3688

ITGB1

integrin, beta 1 (fibronectin receptor, beta polypeptide, antigen CD29 includes MDF2, MSK12);ITGB1;integrin, beta 1 (fibronectin receptor, beta polypeptide, antigen CD29 includes MDF2, MSK12)

glycoprotein IIa|integrin VLA-4 beta subunit|integrin beta-1|very late activation protein, beta polypeptide

10p11.2

protein-coding

To determine how AKT2 might contribute to tumor cell progression, a full-length, wild-type, human AKT2/protein kinase B (PKB)beta cDNA was transfected into a panel of eight human breast and ovarian cancer cells. AKT2 transfectants demonstrated increased adhesion and invasion through collagen IV because of up-regulation of beta1 integrins. In addition, AKT2 cells were more metastatic than control cells in vivo. Increased invasion by AKT2 was blocked by preincubation with an anti-beta1 integrin function blocking antibody, exposure to wortmannin, and by expression of phosphatase and tensin homologue tumor suppressor (PTEN). Confocal microscopy performed on transfected human breast cancer cells showed that unlike AKT1, AKT2 protein predominantly localized adjacent to the collagen IV matrix during cellular attachment. Overexpression of AKT2, but not AKT1 or AKT3, was sufficient to duplicate the invasive effects of phosphoinositide 3-OH kinase (PI3-K) transfected in breast cancer cells. Furthermore, expression of kinase dead AKT2(181 amino acid methionine [M]), and not kinase dead AKT1(179M) or AKT3(177M), was capable of blocking invasion induced by either human epidermal growth factor receptor-2 (HER-2) overexpression or by activation of PI3-K. Taken together, these data indicate that AKT2 mediates PI3-K-dependent effects on adhesion, motility, invasion, and metastasis in vivo.

Integrins regulate cell-cell and cell-matrix adhesion and thereby play critical roles in tumor progression and metastasis. Although work in preclinical models suggests that beta1 integrins may stimulate metastasis of a number of cancers, expression of the beta1 subunit alone has not been shown to be a useful prognostic indicator in human cancer patients. Here we have demonstrated that the alpha2beta1 integrin suppresses metastasis in a clinically relevant spontaneous mouse model of breast cancer. These data are consistent with previous studies indicating high expression of alpha2beta1 integrin in normal breast epithelium and loss of alpha2beta1 in poorly differentiated breast cancer. They are also consistent with our systematic analysis of microarray databases of human breast and prostate cancer, which revealed that decreased expression of the gene encoding alpha2 integrin, but not genes encoding alpha1, alpha3, or beta1 integrin, was predictive of metastatic dissemination and decreased survival. The predictive value of alpha2 expression persisted within both good-risk and poor-risk cohorts defined by estrogen receptor and lymph node status. Thus, the alpha2beta1 integrin functionally inhibits breast tumor metastasis, and alpha2 expression may serve as an important biomarker of metastatic potential and patient survival.

Reduced epithelial cadherin (E-cad) is a hallmark of invasive carcinomas that have acquired epithelial-mesenchymal transition (EMT) phenotypes. Here we show that down-regulated E-cad expression induced by transforming growth factor-beta (TGF-beta) and EMT preceded breast cancer outgrowth in three-dimensional (3D) organotypic assays and in the lungs of mice. Pharmacological inhibitors against focal adhesion kinase prevented metastatic outgrowth of newly seeded organoids, but not that of their fully established counterparts. Interrogating the D2-HAN (hyperplastic alveolar nodule) model of breast cancer dormancy and metastasis showed that dormant D2.OR cells produced branched organoid morphologies in 3D-cultures, and expressed robust quantities of E-cad that was uncoupled from regulation by TGF-beta. In contrast, metastatic D2.A1 organoids were spherical and wholly lacked E-cad expression. Interestingly, D2.A1 cells engineered to re-express E-cad formed branched organoids, down-regulated beta1 integrin expression, and failed to undergo metastatic outgrowth. The tumor-suppressing function of E-cad was inactivated by increased microenvironmental rigidity, and was not recapitulated by expression of an E-cad mutant lacking its extracellular domain. Twist expression, but not that of Snail, reinitiated metastatic outgrowth in dormant D2.OR cells. Our findings show that EMT and its down-regulated expression of E-cad circumvent breast cancer dormancy in part by facilitating beta1 integrin expression necessary for metastatic outgrowth.

BACKGROUND: Although integrins have been implicated in the progression of breast cancer, the exact mechanism whereby they exert this regulation is clearly not understood. To understand the role of integrins in breast cancer, we examined the expression levels of several integrins in mouse breast cancer cell lines by flow cytometry and the data were validated by Western and RT-PCR analysis. The importance of integrins in cell migration and cell invasion was examined by in vitro assays. Further the effect of integrins on metastasis was investigated by in vivo experimental metastasis assays using mouse models. RESULTS: Integrin alpha5 subunit is highly expressed in the nonmetastatic cell line 67NR and is significantly low in the highly invasive cell line 4T1. In contrast, expression levels of integrin alpha6 subunit are high in 4T1 cells and low in 67NR cells. In vitro data indicated that overexpression of alpha5 subunit and knockdown of alpha6 integrin subunit inhibited cell proliferation, migration, and invasion. Our in vivo findings indicated that overexpression of integrin alpha5 subunit and knockdown of alpha6 subunit decreased the pulmonary metastasis property of 4T1 cells. Our data also indicated that overexpression of alpha 5 integrin subunit and suppression of alpha6 integrin subunit inhibited cells entering into S phase by up-regulating p27, which results in downregulation of cyclinE/CDK2 complexes, This suggests that these integrins regulate cell growth through their effects on cell-cycle-regulated proteins. We also found that modulation of these integrins upregulates E2F, which may induce the expression of chk1 to regulate cdc25A/cyclin E/CDK2/Rb in a feedback loop mechanism. CONCLUSION: This study indicates that Integrin alpha5 subunit functions as a potential metastasis suppressor, while alpha6 subunit functions as a metastasis promoter. The modulation of integrins reduces cdc25 A, another possible mechanism for downregulation of CDK2. Taken together we demonstrate a link between integrins and the chk1-cdc25-cyclin E/CDK2-Rb pathway.

BACKGROUND: Phosphatase of regenerating liver-3 (PRL-3) plays a causative role in tumor metastasis, but the underlying mechanisms are not well understood. In our previous study, we observed that PRL-3 could decrease tyrosine phosphorylation of integrin beta1 and enhance activation of ERK1/2 in HEK293 cells. Herein we aim to explore the association of PRL-3 with integrin beta1 signaling and its functional implications in motility, invasion, and metastasis of colon cancer cell LoVo. METHODS: Transwell chamber assay and nude mouse model were used to study motility and invasion, and metastsis of LoVo colon cancer cells, respectively. Knockdown of integrin beta1 by siRNA or lentivirus were detected with Western blot and RT-PCR. The effect of PRL-3 on integrin beta1, ERK1/2, and MMPs that mediate motility, invasion, and metastasis were measured by Western blot, immunofluorencence, co-immunoprecipitation and zymographic assays. RESULTS: We demonstrated that PRL-3 associated with integrin beta1 and its expression was positively correlated with ERK1/2 phosphorylation in colon cancer tissues. Depletion of integrin beta1 with siRNA, not only abrogated the activation of ERK1/2 stimulated by PRL-3, but also abolished PRL-3-induced motility and invasion of LoVo cells in vitro. Similarly, inhibition of ERK1/2 phosphorylation with U0126 or MMP activity with GM6001 also impaired PRL-3-induced invasion. In addition, PRL-3 promoted gelatinolytic activity of MMP2, and this stimulation correlated with decreased TIMP2 expression. Moreover, PRL-3-stimulated lung metastasis of LoVo cells in a nude mouse model was inhibited when integrin beta1 expression was interfered with shRNA. CONCLUSION: Our results suggest that PRL-3s roles in motility, invasion, and metastasis in colon cancer are critically controlled by the integrin beta1-ERK1/2-MMP2 signaling.

AIM: To study the expression levels of E- selectin, integrin beta1 and immunoglobulin superfamily member-intercellular adhesion molecule-1 (ICAM-1) in human gastric carcinoma cells, and to explore the relationship between these three kinds of cell adhesion molecules and gastric carcinoma. METHODS: The serum contents of E-selectin, integrin beta1 and ICAM-1 were detected by enzyme-linked immunosorbent assay (ELISA), in 47 healthy individuals (control group) and in 57 patients with gastric carcinoma (gastric carcinoma group) respectively prior to operation and 7 d after operation. RESULTS: The serum E-selectin, ECAM-1 and integrin beta1 were found to be expressed in both control and gastric carcinoma groups. However, they were highly expressed in patients with gastric carcinoma patients before operation or with unresectable tumours. The expression levels of ICAM-1 and integrin beta1 were significantly higher in gastric carcinoma patients than in controls (P < 0.01). A comparison of the E-selectin levels between the two groups showed statistically insignificant difference (P = 0.64). In addition, the expression levels were all decreased substantially in the postoperative patients subjected to radical resection of the tumours, indicating that the high level expressions of these compounds might be the important factor for predicting the prognosis of these patients. CONCLUSION: Serum E-selectin, ICAM-1 and integrin beta1 expression levels are probably related to the metastasis and relapse of gastric cancer.

OBJECTIVE: The study was to evaluate the association of expression level of alpha5beta1-integrin with clinicopathologic features and prognosis in gastric cancer (GC). METHODS: The expression of alpha5beta1-integrin in normal gastric mucosa and GC tissue was detected with immunohistochemistry. The level of alpha5 and beta1 mRNA in GC tissues and non-neoplastic tissues was evaluated in 48 paired cases by quantitative real-time polymerase chain reaction (qRT-PCR). Survival analysis by the Kaplan-Meier method was performed to assess prognostic significance. RESULTS: The alpha5beta1-integrin expression was detected in 68.3 % (127/186) GC samples, and there was a significant difference on their positive expression rate between GC tissue and normal gastric mucosa (P < 0.001). The positive expression rate of alpha5beta1-integrin in patients with poor histologic differentiation (P = 0.001), lymph node metastasis (P < 0.001), and recurrence (P < 0.001) group was heightened. Using Kaplan-Meier analysis, a comparison of survival curves of low versus high expresser of alpha5beta1-integrin revealed a highly significant difference in human GC tissue (P = 0.002), which suggested that overexpression of alpha5beta1-integrin is associated with a worse prognosis. Multivariate analyses showed that alpha5beta1-integrin expression was independent risk factor predicting overall survival [Hazard ratio (HR) 1.594, 95 % confidence interval (CI) 1.236-2.408, P = 0.006] and disease-free survival [HR 3.952, 95 %

Migration and adhesion of tumor cells are essential prerequisites for the formation of metastases in malignant diseases. Chemokine receptor 7 (CCR7) has been shown to regulate integrin which can then facilitate adhesion of cancer cells to and/or migration through the extracellular matrix (ECM). In order to identify the connection between CCR7 and beta1 integrin, and the influence on cell adhesion and migration in metastatic squamous cell carcinoma of the head and neck (SCCHN). We use adhesion assays, migration assay, immunofluorescence staining, western blotting, and immunohistochemical analysis to find whether beta1 integrin can be activated by CCL19 (CCR7's ligand) and its role in SCCHN. The experiments were performed in the metastatic SCCHN cell line PCI-37B after pre-incubation of the cells with CCL19 and beta1 integrin inhibitors RGD-peptide. Our results demonstrate that CCR7 favours PCI-37B cell adhesion and migration, and induces reorganization of the actin cytoskeleton and up-expression of beta1 integrin protein. beta1 integrin inhibitor RGD-peptide can block all these effects. Taken together, our data indicate that CCR7 regulate cell adhesion and migration via beta1 integrin in metastatic SCCHN, and these results can provide a basis for new strategies in preventing metastases of SCCHN.

PURPOSE: This study aimed to understand the prognostic value of integrin beta1 expression in head and neck squamous cell carcinoma (HNSCC) and the mechanism underlying its association with metastatic HNSCC. EXPERIMENTAL DESIGN: Archival HNSCC tissues including 99 nonmetastatic primary tumors and 101 metastatic primary tumors were examined for the association of integrin beta1 expression with metastasis and disease prognosis by appropriate statistical methods. Fluorescence-activated cell sorting was used to separate the integrin beta1(high/+) cell population from the integrin beta1(low/-) population in HNSCC cell lines. These two populations and integrin beta1 shRNA knockdown HNSCC cells were examined for the effect of integrin beta1 on invasion in vitro and on lymph node and lung metastases in a xenograft mouse model. expression and activation of matrix metalloproteinases (MMP) were examined by zymography. RESULTS: Statistical analysis showed that integrin beta1 expression was significantly higher in the metastatic primary tumors than in the nonmetastatic tumors (42.6% vs. 24.8%, P < 0.0001 and P < 0.0001 by univariate and multivariate analyses, respectively). In patients with lymph node metastasis, integrin beta1 expression was inversely correlated with overall survival (P = 0.035). The integrin beta1 knockdown or integrin beta1(low/-) HNSCC cells showed a significant reduction in lymph node and lung metastases in vivo (P < 0.001 and P < 0.05, respectively). Significantly reduced Matrigel invasion capability was also found in integrin beta1 knockdown or integrin beta1(low/-) HNSCC cells (P < 0.01). Finally, zymography results showed integrin beta1-affected HNSCC invasion by regulating MMP-2 activation. CONCLUSION: These findings indicate that integrin beta1 has a major impact on HNSCC prognosis through its regulation of metastasis.

The invasive and metastatic potentials of hepatocellular carcinoma are positively correlated with the expression level of alpha3beta1 integrin, a high-affinity adhesion receptor for laminin isoforms including laminin-5. In this study, we investigated changes in the adhesive and invasive behaviors of human HCC HepG2 cells after transfection with cDNA for alpha3 integrin in order to elucidate the direct involvement of this integrin in these cellular processes. We introduced cDNA for splice variants of alpha3 integrin (alpha3A and alpha3B) into the cells, and selected two transfectant clones (HepG2-3A and HepG2-3B), which express the alpha3A and alpha3B integrins, respectively. Both transfectant cells adhered almost equally to laminin-5-coated plates in an alpha3 integrin-dependent manner, indicating that transfected alpha3Abeta1 and alpha3Bbeta1 integrins were functionally active in these cells. The migratory and invasive potentials of the transfectant cells were assessed by scratch wound assay and in vitro chemoinvasion assay. The results demonstrated that the migration of HepG2-3A and HepG2-3B cells but not of mock transfectant (HepG2-M) cells was stimulated on the plates coated with laminin-5. Furthermore, HepG2-3A and HepG2-3B cells were found to be more invasive into laminin-5-containing matrices than were HepG2-M cells. These results strongly suggest that enhanced expression of alpha3beta1 integrin on HCC cells is directly involved in their malignant phenotypes such as invasion and metastasis.

Mechanism of HAb18G/CD147 underlying the metastasis process of human hepatoma cells has not been determined. In the present study, we found that integrin alpha3beta1 colocalizes with HAb18G/CD147 in human 7721 hepatoma cells. The enhancing effect of HAb18G/CD147 on adhesion, invasion capacities and matrix metalloproteinases (MMPs) secretion was decreased by integrin alpha3beta1 antibodies (p<0.01). The expressions of integrin downstream molecules including focal adhesion kinase (FAK), phospho-FAK (p-FAK), paxillin, and phospho-paxillin (p-paxillin) were increased in human hepatoma cells overexpressing HAb18G/CD147. Deletion of HAb18G/CD147 reduces the quantity of focal adhesions and rearranges cytoskeleton. Wortmannin and LY294002, specific phosphatidylinositol kinase (PI3K) inhibitors, reversed the effect of HAb18G/CD147 on the regulation of intracellular Ca(2+) mobilization, significantly reducing cell adhesion, invasion and MMPs secretion potential (p<0.01). Together, these results suggest that HAb18G/CD147 enhances the invasion and metastatic potentials of human hepatoma cells via integrin alpha3beta1-mediated FAK-paxillin and FAKPI3K-Ca(2+) signal pathways.

BACKGROUND: HAb18G/CD147 plays pivotal roles in invasion by hepatoma cells, but the underlying mechanism remains unclear. Our previous study demonstrated that overexpression of HAb18G/CD147 promotes invasion by interacting with integrin alpha3beta1. However, it has never been investigated whether alpha3beta1 is solely responsible for this process or if other integrin family members also interact with HAb18G/CD147 in human hepatoma cells. METHODS: Human SMMC-7721 and FHCC98 cells were cultured and transfected with siRNA fragments against HAb18G/CD147. The expression levels of HAb18G/CD147 and integrin alpha6beta1 were determined by immunofluorescent double-staining and confocal imaging analysis. Co-immunoprecipitation and Western blot analyses were performed to examine the native conformations of HAb18G/CD147 and integrin alpha6beta1. Invasion potential was evaluated with an invasion assay and gelatin zymography. RESULTS: We found that integrin alpha6beta1 co-localizes and interacts with HAb18G/CD147 in human hepatoma cells. The enhancing effects of HAb18G/CD147 on invasion capacity and secretion of matrix metalloproteinases (MMPs) were partially blocked by integrin alpha6beta1 antibodies (P < 0.01). Wortmannin, a specific phosphatidylinositol kinase (PI3K) inhibitor that reverses the effect of HAb18G/CD147 on the regulation of intracellular Ca2+ mobilization, significantly reduced cell invasion potential and secretion of MMPs in human hepatoma cells (P < 0.05). Importantly, no additive effect between Wortmannin and alpha6beta1 antibodies was observed, indicating that alpha6beta1 and PI3K transmit the signal in an upstream-downstream relationship. CONCLUSION: These results suggest that alpha6beta1 interacts with HAb18G/CD147 to mediate tumor invasion and metastatic processes through the PI3K pathway.

Arrest of circulating tumor cells in distant organs is required for hematogenous metastasis, but the tumor cell surface molecules responsible have not been identified. Here, we show that the tumor cell alpha3beta1 integrin makes an important contribution to arrest in the lung and to early colony formation. These analyses indicated that pulmonary arrest does not occur merely due to size restriction, and raised the question of how the tumor cell alpha3beta1 integrin contacts its best-defined ligand, laminin (LN)-5, a basement membrane (BM) component. Further analyses revealed that LN-5 is available to the tumor cell in preexisting patches of exposed BM in the pulmonary vasculature. The early arrest of tumor cells in the pulmonary vasculature through interaction of alpha3beta1 integrin with LN-5 in exposed BM provides both a molecular and a structural basis for cell arrest during pulmonary metastasis.

OBJECTIVE: To explore the key regulatory genes associated with lung cancer in order to reduce its occurrence and progress through silencing these key genes. METHODS: To identify the key regulatory genes involved in lung cancer, we performed a combination of gene array and bioinformatics analyses to compare gene transcription profiles in 3 monoclonal cell strains with high, medium or low metastatic abilities, which were separated from the SPC-A-1sci and SPC-A-1 cell lines by limiting dilution monoclone assay. We then analyzed those genes' biological activities by knocking down their expression in SPC-A-1sci cells using siRNA and lenti-viral shRNA vectors, followed by determinations of the invasion and migration capabilities of the resulting cell lines in vitro as well as their potential for inducing occurrence and metastasis of lung cancer in vivo. To examine the clinical relevance of these findings, we analyzed the expression levels of the identified genes in human lung cancer tissues (n = 135) and matched adjacent normal tissues by immunohistochemical (IHC) staining. RESULTS: Three monoclonal cell strains characterized with high, medium or low metastatic abilities were successfully selected. Gene array and bioinformatics analyses implied that osteopontin, LAMB3 and ITGB1 were key genes involved in lung cancer. Knockdown of these genes suppressed human lung cancer cell invasion and metastasis in vitro and in vivo. Clinical sample analyses indicated that osteopontin, LAMB3 and ITGB1 protein expression levels were higher in lung cancer patients, compared to non-cancerous adjacent tissues, and correlated with lymphatic metastasis. CONCLUSIONS: We confirmed that osteopontin, LAMB3 and ITGB1 played important roles in the occurrence and metastasis of lung cancer, thus provided important clues to understanding the molecular mechanism of metastasis and contributing to the therapeutic treatment of lung cancer.

To discover novel lung adenocarcinoma (AdC) biomarkers, isobaric tags for relative and absolute quantitation (iTRAQ)-tagging combined with 2D-LC-MS/MS analysis was used to identify differentially expressed plasma membrane proteins in lung AdC and paired paraneoplastic normal lung tissues (PNLTs) adjacent to tumors. In this study, significant caveolin-1 downregulation and integrin beta1 upregulation was observed in primary lung AdC vs. PNLT. As there has been no report on the association of integrin beta1 with lung AdC, immunohistochemical staining was performed to detect the expression of integrin beta1 in an independent set of archival tissue specimens including 42 cases of PLNT, 46 cases of without lymph node metastasis primary AdC (non-LNM AdC) and 62 cases of LNM AdC; the correlation of their expression levels with clinicopathological characteristics and clinical outcomes were evaluated. Based on the data, upregulation of integrin beta1 was significantly correlated with advanced clinical stage and lymph node metastasis. Integrin beta1 overexpression was significantly associated with advanced clinical stage (P<0.05), lymph node metastasis (P<0.05), increased relapse rate (P<0.05) and decreased overall survival (P<0.05) in AdCs. Cox regression analysis indicated that integrin beta1 overexpression is an independent prognostic factor. The data suggest that integrin beta1 is a potential biomarker for LNM and prognosis of AdC and integrin beta1 upregulation may play an important role in the pathogenesis of AdC.

The capacity of tumor cells to form metastatic foci correlates with their ability to interact with and migrate through endothelial cell layers. This process involves multiple adhesive interactions between tumor cells and the endothelium. Only little is known about the molecular nature of these interactions during extravasation of tumor cells. In human melanoma cells, the integrin alphavbeta3 is involved in transendothelial migration and its expression correlates with metastasis. However, many human melanoma cells do not express beta3 integrins. Therefore, it remained unclear how these cells undergo transendothelial migration. In this study we show that human melanoma cells with different metastatic potency, which do not express beta2 or beta3 integrins, express the VCAM-1 receptor alpha4beta1. VCAM-1 is up-regulated on activated endothelial cells and is known to promote transendothelial migration of leukocytes. Interestingly, despite comparable cell surface levels of alpha4beta1, only the highly metastatic melanoma cell lines MV3 and BLM, but not the low metastatic cell lines IF6 and 530, bind VCAM-1 with high affinity without further stimulation, and are therefore able to adhere to and migrate on isolated VCAM-1. Moreover, we demonstrate that function-blocking antibodies against the integrin alpha4beta1, as well as siRNA-mediated knock-down of the alpha4 subunit in these highly metastatic human melanoma cells reduce their transendothelial migration. These data imply that only high affinity interactions between the integrin alpha4beta1 on melanoma cells and VCAM-1 on activated endothelial cells may enhance the metastatic capacity of human beta2/beta3-negative melanoma cells.

Melanoma cells express and interact with laminins (LMs) and other basement membrane components during invasion and metastasis. In the present study we have investigated the production and migration-promoting activity of laminin isoforms in melanoma. Immunohistochemistry of melanoma specimens and immunoprecipitation/western blotting of melanoma cell lines indicated expression of laminin-111/121, laminin-211, laminin-411/421, and laminin-511/521. Laminin-332 was not detected. In functional assays, laminin-111, laminin-332, and laminin-511, but not laminin-211 and laminin-411, strongly promoted haptotactic cell migration either constitutively or following stimulation with insulin-like growth factors. Both placenta and recombinant laminin-511 preparations were highly active, and the isolated recombinant IVa domain of LMalpha5 also promoted cell migration. Function-blocking antibodies in cell migration assays revealed alpha6beta1 integrin as the major receptor for laminin-111, and both alpha3beta1 and alpha6beta1 integrins for laminin-332 and laminin-511. In contrast, isolated LMalpha5 IVa domain-promoted melanoma cell migration was largely mediated via alphaVbeta3 integrin and inhibited by RGD peptides. Given the ubiquitous expression of alpha5 laminins in melanoma cells and in melanoma-target tissues/anatomical structures, as well as the strong migration-promoting activity of these laminin isoforms, the alpha5 laminins emerge as putative primary extracellular matrix mediators of melanoma invasion and metastasis via alpha3beta1 and other integrin receptors.

Members of the integrin family recognize a variety of spatially-restricted extracellular ligands. Classically, ligation of integrins activates cytoplasmic signals in the integrin-expressing cell and contributes to cell adhesion, migration, proliferation and survival. At least two members of this family, alphavbeta6 and alphavbeta8 perform an additional function, activation of latent complexes of transforming growth factor beta. In effect, this process allows integrins on one cell to activate signals on adjacent (in the case of alphavbeta6) or nearby cells (in the case of alphavbeta8). Integrin-mediated TGFbeta activation has been shown to play important roles in modulating tissue fibrosis, acute lung injury and pulmonary emphysema. Given the important roles that TGFbeta plays in modulating epithelial cell growth, epithelial-to-mesenchymal transformation and tumor invasion and metastasis, integrin-mediated TGFbeta activation is likely to play important roles in tumor growth and metastasis.

Recent studies have shown that lymphangiogenesis or the growth of lymphatic vessels at the periphery of tumors promotes tumor metastasis to lymph nodes. We show here that the fibronectin-binding integrin alpha4beta1 and its ligand fibronectin are novel functional markers of proliferative lymphatic endothelium. tumors and lymphangiogenic growth factors, such as vascular endothelial growth factor-C (VEGF-C) and VEGF-A, induce lymphatic vessel expression of integrin alpha4beta1. Integrin alpha4beta1 then promotes growth factor and tumor-induced lymphangiogenesis, as genetic loss of integrin alpha4beta1 expression in Tie2Cre+ alpha4(loxp/loxp) mice or genetic loss of alpha4 signaling in alpha4Y991A knock-in mice blocks growth factor and tumor-induced lymphangiogenesis, as well as tumor metastasis to lymph nodes. In addition, antagonists of integrin alpha4beta1 suppress lymphangiogenesis and tumor metastasis. Our studies show that integrin alpha4beta1 and the signals it transduces regulate the adhesion, migration, invasion, and survival of proliferating lymphatic endothelial cells. As suppression of alpha4beta1 expression, signal transduction, or function in tumor lymphatic endothelium not only inhibits tumor lymphangiogenesis but also prevents metastatic disease, these results show that integrin alpha4beta1-mediated tumor lymphangiogenesis promotes metastasis and is a useful target for the suppression of metastatic disease.

BACKGROUND: Cellular adhesion molecules (CAMs), which are normally associated with leukocyte trafficking, have also been shown to play an essential role in tumor metastasis to non-CNS sites. However, the role played by CAMs in brain metastasis is largely unexplored. It is known that leukocyte recruitment to the brain is very atypical and that mechanisms of disease in peripheral tissues cannot be extrapolated to the brain. Here, we have established the spatiotemporal expression of 12 key CAMs in the initial phases of tumor seeding in 2 different models of brain metastasis. METHODS: BALB/c or SCID mice were injected intracardially (10(5) cells/100 muL phosphate-buffered saline with either 4T1-GFP or MDA231BR-GFP cells, respectively (n = 4-6/group), and expression of the CAMs was determined by immunohistochemistry and immunofluorescence colocalisation. RESULTS: Endothelial expression of E-selectin, VCAM-1, ALCAM, ICAM-1, VLA-4, and beta4 integrin was markedly increased early in tumor seeding. At the same time, the natural ligands to these adhesion molecules were highly expressed on the metastatic tumor cells both in vitro and in vivo. Two of these ligands showed particularly high tumor cell expression (ALCAM and VLA-4), and consequently their functional role in tumor seeding was determined. Antibody neutralization of either ALCAM or VLA-4 significantly reduced tumor seeding within the brain (>60% decrease in tumor number/mm(2) brain; P < .05-0.01). CONCLUSIONS: These findings suggest that ALCAM/ALCAM and VLA-4/VCAM-1 interactions play an important functional role in the early stages of metastasis seeding in the brain. Moreover, this work identifies a specific subset of ligand-receptor interactions that may yield new therapeutic and diagnostic targets for brain metastasis.

The tumor microenvironment can enhance the invasive capacity of tumor cells. We showed that expression of angiopoietin-like protein 2 (ANGPTL2) in osteosarcoma (OS) cell lines increased and the methylation of its promoter decreased with time when grown as xenografts in mice compared with culture. Compared with cells grown in normal culture conditions, the expression of genes encoding DNA demethylation-related enzymes increased in tumor cells implanted into mice or grown in hypoxic, serum-starved culture conditions. ANGPTL2 expression in OS cell lines correlated with increased tumor metastasis and decreased animal survival by promoting tumor cell intravasation mediated by the integrin alpha5beta1, p38 mitogen-activated protein kinase, and matrix metalloproteinases. The tolloid-like 1 (TLL1) protease cleaved ANGPTL2 into fragments in vitro that did not enhance tumor progression when overexpressed in xenografts. expression of TLL1 was weak in OS patient tumors, suggesting that ANGPTL2 may not be efficiently cleaved upon secretion from OS cells. These findings demonstrate that preventing ANGPTL2 signaling stimulated by the tumor microenvironment could inhibit tumor cell migration and metastasis.

Osteosarcoma, the most common primary bone tumor in children and young adolescents, is characterized by local invasion and distant metastasis. But the detailed mechanisms of osteosarcoma metastasis are not well known. In the present study, we found that betaig-h3 promotes metastatic potential of human osteosarcoma cells in vitro and in vivo. Furthermore, betaig-h3 co-localized with integrin alpha2beta1 in osteosarcoma cells. But betaig-h3 did not change integrin alpha2beta1 expression in Saos-2 cells. Interaction of betaig-h3 with integrin alpha2beta1 mediates metastasis of human osteosarcoma cells. The second FAS1 domain of betaig-h3 but not the first FAS1 domain, the third FAS1 domain or the fourth FAS1 domain mediates human osteosarcoma cells metastasis, which is the alpha2beta1 integrin-interacting domain. We further demonstrated that PI3K/AKT signaling pathway is involved in betaig-h3-induced human osteosarcoma cells metastasis process. Together, these results reveal betaig-h3 enhances the metastasis potentials of human osteosarcoma cells via integrin alpha2beta1-mediated PI3K/AKT signal pathways. The discovery of betaig-h3-mediated pathway helps us to understand the mechanism of human osteosarcoma metastasis and provides evidence for the possibility that betaig-h3 can be a potential therapeutic target for osteosarcoma treatment.

To determine how AKT2 might contribute to tumor cell progression, a full-length, wild-type, human AKT2/protein kinase B (PKB)beta cDNA was transfected into a panel of eight human breast and ovarian cancer cells. AKT2 transfectants demonstrated increased adhesion and invasion through collagen IV because of up-regulation of beta1 integrins. In addition, AKT2 cells were more metastatic than control cells in vivo. Increased invasion by AKT2 was blocked by preincubation with an anti-beta1 integrin function blocking antibody, exposure to wortmannin, and by expression of phosphatase and tensin homologue tumor suppressor (PTEN). Confocal microscopy performed on transfected human breast cancer cells showed that unlike AKT1, AKT2 protein predominantly localized adjacent to the collagen IV matrix during cellular attachment. Overexpression of AKT2, but not AKT1 or AKT3, was sufficient to duplicate the invasive effects of phosphoinositide 3-OH kinase (PI3-K) transfected in breast cancer cells. Furthermore, expression of kinase dead AKT2(181 amino acid methionine [M]), and not kinase dead AKT1(179M) or AKT3(177M), was capable of blocking invasion induced by either human epidermal growth factor receptor-2 (HER-2) overexpression or by activation of PI3-K. Taken together, these data indicate that AKT2 mediates PI3-K-dependent effects on adhesion, motility, invasion, and metastasis in vivo.

The role of the fibronectin receptor, alpha(5)beta(1)-integrin, as an adhesion receptor and in angiogenesis is well established. However, its role in cancer cell invasion and metastasis is less clear. We describe a novel mechanism by which fibronectin regulates ovarian cancer cell signaling and promotes metastasis. Fibronectin binding to alpha(5)beta(1)-integrin led to a direct association of alpha(5)-integrin with the receptor tyrosine kinase, c-Met, activating it in a hepatocyte growth factor/scatter factor (HGF/SF) independent manner. Subsequently, c-Met associated with Src, and activated Src and focal adhesion kinase (FAK). Inhibition of alpha(5)beta(1)-integrin decreased the phosphorylation of c-Met, FAK and Src, both in vitro and in vivo. Independent activation of c-Met by its native ligand, HGF/SF, or overexpression of a constitutively active FAK in HeyA8 cells could overcome the effect of alpha(5)beta(1)-integrin inhibition on tumor cell invasion, indicating that alpha(5)beta(1)-integrin is upstream of c-Met, Src and FAK. Inhibition of alpha(5)beta(1)-integrin on cancer cells in two xenograft models of ovarian cancer metastasis resulted in a significant decrease of tumor burden, which was independent of the effect of alpha(5)beta(1)-integrin on angiogenesis. These data suggest that fibronectin promotes ovarian cancer invasion and metastasis through an alpha(5)beta(1)-integrin/c-Met/FAK/Src-dependent signaling pathway, transducing signals through c-Met in an HGF/SF-independent manner.

Increased expression of cell adhesion molecule CD44v6, integrin-beta1, carbohydrate antigen 199 (CA199), and carcinoembryonic antigen (CEA) are closely associated with the progression and metastasis of numerous cancers. In this study, peripheral blood mononuclear cell (PBMC) and serum samples were collected from 37 pancreatic cancer patients and 12 healthy people. A novel triplex TaqMan real-time reverse transcription polymerase chain reaction assay was used to measure the expression levels of CD44v6 and integrin-beta1 gene in PBMCs, while chemiluminescence and enzyme-linked immunosorbent assay were used to measure the levels of CA199 and CEA expression in serum. The results showed that both the levels of CD44v6 and integrin-beta1 expression had significant correlation with clinical stage, lymph node, and liver metastasis of pancreatic cancer (P < 0.05). Age, tumor size, tumor differentiation, clinical stage, lymph nodes, and liver metastasis were significantly associated with the levels of CA199 and CEA expression (P < 0.05). The levels of CD44v6, integrin-beta1, CA199, and CEA expression in the patients prior cryosurgery and chemotherapy were significantly higher than those in the control group (P < 0.05), whereas no significant difference was found between the patients 1 month post cryosurgery and control group (P > 0.05). The expression levels of CD44v6, integrin-beta1, CA199, and CEA in the patients 1 month post cryosurgery were significantly lower than those in the patients prior cryosurgery (P < 0.05). Interestingly, no significant difference was found for the CD44v6, integrin-beta1, CA199, and CEA levels between the patients prior and post-chemotherapy (P > 0.05). The higher expression of CD44v6, integrin-beta1, CA199, and CEA are closely related to the progression and metastasis of pancreatic cancer and may play a important role in the curative evaluation of cryosurgery of pancreatic cancer.

BACKGROUND: Many previous studies demonstrated that cell adhesion molecules CD44v6 and integrin-beta1 had been extensively investigated as potential prognostic markers of various cancers. However, data in PC are scarce. METHODS: We now investigate CD44v6 and integrin-beta1 mRNA expression in PBMC by a triplex real-time RT-PCR assay and protein expression in plasma by ELISA. All specimens were collected from 54 PC patients who received the treatment of cryosurgery as well as 20 healthy individuals (control). RESULTS: The mRNA and protein expression levels of CD44v6 and integrin-beta1 in patients were significantly increased compared with control group (P<0.05). The high CD44v6 mRNA and protein expression were significantly correlated with clinical stage, tumor differentiation, LNM, liver metastasis and decreased median DFS (P<0.05), while the high integrin-beta1 mRNA and protein expression were significantly correlated with clinical stage, LNM, liver metastasis and decreased median DFS (P<0.05). Clinical stage, LNM, liver metastasis, CD44v6 mRNA and protein expression were the independent predictors of survival in PC patients (P<0.05). Moreover, CD44v6 and integrin-beta1 mRNA and protein expression levels were significantly decreased in patients in 3 months after cryosurgery (P<0.05). No significant difference was found in CD44v6 mRNA and protein expression between patients in 3 months after cryosurgery and control group (P>0.05). CONCLUSION: CD44v6 and integrin-beta1 mRNA and protein expression in blood may serve as biomarkers for the development and metastasis of PC, and as prognostic indicators for PC. They may become useful predictors in assessing outcome of PC patients after cryosurgery. VIRTUAL SLIDES: The virtual slides for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/4035308681009006.

Mechanisms of metastasis, the major complication of prostate cancer, are poorly understood. In this study, we define molecular mechanisms that may contribute to the highly invasive potential of prostate cancer cells. Vascular endothelial growth factor (VEGF), its receptors (VEGFRs), and alpha5beta1 integrin were expressed by prostate cancer cells in vitro and by prostate tumors in vivo, and their expression was elevated at sites of bone metastasis compared to original prostate tumor. VEGF, through interaction with its receptors, regulated adhesive and migratory properties of the cancer cells. Specifically, the highly metastatic prostate cancer cell subline LNCaP-C4-2 showed a decreased adhesive but an enhanced migratory response to fibronectin, a ligand for alpha5beta1 integrin, compared to its nonmetastatic counterpart. A similar pattern was also observed when bone sialoprotein was used as a ligand in migration assays. Increased migration of metastatic prostate cancer cells to fibronectin and bone sialoprotein was regulated by VEGF via VEGFR-2. tumor suppressor PTEN was involved in control of VEGF/VEGFR-2 stimulated prostate cancer cell adhesion as well as proliferation.

BACKGROUND: Androgen receptor (AR) controls male morphogenesis, gametogenesis and prostate growth as well as development of prostate cancer. These findings support a role for AR in cell migration and invasiveness. However, the molecular mechanism involved in AR-mediated cell migration still remains elusive. METHODOLOGY/PRINCIPAL FINDINGS: Mouse embryo NIH3T3 fibroblasts and highly metastatic human fibrosarcoma HT1080 cells harbor low levels of transcriptionally incompetent AR. We now report that, through extra nuclear action, AR triggers migration of both cell types upon stimulation with physiological concentrations of the androgen R1881. We analyzed the initial events leading to androgen-induced cell migration and observed that challenging NIH3T3 cells with 10 nM R1881 rapidly induces interaction of AR with filamin A (FlnA) at cytoskeleton. AR/FlnA complex recruits integrin beta 1, thus activating its dependent cascade. Silencing of AR, FlnA and integrin beta 1 shows that this ternary complex controls focal adhesion kinase (FAK), paxillin and Rac, thereby driving cell migration. FAK-null fibroblasts migrate poorly and Rac inhibition by EHT impairs motility of androgen-treated NIH3T3 cells. Interestingly, FAK and Rac activation by androgens are independent of each other. Findings in human fibrosarcoma HT1080 cells strengthen the role of Rac in androgen signaling. The Rac inhibitor significantly impairs androgen-induced migration in these cells. A mutant AR, deleted of the sequence interacting with FlnA, fails to mediate FAK activation and paxillin tyrosine phosphorylation in androgen-stimulated cells, further reinforcing the role of AR/FlnA interaction in androgen-mediated motility. CONCLUSIONS/SIGNIFICANCE: The present report, for the first time, indicates that the extra nuclear AR/FlnA/integrin beta 1 complex is the key by which androgen activates signaling leading to cell migration. Assembly of this ternary complex may control organ development and prostate cancer metastasis.

Men who die of prostate cancer (PCa) do so because of systemic metastases, the most frequent of which are within the skeleton. Recent data suggest that the colonization of the skeleton is mediated in part by collagen type I, the most abundant protein within the bone. We have shown that enhanced collagen I binding through increased expression of integrin alpha2beta1 stimulated in vitro invasion and promoted the growth of PCa cells within the bone. Accordingly, we sought to determine whether alpha2beta1 integrin is a potential mediator of skeletal metastasis. To examine whether alpha2beta1 integrin mediates PCa metastasis, alpha2 integrin was over-expressed in low-tumorigenic LNCaP PCa cells or selectively knocked-down in highly metastatic LNCaPcol PCa cells. We document that the over-expression of alpha2 cDNA stimulated whereas alpha2 shRNA inhibited the ability of transduced cells to bind to or migrate towards collagen in vitro. Correspondingly, alpha2 integrin knock-down reduced the tumor burden of intra-osseous tumors compared to control-transduced cells. To investigate the clinical significance of alpha2beta1 expression in PCa, alpha2beta1 protein was measured in prostatic tissues and in soft tissue and bone metastases. The data demonstrate that alpha2beta1 protein was elevated in PCa skeletal metastases compared to either PCa primary lesions or soft tissue metastases suggesting that alpha2beta1 contributes to the selective metastasis to the bone. Taken together, these data support that alpha2beta1 integrin is needed for the efficient metastasis of PCa cells to the skeleton.

The molecular mechanisms underlying metastatic dissemination are still not completely understood. We have recently shown that beta(1) integrin-dependent cell adhesion to fibronectin and signaling is affected by a transmembrane molecule, Trop-2, which is frequently upregulated in human carcinomas. Here, we report that Trop-2 promotes metastatic dissemination of prostate cancer cells in vivo and is abundantly expressed in metastasis from human prostate cancer. We also show here that Trop-2 promotes prostate cancer cell migration on fibronectin, a phenomenon dependent on beta(1) integrins. Mechanistically, we demonstrate that Trop-2 and the alpha(5)beta(1) integrin associate through their extracellular domains, causing relocalization of alpha(5)beta(1) and the beta(1)-associated molecule talin from focal adhesions to the leading edges. Trop-2 effect is specific as this molecule does not modulate migration on vitronectin, does not associate with the major vitronectin receptor, alpha(v)beta(3) integrin, and does not affect localization of alpha(v)beta(3) integrin as well as vinculin in focal adhesions. We show that Trop-2 enhances directional prostate cancer cell migration, through modulation of Rac1 GTPase activity. Finally, we show that Trop-2 induces activation of PAK4, a kinase that has been reported to mediate cancer cell migration. In conclusion, we provide the first evidence that beta(1) integrin-dependent migratory and metastatic competence of prostate cancer cells is enhanced by Trop-2.

OBJECTIVE: Tissue transglutaminase (TGM2) up-regulation is involved in the progression and dissemination of carcinomas through beta1 integrin (ITGB1) association. Given that TGM2 interaction with syndecan-4 (SDC4) on the cell surface is important in the activation of ITGB1 and integrin-mediated survival signaling, we investigated the roles of TGM2, ITGB1, and SDC4 in the development and metastasis of renal cell carcinoma (RCC). MATERIAL AND METHODS: expression levels of TGM2, ITGB1, and SDC4 mRNA were analyzed in primary tumor samples (n = 95) and their healthy counterparts in addition to control and RCC epithelial cell lines. TGM2 catalytic activity in 60 randomly selected patient samples was measured by enzyme-linked sorbent plate assay. RESULTS: TGM2 expression ratio showed a significant 2.9-fold decrease in 67 (70.5%) of the primary RCC tumors (P <0.0001) independent of clinical covariates, including tumor node metastasis (TNM) staging and histopathologic grading. For the remaining 28 (29.5%) tumors, a 1.95-fold increase was recorded in the TGM2 expression levels, which also showed a significant increase in ITGB1 and SDC4 expression levels in 82.6% of the overexpression cases (P <0.001). Up-regulation of TGM2 along with ITGB1 and SCD4 was associated with metastasis and a marked decrease in tumor necrosis. Consistently, RCC cell lines exhibited higher levels of TGM2 expression compared with the control epithelial cell line with a significant up-regulation of ITGB1 and SCD4 recorded for the metastatic lines. CONCLUSIONS: Our findings suggest that TGM2 up-regulation along with ITGB1 and SDC4 plays an important role in the development of RCC tumors and advanced RCC with metastasis.

Migration and adhesion of tumor cells are essential prerequisites for the formation of metastases in malignant diseases. Chemokine receptor 7 (CCR7) has been shown to regulate integrin which can then facilitate adhesion of cancer cells to and/or migration through the extracellular matrix (ECM). In order to identify the connection between CCR7 and beta1 integrin, and the influence on cell adhesion and migration in metastatic squamous cell carcinoma of the head and neck (SCCHN). We use adhesion assays, migration assay, immunofluorescence staining, western blotting, and immunohistochemical analysis to find whether beta1 integrin can be activated by CCL19 (CCR7's ligand) and its role in SCCHN. The experiments were performed in the metastatic SCCHN cell line PCI-37B after pre-incubation of the cells with CCL19 and beta1 integrin inhibitors RGD-peptide. Our results demonstrate that CCR7 favours PCI-37B cell adhesion and migration, and induces reorganization of the actin cytoskeleton and up-expression of beta1 integrin protein. beta1 integrin inhibitor RGD-peptide can block all these effects. Taken together, our data indicate that CCR7 regulate cell adhesion and migration via beta1 integrin in metastatic SCCHN, and these results can provide a basis for new strategies in preventing metastases of SCCHN.