The functional consequences of up-regulation of beta-catenin as a transcription factor are complex in different tumors. To clarify roles during squamous differentiation (SqD) of endometrial carcinoma (Em Ca) cells, we investigated expression of beta-catenin, as well as cyclin D1, p53, p21WAF1, and PML (promyelocytic leukemia) in 80 cases of Em Ca with SqD areas, in comparison with cell proliferation determined with reference to Ki-67 antigen positivity. The impact of beta-catenin-T-cell factor (TCF)-mediated transcription was also examined using Em Ca cells. In clinical cases, nuclear beta-catenin accumulation was more frequent in SqD areas, being positively linked with expression of cyclin D1, p53, and p21WAF1, and inversely with Ki-67 and PML immunoreactivity. Significant correlations of nuclear beta-catenin, cyclin D1, p53, and p21WAF1 were noted between SqD and the surrounding carcinoma lesions. The Ishikawa cell line, with stable or tetracycline-regulated expression of mutant beta-catenin, showed an increase in expression levels of cyclin D1, p14ARF, p53, and p21WAF1 but not PML, and activation of beta-catenin-TCF4-mediated transcription determined with TOP/FOP constructs. The cell morphology was senescence-like rather than squamoid in appearance. Moreover, overexpressed beta-catenin could activate transcription from p14ARF and cyclin D1 promoters, in a TCF4-dependent manner. These findings indicate that in Em Cas, nuclear beta-catenin can simultaneously induce activation of the p53-p21WAF1 pathway and overexpression of cyclin D1, leading to suppression of cell proliferation or induction of cell senescence. However, overexpression of beta-catenin alone is not sufficient for development of a squamoid phenotype in Em Ca cells, suggesting that nuclear accumulation is an initial signal for trans-differentiation.
SummaryTo clarify the possible role of aberrant β-catenin expression during endometrial tumorigenesis, a total of 199 cases of endometrial carcinomas (endometrioid type), as well as 37 cases of simple/complex and 32 of atypical hyperplasias, was consecutively investigated for immunohistochemistry, along with 141 normal endometrial samples distant from carcinomas. Of 199 carcinoma cases, 73 tumours as well as 44 normal samples were also analysed using a combination of RT-PCR and Southern blot hybridization, Western blot, and mutation gene assays. Cell membrane β-catenin immunoreactivity showed a stepwise decrease from normal, through atypical hyperplasia, to grade 3 carcinomas. In contrast, the nuclear accumulation in atypical hyperplasias and grade 1 or 2 tumours was higher than in simple/complex hyperplasias. Mutations in exon 3 of the β-catenin gene involving codons 33, 34, 37, 41, and 45 were observed in 16 (22.9%) of 70 endometrial carcinomas, as well as 3 (12.5%) of 24 atypical hyperplasias, the results being significantly related to low membrane and high nuclear immunoreactivity but not relative mRNA expression levels, suggesting that the gene mutations may be closely associated with changes in subcellular distribution. In addition to significant association between β-catenin mutation and low grade histological malignancy (P = 0.048), the mutations were detected in none of 15 and 13 (26%) of 50 tumours with or without lymph node metastasis, the difference being significant (P = 0.027). These findings suggest that β-catenin abnormalities may play an important role in a relatively early event during the endometrial hyperplasia-carcinoma sequence.
Nuclear stabilization of b-catenin and its interaction with TCF/LEF factors are key events in transduction of the Wnt/b-catenin signal pathway. Our previous study indicated that nuclear b-catenin accumulation provides an initial signal for trans-differentiation toward the squamoid phenotype of endometrial carcinoma (Em Ca) cells in a TCF4-dependent manner, which makes this a possible factor for a positive prognosis. However, little is known about regulation of TCF4 expression in Em Cas. We show here that b-catenin can directly induce transcription from the TCF4 promoter, the effect being enhanced by the p300 coactivator. In clinical cases, nuclear b-catenin accumulation was found to frequently overlap with TCF4 immunoreactivity in morules and surrounding glandular carcinoma lesions, showing a significant positive correlation (r ¼ 0.82, Po0.0001), in contrast to areas of squamous metaplasia (SqM) within Em Cas. In cases with coexistence of two squamoid features in transdifferentiated areas, loss of nuclear b-catenin and TCF4 immunoreactivity was closely related to change in the morphology from the morular to the SqM phenotype. The TCF4 promoter contains a single consensus TCFbinding site that is critical for activation by b-catenin. The p300 coactivator, in particular N-terminal residues 1 to 670, appears sufficient to enhance b-catenin-dependent transcription, again with TCF4-dependence. These findings indicate that a positive feedback loop of TCF4 expression mediated by b-catenin/p300 may be important for initial steps during trans-differentiation of Em Ca cells. In addition, its downregulation is associated with induction of a more-differentiated squamoid phenotype.
Uterine carcinosarcomas (UCSs) are considered to represent true examples of the epithelial-mesenchymal transition. Akt plays a key role in the induction of epithelial-mesenchymal transition, but little is known about its involvement in tumorigenesis. Here we examined the functional roles of the Akt/-catenin pathway in UCSs. In clinical samples, phosphoAkt (pAkt) expression was found to be significantly increased in mesenchymal compared with epithelial components, exhibiting both positive and negative correlations with nuclear -catenin and E-cadherin, respectively. Expression levels of the transcription factor Slug were also significantly up-regulated in the mesenchymal components and strongly correlated with both pAkt and nuclear -catenin. In endometrial cancer cell lines, active Akt induced the stabilization of nuclear -catenin through the phosphorylation of GSK-3, and this, in turn, led to the transactivation of Slug, which was mediated by nuclear -catenin. Moreover, Slug overexpression itself caused repression of E-cadherin, with subtle changes in cell morphology. In addition, knockdown of the retinoblastoma gene product (Rb) up-regulated pAkt and repressed E-cadherin, consistent with the in vivo finding of significantly decreased Rb expression in mesenchymal components. These findings suggest that changes in the Akt/-catenin pathway , as well as alterations in Rb expression , may be essential for both the establishment and maintenance of phenotypic characteristics of UCSs , playing key roles in the regulation of E-cadherin through the transactivation of the
Beta-catenin/TCF4/p300 signalling loops play an important role in trans-differentiation towards the morular phenotype of endometrial carcinomas. Crosstalk between NF-kappaB and beta-catenin pathways has been proposed and we focused here on associations between these two pathways during trans-differentiation. In normal endometrium, nuclear phosphorylated p65 (pp65), the active form NF-kappaB subunit, was found to be significantly increased in the secretory phase, correlating positively with vimentin and E-cadherin and inversely with Snail mRNA expression. On transfection of p65, vimentin, E-cadherin, and Snail were transcriptionally altered, indicating possible roles in establishment and maintenance of the secretory phenotype. In endometrial carcinomas with morules, levels of nuclear pp65, Snail mRNA, vimentin, and cytoplasmic TNF-alpha were reduced during trans-differentiation, correlating inversely with nuclear beta-catenin. Nuclear accumulation of GSK-3beta, along with beta-catenin, was observed in morules. In cell lines, overexpression of p65 inhibited beta-catenin/TCF4-mediated transcription, while transfection of GSK-3beta resulted in repression of TNF-alpha-induced NF-kappaB activity. Moreover, nuclear GSK-3beta was increased by overexpression of beta-catenin, as well as induction of G1-cell cycle arrest. These findings provide evidence that a shift from NF-kappaB to beta-catenin signalling pathways through alterations in GSK-3beta expression may be essential for the induction of trans-differentiation of endometrial carcinoma cells, leading to a shut-down of mesenchymal markers.
Nuclear beta-catenin is required for changes in morphology from glandular to morular phenotypes of endometrial carcinoma (Em Ca) cells, with activation of p14(ARF)/p53/p21(Waf1) and alteration of p16(INK4A)/pRb pathways. Having demonstrated previously that the homeodomain transcription factor Cdx2 increases markedly during intestinal epithelial cell differentiation, we have examined its effects in beta-catenin signaling during transdifferentiation of Em Ca cells. In clinical cases, Cdx2 immunoreactivity, along with increased mRNA signals, was found to overlap with nuclear accumulation of beta-catenin and p21(Waf1) in morules, demonstrating an inverse correlation with cell proliferation. In cell lines, over-expression of active form beta-catenin resulted in a significant increase in endogenous Cdx2 expression at both mRNA and protein levels. Furthermore, the Cdx2 promoter was activated by T-cell factor 4 (TCF4) -independent activated beta-catenin, as well as Cdx2 itself, through the region from -39 to +9 bp relative to transcription start site. Cells over-expressing exogenous Cdx2 showed high levels of p21(Waf1) expression due to stabilization of the mRNA status, resulting in significant decrease in the proliferation rate, in contrast to the lack of apparent changes in morphology. Moreover, transfected Cdx2 could inhibit beta-catenin/TCF4-mediated transcriptional activation of target genes, including p14(ARF) and cyclin D1, probably through indirect mechanisms. These data suggest that over-expression of Cdx2 mediated by nuclear beta-catenin and Cdx2 itself can cause an inhibition of Em Ca cell proliferation through up-regulation of p21(Waf1) expression, modulating beta-catenin/TCF4-mediated transcription. We therefore conclude that an association between Cdx2 and beta-catenin signaling may participate in induction of transdifferentiation of Em Ca cells.
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