Mieko Hamano

β-Catenin Simultaneously Induces Activation of the p53-p21WAF1 Pathway and Overexpression of Cyclin D1 during Squamous Differentiation of Endometrial Carcinoma Cells

The functional consequences of up-regulation of β-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 β-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 β-catenin-T-cell factor (TCF)-mediated transcription was also examined using Em Ca cells. In clinical cases, nuclear β-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 β-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 β-catenin, showed an increase in expression levels of cyclin D1, p14ARF, p53, and p21WAF1 but not PML, and activation of β-catenin-TCF4-mediated transcription determined with TOP/FOP constructs. The cell morphology was senescence-like rather than squamoid in appearance. Moreover, overexpressed β-catenin could activate transcription from p14ARF and cyclin D1 promoters, in a TCF4-dependent manner. These findings indicate that in Em Cas, nuclear β-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 β-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.

Upregulation of TCF4 expression as a transcriptional target of β -catenin/p300 complexes during trans -differentiation of endometrial carcinoma cells

Nuclear stabilization of β-catenin and its interaction with TCF/LEF factors are key events in transduction of the Wnt/β-catenin signal pathway. Our previous study indicated that nuclear β-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 β-catenin can directly induce transcription from the TCF4 promoter, the effect being enhanced by the p300 coactivator. In clinical cases, nuclear β-catenin accumulation was found to frequently overlap with TCF4 immunoreactivity in morules and surrounding glandular carcinoma lesions, showing a significant positive correlation (r=0.82, P<0.0001), in contrast to areas of squamous metaplasia (SqM) within Em Cas. In cases with coexistence of two squamoid features in trans-differentiated areas, loss of nuclear β-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 TCF-binding site that is critical for activation by β-catenin. The p300 coactivator, in particular N-terminal residues 1 to 670, appears sufficient to enhance β-catenin-dependent transcription, again with TCF4-dependence. These findings indicate that a positive feedback loop of TCF4 expression mediated by β-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.

Crosstalk between NF‐κB/p65 and β‐catenin/TCF4/p300 signalling pathways through alterations in GSK‐3β expression during trans‐differentiation of endometrial carcinoma cells

β‐Catenin/TCF4/p300 signalling loops play an important role in trans‐differentiation towards the morular phenotype of endometrial carcinomas. Crosstalk between NF‐κB and β‐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‐κB 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‐α were reduced during trans‐differentiation, correlating inversely with nuclear β‐catenin. Nuclear accumulation of GSK‐3β, along with β‐catenin, was observed in morules. In cell lines, overexpression of p65 inhibited β‐catenin/TCF4‐mediated transcription, while transfection of GSK‐3β resulted in repression of TNF‐α‐induced NF‐κB activity. Moreover, nuclear GSK‐3β was increased by overexpression of β‐catenin, as well as induction of G1‐cell cycle arrest. These findings provide evidence that a shift from NF‐κB to β‐catenin signalling pathways through alterations in GSK‐3β expression may be essential for the induction of trans‐differentiation of endometrial carcinoma cells, leading to a shut‐down of mesenchymal markers. Copyright

Role of Estrogen and Estrogen-Related Growth Factor in the Mechanism of Hormone Dependency of Endometrial Carcinoma Cells

The role of estrogen and estrogen-related growth factors in the mechanism of hormone dependency of endometrial adenocarcinoma cells was investigated. The proliferation of hormone-responsive human endometrial adenocarcinoma cells (Ishikawa cells), which possess both estrogen and progesterone receptors, was optimally stimulated by 10 nM estradiol. Both transforming growth factor (TGF)-α and epidermal growth factor (EGF), added to the culture media, stimulated the proliferation of Ishikawa cells in a dose-dependent manner. Anti-TGF-α antibody completely eliminated the stimulatory effects of TGF-α. Anti-EGF receptor antibody inhibited the proliferation of these cells. The production of TGF-α into culture media was 5–40 pg/10 cells/24 h in 9 human endometrial adenocarcinoma cells. Ten nanomoles of estradiol increased the TGF-α production of Ishikawa cells by approximately 2.5-fold of the control level. In contrast, the production of TGF-α in hormone-unresponsive HEC-50 cels was not influenced by estradiol. C-erbB-2 oncoprotein expression of human endometrial adenocarcinoma cells, detected by both immunocytochemical staining and Western blot analysis, was associated with the tumor grade of the original tumor tissues. Ten nanomoles of estradiol clearly increased the c-erbB-2 oncoprotein levels at an optimal incubation period of 72 h, whereas estradiol did not affect the expression in HEC-50 cells.

Transcription factor Egr1 acts as an upstream regulator of β‐catenin signalling through up‐regulation of TCF4 and p300 expression during trans‐differentiation of endometrial carcinoma cells

The β‐catenin/TCF4/p300 pathway is involved in early signalling for trans‐differentiation towards the morular phenotype of endometrial carcinoma cells, but little is known about the upstream regulators. Here we show that transcription factor early growth response 1 (Egr1) acts as an initial mediator through up‐regulating the expression of TCF4 and p300. In an endometrial carcinoma cell line with abundant oestrogen receptor α, Egr1 expression at both mRNA and protein levels was significantly increased by serum and 17β‐oestradiol stimuli. Serum‐stimulated cells also showed increased expression of TCF4 and p300, while inhibition of Egr1 by specific siRNAs resulted in decreased expression. Transfection of Egr1 led to transactivation of TCF4 as well as p300 genes, through specific binding to a promoter region, and thus in turn resulted in nuclear accumulation of β‐catenin mediated by the up‐regulating TCF4. The overexpression also caused inhibition of β‐catenin/TCF4/p300‐mediated transcription, probably through sequestration of p300. Egr1 promoter activity was increased by serum but not 17β‐oestradiol, in contrast to the marked repression associated with TCF4, p300, and Egr1 itself, indicating that the regulation involves several feedback loops. In clinical samples, cells immunopositive for nuclear Egr1, as well as β‐catenin and TCF4, were found to be sporadically distributed in glandular components of endometrial carcinoma with morules. A significant positive correlation between nuclear β‐catenin and TCF4 was observed, but no such link was evident for Egr1, probably due to the existence of negative feedback regulation. Together, these data indicate that Egr1 may participate in modulation of the β‐catenin/TCF4/p300 signalling pathway as an initial event during trans‐differentiation of endometrial carcinoma cells, through its impact on several signalling networks. Copyright

Induction of p16INK4A mediated by β‐catenin in a TCF4‐independent manner: Implications for alterations in p16INK4A and pRb expression during trans‐differentiation of endometrial carcinoma cells

Excessive β‐catenin is considered to contribute to tumor progression by inducing transcription of cell cycle‐related genes such as cyclin D1 and c‐myc. In contrast, our recent studies demonstrated that β‐catenin could inhibit cell proliferation through activation of p14ARF/p53/p21WAF1 pathway during trans‐differentiation toward morular phenotype of endometrial carcinoma (Em Ca) cells. Here, we focused on associations with alterations in p16INK4A and pRb expression during this process. In clinical cases, p16INK4A immunoreactivity was found to frequently overlap with nuclear β‐catenin accumulation in small‐sized morules and surrounding glandular carcinomas (Sur‐Ca), demonstrating a significant positive correlation (r = 0.447, p < 0.0001) overall, while the immunoreactions showed stepwise decrease in enlarged morules, despite persistent accumulation of β‐catenin and p21WAF1 in nuclei. Immunoreactivity for both total pRb and its phosphorylated form was apparently decreased in all morules as compared to Sur‐Ca lesions, with a significantly positive correlation. In cell lines, transcriptional activation of p16INK4A promoter by active form β‐catenin, as well as p21WAF1, occurred through the region from −385 to −280 bp relative to the translation start site, in a TCF4‐independent manner. Moreover, cell proliferation was accompanied with phosphorylation of pRb and increased p16INK4A expression, while its inhibition by serum starvation caused decreased expression of total pRb but not p16INK4A, resulting in high relative amounts of the latter. These findings indicate that induction of p16INK4A mediated by nuclear β‐catenin and p21WAF1, along with loss of pRb expression, may be important for initial steps during trans‐differentiation of Em Ca cells. In addition, its down‐regulation is associated with progression of lesions.

Establishment and characterization of the cell-line of a human edonmetrial adenoacanthoma

Abstract A new cell-line of a human endometrial adenoacanthoma, designated as HEC-6, has been established and propagates well with a permanent stability. The population doubling time of the line is calculated as 52 hr. The in vitro morphology reveals the features of a well-differentiated adenocarcinoma such as a jig-saw puzzle-like arrangement and the formation of an acinous cell-free space in the monolayer culture. Each cell consists of a round atypical nucleus with 2 or 3 marked nucleoli and a vacuolated cytoplasm. A piling-up tendency is a prominent feature and forms an obvious palisade-like or glandular structure. The karyotype of the stem cell is a pseudo-diploid in which variant chromosomes occur at random in various groups. When transplanted into a hamster cheek pouch, line HEC-6 demonstrated an adenocarcinoma in addition to a stratified epithelial component. The original characteristics of an adenoacanthoma of the corpus uteri have been maintained in the culture system.

Effect of p53 gene transfection on vascular endothelial growth factor expression in endometrial cancer cells.

It has been reported that tumor suppressor gene p53 regulates vascular endothelial growth factor (VEGF) expression, but the relation between them in endometrial carcinoma remains unclear. We investigated VEGF expression in 11 endometrial carcinoma cell lines and the effect of p53 gene transfection on VEGF expression in the p53-mutated endometrial carcinoma cell line, HEC-50B. Immunoblotting for detecting VEGF, p53, and beta-actin was performed. Wild type p53 gene was transfected using the SuperFect method. The mean VEGF value of 0.8 +/- 0.3 (n = 6) in p53 wild-type group was significantly lower than the 1.6 +/- 0.8 (n = 5) that was found in the p53 mutant group (P < 0.05). Levels of VEGF in the culture medium were measured by enzyme immunoassay (EIA). VEGF levels in the p53 gene-transfected HEC-50B cells and the conditioned medium were decreased at 48 h after p53 gene transfection. VEGF expression was downregulated by p53 in endometrial carcinoma cells.

HEC-1 cells

HEC-1 cell line was the first in vitro cell line of a human endometrial adenocarcinoma which enabled us to perform research work on the endometrium and endometrial carcinoma at a simplified cellular system, contributing cell and molecular biological studies on endometrial carcinoma Once a cell line is established, it provides a stable experimental system that facilitates and progresses in the study of the tissues and/or neoplasias from which they are derived. In this article we report how HEC-1 cells have been established and cleared the proposed requirements to characterize the established cell line. Also to show the usefulness of the cell line for research work, once it was established, we illustrate these concepts by recalling results obtained with HEC-1 cells and reviewing the literature on what has been achieved by using these cells.

Up-regulation and nuclear localization of β-catenin in endometrial carcinoma in response to progesterone therapy

Ovarian hormones are considered to be capable of regulating expression of β‐catenins. A possible role of β‐catenin in alteration of cell morphology has been proposed, but little is known about β‐catenin expression during changes in the tumor morphology of endometrial carcinomas induced by progesterone therapy. To clarify changes in expression of β‐catenin and their relation to morphological alteration, expression of hormone receptors and several cell kinetic markers, sequential biopsy and hysterectomy specimens of 23 endometrial carcinoma and 6 complex hyperpla‐sia with atypia (atypical hyperplasia) cases receiving progesterone therapy were investigated. In vitro assay was also conducted using two endometrial carcinoma cell lines (HEC265 and Ishikawa) expressing progesterone receptors (PRs). An increase of nuclear β‐catenin accumulation was evident during progesterone therapy in endometrial carcinomas and atypical hyperplasias. The nuclear labeling indices were significantly associated with gene mutations and alteration in morphological features in response to progesterone, independently of the status of Ki‐67, p21WAF1 and p27Kip1, and hormone receptors. In HEC265 having a β‐catenin gene mutation (A32V), cytoplasmic p‐catenin levels were elevated by progesterone treatment, linked to down‐regulation of PR expression, but such changes were relatively minor in Ishikawa without the gene alterations. These findings demonstrate a possible role of progesterone in regulation of β‐catenin expression in endometrial tumors. Moreover, nuclear β‐catenin accumulation, like gene abnormalities, is associated with the alteration of tumor morphology due to progesterone, indicating that β‐catenin may be a clinically useful marker of hormone therapeutic effects. (Cancer Sci 2003; 94: 103‐111)