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Invasion is a defining event in carcinoma progression. In general, invasive carcinoma is characterized by an epithelial-fibroblastoid conversion associated with loss of cell-cell adhesion receptors such as E-cadherin and beta-catenin. We report here that TGF-beta1 promotes the invasiveness by modulating the alterations of cellular plasticity including a loss of cell-cell contact in Ras-transformed epithelial cells. In order to examine the role of TGF-beta1 in the Ras-induced responses, intestinal epithelial cells expressing a conditionally activated Ha-Ras(Val12) (RIE-iRas cells) were used in this study. Induced expression of activated Ha-Ras(Val12) caused morphologic transformation of the RIE-iRas cells with an increase in vimentin expression and a decrease of E-cadherin levels. There was also redistribution of beta-catenin from the cytoplasm to the nucleus after the induction of Ras. TGF-beta1 treatment enhanced both the decrease in E-cadherin levels and the redistribution of beta-catenin. Interestingly, the activation of Ras markedly decreased the level of TGF-beta receptor type II (TbetaRII) in RIE-iRas cells. However, the expression of plasminogen activator inhibitor-1, which is known to be transcriptionally induced by TGF-beta1, was strongly induced by TGF-beta1 despite the marked downregulation of TbetaRII. The induction of Ha-Ras(Val12) markedly increased the invasiveness in RIE-iRas cells, as evaluated by a collagen type I-coated Boyden-chamber assay, and the Ras-mediated invasiveness was significantly enhanced by TGF-beta1 treatment. Expression of a dominant-negative form of TbetaRII in the RIE-iRas cells abrogated both growth-inhibitory and invasion responses to TGF-beta1. Collectively, these results suggest that TGF-beta1 and oncogenic Ras collaborate in promoting cellular invasiveness in intestinal epithelial cells. The enhancement of invasiveness was correlated with decreased E-cadherin levels and subcellular distribution of beta-catenin. The enhancement of oncogenic Ras-mediated cell transformation by TGF-beta1 occurs via TbetaRII.
Copyright 2001 Academic Press.
The matrix metalloproteinase matrilysin (MMP-7) is expressed in the tumor cells of a majority of mouse intestinal and human colonic adenomas. We showed previously that matrilysin is a target gene of beta-catenin-Tcf, the transcription factor complex whose activity is thought to play a crucial role in the initiation of intestinal tumorigenesis. Here we report that overexpression of a stable mutant form of beta-catenin alone was not sufficient to effect expression of luciferase from a matrilysin promoter-luciferase reporter plasmid. However, cotransfection of the reporter with an expression vector encoding the PEA3 Ets transcription factor, or its close relatives ER81 and ERM, increased luciferase expression and rendered the promoter responsive to beta-catenin-LEF-1 as well as to the AP-1 protein c-Jun. Other Ets proteins could not substitute for the PEA3 subfamily. Luciferase activity was induced up to 250-fold when PEA3, c-Jun, beta-catenin, and LEF-1 were coexpressed. This combination of transcription factors was also sufficient to induce expression of the endogenous matrilysin gene. Furthermore, all matrilysin-expressing benign intestinal tumors of the Min mouse expressed a member of the PEA3 subfamily, as did all human colon tumor cell lines examined. These data suggest that the expression of members of the PEA3 subfamily, in conjunction with the accumulation of beta-catenin in these tumors, leads to coordinate upregulation of matrilysin gene transcription, contributing to gastrointestinal tumorigenesis.
Regulation of beta-catenin degradation by intracellular components of the wnt pathway was reconstituted in cytoplasmic extracts of Xenopus eggs and embryos. The ubiquitin-dependent beta-catenin degradation in extracts displays a biochemical requirement for axin, GSK3, and APC. Axin dramatically accelerates while dishevelled inhibits beta-catenin turnover. Through another domain, dishevelled recruits GBP/Frat1 to the APC-axin-GSK3 complex. Our results confirm and extend models in which inhibition of GSK3 has two synergistic effects: (1) reduction of APC phosphorylation and loss of affinity for beta-catenin and (2) reduction of beta-catenin phosphorylation and consequent loss of its affinity for the SCF ubiquitin ligase complex. Dishevelled thus stabilizes beta-catenin, which can dissociate from the APC/axin complex and participate in transcriptional activation.
RPTPmu is a prototypic receptor-like protein-tyrosine phosphatase (RPTP) that mediates homotypic cell-cell interactions. Intracellularly, RPTPmu consists of a relatively large juxtamembrane region and two phosphatase domains, but little is still known about its substrate(s). Here we show that RPTPmu associates with the catenin p120(ctn), a tyrosine kinase substrate and an interacting partner of cadherins. No interaction is detectable between RPTPmu and beta-catenin. Furthermore, we show that tyrosine-phosphorylated p120(ctn) is dephosphorylated by RPTPmu both in vitro and in intact cells. Complex formation between RPTPmu and p120(ctn) does not require tyrosine phosphorylation of p120(ctn). Mutational analysis reveals that both the juxtamembrane region and the second phosphatase domain of RPTPmu are involved in p120(ctn) binding. The RPTPmu-interacting domain of p120(ctn) maps to its unique N terminus, a region distinct from the cadherin-interacting domain. A mutant form of p120(ctn) that fails to bind cadherins can still associate with RPTPmu. Our findings indicate that RPTPmu interacts with p120(ctn) independently of cadherins, and they suggest that this interaction may serve to control the tyrosine phosphorylation state of p120(ctn) at sites of cell-cell contact.
Matrilysin is a matrix metalloproteinase expressed in the tumor cells of greater than 80% of intestinal adenomas. The majority of these intestinal tumors are associated with the accumulation of beta-catenin, a component of the cadherin adhesion complex and, through its association with the T Cell Factor (Tcf) DNA binding proteins, a regulator in the Wnt signal transduction pathway. In murine intestinal tumors, matrilysin transcripts show striking overlap with the accumulation of beta-catenin protein. The matrilysin promoter is upregulated as much as 12-fold by beta-catenin in colon tumor cell lines in a manner inversely proportional to the endogenous levels of beta-catenin/Tcf complex and is dependent upon a single optimal Tcf-4 recognition site. Coexpression of the E-cadherin cytoplasmic domain blocked this induction and reduced basal promoter activity in every colon cancer cell line tested. Inactivation of the Tcf binding site increased promoter activity and overexpression of the Tcf factor, LEF-1, significantly downregulated matrilysin promoter activity, suggesting that beta-catenin transactivates the matrilysin promoter by virtue of its ability to abrogate Tcf-mediated repression. Because genetic ablation of matrilysin decreases tumor formation in multiple intestinal neoplasia (Min) mice, we propose that regulation of matrilysin production by beta-catenin accumulation is a contributing factor to intestinal tumorigenesis.
p120(ctn) binds to the cytoplasmic domain of cadherins but its role is poorly understood. Colo 205 cells grow as dispersed cells despite their normal expression of E-cadherin and catenins. However, in these cells we can induce typical E-cadherin-dependent aggregation by treatment with staurosporine or trypsin. These treatments concomitantly induce an electrophoretic mobility shift of p120(ctn) to a faster position. To investigate whether p120(ctn) plays a role in this cadherin reactivation process, we transfected Colo 205 cells with a series of p120(ctn) deletion constructs. Notably, expression of NH2-terminally deleted p120(ctn) induced aggregation. Similar effects were observed when these constructs were introduced into HT-29 cells. When a mutant N-cadherin lacking the p120(ctn)-binding site was introduced into Colo 205 cells, this molecule also induced cell aggregation, indicating that cadherins can function normally if they do not bind to p120(ctn). These findings suggest that in Colo 205 cells, a signaling mechanism exists to modify a biochemical state of p120(ctn) and the modified p120(ctn) blocks the cadherin system. The NH2 terminus-deleted p120(ctn) appears to compete with the endogenous p120(ctn) to abolish the adhesion-blocking action.
PURPOSE - In diabetic retinopathy and macular edema, the blood-retinal barrier fails to function properly, and there is transvascular leakage of proteins and solutes. The tight junction protein occludin and the adherens junction protein cadherin-5 have been shown to be critical to maintaining the endothelial barrier and regulating paracellular transport of large vessel endothelia. However, the expression and distribution of these junction proteins in the retinal endothelium is not well characterized.
METHODS - Human and bovine retinal endothelial cells were isolated as described previously. Western blot analysis and flow cytometry techniques were used to assay for the presence of occludin, zonula occludens-1 (ZO-1), cadherin-5, and beta-catenin. The subcellular localization of the proteins was visualized by immunohistochemistry performed on cultured human retinal endothelial cells and cryosections of bovine retina.
RESULTS - Western blot analysis and flow cytometry techniques found occludin, ZO-1, cadherin-5, and beta-catenin in cultured human retinal endothelial cells. Immunofluorescence staining of cultured retinal endothelial cells and cryosections of bovine retina showed junctional localization of occludin, ZO-1, cadherin-5, and beta-catenin.
CONCLUSIONS - This report demonstrates the expression of occludin and cadherin-5 in retinal endothelial cells and their localization to sites of cell-cell contact. Expression of their respective regulatory proteins, ZO-1 and beta-catenin, at sites of cell-cell contact suggests that occludin and cadherin-5 play a role in maintaining the retinal endothelial barrier.
Loss of expression and function of the E-cadherin/catenin membrane complex can result in loss of cell adhesion and contribute to invasive or metastatic potential in carcinomas. The aim of this study was to examine the expression of alpha- and beta-catenin and E-cadherin in Barrett's esophagus with and without dysplasia and in esophageal adenocarcinomas and to identify any relationship with tumor growth pattern and clinical outcome. Immunoperoxidase staining for alpha- and beta-catenin and E-cadherin was performed on specimens of Barrett's esophagus with and without dysplasia and on 54 esophageal adenocarcinoma specimens. Membranous staining for all of the components was seen in normal gastric and esophageal mucosa. Abnormal expression of beta-catenin, alpha-catenin, and E-cadherin was significantly associated with higher degrees of dysplasia in Barrett's esophagus. Fourteen of 16 cases of high grade dysplasia and 7 of 7 cases of intramucosal carcinoma showed abnormal expression of beta-catenin, compared with 3 of 6 cases indefinite for dysplasia and 11 of 17 cases with low grade dysplasia (P = 0.022). Similar results were seen for expression of alpha-catenin (P < .01) and E-cadherin (P = .049). In esophageal adenocarcinomas, preserved expression of these proteins occurred more frequently in well-differentiated tumors; abnormal expression was more common in diffusely infiltrative poorly differentiated tumors that did not form glands. Focal nuclear staining for beta-catenin was present in two high-grade dysplasias, two intramucosal carcinomas, and five adenocarcinomas. No survival advantage was demonstrated for patients whose tumors retained expression of these cell adhesion components. In conclusion, abnormal expression of the E-cadherin/catenin membrane complex is common in esophageal adenocarcinoma and occurs early in the dysplasia/carcinoma sequence in Barrett's esophagus, indicating that disturbances in this cell adhesion complex might be important in tumorigenesis and tumor progression in this disorder.
The physical interaction between beta-catenin and the adenomatous polyposis coli (APC) gene, and the ability of APC to regulate cytoplasmic levels of beta-catenin suggest a role for beta-catenin in colorectal carcinogenesis. In this study, we found that beta-catenin immunoreactivity was detected exclusively in the cell membrane and cytoplasm of morphologically normal intestinal epithelial cells with predominant distribution in the differentiated nonproliferative cell population. In contrast, beta-catenin was localized predominantly in the nucleus of adenomas from Min/+ mice and transgenic mice expressing a mutant truncated form of the APC gene (Apc(delta716) mice). Beta-catenin was expressed predominantly at the cell membrane and cytoplasm of the nontransformed rat intestinal epithelial (RIE-1) cells in culture, whereas predominantly nuclear localization of beta-catenin was observed in the human colon cancer cell line SW480. In the azoxymethane (AOM) treated rats, overexpression and nuclear localization of beta-catenin was observed in all adenomas. Previous studies have indicated the incidence of APC mutations amongst AOM-induced tumors to be 15% or less. These results demonstrate that nuclear localization of beta-catenin is a common event in colorectal tumorigenesis.
Several studies have reported loss or alteration of expression of E-cadherin in breast cancer and more recently changes in levels of expression of the catenins. We used immunofluorescence to examine E-cadherin, alpha-catenin, beta-catenin, and p120ctn (formerly p120CAS) expression in 91 cases of invasive ductal carcinoma. As expected, all four proteins co-localize to the junctional regions of the cells. Although nuclear localization has been described for beta-catenin in colonic polyps, no examples were found in these breast cancer cases. We found that, although alteration is common in the catenins and E-cadherin, complete loss, as exemplified by E-cadherin in lobular carcinoma (where E-cadherin is frequently mutated), is rarely seen. In contrast, the catenin-related protein p120ctn shows an expression pattern that is significantly unrelated to the other catenins (or E-cadherin), including complete loss of expression in approximately 10% of the cases. No statistically significant correlations with traditional prognostic indicators were observed with any of these proteins. We conclude 1) that expression of E-cadherin and alpha- and beta-catenin are generally retained at the membrane although frequently reduced or altered, 2) that complete loss of p120ctn expression is seen in approximately 10% of the cases, and 3) that there is a significant correlation in the expression of E-cadherin and the catenins but no correlation between these molecules and p120ctn, suggesting an absence of coordinate regulation.