Oncogene database

1499

CTNNB1

catenin (cadherin-associated protein), beta 1, 88kDa;CTNNB1;catenin (cadherin-associated protein), beta 1, 88kDa

catenin beta-1

3p21

protein-coding

The activation of beta-catenin to an oncogenic state can result from the inactivation of the tumor suppressor adenomatous polyposis coli (APC), by direct mutation in the beta-catenin gene, or by the activation of wnt receptors. Once activated, beta-catenin most likely promotes tumor progression through its persistent interaction with one or more of its numerous downstream targets.

Activation of beta-catenin-mediated transcription is the nuclear end point of organ-specific Wnt signaling. In the developing kidney, Wnt-4, a secreted glycoprotein, acts as an autoinducer of the mesenchymal to epithelial transition that underlies normal nephron development. Dysregulation of this epithelial transformation process may lead to Wilms tumors (WTs). In this study, we investigated the potential role of the beta-catenin proto-oncogene, a candidate downstream target molecule of Wnt-4 signaling, in the development of WTs. In 6 of 40 tumors (15%), mutation analysis revealed heterozygous missense mutations or small deletions that result in the loss of important regulatory phosphorylation sites within the beta-catenin protein. These findings indicate that activating beta-catenin mutations may play a significant role in the development of WTs and establish a direct link between Wilms tumorigenesis and the Wnt signal transduction pathway governing normal kidney development.

beta-Catenin and gamma-catenin (plakoglobin), vertebrate homologs of Drosophila armadillo, function in cell adhesion and the Wnt signaling pathway. In colon and other cancers, mutations in the APC tumor suppressor protein or beta-catenin s amino terminus stabilize beta-catenin, enhancing its ability to activate transcription of Tcf/Lef target genes. Though beta- and gamma-catenin have analogous structures and functions and like binding to APC, evidence that gamma-catenin has an important role in cancer has been lacking. We report here that APC regulates both beta- and gamma-catenin and gamma-catenin functions as an oncogene. In contrast to beta-catenin, for which only amino-terminal mutated forms transform RK3E epithelial cells, wild-type and several amino-terminal mutated forms of gamma-catenin had similar transforming activity. gamma-Catenin s transforming activity, like beta-catenin s, was dependent on Tcf/Lef function. However, in contrast to beta-catenin, gamma-catenin strongly activated c-Myc expression and c-Myc function was crucial for gamma-catenin transformation. Our findings suggest APC mutations alter regulation of both beta- and gamma-catenin, perhaps explaining why the frequency of APC mutations in colon cancer far exceeds that of beta-catenin mutations. Elevated c-Myc expression in cancers with APC defects may be due to altered regulation of both beta- and gamma-catenin. Furthermore, the data imply beta- and gamma-catenin may have distinct roles in Wnt signaling and cancer via differential effects on downstream target genes.

The intronless N-myc2 gene was originally identified as the major target of hepatitis virus insertion in woodchuck liver tumors. Here we report that transgenic mice carrying the N-myc2 gene controlled by woodchuck hepatitis virus (WHV) regulatory sequences are highly predisposed to liver cancer. In a WHV/N-myc2 transgenic line, hepatocellular carcinomas or adenomas arose in over 70% of mice, despite barely detectable expression of the methylated transgene in liver cells. Furthermore, a transgenic founder carrying unmethylated transgene sequences succumbed to a large liver tumor by the age of two months, demonstrating the high oncogenicity of the woodchuck N-myc2 retroposon. Stabilizing mutations or deletions of beta-catenin were found in 25% of liver tumors and correlated with reduced tumor latency (P<0.05), confirming the important role of beta-catenin activation in Myc-induced tumorigenesis. The ability of the tumor suppressor gene p53 to cooperate with N-myc2 in liver cell transformation was tested by introducing a p53-null allele into WHV/N-myc2 transgenic mice. The loss of one p53 allele in transgenic animals markedly accelerated the onset of liver cancer (P=0.0001), and most tumors of WHV/N-myc2 p53+/Delta mice harbored either a deletion of the wt p53 allele or a beta-catenin mutation. These findings provide direct evidence that activation of N-myc2 and reduction of p53 levels act synergistically during multistage carcinogenesis in vivo and suggest that different genetic pathways may underlie liver carcinogenesis initiated by a myc transgene. oncogene (2000).

Inappropriate activation of the Wnt/beta-catenin signaling, resulting mainly from activating mutations of the beta-catenin gene, has been implicated recently in the development of hepatocellular carcinoma (HCC). We have generated transgenic mice expressing an oncogenic form of beta-catenin in their hepatocytes to analyze the effect of deregulated beta-catenin signaling on liver homeostasis. These mice rapidly developed hepatomegaly soon after birth, with livers three to four times heavier than those of nontransgenic littermates. The liver cell hyperplasia resulted from increased cell proliferation without any compensatory apoptosis. Although the genes encoding c-myc and cyclin D1 are potential targets of the beta-catenin signaling pathway, neither of them was overexpressed in the hyperplastic livers of beta-catenin transgenic mice. Thus, the key target genes of the beta-catenin signaling pathway in the liver remain to be identified.

beta-Catenin is an oncogenic protein involved in regulation of cell-cell adhesion and gene expression. Accumulation of cellular beta-catenin occurs in many types of human cancers. Four mechanisms are known to cause increases in beta-catenin: mutations of beta-catenin, adenomatous polyposis coli, or axin genes and activation of Wnt signaling. We report a new cause of beta-catenin accumulation involving oncogenic mutants of RON and MET receptor tyrosine kinases (RTKs). Cells transfected with oncogenic RON or MET were characterized by beta-catenin tyrosine phosphorylation and accumulation; constitutive activation of a Tcf transcriptional factor; and increased levels of beta-catenin/Tcf target oncogene proteins c-myc and cyclin D1. Interference with the beta-catenin pathway reduced the transforming potential of mutated RON and MET. Activation of beta-catenin by oncogenic RON and MET constitutes a new pathway, which might lead to cell transformation by these and other mutant growth factor RTKs.

Constitutive activation of the wnt-signaling pathway plays an important role during both human and rat colon carcinogenesis and can be brought through mutations in either the adenomatous polyposis coli or the beta-catenin gene. mutations found in the beta-catenin gene typically affect one out of four regulatory phosphorylation sites near the N-terminus of the beta-catenin protein. Whereas in human colon cancers, however, the majority of beta-catenin mutations directly alter threonine 41 or serine 45; the beta-catenin mutations found in chemically induced rat colon tumors seemed to cluster around codon 33 instead. Unlike previous studies, that have used relatively short-term (2-5 weeks) treatment with one of the alkylating agents 1,2,-dimethylhydrazine (DMH) or azoxymethane, we have investigated the mutational spectrum of the beta-catenin gene in a panel of rat colon tumors induced by long-term (20 weeks) DMH-treatment. We detected beta-catenin mutations in 12 of 33 (36%) tumors. Interestingly, only one of the beta-catenin mutations found affected the previously implicated codon 33 cluster region (Asp32Asn), whereas 11 of 12 (>90%) mutations represented identical C-->T transitions within codon 41 resulting in the common replacement of threonine by isoleucine. We propose a model in which codon 41 mutations bear higher oncogenic potential but are induced by DMH less frequently than mutations in the codon 33 cluster region. Consequently, only after sustained carcinogenic treatment, as is achieved in the long-term DMH-protocol, codon 41 mutations will be induced frequently enough to be present in ALL developing malignant lesions and, then, because of their higher oncogenic potential, these are selected for.

Development of strategies for prevention of breast cancer development requires an understanding of the effects of mammary oncogenes on mammary cells at early stages in neoplastic transformation. As mammary oncogenes wnt-1 and int-2 affect different signal transduction pathways, we investigated their effects on established mouse mammary epithelial cell lines (MMECLs) reflecting early stages in tumorigenesis. Normal interactions between beta-catenin and E-cadherin were abrogated in ALL three immortalized MMECLs and the cells lacked beta-catenin-mediated transactivation activity, detectable using a reporter assay, suggesting that alterations in cell adhesion may be very early events in mammary tumorigenesis. Immortalized FSK4 and EL12 cells and hyperplastic TM3 cells were stably transfected with expression vectors encoding wnt-1 or int-2 or the control vector, and drug-selected pooled cells from each line were confirmed by reverse transcription-polymerase chain reaction to express the transfected oncogene; this expression persisted in the cells analysed in vitro and in vivo. Resultant phenotypic changes depended both on the oncogene and the target mammary cell line. In FSK4 cells, expression of wnt-1 or int-2 resulted in proliferative changes in vitro, including reduced contact inhibition, increased beta-catenin expression, and decreased p53 transcriptional activity, but neither oncogene conferred upon those cells the ability to produce tumors in vivo. EL12 cells were highly refractory to the effects of both oncogenes, with the only measurable changes being increased E-cadherin levels induced by both oncogenes and increased proliferation of the int-2-transfected cells in the absence of serum. Parental TM3 cells were phenotypically similar to wnt-1- or int-2-transfected FSK4 cells and displayed an increased rate of proliferation in vitro and markedly increased tumorigenicity in vivo following transfection with int-2 but not with wnt-1. These results suggest that wnt-1 signaling is redundant in the hyperplastic TM3 cells and indicate that wnt-1-induced effects in the immortalized FSK4 and EL12 cells were not sufficient to mediate a tumorigenic phenotype. This study showed that the wnt-1 and int-2 oncogenes have similar but distinguishable effects on immortalized MMECLs and that the genetic background of the mammary cells greatly influences the consequences of oncogene expression at early stages of cell transformation.

BACKGROUND & AIMS: Recent in vitro studies showed that beta-catenin translocated into the tumor cell nucleus functions as an oncogene by transactivating oncogenes, including MMP-7. We conducted a large-scale analysis of beta-catenin and MMP-7 expression in human colon cancer to determine the potential clinical importance of these molecules. METHODS: In 202 colon cancer patients with known postoperative outcomes, we determined the expression of beta-catenin and MMP-7 in the tumors immunohistochemically and correlated the findings with the patients clinicopathological characteristics and survival. RESULTS: We found 2 distinct patterns of beta-catenin nuclear accumulation (NA) in the colon cancers: diffuse NA (NAd) in 89 cases (44%) and selective NA at the invasion front (NAinv) in 18 cases (9%). The presence of the NAinv pattern was significantly correlated with advanced Dukes stage (P = 0.0187) and tumor recurrence (P = 0.0005) as well as with MMP-7 expression in the tumor invasion front (P = 0.0025), resulting in extremely unfavorable clinical outcomes. A multivariate analysis determined that the NAinv expression pattern and Dukes C stage were independent prognostic factors. CONCLUSIONS: Oncogenic activation of beta-catenin in the tumor invasion front, as represented by its NAinv pattern of expression, may be an independent and reliable indicator of membership in a subset of colon cancer patients who are highly susceptible to tumor recurrence and have a less favorable survival rate.

Invasion and dissemination of well-differentiated carcinomas are often associated with loss of epithelial differentiation and gain of mesenchymal-like capabilities of dedifferentiated tumor cells at the invasive front. However when analysing central areas of metastases of colorectal carcinomas one finds a regain of the differentiated epithelial growth patterns like in the primary tumor. More than 80% of these tumor have loss of function mutations in the APC tumor suppressor gene, leading to an overexpression of beta-catenine. In its nuclear pool beta-catenine acts as a transcription factor and is now considered as one of the main oncogenic proteins in colorectal carcinogenesis. We could define several molecules important for the processes of invasion and dissemination, like MMP-7, uPA, laminin-5, as target genes activated by nuclear beta-catenine. Moreover the characteristic phenotypic changes during tumor progression were associated with distinct expression patterns of beta-catenine and E-cadherin. Nuclear beta-catenine was found in dedifferentiated mesenchyme-like tumor cells at the invasive front, but strikingly, like in central areas of the primary tumors, was localized to the membrane and cytoplasm in polarized epithelial tumor cells in the metastases. This was accompanied by changes in the proliferative activity. Based on these data, we postulate that an important driving force for progression of well-differentiated colorectal carcinomas is the specific environment, initiating two transient phenotypic transition processes by modulating intracellular beta-catenine distribution in the tumor cells.

The homeobox gene Cdx1 is a regulator of intestinal epithelial cell proliferation and differentiation. Using a transfection approach, we showed here that the oncogenic activation of the beta-catenin pathway stimulates the endogenous expression of the Cdx1 mRNA as well as the activity of the Cdx1 promoter in cancer cells of the human colon. Reciprocally, the paralogue homeobox gene Cdx2 exerts an inhibitory effect on the basal and on the beta-catenin-stimulated activity of the Cdx1 promoter. The inhibitory effect of CDX2 requires the intact homeodomain. It is not dependent on canonical CDX binding sites in the Cdx1 promoter nor on the cis-elements specifically targeted by the beta-catenin/Tcf complex. We conclude that the oncogenically activated beta-catenin and CDX2 have opposite and independent effects on the Cdx1 homeobox gene.

Somatic cell gene targeting was used to create an isogenic set of human colon cancer cells that differs only in the presence or absence of their endogenous activated beta-catenin oncogene. Affymetrix Genechip expression profiling of parental cells and gene-targeted derivatives identified numerous novel genes whose expression was dependent on the presence of oncogenic beta-catenin. The transforming growth factor-beta family member bone morphogenetic protein 4 (BMP4), whose receptor is mutated in a rare inherited gastrointestinal cancer predisposition syndrome, was the most highly differentially expressed gene. Additional experiments revealed that the oncogenic allele of beta-catenin specifically is absolutely required for BMP4 expression and secretion by human cancer cells and that BMP4 is overexpressed and secreted by human colon cancer cells with mutant adenomatous polyposis coli genes. These data identify the presence of regulatory interactions between the Wnt and BMP signaling pathways in cancer pathogenesis, providing an intriguing connection between the sporadic and inherited forms of a common human malignancy.

Genetic analysis of beta-catenin-induced oncogenic transformation in chicken embryo fibroblasts (CEF) revealed the following prerequisites for oncogenicity: (1) removal of the N terminal phosphorylation sites targeted by glycogen synthase kinase 3beta (GSK3beta), (2) retention of the N terminal transactivation domain, and (3) retention of the armadillo repeats. The C terminal transactivation domain played an ancillary role in the transformation of CEF. There was a rough correlation between the transforming activity of various beta-catenin constructs and their transactivation of the TOPFLASH reporter. expression levels of the candidate target genes of beta-catenin-LEF, cyclin D1 and myc were not correlated with each other or with the transforming activity of beta-catenin constructs. A new target gene, coding for inositol hexakisphosphate kinase 2 (IP6K2) was identified. Its expression showed concordance with the transforming activity of beta-catenin constructs.

Activation of beta-catenin is a critical step in the pathogenesis of many common human cancers and is the initiating event in adenocarcinoma of the colon. Because activation of beta-catenin provides a gain-of-function, it is tempting to speculate that specific pharmacological inhibition of activated beta-catenin might reverse the tumorigenic properties of human cancer cells and therefore form the basis of an effective anticancer strategy. In an effort to provide proof-of-principle for such a strategy, we used a novel clonal growth assay based on human somatic cell gene targeting to determine whether activated beta-catenin remains a necessary oncogenic stimulus in advanced human cancer cells. Using this approach, we demonstrate that beta-catenin is a necessary oncogene in human SW48 and DLD1 colon cancer cells but not in HCT116 cells. These data indicate that activated beta-catenin can remain a critical oncogenic stimulus throughout the progression of human colon cancer and suggest that the small molecule inhibitors of activated beta-catenin currently under development will be effective anticancer therapeutics in a subset of malignant colon cancers.

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.

Key molecular lesions in colorectal and other cancers cause beta-catenin-dependent transactivation of T cell factor (Tcf)-dependent genes. Disruption of this signal represents an opportunity for rational cancer therapy. To identify compounds that inhibit association between Tcf4 and beta-catenin, we screened libraries of natural compounds in a high-throughput assay for immunoenzymatic detection of the protein-protein interaction. Selected compounds disrupt Tcf/beta-catenin complexes in several independent in vitro assays and potently antagonize cellular effects of beta-catenin-dependent activities, including reporter gene activation, c-myc or cyclin D1 expression, cell proliferation, and duplication of the Xenopus embryonic dorsal axis. These compounds thus meet predicted criteria for disrupting Tcf/beta-catenin complexes and define a general standard to establish mechanism-based activity of small molecule inhibitors of this pathogenic protein-protein interaction.

Beta-Catenin, a member of the Wnt signaling pathway, is downregulated by glycogen synthase kinase-3beta (GSK-3beta)-dependent phosphorylation of Ser/Thr residues in the N-terminus of the protein, followed by ubiquitination and proteosomal degradation. In human and rodent cancers, mutations that substitute one of the critical Ser/Thr residues in the GSK-3beta region of beta-catenin stabilize the protein and activate beta-catenin/TCF/LEF target genes. This study examined three oncogenic beta-catenin mutants from rat colon tumors containing substitutions adjacent to amino-acid residue Ser33, a key target for phosphorylation by GSK-3beta. Compared with wild-type beta-catenin (WT), the beta-catenin mutants D32G, D32N, and D32Y strongly activated TCF-4-dependent transcription in HEK293 cells, and there was accumulation of beta-catenin in the cell lysates. Immunoblotting with phosphospecific antibodies indicated that there was little if any effect on the phosphorylation of Ser37, Thr41 or Ser45; however, the phosphorylation of Ser33 appeared to be affected in the beta-catenin mutants. Specifically, antiphospho-beta-catenin 33/37/41 antibody identified high, intermediate and low expression levels of phosphorylated beta-catenin in cells transfected with D32G, D32N and D32Y, respectively. Experiments with the proteosome inhibitor N-acetyl-Leu-Leu-norleucinal (ALLN) revealed ubiquitinated bands on ALL three mutant beta-catenins, as well as on WT beta-catenin. The relative order of ubiquitination was WT>D32G>D32N>D32Y, in parallel with findings from the phosphorylation studies. These results are discussed in the context of previous studies, which indicated that amino-acid residue D32 lies within the ubiquitination recognition motif of beta-catenin.

The peptidyl-proplyl-isomerase, PIN1, upregulates beta-catenin by inhibiting its interaction with APC. beta-catenin accumulation occurs in about 70% of hepatocellular carcinoma (HCC), of which only 20% are due to beta-catenin mutations. The role of PIN1 in beta-catenin upregulation in HCC was investigated. PIN1 was shown to be overexpressed in more than 50% of HCC. ALL cases with PIN1 overexpression also showed beta-catenin accumulation, with 68% of cases showing concomitant beta-catenin and cyclin D1 accumulation. PIN1 was shown to contribute to beta-catenin and cyclin D1 overexpression directly by in vitro cell-line transfection experiments. Finally, we showed that PIN1 overexpression and beta-catenin gene mutations appeared to be mutually exclusive events, leading to beta-catenin accumulation in HCC. These results showed that PIN1 overexpression leading to beta-catenin accumulation might be a critical event in hepatocarcinogenesis, and that PIN1 is a potential target for therapeutic intervention in HCC.

Beta-Catenin plays an essential role in the Wingless/Wnt signaling cascade. Phosphorylation of beta-Catenin in its N-terminal region by the kinase GSK-3beta is required for the interaction with the SCF-beta-TrCP protein complex that targets beta-Catenin for proteasome degradation. In the present work, we used two peptides of 32 amino acids referred to beta-Cat17-48 and P-beta-Cat17-48 for the phosphorylated peptide at the two sites Ser33 and Ser37. Circular dichroism and NMR techniques were used to assess the influence of the phosphorylation. The spectra of the peptides at pH 7.2 were completely assigned. Analysis of the medium-range NOE connectivities indicated that beta-Cat17-48 seems to be only poorly folded. These data are in agreement with the result of structure calculations. P-beta-Cat17-48 possesses two helical segments around the DpSGXXpS motif, which forms a large bent with the phosphate groups pointing out of the structure. On the contrary, beta-Cat17-48 shows less well-defined secondary structures and appears as a more flexible peptide, but adopts in the motif DSGXXS a more compact conformation than P-beta-Cat17-48. Differences in this molecular region suggest that conformational changes of phosphorylated beta-Catenin play an important role for the interaction with the SCF-beta-TrCP protein complex.

Homo-oligomerization via a coiled-coil (C-C) domain has been shown to be necessary for the promyelocytic leukemia (PML)-retinoic acid receptor-alpha (RARalpha) fusion protein to acquire oncogenic potential in acute promyelocytic leukemia. We show here that PML(DeltaC-C)-RARalpha, which contains a deletion in its C-C domain, is neither localized as characteristic microspeckles nor modified by small ubiquitin-like modifiers (SUMO). The absence of sumoylation of the DeltaC-C mutant was due to the lack of binding to Ubc9, a SUMO conjugation enzyme. The integrity of RING finger domain was also needed for both sumoylation and microspeckle formation. In GAL4-DNA tethering assays, the DeltaC-C mutant completely lost the inhibitory effect on retinoic acid (RA)-mediated transactivation. Furthermore, the expression of CD14 in U937 cells expressing the DeltaC-C mutant in response to vitamin D3 was markedly higher than in cells expressing PML-RARalpha. However, the RA-mediated induction of C/EBPbeta in cells expressing the DeltaC-C mutant was comparable to that of control cells. Thus, our results suggest that the C-C domain-associated functions of sumoylation, localization as microspeckles, and the inhibition of monocyte differentiation ALL contribute to the oncogenic activity of PML-RARalpha.

Recent studies suggest that lysophosphatidic acid (LPA) and its G protein-coupled receptors (GPCRs) LPA(1), LPA(2), or LPA(3) may play a role in the development of several types of cancers, including colorectal cancer. However, the specific receptor subtype(s) and their signal-transduction pathways responsible for LPA-induced cancer cell proliferation have not been fully elucidated. We show by specific RNA interference (RNAi) that LPA(2) and LPA(3) but not LPA(1) are targets for LPA-induced proliferation of HCT116 and LS174T colon cancer cells. We determined that LPA-induced colon cancer cell proliferation requires the beta-catenin signaling pathway, because knockdown of beta-catenin by RNAi abolished LPA-induced proliferation of HCT116 cells. Moreover, LPA activates the main signaling events in the beta-catenin pathway: phosphorylation of glycogen synthase kinase 3beta (GSK3beta), nuclear translocation of beta-catenin, transcriptional activation of T cell factor (Tcf)/lymphoid-enhancer factor (Lef), and expression of target genes. Inhibition of conventional protein kinase C (cPKC) blocked the effects, suggesting its involvement in LPA-induced activation of the beta-catenin pathway. Thus, LPA(2) and LPA(3) signal the proliferation of colon cancer cells through cPKC-mediated activation of the beta-catenin pathway. These results link LPA and its GPCRs to cancer through a major oncogenic signaling pathway.

beta-TrCP is the F-box protein component of an Skp1/Cul1/F-box (SCF)-type ubiquitin ligase complex. Biochemical studies have suggested that beta-TrCP targets the oncogenic protein beta-catenin for ubiquitination and followed by proteasome degradation. To further elucidate the basis of this interaction, a complex between a 32-residue peptide from beta-catenin containing the phosphorylated motif DpSGXXpS (P-beta-Cat17-48) and beta-TrCP was studied using Saturation Transfer Difference (STD) Nuclear Magnetic Resonance (NMR) experiments. These experiments make it possible to identify the binding epitope of a ligand at atomic resolution. An analysis of STD spectra provided clear evidence that only a few of the 32 residues receive the largest saturation transfer. In particular, the amide protons of the residues in the phosphorylated motif appear to be in close contact to the amino acids of the beta-TrCP binding pocket. The amide and aromatic protons of the His24 and Trp25 residues also receive a significant saturation transfer. These findings are in keeping with a recently published x-ray structure of a shorter beta-catenin fragment with the beta-TrCP1-Skp1 complex and with the earlier findings from mutagenesis and activity assays. To better characterize the ligand-protein interaction, the bound conformation of the phosphorylated beta-catenin peptide was obtained using TRansfer Nuclear Overhauser Effect SpectroscopY (TRNOESY) experiments. Finally, we obtained the bound structure of the phosphorylated peptide showing the protons identified by STD NMR as exposed in close proximity to the molecule surface.

BACKGROUND/AIMS: The molecular mechanisms of hepatocarcinogenesis remain largely unknown. Previous studies suggest that activation of the Wnt/beta-catenin pathway is important during hepatocyte transformation but the role of Frizzled receptor (FZD) in this process has not been defined. Here we investigate activation of this pathway by FZD using transgenic hepatocellular carcinoma (HCC) murine models. METHODS: We employed single (c-myc, SV40-Tag) and established double [insulin receptor substrate-1 (IRS-1/c-myc) and hepatitis Bx protein (X/c-myc)] transgenic lines and ALL developed HCC. expression of 9 FZD was measured by real time RT-PCR and Western blot analysis. Phosphorylation and cellular accumulation of beta-catenin were assessed in both dysplastic tissue and tumors. We investigated the effect of a dominant negative (DN) FZD7 on TCF transcriptional activity in a SV40 derived HCC cell line. RESULTS: FZD7 was highly overexpressed at the mRNA and protein level(s) in HCC and occurred in dysplasia. Upregulation of FZD7 was associated with reduced phosphorylation of beta-catenin and led to nuclear accumulation in HCC tumors. Ectopic expression of a DN FZD7 construct decreased TCF transcriptional activity in tumor cells. CONCLUSIONS: These observations suggest that upregulation of FZD7 receptors in association with activation of the canonical Wnt/beta-catenin pathway is a common molecular event in HCC.

While Wnt and Ras signaling pathways are activated during progression of colorectal cancers, many of their important downstream targets remain to be elucidated. The gastrin gene encodes for a family of peptide growth factors that are commonly upregulated in colorectal neoplasia. Previously, we showed that the Wnt signaling pathway moderately stimulates the gastrin promoter. To determine whether Ras signaling can cooperate with Wnt signaling in transcriptional regulation of gastrin gene expression, we have analyzed the response of murine gastrin promoter-reporter gene constructs to combinations of oncogenic stimulation in transient transfection assays. We found a strong (25- to 40-fold) synergistic stimulation of the gastrin promoter by the combination of oncogenic beta-catenin and K-ras overexpression. Deletion analysis localized the response element to an area between -140 and -110bp upstream in the murine gastrin promoter. Electrophoretic mobility shift assays detected a complex containing beta-catenin/TCF, AP1, and SMAD3/4 transcription factors that bound to a DNA element through AP1 and SMAD binding sites. Gastrin promoter activation could be further enhanced or suppressed by the co-expression of wild type SMAD4 or dominant negative mutant of SMAD4, respectively, and abrogated by the PI3K inhibitor, LY20004, but not by the MEK inhibitor, PD98059. Taken together, our data strongly suggest that oncogenic Wnt and Ras signaling pathways can synergistically induce gastrin expression, possibly contributing to neoplastic progression.

BACKGROUND & AIMS: beta-Catenin is a downstream effector of the Wnt signaling pathway and is believed to exert its oncogenic function by activating T-cell factor (TCF)/lymphoid enhancer factor (LEF) family transcriptional factors. However, it is still uncertain whether the diverse effects of beta-catenin are caused solely by aberrant gene transactivation. In this study, we used a proteomics approach to obtain further insight into the functional properties of nuclear beta-catenin. METHODS: The protein assembly of a native beta-catenin-containing complex in nuclear extracts from a colorectal cancer cell line, DLD-1, was identified using immunoprecipitation and mass spectrometry. RESULTS: beta-Catenin physically interacted with fusion (FUS)/translocated in liposarcoma (TLS) and various RNA-binding proteins. The expression of FUS/TLS was closely associated with the accumulation of beta-catenin and with the undifferentiated status of intestinal epithelial cells. The transient transfection of FUS suppressed beta-catenin-evoked gene transactivation of TCF/LEF, and beta-catenin transfection affected the splicing pattern of the E1A minigene and induced a novel splicing variant of estrogen receptor (ER)-beta exerting a dominant-negative activity. CONCLUSIONS: Human cancer expresses a large variety of alternatively spliced messenger RNA (mRNA), but the precise molecular mechanisms responsible for cancer-related alternative splicing are largely unknown. In this study, we demonstrated the interaction of beta-catenin with FUS/TLS and other RNA-binding proteins involved in the regulation of pre-mRNA splicing. Certain mRNA splicing abbreviations seen in human cancers may be induced by the activation of the Wnt signaling pathway.

Beta-catenin has two distinct functions, namely, maintaining cell-to-cell adhesion and mediating the Wnt/beta-catenin signal transduction pathway, which plays pivotal roles in embryogenesis and in malignant transformation of cells. The oncogenic properties of Wnt/beta-catenin signaling stem from alteration in phosphorylation-dependent protein degradation and subcellular localization of beta-catenin from cell membrane to the nucleus, where it binds to T-cell factor (Tcf) to form a bipartite transcription factor. The beta-catenin/Tcf complex facilitates transcription of target genes that encode effectors for activation of cell proliferation and invasion and inhibition of apoptosis, leading to colorectal cancer development. In addition, in the tumor invasion front, stabilized and activated beta-catenin interacts with other molecular pathways to facilitate tumor progression. This review highlights the beta-catenin-dependent oncogenic signaling network involved in the multi-step process of colorectal tumorigenesis. Wnt signaling evidently regulates stem cells, leading them to differentiate or self-renew. We address roles of oncogenic beta-catenin signaling in the microenvironment of the tumor-host interface that determine the individual tumor s malignant potential and in regulation of putative cancer stem or progenitor cells that represent plausible targets for cancer eradication.

The protein levels of beta-catenin are tightly regulated by the ubiquitin/proteasome system. We provide evidence that two distinct ubiquitin-dependent degradation pathways for beta-catenin are active in the same Burkitt s lymphoma cells: Along with the classical glycogen-synthase kinase 3beta-dependent destruction machinery, degradation of beta-catenin through seven in absentia homolog 1 (Siah-1) ubiquitin ligase is functional in these cells. We show that inhibition of endogenous Siah-1 stabilizes and activates beta-catenin in B cells. The principal Epstein-Barr virus oncoprotein, latent membrane protein 1, is involved in beta-catenin up-regulation, and expression of latent membrane protein 1 in B lymphoma cells is associated with decreased Siah-1 RNA and protein levels. Thus, we demonstrate the significance of the endogenous Siah-1-dependent ubiquitin/proteasome pathway for beta-catenin degradation in malignant human cells and its regulation by a viral oncogene.

The oncogenic beta-catenin/T-cell factor (TCF) signal is a common trigger inducing expressions of various cancer-related genes and is activated in various types of human malignancy. The aim of this study was to create an effective double-stranded DNA decoy that would interfere with endogenous TCF hyperactivity in tumor cells. We first established the TCF-activated model using nontumor human embryonic kidney 293 (HEK293) cells by introducing a beta-catenin cDNA. Based on a consensus TCF-binding sequence in the cyclin D1 and c-myc promoters, several double-stranded oligodeoxynucleotides were designed and tested for their ability to inhibit TCF activity in the HEK293 model. Among them, the 18-mer oligodeoxynucleotide stably formed double-stranded DNA and efficiently inhibited TCF activity. FITC-labeled oligodeoxynucleotide was efficiently incorporated into the nucleus at 6 hours and remained within cells for up to 72 to 96 hours. When compared with scrambled oligodeoxynucleotide, we found that the 18-mer TCF decoy significantly inhibited TCF activity and promoter activities of the downstream target genes, such as cyclin D1, c-myc, and matrix metalloproteinase 7 in HCT116 colon cancer cells. Reverse transcription-PCR assays indicated that mRNA expression of these genes decreased with treatment of the TCF decoy. Proliferation assay showed that the TCF decoy significantly inhibited growth of HCT116 tumor cells, but not of nontumor HEK293 cells. Our data provide evidence that the TCF decoy reduced both TCF activity and transcriptional activation of downstream target genes. Thus, this TCF decoy is potentially an efficient and nontoxic molecular targeting therapy for controlling malignant properties of cancer cells.

The interaction of the P-beta-Cat(19-44) peptide, a 26 amino acid peptide (K(19)AAVSHWQQQSYLDpSGIHpSGATTTAP(44)) that mimics the phosphorylated beta-Catenin antigen, has been studied with its monoclonal antibody BC-22, by transferred nuclear Overhauser effect NMR spectroscopy (TRNOESY) and saturation transfer difference NMR (STD NMR) spectroscopy. This antibody is specific to diphosphorylated beta-Catenin and does not react with the non-phosphorylated protein. Phosphorylation of beta-Catenin at sites Ser33 and Ser37 on the DSGXXS motif is required for the interaction of beta-Catenin with the ubiquitin ligase SCF(beta-TrCP). beta-TrCP is involved in the ubiquitination and proteasome targeting of the oncogenic protein beta-Catenin, the accumulation of which has been implicated in various human cancers. The three-dimensional structure of the P-beta-Cat(19-44) in the bound conformation was determined by TRNOESY NMR experiments; the peptide adopts a compact structure in the presence of mAb with formation of turns around Trp25 and Gln26, with a tight bend created by the DpS(33)GIHpS(37) motif; the peptide residues (D32-pS37) forming this bend are recognized by the antibody as demonstrated by STD NMR experiments. STD NMR studies provide evidence for the existence of a conformational epitope containing tandem repeats of phosphoserine motifs. The peptide s epitope is predominantly located in the large bend and in the N-terminal segment, implicating bidentate association. These findings are in excellent agreement with a recently published NMR structure required for the interaction of beta-Catenin with the SCF(beta-TrCP) protein.

Activated beta-catenin regulates the transcription of oncogenic target genes and is critical for tumorigenesis. Because nuclear functions are frequently coupled, we investigated whether it also has a role in alternative splicing of oncogenic genes. We showed that stabilized beta-catenin caused alternative splicing of estrogen receptor-beta pre-mRNA in colon cancer cells. To establish a direct role of beta-catenin in regulated splicing, we selected a high-affinity RNA aptamer that associated with beta-catenin in vivo. Nuclear localized aptamer inhibited beta-catenin-dependent transcription of cyclin D1 and c-myc in colon cancer cells; thus, cells stably expressing the aptamer exhibited cell cycle arrest and reduced tumor forming potential. Most significantly, the aptamer prevented the alternative splicing induced by stabilized beta-catenin. Taken together, our results establish that beta-catenin has an important role in both transcription and splicing, and that its action can be modulated by a high-affinity RNA aptamer. The RNA aptamer could be further developed as a specific inhibitor for cancer therapeutics.

PURPOSE: Protein-bound polysaccharide K is an immunotherapeutic agent that promotes apoptosis by inhibiting nuclear factor-kappaB activation in cancer cells. We previously showed that oncogenic beta-catenin activates nuclear factor-kappaB and inhibits apoptosis by up-regulating beta-transducin repeat-containing protein. We investigated whether the activation state of beta-catenin in the primary tumor is associated with differences in survival rates of patients with colon cancer undergoing immunochemotherapy with 5-fluorouracil plus polysaccharide K vs. chemotherapy with 5-fluorouracil alone. METHODS: We assessed the activation states of beta-catenin and nuclear factor-kappaB in the primary tumors of 202 colon cancer patients, and analyzed the data in terms of the clinicopathologic characteristics and survival of patients undergoing the two forms of adjuvant therapy. RESULTS: We found two distinct patterns of nuclear accumulation of activated beta-catenin in the tumor cells: diffuse nuclear accumulation in 89 cases (44 percent) and selective nuclear accumulation at the tumor invasion front in 18 cases (9 percent). Nuclear factor-kappaB activation was found in 64 cases (32 percent). In patients with diffuse nuclear accumulation-type beta-catenin activation, immunochemotherapy significantly improved recurrence-free survival, cancer death survival, and overall survival rates compared with patients receiving chemotherapy alone. No survival benefit was found in cases with nuclear accumulation at the tumor invasion front-type beta-catenin activation or no activation. Similarly, immunochemotherapy favored the survival of patients with nuclear factor-kappaB activation. Multivariate analysis established the TNM stage and administration of polysaccharide K as independent prognostic factors in the patients with diffuse nuclear accumulation-type beta-catenin activation. CONCLUSIONS: The presence of diffuse nuclear accumulation-type beta-catenin activation identifies patients with colon cancer who respond better to immunotherapy with polysaccharide K.

Plakoglobin (Pg) and beta-catenin are homologous proteins that function in cell-cell adhesion and signaling. The cadherin-associated form of these proteins mediates adhesion, whereas the cytosolic/nuclear form has a signaling role. Despite their interactions with common cellular partners, beta-catenin has a well-documented oncogenic potential while Pg has a less characterized tumor suppressor activity. We showed previously that Pg overexpression in Pg-deficient SCC9 cells (SCC9-Pg-WT) induced Bcl-2 expression and inhibited apoptosis. To assess the exact role of Pg in Bcl-2 expression, we generated and characterized SCC9 transfectants expressing Pg with a restricted cytoplasmic (Pg-NES) or nuclear (Pg-NLS) distribution. We show that Bcl-2 was expressed regardless of Pg localization, although its level was substantially lower in SCC9-Pg-NLS cells. Bcl-2 expression coincided with increased nuclear beta-catenin levels (Pg-NES) or a decrease in the level of total and nuclear beta-catenin associated with N-cadherin and alpha-catenin (Pg-WT and -NLS) cells. Bcl-2 expression also was induced in SCC9 cells overexpressing beta-catenin. In contrast, SCC9 cells expressing mutant Pg proteins, unable to interact with N-cadherin and alpha-catenin, had noticeably lower Bcl-2 levels. Our data suggest that Bcl-2 expression is induced by beta-catenin and modulated by Pg. We show that the inhibition of beta-catenin-dependent TCF transactivation had no effect on Bcl-2 levels, suggesting that induction of Bcl-2 expression by beta-catenin and its modulation by Pg may involve factors other than, or in addition, to, TCF. These results provide a possible mechanism for the tumor suppressor activity of Pg via its role as a regulator of the oncogenic potential beta-catenin.

Gain-of-function mutations in the proto-oncogene c-kit that induce constitutive kinase activity of its product, KIT protein, are characteristic of human mast cell disease and are believed to play a central role in mast cell leukemia oncogenesis, proliferation and survival. Nuclear overexpression of the Wnt effector beta-catenin and deregulated beta-catenin nuclear signaling can promote malignant transformation in solid tumors and hematologic malignancies. However, a role for beta-catenin in mast cell leukemia has not been described. Nuclear accumulation of beta-catenin is upregulated by its tyrosine phosphorylation, a process that can be exacerbated by deregulated expression of oncogenic tyrosine kinases. Here, we investigated the relationship between activated KIT and beta-catenin signaling in mast cell leukemia. Beta-catenin was tyrosine-phosphorylated in cells with KIT activated by either gain-of-function mutation or incubation with the KIT ligand stem cell factor. Beta-catenin tyrosine phosphorylation depended on KIT activity but not on PI3K-AKT activation. Tyrosine phosphorylation of beta-catenin was associated with its nuclear localization and enhanced transcription of target genes c-myc and cyclin D1. Endogenous KIT and beta-catenin were found to associate in mast cell leukemia cells, and in vitro kinase assay demonstrated that active KIT phosphorylates tyrosine residues of beta-catenin directly. Aberrant beta-catenin-driven transcription caused by deregulated KIT may represent a significant new target for treatment of mast cell leukemia.

Although the oncogenic role of the Wnt/beta-catenin pathway is well defined, it remains unclear how this pathway is aberrantly activated in lung cancer. We found that Dickkopf (Dkk)-3, a member of Dkk family of Wnt antagonists, is frequently inactivated in lung cancer and plays a role in suppressing lung cancer cell growth through inhibition of beta-catenin/T-cell factor (TCF)-4 signaling. Dkk3 is the only Dkk family member abundantly expressed in normal lung, but silenced by promoter hypermethylation in a large fraction of lung cancer cell lines and lung tumors. Downregulation of Dkk3 was correlated with tumor progression and expression of nuclear beta-catenin in lung tumors. Ectopic expression of Dkk3 in lung cancer cells with Dkk3 hypermethylation induced apoptosis and inhibited TCF-4 activity as well as nuclear accumulation of beta-catenin and expression of TCF-4 targets c-Myc and cyclin D1. Furthermore, small interference RNA knock down of Dkk3 in cells lacking Dkk3 hypermethylation was sufficient to promote cell proliferation, beta-catenin nuclear translocation and expression of c-Myc. These observations suggested that epigenetic inactivation of Dkk3 activates the Wnt/beta-catenin pathway, thereby promoting the growth of lung cancer cells.

Previous reports showed that receptor-type protein-tyrosine phosphatase PTPRK co-localizes with beta-catenin at adherens junctions, and in vitro experiments suggested that beta-catenin could be substrate of PTPRK-mediated phosphatase activity. beta-catenin is a molecule endowed with a dual function being involved both in cell adhesion and in Wnt signaling pathway. Here we provide evidence for the role of PTPRK in negatively regulating the beta-catenin transcriptional activity by modulating its intracellular and membrane distribution. expression of PTPRK protein in HEK293 cells and in PTPRK-null melanoma cell lines, one of which harbors a mutated oncogenic beta-catenin, impairs nuclear accumulation of wild type and oncogenic forms of beta-catenin, limits cytosolic levels of tyrosine-phosphorylated beta-catenin, and leads to re-localization of E-cadherin/beta-catenin complexes in ordered membrane phase along cell-cell contacts. This re-modulation of beta-catenin cellular distribution results in the inhibition of cyclin D1 and c-myc protein expression, whose genes are targets of beta-catenin. tumor cells upon re-expression of PTPRK have a reduced proliferative and migration capacity. Moreover we show that PTPRK is also active in negatively regulating the transactivating function of beta-catenin in normal melanocytes as confirmed by experiments with silenced PTPRK by specific siRNA. Our data show that PTPRK influences transactivating activity of beta-catenin in non-tumoral and neoplastic cells by regulating the balance between signaling and adhesive beta-catenin, thus providing biochemical basis for the hypothesis of PTPRK as a tumor suppressor gene.

The Wnt/beta-catenin signaling pathway plays diverse roles in embryonic development and in maintenance of organs and tissues in adults. Activation of this signaling cascade inhibits degradation of the pivotal component beta-catenin, which in turn stimulates transcription of downstream target genes. Over the past two decades, intensive worldwide investigations have yielded considerable progress toward understanding the cellular and molecular mechanisms of Wnt signaling and its involvement in the pathogenesis of a range of human diseases. Remarkably, beta-catenin signaling is aberrantly activated in greater than 70% of colorectal cancers and to a lesser extent in other tumor types, promoting cancer cell proliferation, survival and migration. Accordingly, beta-catenin has gained recognition as an enticing molecular target for cancer therapeutics. Disruption of protein-protein interactions essential for beta-catenin activity holds immense promise for the development of novel anti-cancer drugs. In this review, we focus on the regulation of beta-catenin-dependent transcriptional activation and discuss potential therapeutic opportunities to block this signaling pathway in cancer.

Loss of alpha-catenin is one of the characteristics of prostate cancer. The catenins (alpha and beta) associated with E-cadherin play a critical role in the regulation of cell-cell adhesion. Tyrosine phosphorylation of beta-catenin dissociates it from E-cadherin and facilitates its entry into the nucleus, where beta-catenin acts as a transcriptional activator inducing genes involved in cell proliferation. Thus, beta-catenin regulates cell-cell adhesion and cell proliferation. Mechanisms controlling the balance between these functions of beta-catenin invariably are altered in cancer. Although a wealth of information is available about beta-catenin deregulation during oncogenesis, much less is known about how or whether alpha-catenin regulates beta-catenin functions. In this study, we show that alpha-catenin acts as a switch regulating the cell-cell adhesion and proliferation functions of beta-catenin. In alpha-catenin-null prostate cancer cells, reexpression of alpha-catenin increased cell-cell adhesion and decreased beta-catenin transcriptional activity, cyclin D1 levels, and cell proliferation. Further, Src-mediated tyrosine phosphorylation of beta-catenin is a major mechanism for decreased beta-catenin interaction with E-cadherin in alpha-catenin-null cells. alpha-Catenin attenuated the effect of Src phosphorylation by increasing beta-catenin association with E-cadherin. We also show that alpha-catenin increases the sensitivity of prostate cancer cells to a Src inhibitor in suppressing cell proliferation. This study reveals for the first time that alpha-catenin is a key regulator of beta-catenin transcriptional activity and that the status of alpha-catenin expression in tumor tissues might have prognostic value for Src targeted therapy.

There has been a surge of interest in the therapeutic targeting of the Wnt pathway following the demonstration that it is activated in a wide variety of tumors and that blocking aberrant signaling promoted tumor cell apoptosis or differentiation. This review describes recent therapeutic approaches and discusses potential opportunities for intervention at multiple levels within the Wnt pathway.

Previously we showed that end-binding protein 1 (EB1) may promote cellular growth by activating beta-catenin/T-cell factor (TCF) pathway. To further investigate the role of EB1 in regulating cellular growth, we established an EB1-inducible expression system in which the protein level of EB1 was significantly upregulated upon doxycycline induction. We found that EB1 promoted cellular growth and resulted in a significant increase in colony formation. In addition, EB1 could induce tumor formation in nude mice, activate beta-catenin-dependent gene expression and upregulate the transcriptional activity of c-myc. We also showed that EB1 in this manner inhibited apoptosis of 293-T-REx cells upon cisplatin and upregulated expression of Bcl-2, whereas DeltaN TCF4, an inhibitor of beta-catenin/TCF pathway, could completely or partially abolish the effects of EB1 on the promotion of cell growth and the inhibition of apoptosis activity. Moreover, knockdown of c-myc by RNAi could abrogate upregulation of EB1-dependent induction of Bcl-2 expression. Overall, EB1 acts as a potential oncogene via activating beta-catenin/TCF pathway to promote cellular growth and inhibit apoptosis.

Pancreatic cancer is a deadly disease characterized by late diagnosis and resistance to therapy. Much progress has been made in defining gene defects in pancreatic cancer, but a full accounting of its molecular pathogenesis remains to be provided. Here, we show that expression of the ataxia-telangiectasia group D complementing gene (ATDC), also called TRIM29, is elevated in most invasive pancreatic cancers and pancreatic cancer precursor lesions. ATDC promoted cancer cell proliferation in vitro and enhanced tumor growth and metastasis in vivo. ATDC expression correlated with elevated beta-catenin levels in pancreatic cancer, and beta-catenin function was required for ATDC s oncogenic effects. ATDC was found to stabilize beta-catenin via ATDC-induced effects on the Disheveled-2 protein, a negative regulator of glycogen synthase kinase 3beta in the Wnt/beta-catenin signaling pathway.

BACKGROUND: oncogene HCCR-1 functions as a negative regulator of the p53 and contributes to tumorigenesis of various human tissues. However, it is unknown how HCCR-1 contributes to the cellular and biochemical mechanisms of human tumorigenesis. RESULTS: In this study, we showed how the expression of HCCR-1 is modulated. The luciferase activity assay indicated that the HCCR-1 5 -flanking region at positions -166 to +30 plays an important role in HCCR-1 promoter activity. Computational analysis of this region identified two consensus sequences for the T-cell factor (TCF) located at -26 to -4 (Tcf1) and -136 to -114 (Tcf2). mutation at the Tcf1 site led to a dramatic decrease in promoter activity. Mobility shift assays (EMSA) revealed that nuclear proteins bind to the Tcf1 site, but not to the Tcf2 site. LiCl, Wnt signal activator by GSK-3beta inhibition, significantly increased reporter activities in wild-type Tcf1-containing constructs, but were without effect in mutant Tcf1-containing constructs in HEK/293 cells. In addition, endogenous HCCR-1 expression was also increased by treatment with GSK-3beta inhibitor, LiCl or AR-A014418 in HEK/293 and K562 cells. Finally, we also observed that the transcription factor, TCF, and its cofactor, beta-catenin, bound to the Tcf1 site. CONCLUSION: These findings suggest that the Tcf1 site on the HCCR-1 promoter is a major element regulating HCCR-1 expression and abnormal stimulation of this site may induce various human cancers.

Myelodysplastic syndromes (MDS) are a group of heterogeneous clonal stem cell diseases with a tendency to progress to leukemic transformation. The cytogenetic and molecular pathogenesis of MDS has not been well understood. SALL4, a newly identified oncogene, modulates stem cell pluripotency and self-renewal capability in embryonic development and also plays a role in leukemogenesis. Overexpression of SALL4 induces MDS-like features and subsequent leukemic progression in transgenic mice. Here, we examined SALL4 expression levels in bone marrow mononuclear cells from MDS patients, acute myeloid leukemia (AML) patients, and normal control subjects using a semiquantitative reverse transcription polymerase chain reaction. Higher levels of SALL4 expression were seen in MDS and AML samples than in control samples. The expression level of SALL4 positively correlated with those of MYC and CCND1, both of which are downstream target genes in the Wnt/beta-catenin pathway. We therefore propose that SALL4 plays a critical role in the pathogenesis of MDS by causing the aberrant activation of the Wnt/beta-catenin pathway.

tumours of the Ewing family, which comprise Ewing s sarcoma and peripheral primitive neuroectodermal tumours, are highly aggressive and mostly affect children and adolescents. They are characterized by chromosomal translocations leading to the generation of fusion proteins between EWS (or very rarely FUS) and members of the E-twenty-six (ETS) family of transcription factors that are capable of transforming cells. EWS/FLI1, the most frequent fusion, is thought to cause transformation through activation or repression of specific target genes. We present evidence demonstrating that the Wnt inhibitor and beta-catenin/T-cell factor (TCF)-responsive gene DICKKOPF-1 (DKK-1) is a transcriptional target of EWS/FLI1, which can inhibit both basal and beta-catenin-induced transactivation of the DKK-1 promoter. Moreover, our data indicate that EWS/FLI1 has a more general effect on beta-catenin/TCF-mediated transcription since it can block transactivation of a consensus beta-catenin/TCF reporter construct. Consistently, Ewing tumour cells expressing different EWS/ETS translocations cannot engage beta-catenin/TCF-dependent transcription, whereas silencing of EWS/FLI1 restores beta-catenin responsiveness in A673 and RD-ES Ewing tumour cells. Accordingly, gene set enrichment analysis shows that beta-catenin/TCF target genes are significantly enriched among genes downregulated by EWS/FLI1 in the Ewing cell line A673. Mechanistically, the inhibitory effect of EWS/FLI1 can be overcome by a constitutively active TCF4 protein (TCF4-VP16). Moreover, EWS/FLI1 binds lymphoid enhancer factor 1, a TCF family member, and interferes with its binding to beta-catenin, which could explain its negative effect on beta-catenin/TCF-mediated transcription. Our results show that EWS/FLI1 inhibits both DKK-1 expression as well as beta-catenin/TCF-dependent transcription, which could contribute to progression of tumours of the Ewing family.

MicroRNAs have been implicated in regulating diverse cellular pathways. Emerging evidence indicate that the miR-17-92 cluster may have a causal role in breast cancer tumorigenesis as a novel class of oncogenes, but the role of these miRNAs in breast cancer invasion and migration remains unexplored. The aims of this study were to verify the effect of miR-17-5p (an important member of the miR-17-92 cluster) on the invasive and migratory ability of breast cancer cells. The matching of miR-17-5p and HMG box-containing protein 1 (HBP1) was predicted by TargetScan and confirmed by DNA constructs and luciferase target assay. The expression levels of miR-17-5p and its candidate target-HBP1 in MCF7 and MDA-MB-231 breast cancer cells were measured by real-time PCR and western blotting. Effects of miR-17-5p in cell cycle progression, proliferation, invasion and migration were evaluated by flow cytometry assay, 3-(4,-dimethy -lthiazol-2-yl)-2,-diphenyl -tetrazoliumbromide assay, soft-agar colony formation assay, and transwell invasive and migratory assay, respectively. The results showed that miR-17-5p was highly expressed in high-invasive MDA-MB-231 breast cancer cells but not in low-invasive MCF-7 breast cancer cells. Over-expression of miR-17-5p in MCF-7 cells rendered them the invasive and migratory abilities by targeting HBP1/beta-catenin pathway. On the other hand, down-regulation of endogenous miR-17-5p suppressed the migration and invasion of MDA-MB-231 cells in vitro. These findings suggest that miR-17-5p plays an important role in breast cancer cell invasion and migration by suppressing HBP1 and subsequent activation of Wnt/beta-catenin.

In cancer, Wnt/beta-catenin signaling is ubiquitously referred to as an "oncogenic" pathway that promotes tumor progression. This review examines how the regulation and downstream effects of Wnt/beta-catenin signaling in cancer varies depending on cellular context, with a focus on malignant melanoma. We emphasize that the cellular homeostasis of Wnt/beta-catenin signaling may represent a more appropriate concept than the simplified view of the Wnt/beta-catenin pathway as either oncogenic or tumor-suppressing. Ultimately, a more refined understanding of the contextual regulation of Wnt/beta-catenin signaling will be essential for addressing if and how therapeutic targeting of this pathway could be leveraged for patient benefit.

BACKGROUND: Beta-catenin is a multifunctional oncogenic protein that contributes fundamentally to cell development and biology. Elevation in expression and activity of beta-catenin has been implicated in many cancers and associated with poor prognosis. Beta-catenin is degraded in the cytoplasm by glycogen synthase kinase 3 beta (GSK-3beta) through phosphorylation. Cell growth and proliferation is associated with beta-catenin translocation from the cytoplasm into the nucleus. This laboratory was the first to demonstrate that selenium-containing compounds can enhance the efficacy and cytotoxicity of anticancer drugs in several preclinical xenograft models. These data provided the basis to identify mechanism of selenium action focusing on beta-catenin as a target. This study was designed to: (1) determine whether pharmacological doses of methylseleninic acid (MSeA) have inhibitory effects on the level and the oncogenic activity of beta-catenin, (2) investigate the kinetics and the mechanism of beta-catenin inhibition, and (3) confirm that inhibition of beta-catenin would lead to enhanced cytotoxicity of standard chemotherapeutic drugs. RESULTS: In six human cancer cell lines, the inhibition of total and nuclear expression of beta-catenin by MSeA was dose and time dependent. The involvement of GSK-3beta in the degradation of beta-catenin was cell type dependent (GSK-3beta-dependent in HT-29, whereas GSK-3beta-independent in HCT-8). However, the pronounced inhibition of beta-catenin by MSeA was independent of various drug treatments and was not reversed after combination therapy.Knockout of beta-catenin by ShRNA and its inhibition by MSeA yielded similar enhancement of cytotoxicity of anticancer drugs.Collectively, the generated data demonstrate that beta-catenin is a target of MSeA and its inhibition resulted in enhanced cytotoxicity of chemotherapeutic drugs. CONCLUSIONS: This study demonstrates that beta-catenin, a molecule associated with drug resistance, is a target of selenium and its inhibition is associated with increased multiple drugs cytotoxicity in various human cancers. Further, degradation of beta-catenin by GSK-3beta is not a general mechanism but is cell type dependent.

Breast cancer is a major cause of cancer-related deaths in American women; therefore, the identification of novel breast cancer-related molecules for the discovery of new markers and drug targets remains essential. The human DEK gene, which encodes a chromatin-binding protein and DNA topology regulator, is upregulated in many types of cancer. DEK has been implicated as an oncogene in breast cancer based on mRNA expression studies, but its functional significance in breast cancer growth and progression has not yet been tested directly. We demonstrate that DEK is highly expressed in breast cancer cells compared with normal tissue, and functionally important for cellular growth, invasion and mammosphere formation. DEK overexpression in non-tumorigenic MCF10A cells resulted in increased growth and motility, with a concomitant downregulation of E-cadherin. Conversely, DEK knockdown in MCF7 and MDA-MB-468 breast cancer cells resulted in decreased growth and motility with upregulation of E-cadherin. The use of DEK-proficient and -deficient breast cancer cells in orthotopic xenografts provided further in vivo evidence that DEK contributes to tumor growth. Activation of the beta-catenin signaling pathway is important for normal and cancer stem cell character, growth and metastasis. We show that DEK expression stimulated, and DEK knockdown repressed beta-catenin nuclear translocation and activity. Importantly, the expression of constitutively active beta-catenin rescued breast cancer invasion defects of DEK knockdown cells. Together, our data indicate that DEK expression stimulates the growth, stem cell character and motility of breast cancer cells, and that DEK-dependent cellular invasion occurs at least in part via beta-catenin activation.

BACKGROUND: Colon cancers are the frequent causes of cancer mortality worldwide. Recently bacterial toxins have received marked attention as promising approaches in the treatment of colon cancer. Thermostable direct hemolysin (TDH) secreted by Vibrio parahaemolyticus causes influx of extracellular calcium with the subsequent rise in intracellular calcium level in intestinal epithelial cells and it is known that calcium has antiproliferative activity against colon cancer. KEY RESULTS: In the present study it has been shown that TDH, a well-known traditional virulent factor inhibits proliferation of human colon carcinoma cells through the involvement of CaSR in its mechanism. TDH treatment does not induce DNA fragmentation, nor causes the release of lactate dehydrogenase. Therefore, apoptosis and cytotoxicity are not contributing to the TDH-mediated reduction of proliferation rate, and hence the reduction appears to be caused by decrease in cell proliferation. The elevation of E-cadherin, a cell adhesion molecule and suppression of beta-catenin, a proto-oncogene have been observed in presence of CaSR agonists whereas reverse effect has been seen in presence of CaSR antagonist as well as si-RNA in TDH treated cells. TDH also triggers a significant reduction of Cyclin-D and cdk2, two important cell cycle regulatory proteins along with an up regulation of cell cycle inhibitory protein p27(Kip1) in presence of CaSR agonists. CONCLUSION: Therefore TDH can downregulate colonic carcinoma cell proliferation and involves CaSR in its mechanism of action. The downregulation occurs mainly through the involvement of E-cadherin-beta-catenin mediated pathway and the inhibition of cell cycle regulators as well as upregulation of cell cycle inhibitors.

BACKGROUND: The Hepatitis C virus (HCV) core protein has been implicated as a potential oncogene or a cofactor in HCV-related hepatocellular carcinoma (HCC), but the underlying mechanisms are unknown. Overactivation of the Wnt/beta-catenin signaling is a major factor in oncogenesis of HCC. However, the pathogenesis of HCV core-associated Wnt/beta-catenin activation remains to be further characterized. Therefore, we attempted to determine whether HCV core protein plays an important role in regulating Wnt/beta-catenin signaling in HCC cells. METHODOLOGY: Wnt/beta-catenin signaling activity was investigated in core-expressing hepatoma cells. Protein and gene expression were examined by Western blot, immunofluorescence staining, RT-qPCR, and reporter assay. PRINCIPAL FINDINGS: HCV core protein significantly enhances Tcf-dependent transcriptional activity induced by Wnt3A in HCC cell lines. Additionally, core protein increases and stabilizes beta-catenin levels in hepatoma cell line Huh7 through inactivation of GSK-3beta, which contributes to the up-regulation of downstream target genes, such as c-Myc, cyclin D1, WISP2 and CTGF. Also, core protein increases cell proliferation rate and promotes Wnt3A-induced tumor growth in the xenograft tumor model of human HCC. CONCLUSIONS/SIGNIFICANCE: HCV core protein enhances Wnt/beta-catenin signaling activity, hence playing an important role in HCV-associated carcinogenesis.

Liposarcoma can be an aggressive, debilitating, and fatal malignancy. In this study, we identifed miRNAs associated with the differentiation status of liposarcoma to gain insight into the basis for its progression. miRNA expression profiles determined in human tumors and normal fat specimens identified a dedifferentiated tumor expression signature consisting of 35 miRNAs. Deregulated miRNA expression was confirmed in a second independent sample cohort. The miR-155 was the most overexpressed miRNA and functional investigations assigned an important role in the growth of dedifferentiated liposarcoma cell lines. Transient or stable knockdown of miR-155 retarded tumor cell growth, decreased colony formation, and induced G(1)-S cell-cycle arrest in vitro and blocked tumor growth in murine xenografts in vivo. We identified casein kinase 1alpha (CK1alpha) as a direct target of miR-155 control which enhanced beta-catenin signaling and cyclin D1 expression, promoting tumor cell growth. In summary, our results point to important functions for miR-155 and beta-catenin signaling in progression of liposarcoma, revealing mechanistic vulnerabilities that might be exploited for both prognostic and therapeutic purposes.

SOX (high mobility group) genes play an important role in a number of developmental processes. Potential roles of SOXs have been demonstrated in various neoplastic tissues as tumor suppressors or promoters depending on tumor status and types. The aim of this study was to investigate the function role of SOXs in the human hepatocellular carcinoma (HCC). The gene expression changes of SOXs in HCC tissues compared with those in noncancerous hepatic tissues were detected using real-time quantitative reverse transcriptase polymerase chain reaction (QRT-PCR) analysis and immunohistochemistry. In addition, we identified the gene SOX10 that was significantly upregulated in HCC by QRT-PCR analysis and immunohistochemistry. Furthermore, we discovered that SOX10 promoted cancer cell proliferation in vitro, and SOX10 expression correlated with elevated beta-catenin levels in HCC, and beta-catenin function was required for SOX10 s oncogenic effects. Mechanistically, SOX10 facilitates TCF4 to bind to beta-catenin and form a stable SOX10/TCF4/beta-catenin complex and trans-activate its downstream target gene. SOX10 mutations that disrupt the SOX10-beta-catenin interaction partially prevent its function in tumor cells. ALL in all, SOX10 is a commonly activated tumor promoter that activates Wnt/beta-catenin signaling in cancer cells of HCC.

BACKGROUND: Overexpression of the c-erbB-2 oncogene has been implicated in the development and/or prognosis of several human carcinomas, including that of the prostate. Recently, c-erbB-2 protein was found to be released in the circulation. The present study was undertaken to study the significance of serum c-erbB-2 protein determination in men with prostate cancer. METHODS: Serum c-erbB-2 protein determination was performed via immunoradiometric assay using two monoclonal antibodies that react with the extracellular domain of the protein. The study population consisted of 71 untreated prostate cancer patients. Of those, 33 with stage D2 disease entered a follow-up study. As control, serum c-erbB-2 protein levels were determined in 92 patients with benign prostatic hypertrophy. In addition, elevations of c-erbB-2 protein were examined in patients with various disease statuses: clinically well controlled (28 patients), disease progression (24 patients), and end-stage disease (17 patients). RESULTS: Elevation of serum c-erbB-2 protein level was observed in patients in advanced stages, such as stage D2 disease (30%), disease progression (42%), and end-stage disease (82.4%). In the follow-up study, patients with an elevated c-erbB-2 level had a significantly shorter interval to disease progression than did those with a normal level. CONCLUSIONS: The results suggest that c-erbB-2 can be used as a biomarker to identify a malignant subgroup in prostate cancer.

The beta-catenin, glycogen synthase kinase 3beta (GSK-3beta), and adenomatous polyposis coli (APC) gene products interact to form a network that influences the rate of cell proliferation. Medulloblastoma occurs as part of Turcot s syndrome, and patients with Turcot s who develop medulloblastomas have been shown to harbor germ-line APC mutations. Although APC mutations have been investigated and not identified in sporadic medulloblastomas, the status of the beta-catenin and GSK-3beta genes has not been evaluated in this tumor. Here we show that 3 of 67 medulloblastomas harbor beta-catenin mutations, each of which converts a GSK-3beta phosphorylation site from serine to cysteine. The beta-catenin mutation seen in the tumors was not present in matched constitutional DNA in the two cases where matched DNA was available. A loss of heterozygosity analysis of 32 medulloblastomas with paired normal DNA samples was performed with four microsatellite markers flanking the GSK-3beta locus; loss of heterozygosity with at least one marker was identified in 7 tumors. Sequencing of the remaining GSK-3beta allele in these cases failed to identify any mutations. Taken together, these data suggest that activating mutations in the beta-catenin gene may be involved in the development of a subset of medulloblastomas. The GSK-3beta gene does not appear to be a target for inactivation in this tumor.

Sixty to eighty percent of ALL colorectal cancers are characterized by mutations in the APC tumor suppressor gene. Recently, it was shown that these mutations lead to a nuclear overexpression of beta-Catenin by disruption of the wingless/WNT signal pathway. Since nuclear beta-Catenin functions as a transcriptional activator of hitherto unknown tumor genes, this form of beta-Catenin is now considered a major oncoprotein in colorectal cancer. Using immunohistochemistry, we investigated the distribution of overexpressed beta-Catenin within individual colorectal carcinomas. In the majority of the tumors, we found no homogeneous staining, but a strong nuclear expression of beta-Catenin predominantly localized at the invasion front with strongest nuclear staining of isolated, scattered tumor cells. In contrast, cells in the tumor center often showed no nuclear staining, but retained a membranous expression of beta-Catenin, comparable to normal colon epithelium. It is, therefore, likely that in addition to the overexpression of beta-Catenin caused by defects in the APC locus, regulatory events in the tumor itself lead to a different distribution of this oncoprotein. Possibly, surrounding tissue at the invasion front can give signals to the tumor cells, leading to a nuclear translocation of beta-Catenin, where it may play a direct role in tumor invasion processes.