific probe, demonstrating that FBR-provirus contains a c-fos-derived onc-gene.

The proto-oncogene product, Cbl, is a 120-kDa protein present in lymphocytes that contains numerous PXXP motifs in its COOH-terminal region and constitutively binds the SH3-containing adaptor protein Grb2. Cross-linking of CD3 and CD4 receptors in Jurkat T cells causes tyrosine phosphorylation of Cbl and its association with phosphatidylinositol 3 -kinase (Meisner, H., Conway, B., Hartley, D., and Czech, M. P. (1995) Mol. Cell. Biol. 15, 3571-3578). Here we demonstrate that Cbl is also present in nonlymphoid cells, and that epidermal growth factor (EGF) elicits its rapid tyrosine phosphorylation in human embryonic 293 cells. Immunoprecipitates of Cbl from lysates of these cells contain Grb2 in the basal state, while EGF stimulation causes co-precipitation of tyrosine-phosphorylated EGF receptors. Similarly, EGF receptor immunoprecipitates from EGF-treated 293 cells contain Cbl and Grb2. Both Grb2 and EGF receptors are released from Cbl in the presence of a proline-rich peptide that binds the NH2-terminal SH3 domain of Grb2. These results indicate that autophosphorylated EGF receptors associate with the SH2 domain of Grb2, which is complexed through its SH3 domain with proline-rich regions of Cbl. Such recruitment of Cbl to EGF receptors may reflect an important mechanism for its tyrosine phosphorylation and for assembling signaling components that mediate or modulate EGF actions.

AIMS: To examine thymomas for proteins encoded by oncogenes and to determine whether their presence correlates with tumour growth and associated myasthenia gravis. METHODS: Sections of 24 thymomas were incubated with anti-EGF receptor (EGF-R), anti-Ki67 antigen, anti-p53, and anti-bcl-2 antibodies, and then stained using the alkaline phosphatase/anti-alkaline phosphatase (APAAP) technique. Cell suspensions and epithelial cell cultures from some of the tumours were also studied. RESULTS: Whereas EGF-R expression was not detected in any of the controls (but only in a 20 week old fetus), it was detected in neoplastic epithelial cells of ALL thymomas, and was most strongly expressed in metastases and in samples from donors with severe myasthenia gravis. Ki67 labelling was also increased, especially in the larger thymomas. Epithelial expression of both of these markers was confirmed in fresh cell suspensions and monolayer cultures from the five available cases. In contrast, p53 and bcl-2 were not detected in the neoplastic cells, but bcl-2 was present in the intermingling thymocytes. CONCLUSIONS: Neoplastic thymoma cells express EGF-R and Ki67, but there is no concomitant increase in the expression of p53 and bcl-2 proteins. Increased EGF-R expression may result in increased proliferation of neoplastic cells and also in myasthenia gravis. Measurement of EGF-R concentrations may be of prognostic value. The bcl-2 staining pattern in T lymphocytes illustrates the broad spectrum of maturational stages in thymoma lymphocytes.

The murine retroviral oncogene v-cbl induces pre-B cell lymphomas and myelogenous leukemias. The protein product of the mammalian c-cbl proto-oncogene is a widely expressed cytoplasmic 120-kDa protein (p120cbl) whose normal cellular function has not been determined. Here we show that upon stimulation of human epidermal growth factor (EGF) receptor, p12ocbl becomes strongly tyrosine-phosphorylated and associates with activated EGF receptor in vivo. A GST fusion protein containing amino acids 1-486 of p120cbl, including a region highly conserved in nematodes, binds directly to the autophosphorylated carboxyl-terminal tail of the EGF receptor. Platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), or nerve growth factor (NGF) stimulation also results in tyrosine phosphorylation of p120cbl. Recent genetic studies in Caenorhabditis elegans indicate that Sli-1, a p120cbl homologue, plays a negative regulatory role in control of the Ras signaling pathway initiated by the C. elegans EGF receptor homologue. Our results indicate that p120cbl is involved in an early step in the EGF signaling pathway that is conserved from nematodes to mammals.

Cytokeratins 8 and 18 (Endo A and B) are among the earliest expressed embryonic genes and the major components of the cytoskeleton in simple epithelia of the adult. Recent data indicate that these cytokeratins are aberrantly expressed in several epithelial tumor types and that expression in cultured mouse keratinocytes is linked to activation of the rasHa oncogene. Furthermore, up-regulation of K8/K18 in keratinocytes is associated with reciprocal suppression of K1. We now show that the aberrant expression of K8 and K18 and suppression of K1 in cultured keratinocytes transduced with the v-rasHa gene are mediated by a factor secreted into the culture medium. Furthermore, transforming growth factor alpha (TGF-alpha) and epidermal growth factor elicit an identical pattern of K8/K18 expression and K1 suppression in normal keratinocytes. The factor in medium from v-rasHa keratinocytes is TGF-alpha, as a specific blocking antibody for rat and mouse TGF-alpha prevents the expression of K8 and restores expression of K1. The tyrosine kinase inhibitor genistein also prevents K8 induction in v-rasHa keratinocytes and in normal keratinocytes treated with TGF-alpha- or v-rasHa-conditioned medium. However, simply stimulating proliferation of keratinocytes by cholera toxin does not result in expression of K8 or suppression of K1. Finally, tumor grafts from neoplastic epidermal cells overexpressing TGF-alpha via retroviral transduction of human TGF-alpha complementary DNA in vitro show coordinate expression of K8 and human TGF-alpha. These studies indicate that K8 expression in keratinocytes, and derivative neoplastic cells, in vivo and in vitro is regulated by epidermal growth factor receptor ligands. Since the expression of cytokines and K8/K18 in early embryogenesis is often coincident, cytokines may be the physiological mediators of K8/K18 expression in embryonic cells.

mutations in the receptor for the epidermal growth factor provide valuable insight into mechanisms of growth control. Oncogenic mutants of this receptor tyrosine kinase cause erythroid leukemia, fibrosarcoma, angiosarcoma, glioblastoma, and melanoma. mutations in the avian protooncogene occur by retroviral mechanisms. Deletion of the ligand-binding domain results in erythroblastosis, while additional mutations in cytoplasmic structures broaden the disease potential to other cell types. A carboxyl-terminal structure of erbB oncogenes modulates growth responses in a complex, cell-specific manner; this tissue-specificity region appears to promote growth in erythroblasts and to produce trans-dominant inhibition in fibroblasts. Human glioblastoma multiforme frequently contains receptor mutations that are reminiscent of avian oncogenes. In hereditary melanoma of Xiphophorus, aberrant regulation of transcription by a recombinant promoter determines tissue-specific tumorigenesis. The diversity of oncogenic mutations raises important questions concerning the roles of several receptor structures. The extracellular domain inhibits the receptor when unoccupied by ligand, for example, through a mechanism that is unknown. The auto-phosphorylation sites are dispensable for transformation, so their function in neoplastic growth is unclear. The carboxyl-terminal region promotes or blocks transformation in different tissues, suggesting complex regulation by unknown cellular factors. These issues are critical to understanding of the mechanisms of receptor activation and tissue tropism for this family of oncogenes.

The influence of cytostatically active alkyllysophospholipid analogs with different chemical structure on IP3 (inositol-1,4,5-trisphosphate) formation and intracellular Ca++ concentration was studied in human mammary epithelial cells before and after transfection with v-erb B oncogene DNA. Transformed cells showed an increased IP3 formation compared with normal cells. In the presence of ALP (alkyllysophospholipid) analogs IP3 formation is inhibited more strongly in transformed cells than in normal cells, dependent on the structure of ALP analogs. Furthermore, these compounds increased [Ca++]i (intracellular Ca++ concentration) in transformed cells much more strongly than in normal cells. From these results, obtained in pursuit of an oncogene-related cancer treatment, it follows that ALP analogs may inhibit processes in signal transduction in cells more strongly after transfection with a defined oncogene than before.

C-erbB-2-oncoprotein and the epidermal growth factor receptor (EGFR) protein are transmembrane glycoproteins with an external ligand-binding domain and a nearly homologous internal tyrosine kinase domain. In the present study it was investigated in 63 astrocytic tumors (9 astrocytomas G1, 18 astrocytomas G2, 17 anaplastic astrocytomas G3 and 19 glioblastomas G4) whether the structural homology of both glycoproteins correlated with the coexpression in astrocytic tumor cells. The immunoreactive products were identified by a computerized image analysis. There was no expression of the EGFR-protein in low grade astrocytomas (G1, G2) measured by density of gray level. The immunoreactivity increased remarkably in anaplastic and malignant gliomas. The number of the c-erbB2-oncoprotein-reactive tumor cells increased with the progression and dedifferentiation of tumors. Significant differences could be found between low grade anaplastic astrocytoma as well as glioblastoma. The correlative analysis resulted in a significant positive homology with increasing grading level between the expression of EGFR- and c-erbB-2 protein. The trend goes in the same direction. The results emphasize that EGFR- and c-erbB-2 protein were expressed in astrocytic tumors with increased malignancy and dedifferentiation.

Differential PCR has been used to detect the variance of c-erbB-2 gene copies in 23 colon carcinoma tissues. It was found that amplification of c-erbB-2 gene existed in 7 out of 23 (30%) cases. ALL of the three high-middle differentiated colon carcinomas had amplification of c-erbB-2 gene in our specimens. It was higher than that of the middle differentiated colon carcinomas (P < 0.05). The rate of amplification of c-erbB-2 gene in patients with positive lymph node was approximately two times as high as that in negative nodo patients, but this difference was not significant (P > 0.20).

Altered expression of protooncogenes/oncogenes is believed to be involved in hepatocarcinogenesis of the chemically induced, transplantable Morris hepatoma 7777. We compared the mRNA expression of c-N-ras and v-erb B mRNA of normal rat liver with that of Morris hepatoma 7777 using Northern blot analysis and in situ hybridization. Northern blot analysis revealed a strong overexpression of the v-erb B related mRNA, while the c-N-ras mRNA was only slightly increased. In situ hybridization using a c-N-ras mRNA probe also showed only a slightly increased number of silver grains in the hepatoma cells compared with normal rat liver. On the other hand, the v-erb B related mRNA was strongly overexpressed in the hepatoma cells, while the connective-tissue capsule, the blood vessels, blood cells and the necrotic foci did not show an elevated v-erb B related gene mRNA expression. Similar results were obtained in liver metastases. The detectable v-erb B hybridization signal was lost by pretreatment with RNase A. We conclude that the c-N-ras gene is of minor importance in the chemically induced, transplantable Morris hepatoma 7777, while the increased expression of the v-erb B related mRNA is due to a selection of ligand-independent tyrosine kinase activity.

The aim of this study was to evaluate the immunohistochemical expression of epidermal growth factor (EGFR) and c-erb-B-2 oncoprotein in a series of 71 hepatocellular carcinomas as well as in the adjacent hepatic tissue and to assess any correlation with HBsAg expression. The total of the 71 hepatocellular carcinomas (HCCs) was classified into 17 low grade and 54 high grade cases with adjacent non-neoplastic liver parenchyma, observed in 14 and 28 cases respectively. Coexisting cirrhosis or fibrosis was noticed in the adjacent non-neoplastic parenchyma in 12 cases of low grade and 22 cases of high grade HCC. The immunohistochemical avidin-biotin-peroxidase complex (ABC) method was performed on formalin-fixed paraffin sections for the detection of EGFR, c-erb-B-2 oncoprotein and HBsAg using monoclonal antibodies. The expression of c-erb-B-2 was observed in 29.5% (21/71) of the HCCs showing no statistically significant correlation with histological grade. The c-erb-B-2 was also detected in the adjacent non-neoplastic parenchyma in 7/14 low grade HCCs, and in 9/28 high grade HCCs. No statistically significant differences in c-erb-B-2 oncoprotein expression were observed between the HCCs and the adjacent non-neoplastic parenchyma. In addition, HBsAg was detected in 10/42 examined cases of HCC with adjacent non-neoplastic parenchyma, while only 4 cases of HCCs were simultaneously positive for c-erb-B-2 and HBsAg. EGFR was detected in only 3/71 cases of HCC, while the antigen was not detected at ALL in the adjacent non neoplastic parenchyma. HBsAg expression was not observed in any of the EGFR-positive HCCs. Our results suggest that both c-erb-B-2-oncoprotein and EGFR do not seem to be predominantly involved in the transformation of hepatocytes to the malignant phenotype.

Fifty-six specimens of gastric carcinoma were examined for the localization of HER-2/neu oncoprotein (HER-2/neu) and epidermal growth factor receptor (EGFR) by immunohistochemistry using polyclonal antibodies on paraffin-embedded material. Strong membrane staining for HER-2/neu was noted in 14 cases (25%), ALL of which were of the intestinal type. Only cytoplasmic staining was found in an additional 21 cases (37.5%), including seven diffuse tumors. Twenty-four cases (nine diffuse and 15 intestinal) showed cytoplasmic staining with accentuation on the cell membrane for EGFR. Patchy staining was common for HER-2/neu, while EGFR immunoreactivity was always diffuse. Twenty cases (35.7%) showed positive staining for both, 15 cases (26.8%) for HER-2/neu only, four cases (7.1%) for EGFR only, and 17 cases (30.4%) for neither. expression of HER-2/neu was more commonly associated with intermediate-grade and high-stage tumors. Cases with positive (either membrane or cytoplasmic) staining for HER-2/neu showed poorer overall mean survival (308 days) than cases that failed to stain (763 days). The EGFR-positive cases showed shorter mean survival (387 days) than the negative cases (547 days), but this difference did not reach statistical significance. The EGFR positivity did not further reduce survival in HER-2/neu-positive cases (362 days). The results of this study support the hypothesis that the expression of HER-2/neu may be a significant predictor of prognosis in patients with gastric carcinoma. Our findings also suggest that expression of these two closely related protooncogenes in malignant and benign gastric tissues is independent of each other and that EGFR does not potentiate the oncogenic effect of HER-2/neu.

The expression of epidermal growth factor receptor (EGFR) was studied immunohistochemically in 234 cases of transitional cell bladder cancer. EGFR was overexpressed in 35% of cases and distinct nuclear localisation of EGFR positivity was found in 31% of the tumours. Overexpression was related to invasive growth, grade 2-3 histology, non-papillary type, DNA aneuploidy and high proliferation rate of cancer cells. The expressions of p53 and EGFR were interrelated, while expression of c-erbB-2 was independent of EGFR expression. Progression of superficial tumours, recurrence-free survival and survival were independently related to overexpression of EGFR in multivariate analysis. T category, S-phase fraction and non-papillary type included ALL the available prognostic information when the entire cohort was analysed by multivariate methods. The results show that overexpression of EGFR is related to several malignant features and prognosis in superficial bladder cancer. Moreover, the results suggest that overexpression of EGFR is usually a late event in bladder cancer development related to genetic instability rather than an early event in malignant transformation. Further studies are still needed to establish whether the direct measurement of cell proliferation or analysis of growth factor receptors and other oncoproteins gives more accurate prognostic information in bladder cancer.

The expression of epidermal growth factor receptor (EGF-R), c-erbB-2 proto-oncogene, and estrogen receptor (ER) was studied immunohistochemically in a series of 97 human papillomavirus (HPV) lesions of the uterine cervix, with special emphasis on their association with the HPV type, grade of intraepithelial neoplasia (CIN), and the natural history of the disease. EGF-R expression was found in 95 of 97 (98%) specimens, mainly in basal and parabasal cells. Diffuse nuclear and cytoplasmic staining was detected in 36 of 97 (37%) samples, of which 29 were HPV positive. This staining pattern was most prominent in HPV 18-positive and in CIN lesions. Weak or moderate c-erbB-2 expression was found in 26 of 97 (27%) specimens. Estrogen receptor expression was observed in 28 of 77 (36%) samples, epithelial staining was seen in 11 of 77 (14%), and stromal staining occurred in 24 of 77 (31%) specimens. No clear-cut associations were established between the EGF-R, c-erbB-2, or ER expression and HPV type, nor in CIN or the clinical course of HPV infections. This failure for EGF-R, c-erbB-2, and ER to be associated with the specific HPV types, grade of CIN, or the clinical course of cervical HPV lesions suggests that the assessment of these factors is of limited value in explaining the development of HPV-associated CIN and in predicting the prognosis of this disease.

Amplifications of cellular oncogenes and growth factor genes have previously been reported in gliomas. Here we have evaluated 21 gliomas for amplification of tumor related genes including NMYC, EGFR, TGFalpha, MET, CMYC, SRC, HRAS, NRAS, SEC, ROS1, JUN, and WNT1. Five amplifications were observed. The epidermal growth factor receptor (EGFR) gene was amplified in 4 glioblastomas. The oncogene MET was amplified in a glioblastoma which showed no EGFR gene amplification. Importantly, both genes are located on chromosome 7 and belong to a family with tyrosine kinase activity. There was no amplification found for TGFalpha which was previously reported to be amplified in gliomas. The finding of MET and EGFR independently amplified in glioma lends further support to a crucial role of chromosome 7 in the development of gliomas.

Oncogenicity tests have revealed that a nonmutated erbB oncogene induces renal adenocarcinoma in addition to erythroblastosis. The erbB oncogene is a truncated form of the chicken epidermal growth factor receptor that lacks the extracellular ligand-binding domain. Previously, the nonmutated erbB oncogene has been reported to cause only erythroblastosis. The expansion of the disease potential of erbB to additional neoplasms has been associated with mutations (truncations, deletions, and point mutations) within the erbB gene. Our results indicate that a nonmutated virally expressed erbB oncogene (REB-c) causes a 100% incidence of renal neoplasia.

The evolution of oncogene-transducing retroviruses was followed by studying the genomes of five new, erbB carrying retroviruses. These viruses, isolated from cells of one chicken infected with Rous Associated virus 1 (RAV-1), had captured c-erbB sequences as a consequence of RAV-1 integration into the host genome. Their genome structures were distinct; however, their v-erbB genes had sustained identical 5 and 3 deletions and the v-erbB-env junctions were identical at the nucleotide level. The results therefore strongly suggest that ALL five viruses originate from the same capture event. Sequence analyses of the v-erbB genes from three of these viruses revealed that one of them had undergone no further mutation and lacked detectable capacity to transform cells, therefore probably representing an early form of transducing virus. The two other v-erbB genes contained distinct mutations and differed in their potential to induce fibroblast- and erythroblast transformation; they therefore probably represent later derivatives of the virus that captured the erbB oncogene. The data suggest that the initial retrovirus rapidly underwent many alterations after capture of c-erbB sequences, already in the RAV-1 infected bird as well as during subsequent in vitro isolation procedures. The changes involve both major rearrangements of the genome as well as point mutations that activated the erbB oncogene.

Western blotting analysis of the p21 ras oncoprotein was performed in seven normal laryngeal mucosa specimens and 43 primary laryngeal cancers. Varying p21 levels, expressed as optical density (OD), were found in normal mucosa (median 1.94 OD, range 0.90-2.17 OD) and in primary laryngeal tumours (median 1.74 OD, range 0.30-6.37 OD). When p21 expression in laryngeal cancer was compared with the normal counterpart, higher levels were found in neoplastic than in normal laryngeal tissue (median 2.54 OD, range 1.76-6.37 OD, vs median 1.94 OD, range 0.90-2.17 OD) (P = 0.023). Immunohistochemical analysis demonstrated that most of the tumour cells (more than 70%) were immunostained while the stromal component was unreactive. No correlation between p21 expression and tumour location, stage and histopathological grade was observed. The correlation between ras p21 protein expression and epidermal growth factor receptor (EGFR) levels was also investigated. EGFR-positive cases did not show any difference in p21 expression with respect to EGFR-negative cases (median 1.52 OD, range 0.30-6.37 OD, vs median 1.84 OD, range 0.93-3.71 OD). Our findings suggest that overexpression of p21 protein is associated with a malignant phenotype in laryngeal cancer. Further studies should be undertaken to evaluate whether the assessment of p21 protein expression may have clinical significance in laryngeal cancer.

A series of 119 patients with 122 gastric carcinomas treated by gastrectomy, with a follow up of more than 5 years is presented. There were 80 (67.2%) men and 39 (32.8%) women with a mean age of 66.4 years. In 101 (84.9%) the diagnosis of cancer was endoscopic. Pain was the most frequent symptom (55.5%). Seventy two (59%) were localized in the antrum; 26 (21%) were cardial, and 24 (19.7%) were in the body. In 43.4% the size was smaller than 5 cm and in 56.6% it measured 5 cm or more. Eighty nine (73%) were of the intestinal type, 15 (12.3%) were diffuse and 18 (14.8%) were mixed. Eighty (65.6%) were low histological grade and 42 (34.4%) were high grade. Ten (9.2%) were early carcinomas and 112 (91.8%) advanced carcinomas. An amplification of the c-erbB-2 oncogene was associated with tumors that were smaller than 5 cm (p = 0.05) and with histological low grade (p = 0.005). A five years survival correlated with tumors smaller than 5 cm (p = 0.02), with parietal infiltration not surpassing the muscular layer (p = 0.001), and without lymph node metastases (p = 0.001). There was no association between survival and amplification of c-erbB-2 oncogene.

To determine the function of the E5 open reading frame (ORF) of the human papillomaviruses (HPVs), rodent fibroblast cell lines were transfected with the E5 ORF of HPV type 6 (HPV-6) and HPV-16 expressed from an exogenous promoter. Transfected fibroblasts were transformed to colony formation in soft agar, and the transformation frequency was increased by epidermal growth factor (EGF) but not by platelet-derived growth factor. In a transitory assay, the E5 ORFs from both HPV-6 and HPV-16 were mitogenic in primary human foreskin epithelial cells (keratinocytes) and acted synergistically with EGF. Investigation of keratinocytes expressing HPV-16 E5 showed that the number of endogenous EGF receptors (EGFRs) per cell was increased two- to fivefold. Immunofluorescence microscopy of HPV-16 E5-expressing keratinocytes indicated that there was an apparent delay in the internalization and degradation of EGFRs compared with controls. Kinetic studies with [125I]EGF showed that the ligand underwent normal internalization and degradation in both HPV-16 E5-expressing and control keratinocytes, but in E5-expressing cells, a greater number of receptors recycled back to the cell surface within 1 to 6 h of ligand binding. Finally, ligand-stimulated phosphorylation of the EGFR on tyrosine, an indication of receptor kinase activity, was of greater magnitude in the HPV-16 E5-expressing keratinocytes than in control cells, although the basal level of receptor phosphorylation was similar.

The bovine papillomavirus E5 transforming protein appears to activate both the epidermal growth factor receptor (EGF-R) and the platelet-derived growth factor receptor (PDGF-R) by a ligand-independent mechanism. To further investigate the ability of E5 to activate receptors of different classes and to determine whether this stimulation occurs through the extracellular domain required for ligand activation, we constructed chimeric genes encoding PDGF-R and EGF-R by interchanging the extracellular, membrane, and cytoplasmic coding domains. Chimeras were transfected into NIH 3T3 and CHO(LR73) cells. ALL chimeras expressed stable protein which, upon addition of the appropriate ligand, could be activated as assayed by tyrosine autophosphorylation and biological transformation. Cotransfection of E5 with the wild-type and chimeric receptors resulted in the ligand-independent activation of receptors, provided that a receptor contained either the transmembrane domain of the PDGF-R or the cytoplasmic domain of the EGF-R. Chimeric receptors that contained both of these domains exhibited the highest level of E5-induced biochemical and biological stimulation. These results imply that E5 activates the PDGF-R and EGR-R by two distinct mechanisms, neither of which specifically involves the extracellular domain of the receptor. Consistent with the biochemical and biological activation data, coimmunoprecipitation studies demonstrated that E5 formed a complex with any chimera that contained a PDGF-R transmembrane domain or an EGF-R cytoplasmic domain, with those chimeras containing both domains demonstrating the greatest efficiency of complex formation. These results suggest that although different domains of the PDGF-R and EGF-R are required for E5 activation, both receptors are activated directly by formation of an E5-containing complex.

In vitro studies have shown that growth factors may mediate the growth stimulatory effect of estrogen in hormone-dependent human breast carcinomas while the constitutive expression of same growth factors might by-pass the need for estrogenic stimulus in hormone-independent neoplasms. We have performed immunocytochemical analysis of the expression of epidermal growth factor receptor (EGF-R or proto-oncogen c-erbB-1) and estrogen receptor (ER) in 70 cases of human breast carcinoma. We found an inverse relationship between the expression of EGF-R and ER (Kendall s tau b = -0.1997, P < 0.03), which prompts us to conclude that ER(-) breast carcinomas may grow in a hormone-independent manner through the over-expression of the proto-oncogene c-erbB-1, which is the receptor for epidermal (EGF) and alpha transforming (TGFalfa) growth factors.

Genomic DNAs of cattle, horses, pigs, dogs, cats and chickens were surveyed using Southern blot hybridization analysis, with a human EGFR cDNA fragment. Several bands with different numbers and molecular weights were observed under the condition of low stringency in the individual animal species. The bands showing DNA polymorphism were observed among bovine genomic PstI-digested DNAs from 4 individuals and EcoRI-digested genomic DNAs from 4 chickens. These results may provide basic data which are useful for analysis of tumorigenetic mechanisms in domestic animals.

Forty-six primary vaginal carcinomas were examined immunohistochemically for expression of retinoblastoma (RB) protein, epidermal growth factor receptor (EGFR), and c-erbB-2 oncoprotein. The results demonstrated that RB protein was not lost in any of the cases, suggesting that structural abnormalities of the RB gene may not play an important role in the pathogenesis of vaginal carcinoma. Fifteen and 11% of the cases showed increased expression of EGFR and c-erbB-2 oncoprotein, respectively, indicating that these oncoproteins may be involved in the neoplastic process of a minority of vaginal carcinomas. Overexpression of EGFR and c-erbB-2 oncoprotein had no prognostic significance in vaginal carcinomas.

We have previously demonstrated that two mouse cell lines that are poorly infectible by reovirus become highly susceptible upon transfection with the gene encoding the epidermal growth factor receptor (EGFR) (J. E. Strong, D. Tang, and P. W. K. Lee, Virology 197:405-411, 1993). This enhancement of infection efficiency requires a functional EGFR, since such an enhancement is not observed in cells expressing a mutated (kinase-inactive) EGFR. The additional finding that reovirus is capable of directly binding to the N-terminal ectodomain of the EGFR (D. Tang, J. E. Strong, and P. W. K. Lee, Virology 197:412-414, 1993) has led us to question whether this interaction is required for the activation of a signalling cascade that somehow augments the ensuing infection process. In the present study, we address this question, using cells transfected with the v-erbB oncogene, which encodes a protein structurally related to the EGFR but lacking a large portion of the N-terminal ligand-binding domain. The v-erbB protein also possesses ligand-independent, constitutive tyrosine kinase activity. Control NIH 3T3 cells, which are poorly infectible by reovirus (serotype 3, strain Dearing), and NIH 3T3 cells transfected with the v-erbB oncogene (THC-11) were assayed for their susceptibilities to reovirus infection. Infectivity was determined by immunofluorescent detection of viral proteins, sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of radiolabeled cells, and plaque titration. ALL three assays demonstrated a drastically higher degree of susceptibility to infection in the THC-11 cell line. This enhanced susceptibility was found to be abrogated by treatment of the cells with genistein, an inhibitor of tyrosine protein kinases, but only partially by treatment with daidzein, an inactive analog of genistein. We propose that the mechanism of enhancement of infection efficiency conferred by EGFR and v-erbB is through the opportunistic utilization by the virus of an already activated signal transduction pathway.

The proliferative capacity of breast carcinomas has prognostic significance as measured by S-phase fraction (SPF), yet the molecular parameters that influence the proliferative capacity of breast carcinomas have not been fully elucidated. Ninety-three cases of invasive ductal breast carcinomas, not otherwise specified, were studied by immunohistochemistry and flow cytometry to determine what correlations exist between nuclear grade (NG), SPF, proliferating cell nuclear antigen and the overexpression of p53, epidermal growth factor receptor (EGFR), and c-erb-B-2 in formalin-fixed tissues by the immunoperoxidase method. NG predicted elevated SPF in 78% of cases and was associated with high proliferating cell nuclear antigen score. The presence of p53 was detectable in 13% of cases, and, in each case, the NG was high (Grade 3), with nine aneuploid tumors and three diploid tumors. The SPFs for ALL p53-positive cases were markedly elevated, with 77.8% of the cases with SPF > 15%. EGFR was present in 20.4% of ALL tumors, including 77.8% of tumors positive for p53 and 14% of tumors negative for p53 (P < .001). The mean SPF for p53-positive EGFR-positive tumors was 17.5% versus 21.1% for p53-positive EGFR-negative tumors. The mean SPF for p53-negative tumors was significantly less, regardless of the presence of EGFR. The gene c-erb-B-2 was found in 28% of tumors, ALL of which were p53 negative. These data clearly show a close relationship between high NG and elevated SPF. As determined by flow cytometry, SPF is more consistent and more reliably related to NG than proliferating cell nuclear antigen.(ABSTRACT TRUNCATED AT 250 WORDS)FAU - Dabbs, D J

BACKGROUND: Hormone receptors and oncoproteins are receiving increased attention as possible prognostic factors in different carcinomas. Few data are available regarding quantification of their levels of expression in gynecologic malignancies. METHODS: Epidermal growth factor (EGF) receptor specific binding capacities and affinities were measured by ligand binding assay using [125I]EGF in a competition mode with Accufit software (Lundon Software, Inc., Middlefield, OH). HER-2/neu oncoprotein was extracted from membranes and measured using an enzyme-linked immunosorbent assay. Cathepsin D was measured by an immunoradiometric assay using cytosols for steroid receptor analyses. RESULTS: EGF receptors in 23 nonmalignant uteri ranged from undetectable to 50 fmol/mg membrane protein (median, 0), with dissociation constant values of 1.2 x 10(-9) M to 8.5 x 10(-10) M, compared with EGF receptors in 76 endometrial cancers that ranged from undetectable to 7674 fmol/mg (median, 52). HER-2/neu oncoprotein ranged from undetectable to 2.9 HER-2/neu units (HNU)/microg protein (median, 0.6) in 41 nonmalignant uteri and from undetectable to 5.8 HNU/microg protein (median, 2.5) in endometrial cancers (n = 53). Cathepsin D ranged from 5 to 32 pmol/mg cytosol protein (median, 11) in 42 nonmalignant uteri and 18 to 144 pmol/mg protein (median, 42) in 29 endometrial cancers. CONCLUSIONS: Determination of the frequency and levels of EGF receptors, HER-2/neu protein, and cathepsin D in uteri with and without cancer and the availability of reference materials developed in our laboratory, will allow evaluation of their prognostic value in cancers of the uterus.

BACKGROUND: Epidermal growth factor receptor (EGFR) and c-erbB-2 (also known as HER-2/neu) oncoprotein (p185erbB-2) are members of the subfamily of tyrosine kinase, transmembrane receptors often implicated in human carcinogenesis. We hypothesize that expression of EGFR and p185erbB-2 adds useful prognostic and histogenetic information regarding female genital tract carcinosarcomas (FGTCSs). METHODS: Paraffin sections from 82 FGTCS (61 endometrium, 14 ovary, 5 cervix, and 2 fallopian tube), 56% of which exhibited heterologous elements, were stained using anti-EGFR (clone 31G7, Triton Diagnostics, Alameda, CA) and anti-p185erbB-2 (clone CB11, Novocastra Labs, UK). RESULTS: EGFR reactivity was present in 11 (13.4%) FGTCSs (55% carcinomatous component [CC] only, 18% sarcomatous component [SC] only, and 27% in both). EGFR was associated with adenosquamous histology of the CC (P < 0.05) and heterologous rhabdomyosarcomatous differentiation in the SC (P < 0.05); no other histopathologic features were correlated. p185erbB-2 reactivity was present in 79 (87.8% strong [S], 78% membrane [M], and 8.5% weak) FGTCSs (1% CC only, 0% SC only, and 99% in both). p185erbB-2 did not correlate with histopathologic features or EGFR. Seventy-seven patients had clinical follow-up for longer than 12 months. Approximately 49.3% and 72.3% of patients had recurrent disease by 12 and 80 months, respectively; ALL but 1 were dead from disease. 27% of patients were disease free after 15 to 307 months (median, 77 months; mean, 92 months). EGFR, but not p185erbB-2 expression predicted disease recurrence (P < 0.05). Recurrent disease was associated with Stage greater than I (P < 0.0001), vascular space invasion in resection specimens (P < 0.01), and deep myometrial invasion in hysterectomies (P < 0.05). EGFR was associated with Stage greater than I and did not help predict recurrence in good prognosis groups. CONCLUSIONS: p185erbB-2 overexpression in both CC and SC of FGTCS suggests a common carcinogenic mechanism for both components and supports the conversion-histogenesis hypothesis implicating a dominant role for the CC with the SC arising as a metaplastic change from the CC. EGFR may be expressed in either component and indicates aggressive biologic behavior; however, its prognostic utility is limited by its low predictive value for recurrence (40.3%), inability to foretell recurrence in good prognosis groups, and dependence on stage. High frequency of overexpression and dismal prognosis make FGTCS patients good candidates for trials of therapeutic strategies involving the p185erbB-2 receptor manipulations.

Mutant v-erbB products of avian c-erbB1 have previously been used to correlate structural domains of the receptor encoded by this proto-oncogene with tissue-specific transformation potential. In these studies, deletion of the ligand-binding domain of the receptor has been shown to be required for transformation of erythroblasts, fibroblasts, and endothelial cells. It has, therefore, been postulated that deletion of this domain results in an allosteric change in the receptor analogous to the ligand-bound state of the epidermal growth factor receptor; i.e., it induces a receptor conformation that is constitutively active with respect to mitogenic signaling. While oncogenic v-erbB products have been shown to be expressed on the cell surface of both fibroblasts and erythroblasts, no comprehensive analysis of the oligomeric potential of these products has been conducted. Since the first event known to follow epidermal growth factor binding to its receptor is oligomerization, and receptor dimerization has been correlated with mitogenic signaling, we have carefully analyzed the ability of several v-erbB products to oligomerize in the three target cell types transformed by these oncogenes. In this report, we demonstrate the v-erbB products can efficiently homodimerize in ALL three target tissues, that this dimerization is ligand independent and occurs at the cell surface, and that there is no apparent correlation between v-erbB dimerization and transformation of avian fibroblasts. Furthermore, both oncogenic and nononcogenic v-erbB products can heterodimerize with the native c-erbB1 product in chicken embryo fibroblasts, suggesting that heterodimerization between v-erB and native c-erbB1 is not sufficient to result in c-erbB1-mediated sarcomagenesis.

v-erbA is the oncogenic form of the c-erbA proto-oncogene, which encodes the receptor for thyroid hormones. The expression of the v-erbA oncogene in thyroid differentiated cells, PC Cl 3, inhibits iodide uptake and thyrotropin-dependent growth, whereas it has no effect on the expression of the other thyroid specific markers, i.e. thyroglobulin, thyroperoxidase and thyrotropin receptor. The activity of transcription factor AP-1, evaluated by a specific DNA binding assay and by transcription of AP-induced promoter (TRE) is enhanced in PC v-erbA cells. v-erbA mutants in the DNA binding domain do not affect the iodide uptake of thyroid cells nor AP-1 activity. We suggest that this transcriptional activation mediates the selective effects of v-erbA on the expression of thyroid specific markers.

The prognostic role of the epidermal growth factor receptor (EGFR) and the related receptor p185HER-2 in lung cancer is as yet undefined. We investigated the immunohistochemical expression of EGFR (monoclonal antibody R1; Amersham) and p185HER-2 (polyclonal antibody A485; Dako) in cryosections. A total of 186 unselected and systemically untreated patients with non-small-cell lung cancer (NSCLC) diagnosed and treated at Odense University Hospital, Denmark, were included. Median follow-up period was 66 months. EGFR and p185HER-2 was highly expressed in 55% and 26% of cases respectively. expression of EGFR was independent of p185HER-2 expression. The expression of EGFR was higher in squamous cell carcinomas whereas the level of p185HER-2 staining was higher in adenocarcinomas. expression of either or both receptors was not correlated with age, histological grading, stage and prognosis. We conclude that immunohistochemical detection of these growth factor receptors failed to demonstrate a prognostic significance in patients operated on for NSCLC.

The expression of c-erbB-2 oncoprotein, epidermal growth factor receptor (EGFR) and estrogen receptor (ER) was evaluated by the immunoperoxidase technique (PAP) in ductal breast carcinomas. The relationship between these cell growth regulatory factors was considered and compared with tumor grading, tumor size, lymph node involvement and age of patients. Stratifying of patients on the basis of c-erbB-2, EGFR and ER status indicated that the combination of c-erbB-2 overexpression accompanied by high EGFR value and undetectable ER, identified poorly differentiated tumors and patients with high incidence of axillary lymph node metastases, while high EGFR expression and negative c-erbB-2 staining was connected only with poor tumor grade. The undetectability of molecular markers was associated with higher histological grade and lack of lymph node involvement. Our results indicate that the comparison of c-erbB-2, EGFR and ER status seems to be a powerful tool in discriminating breast carcinomas with different biological phenotypes.

The c-neu oncoprotein, p185c-neu, is a transmembrane tyrosine kinase that shares structural similarities with the receptor for epidermal growth factor (EGFr). We used immunoblots, immunoprecipitation, and immunohistochemistry 1) to test the hypothesis that p185c-neu and EGFr are coordinately expressed in central nervous system tissue and 2) to assess the spatiotemporal expression of both the c-neu oncoprotein and EGFr in the rostral cerebral cortex. In nondenaturing gels, anti-c-neu antibody identified high molecular weight proteins (about 300-400 kDa) that were reduced by EDTA to a molecular weight of 180-200 kDa. Sodium dodecylsulfate polyacrylamide gel electrophoresis broke down this protein into an array of smaller peptides, which were expressed prenatally, transiently during the first three postnatal weeks, or in the adult. Perinatally, c-neu immunoreactivity was evident in subplate neurons, ascending processes of neurons in the cortical plate, and ventricular zone cells. During the second postnatal week, cells throughout cortex expressed somatodendritic immunostaining, but, in the adult, c-neu immunoreactivity was expressed only by pyramidal neurons in layer V and by glia in the white matter and ependyma. EGFr-positive proteins behaved in the nondenaturing gels as did c-neu-positive oncoproteins, suggesting that both proteins naturally formed dimers. This contention was supported by the EGFr-or c-neu immunolabeling of tissue that was previously immunoprecipitated with anti-c-neu or anti-EGFr, respectively. The pattern of EGFr immunolabeling in the developing and mature cortex was virtually identical to that described for c-neu immunoreactivity. Cortical neurons express the c-neu oncoprotein and EGFr, probably as heterodimers. The specific immunolabeling of layer V neurons in the adult cortex with anti-c-neu and anti-EGFr suggests that the p185c-neu ligand and EGF regulate the activity of corticofugal systems. The expression of different c-neu- and EGFr-positive peptides is developmentally defined and may be related to specific ontogenetic events.

Two new immunoenzymatic assays for c-erbB-2 oncoprotein and epidermal growth factor receptor (EGF-R) (Oncogene Science) in human breast cancer were validated. Correlations between these assays and some clinical and biological parameters were also studied. The repeatability and reproducibility of standard curves for the two methods gave a coefficient of variation (CV) of less than 4% and about 10% respectively. The accuracy of c-erbB-2 oncoprotein and EGF-R assays was examined by using dilution and recovery tests throughout the standard curves. The linear relations between theoretical and measured values, for these tests, had slopes close to 1 and an intercept near 0. The median value for EGF-R, measured on solubilized membranes of 290 primary tumors, was 0.12 fmol/micrograms protein, the mean value was 0.37 (range 0 to 35.7). For c-erbB-2 oncoprotein, the median value, measured using the same population, was 2.75 human neu unit/micrograms protein, the mean value was 7.85 (range 1 to 125). There was an inverse relationship between EGF-R values and those for the estrogen receptor (ER), progesterone receptor and pS2 protein as well as menopausal status. C-erbB-2 oncoprotein concentrations were positively correlated with ER, pS2 protein and cathepsin D. Furthermore, a significant positive correlation was observed between EGF-R levels and c-erbB-2 oncoprotein levels. In conclusion, immunoenzymatic assays of EGF-R and c-erbB-2 oncoprotein are easy to use, sensitive and reliable. The accurate standardisation of immunoenzymatic assays could contribute to the clinical use of EGF-R and c-erbB-2 oncoprotein as prognostic factors in breast cancer.

Many growth factors including epidermal growth factor (EGF) induce tyrosine phosphorylation of the c-Cbl proto-oncogene product, whose function, however, remains unclear. Recently, Sli-1, a Caenorhabditis elegans homologue of c-Cbl, was found to be a negative regulator of let-23-mediated vulval induction pathway, suggesting that c-Cbl may negatively regulate EGF receptor (EGFR)-mediated signaling. In this study, by an antisense RNA approach, we examined the effects of expression level of c-Cbl on EGFR signaling and showed that overexpression of c-Cbl reduces and antisense repression of c-Cbl enhances autophosphorylation of EGF receptors and activation of the JAK-STAT pathway. However, in contrast to the Sli-1 protein, the expressed amount of c-Cbl does not affect activation of the Ras pathway, suggesting that the EGFR-mediated signaling pathways are differently regulated by c-Cbl among nematodes and mammals.

We have shown recently that a retrovirus vector expressing a natural mutant form of the PML-RAR alpha protein characteristic of human acute promyelocytic leukaemia can transform early chicken hematopoietic progenitors (Altabef et al., 1996). Neither truncated PML nor truncated RAR alpha alone could induce transformation which suggest that the two domains should cooperate for the oncogenicity of the fusion product. To further investigate the mechanisms of this co-operation, we have tested whether a truncated RAR alpha could cooperate with the v-erbB oncogene. This oncogene has previously been shown to co-operate with the rearranged thyroid hormone receptor, v-erbA, to transform erythrocytic progenitors. We show that v-erbB and a truncated RAR alpha co-operate when expressed simultaneously as independent products to transform very early chicken haematopoietic cells close to pluripotent stage. In addition, we show that v-erbB alters transcriptional abilities of RAR alpha by both enhancing its effects on RARE and reducing those on AP-1. Therefore, RAR alpha is able to co-operate with different kinds of proteins to induce transformation of early haematopoietic cells. This strongly suggests that RAR alpha are involved in the differentiation commitment of early haematopoietic progenitors during the normal process of haematopoietic differentiation. These data bring new insights in the mechanisms of oncogenic transformation by rearranged RAR alpha.

Epidermal growth factor receptor (EGF-R) and the oncogene product of c-erbB-2 have been shown to be expressed in human tumors and in some cases relate to clinical outcome. This study investigates the correlation between the presence of these receptor proteins and various histological grades of cervical tissues. Immunocytochemical analysis of benign, premalignant and malignant cervical tissues showed a highly significant correlation between the expression of the two proteins and the histological grade of the lesions. Moreover, the overall frequency of coexpression of these proteins was similar. This study suggests that these proteins may be involved in cellular proliferation and have a significant role in the progression of cervical cancer.

The Crk proto-oncogene product is an SH2 and SH3 domain-containing adaptor protein. We have previously demonstrated that Crk-II becomes rapidly tyrosine-phosphorylated in response to stimulation with insulin-like growth factor I (IGF-I) and might be involved in the IGF-I receptor signalling pathway. To determine whether this involvement includes the direct interaction of Crk-II with the cytoplasmic region of the receptor, studies were performed in vitro with glutathione S-transferase (GST) fusion proteins containing various domains of Crk-II. The kinase assay in vitro showed that activated IGF-I receptors efficiently phosphorylated the GST-Crk-II fusion protein. This phosphorylation was dependent on the presence of the SH2 domain and Tyr-221 located in the spacer region between the two SH3 domains. mutation of Tyr-221 not only prevented phosphorylation of GST-Crk in vitro, but also significantly increased the ability of GST-Crk proteins to co-precipitate activated IGF-I receptors from total cell lysates. Additional binding experiments in vitro showed that Crk-II might interact with the phosphorylated IGF-I receptor through its SH2 domain. To elucidate which region of the IGF-I receptor interacts with Crk-II, a peptide association assay was used in vitro. Different domains of the IGF-I receptor were expressed as (His)6-tagged fusion peptides, phosphorylated with activated wheat germ agglutinin-purified IGF-I receptors and tested for association with GST-Crk-II fusion proteins. Using wild-type as well as mutated peptides, we showed that the SH2 domain of Crk-II preferentially binds the peptide encoding the juxtamembrane region of the IGF-I receptor. Phosphorylation of Tyr-950 and Tyr-943 of the receptor is important for this interaction. These findings allow us to propose a model of direct interaction of Crk-II and the IGF-I receptor in vivo. On activation of the IGF-I receptor, Crk-II binds to phosphorylated tyrosine residues, especially in the juxtamembrane region. As a result of this binding, the IGF-I receptor kinase phosphorylates Tyr-221 of Crk-II, resulting in a change in intramolecular folding and binding of the SH2 domain to the phosphorylated Tyr-221, which causes rapid disassociation of the Crk-II-IGF-I receptor complex.

The exact role of oncogenes and proto-oncogenes in the development of squamous cell carcinoma of the head and neck (SCCHN) is still debatable. The expression of the c-erbB-2, c-erbB-3 and c-erbB-4 members of the epidermal growth factor receptor family was examined in 16 fresh frozen tissue specimens of SCCHN using avidin-biotin complex immunohistochemistry, with monoclonal and/or polyclonal antibodies directed against each. Eight fresh frozen tissue specimens of normal oral mucosa were included as controls. Of the SCCHN examined, mixed membrane/cytoplasmic staining (moderate to intense) of c-erbB-2 was found in 14/16 cases (88%). When present in the specimen, immunopositive staining of c-erbB-2 was seen in some of the oral surface epithelial cell layers (basal, intermediate and/or superficial) as well as the tumour islands. Weak cytoplasmic staining of c-erbB-3 and c-erbB-4 was found in 13/16 (81%) and 11/16 (69%) cases respectively. When present in the specimen, c-erbB-3 and cerbB-4 immunopositive staining was seen in some of the oral surface epithelial cell layers (basal, intermediate and/or superficial) as well as the tumour islands. For the positive carcinomas for c-erbB-2, c-erbB-3 and c-erbB-4, the epithelium located near the carcinomas showed weak mixed membrane/cytoplasmic staining of c-erbB-2 in 5/14 cases (36%), weak cytoplasmic staining of c-erbB-3 in 7/13 cases (54%) and of c-erbB-4 in 3/11 cases (27%). ALL the normal control oral mucosa showed the same pattern of staining for c-erbB-2, c-erbB-3 and c-erbB-4 found in the epithelium located near the carcinomas. Only expression of c-erbB-2 was found to correlate with the increase in the tumour stage, while co-expression of c-erbB-2, c-erbB-3 and c-erbB-4 was found to correlate with the patient survival time in 25% of the carcinomas examined. The present study shows that a) expression of c-erbB-2, but not c-erbB-3 and c-erbB-4 correlates with the increase of the tumour stage b) co-expression of c-erbB-2, c-erbB-3 and c-erbB4 correlates with decreased survival time in some of the cases of SCCHN, but not the majority c) co-expression of the c-erbB family in normal oral mucosa as well as in the carcinoma may question whether the increased tendency for development of the disease is due to co-expression of c-erbB proto-oncogenes in head and neck lesions.

EGFRvIII is a ligand-independent, constitutively active variant of the epidermal growth factor receptor (EGFR) that is specifically expressed in gliomas and various other human malignancies and has been proposed as a target for directed tumor therapy. We have recently constructed a highly potent single-chain antibody-toxin, scFv(14E1)-ETA, which consists of the variable domains of the antibody 14E1 specific for human full-length EGFR genetically fused to a truncated form of Pseudomonas exotoxin A. We demonstrate here binding of 14E1 antibody to both full-length and variant EGFR. In contrast to a recombinant toxin containing transforming growth factor-alpha (TGF-alpha) as a cell targeting domain, scFv(14E1)-ETA was highly active on cells expressing EGFRvIII. Surprisingly, scFv(14E1)-ETA displayed cell killing activity on EGFRvIII-expressing cells that was up to 100-fold higher than on control cells expressing full-length EGFR. No differences in the binding affinities of scFv(14E1)-ETA to full-length EGFR or EGFRvIII were observed, suggesting that events downstream of immunotoxin binding are responsible for the increased sensitivity of EGFRvIII-expressing cells. This might have implications for the development of therapeutic reagents simultaneously targeting different forms of the EGFR.

Matrix metalloproteinases (MMPs) are a group of enzymes thought to be responsible for both normal connective tissue matrix remodelling and accelerated breakdown associated with tumour development. The current study aimed to investigate the immunohistochemical expression of matrix metalloproteinase 3 (MMP-3, stromelysin-1) in correlation with the expression of Basement Membrane (BM) antigen (type IV collagen, laminin), fibronectin, cathepsin D, p53, c-erbB-2, proliferative activity (Ki-67, PCNA), steroid receptor content as well as to the other conventional clinicopathological parameters in breast cancer. This study was performed on a series of frozen and paraffin sections from 84 breast cancer specimens by immunohistochemistry using the monoclonal antibody MMP-3 (Ab-1). Stromelysin-1 (ST1) was observed in about 10% of epithelial cells in the control groups (cases of fibrocystic and benign proliferative breast disease), while expression (> 10% of expression) was detected in 89.7% of tumours. The expression of ST1 in carcinoma cells was strongly associated with its presence in the stroma (p < 0.001). A significantly positive correlation was found between ST1 expression, and p53 tumour suppressor gene product (p = 0.004), and a relationship with c-erbB-2 protein and progesterone receptor status was also indicated. These findings suggest that ST1 expression in breast cancer tissue is irrespective of the expression of the extracellular matrix component, the proteolytic enzyme cathepsin D and the growth fraction of the tumour, and that it could be a potential new prognostic marker in breast cancer.

C3H10T1/2 fibroblasts transformed by the minimal expression of oncogenic Ha-Ras (V12H10 cells) or N-Ras (K61N10 cells) have constitutive mitogen-activated protein kinase (MAPK) activity and proliferate in serum-free medium. The constitutive MAPK activity and serum-independent proliferation of V12H10 cells are sensitive to the growth factor antagonist, suramin (Hamilton, M., and Wolfman, A. (1998) oncogene 16, 1417-1428), suggesting that Ha-Ras-mediated regulation of the MAPK cascade is dependent upon the action of an autocrine factor. Serum-free medium conditioned by V12H10 cells contains an activity that stimulates MAPK activity in quiescent fibroblasts. This MAPK stimulatory activity could be specifically blocked by the epidermal growth factor receptor (EGFR) inhibitors, PD153035 and PD158780. These inhibitors also blocked the serum-independent proliferation of V12H10 cells. Immunodepletion of conditioned medium with antibodies to transforming growth factor alpha and EGF significantly inhibited its ability to stimulate MAPK activity. Stable transfection of EGFR-negative NR6 and EGFR-positive Swiss3T3 cells with oncogenic (G12V)Ha-Ras demonstrated that only the Ha-Ras-transfected Swiss 3T3 cells possessed constitutive MAPK activity, and this activity was sensitive to PD153035. These data suggest that autocrine activation of the EGFR is required for the regulation of the MAPK cascade in cells minimally expressing oncogenic Ha-Ras.

Human cancers arise from a series of mutations, many of which direct the expression of mutant proteins with altered functions. These aberrant proteins are attractive targets for new therapeutic agents. One such protein is a mutant epidermal growth factor receptor (EGFRvIII) that has an in-frame deletion near the NH2 terminus of its extracellular domain. This protein was first identified in human gliomas, but has also been shown to be present in lung and breast carcinomas. The deletion results in a receptor with constitutive tyrosine kinase activity that enhances the tumorigenicity of glioblastomas in vivo. The deletion also creates a tumor-specific cell-surface sequence at the deletion junction. Three specific anti-EGFRvIII mAbs have been isolated following immunization with a mixture of a deletion junction synthetic peptide and EGFRvIII as present on cell membranes. We have constructed immunotoxins by conjugating a modified version of Pseudomonas exotoxin A to these mAbs. Immunotoxins were tested on cells that had been transfected with cDNA for the EGFRvIII receptor and expressed receptor protein at 5 x 10(5) receptors/cell. ALL three immunotoxins were cytotoxic to these cells, with 50% inhibition of protein synthesis occurring in a 15-50 pM range. The immunotoxins specifically targeted EGFRvIII, as their cytotoxicity could be blocked by their respective free antibody. They showed little or no cytotoxicity to cells expressing high levels of normal epidermal growth factor receptors, demonstrating that they are able to discriminate between cells expressing the mutant receptor and those expressing the wild-type receptor. Immunotoxins targeted to mutant epidermal growth factor receptors are promising candidates for further development as tumor cell-specific therapeutic agents.