Em Ca Research Articles

A Complex Interplay between Notch Effectors and β-Catenin Signaling in Morular Differentiation of Endometrial Carcinoma Cells

Notch signaling contributes to tissue development and homeostasis, but little is known about its roles in morular differentiation of endometrial carcinoma (Em Ca) cells. The current study focused on crosstalk between Notch and β-catenin signaling in Em Ca with morules. Promoters of hairy and enhancer of split 1 (Hes1) and mastermind-like 2 (MAML2) were activated by Notch intracellular domain 1 (NICD1) but not β-catenin; we also observed a positive feedback loop between Hes1 and MAML2. Immunoreactivities for nuclear β-catenin, Hes1, and MAML2, as well as the interaction between β-catenin and Hes1 or MAML2, were significantly higher in morular lesions as compared to surrounding carcinoma (Sur Ca) in Em Ca. Inhibition of glycogen synthase kinase-3β (GSK-3β) increased expression of total nuclear and cytoplasmic GSK-3β and its phosphorylated forms, as well as NICD1, Hes1, and active β-catenin. GSK-3β inhibition also decreased proliferation and migration, consistent with the response of cells stably overexpressing Hes1. Finally, the nuclear/cytoplasmic GSK-3β score was significantly higher in morules compared to Sur Ca in Em Ca, and was positively correlated with nuclear β-catenin, Hes1, and MAML2 scores. This complex interplay between Notch effectors and β-catenin signaling through GSK-3β inhibition contributes to the establishment and maintenance of β-catenin-mediated morular differentiation, which is in turn associated with reduced proliferation and inhibition of migration in Em Ca.

Anaplastic lymphoma kinase overexpression enhances aggressive phenotypic characteristics of endometrial carcinoma

BackgroundAlthough anaplastic lymphoma kinase (ALK) is overexpressed in several primary solid tumor types, its role in endometrial carcinoma (Em Ca) remains unclear.MethodsWe evaluated expression of ALK and its related molecules in clinical samples consisting of 168 Em Ca tissues. We also used Em Ca cell lines to evaluate the functional role of ALK.ResultsCytoplasmic ALK immunoreactivity in the absence of chromosomal rearrangement was positively correlated with ALK mRNA expression, and was significantly higher in Grade (G) 3 Em Ca than in G1 or G2 tumors. ALK immunoreactivity was also significantly associated with expression of cancer stem cell (CSC)-related molecules (cytoplasmic CD133, ALDH1, Sox2) and neuroendocrine markers (CD56 and synaptophysin). Although the proliferative index was significantly higher in ALK-positive Em Ca when compared to ALK- negative malignancies, there was no association between ALK expression and other clinicopathological factors in this disease. In Em Ca cell lines, full-length ALK overexpression increased proliferation, decreased susceptibility to apoptosis, enhanced cancer stem cell features, and accelerated cell mobility, whereas these phenotypes were abrogated in ALK-knockdown cells. Finally, patients with tumors harboring either wild-type ALK or high ALK mRNA expression had a poorer prognosis than those with either mutant ALK or low ALK mRNA expression.ConclusionFull-length ALK overexpression occurs in a subset of Em Ca, particularly in G3 tumors, and contributes to the establishment and maintenance of aggressive phenotypic characteristics through modulation of several biological processes.

Nucleobindin-2 Mediates Transforming Growth Factor-β1–Driven Phenotypes in Zinc Finger E-Box Binding Homeobox 1–High Uterine Carcinosarcoma

Epithelial-mesenchymal transition is a hallmark of uterine carcinosarcoma (UCS). Here, shotgun proteomics analysis used to identify biomarkers associated with blebbistatin-mediated epithelial-mesenchymal transition in UCS indicated up-regulation of nucleobindin-2 (NUCB2) in endometrial carcinoma (Em Ca) cells. Expression of N-cadherin, Snail, Slug, and ZEB1 was reduced in NUCB2 knockout Em Ca cells, whereas ZEB1, Twist1, and vimentin were up-regulated in NUCB2-overexpressing Em Ca cells. NUCB2 knockout reduced cell proliferation and migration, whereas NUCB2 overexpression had the opposite effect. Treatment of Em Ca cells with transforming growth factor (TGF)-β1 dramatically altered morphology toward a fibroblastic appearance; concomitantly, expression of NUCB2 and ZEB1 increased. The NUCB2 promoter was also activated by transfection of Smad2. In UCS tissues, NUCB2 expression was significantly higher in sarcomatous compared with carcinomatous components, which was consistent with increased TGF-β1 mRNA expression in stromal and sarcomatous components compared with carcinomatous components. In addition, NUCB2 score correlated positively with ZEB1 and vimentin scores, whereas ZEB1 score correlated positively with Slug and vimentin scores and inversely with the E-cadherin score. Collectively, these data indicate that TGF-β-dependent up-regulation of NUCB2 and ZEB1 contributes to the phenotypic characteristics of sarcomatous components in UCS.

PTEN overexpression and nuclear β-catenin stabilization promote morular differentiation through induction of epithelial–mesenchymal transition and cancer stem cell-like properties in endometrial carcinoma

BackgroundAlthough a lack of functional PTEN contributes to tumorigenesis in a wide spectrum of human malignancies, little is known about the functional role of its overexpression in the tumors. The current study focused on PTEN overexpression in endometrial carcinoma (Em Ca).MethodsThe functional impact of PTEN overexpression was assessed by Em Ca cell lines. Immunohistochemical analyses were also conducted using 38 Em Ca with morular lesions.ResultsEm Ca cell lines stably overexpressing PTEN (H6-PTEN) exhibited epithelial–mesenchymal transition (EMT)-like features, probably through β-catenin/Slug-meditated suppression of E-cadherin. PTEN overexpression also inhibited cell proliferation, accelerated cellular senescence, increased apoptotic features, and enhanced migration capability. Moreover, H6-PTEN cells exhibited cancer stem cell (CSC)-like properties, along with high expression of aldehyde dehydrogenase 1 and CD44s, a large ALDH 1high population, enriched spheroid formation, and β-catenin-mediated upregulation of cyclin D2, which is required for persistent CSC growth. In clinical samples, immunoreactivities for PTEN, as well as CSC-related molecules, were significantly higher in morular lesions as compared to the surrounding carcinomas. PTEN score was positively correlated with expression of nuclear β-catenin, cytoplasmic CD133, and CD44v6, and negatively with cell proliferation. Finally, estrogen receptor-α (ERα)-dependent expression of Ezrin-radixin-moesin-binding phophoprotein-50 (EBP50), a multifunctional scaffolding protein, acts as a negative regulator of morular formation by Em Ca cells through interacting with PTEN and β-catenin.ConclusionIn the abscess of ERα/EBP50 expression, PTEN overexpression and nuclear β-catenin stabilization promote the establishment and maintenance of morular phenotype associated with EMT/CSC-like features in Em Ca cells.BZqz53M46AEhaXf_YNaSmyVideo

Electromagnetic-thermal two-way coupling analysis and application on helium-cooled solid blanket

The blanket plays an important role in fusion reactor and stands extremely high thermal and electromagnetic loads during operation situation and plasma disruption event, brings the need for precise thermal and electromagnetic analysis. Since the thermal field and EM field interact with each other nonlinearly, we develop a method of electromagnetic-thermal two-way coupling by using finite element software COMSOL. The coupling analyses of blanket under steady state and MD event are implemented and the results are analyzed. For steady state, the influences of coupling effects are relatively small but still recommended to be considered for a high precision analysis. The influence of thermal field on EM field can't be ignored under MD events. The variation of force density could cause a significant change in stress in certain parts of blanket. The influence of Joule heat during MD event is negligible, yet the potential temperature rise caused by induced current after MD event still needs to be researched.

Functional interaction between S100A1 and MDM2 may modulate p53 signaling in normal and malignant endometrial cells

BackgroundS100A1 expression is deregulated in a variety of human malignancies, but its role in normal and malignant endometrial cells is unclear.MethodsWe used endometrial carcinoma (Em Ca) cell lines to evaluate the physical and functional interaction of S100A1 with p53 and its negative regulator, mouse double minute 2 (MDM2). We also evaluated the expression of S100A1, p53, and MDM2 in clinical samples consisting of 89 normal endometrial and 189 Em Ca tissues.ResultsS100A1 interacted with MDM2 but not p53 in Em Ca cell lines. Treatment of cells stably overexpressing S100A1 with Nutlin-3A, an inhibitor of the p53/MDM2 interaction, increased expression of p53-target genes including p21waf1 and BAX. S100A1 overexpression enhanced cellular migration, but also sensitized cells to the antiproliferative and proapoptotic effects of Adriamycin, a genotoxic agent; these phenotypes were abrogated when S100A1 was knocked down using shRNA. In clinical samples from normal endometrium, S100A1 expression was significantly higher in endometrial glandular cells of the middle/late secretory and menstrual stages when compared to cells in the proliferative phases; high S100A1 was also positively correlated with expression of MDM2 and p21waf1 and apoptotic status, and inversely correlated with Ki-67 scores. However, such correlations were absent in Em Ca tissues.ConclusionThe interaction between S100A1 and MDM2 may modulate proliferation, susceptibility to apoptosis, and migration through alterations in p53 signaling in normal- but not malignant-endometrial cells.

A functional role of LEFTY during progesterone therapy for endometrial carcinoma

BackgroundThe left-right determination factor (LEFTY) is a novel member of the TGF-β/Smad2 pathway and belongs to the premenstrual/menstrual repertoire in human endometrium, but little is known about its functional role in endometrial carcinomas (Em Cas). Herein, we focused on LEFTY expression and its association with progesterone therapy in Em Cas.MethodsRegulation and function of LEFTY, as well as its associated molecules including Smad2, ovarian hormone receptors, GSK-3β, and cell cycle-related factors, were assessed using clinical samples and cell lines of Em Cas.ResultsIn clinical samples, LEFTY expression was positively correlated with estrogen receptor-α, but not progesterone receptor (PR), status, and was inversely related to phosphorylated (p) Smad2, cyclin A2, and Ki-67 levels. During progesterone therapy, expression of LEFTY, pSmad2, and pGSK-3β showed stepwise increases, with significant correlations to morphological changes toward secretory features and decreased Ki-67 values. In Ishikawa cells, an Em Ca cell line that expresses PR, progesterone treatment reduced proliferation and induced increased expression of LEFTY and pGSK-3β, although LEFTY promoter regions were inhibited by transfection of PR. Moreover, inhibition of GSK-3β resulted in increased LEFTY expression through a decrease in its ubiquitinated form, suggesting posttranslational regulation of LEFTY protein via GSK-3β suppression in response to progesterone. In addition, overexpression or knockdown of LEFTY led to suppression or enhancement of Smad2-dependent cyclin A2 expression, respectively.ConclusionUpregulation of LEFTY may serve as a useful clinical marker for the therapeutic effects of progesterone for Em Cas, leading to inhibition of tumor cell proliferation through alteration in Smad2-dependent transcription of cyclin A2.

Cooperation of Sox4 with β-catenin/p300 complex in transcriptional regulation of the Slug gene during divergent sarcomatous differentiation in uterine carcinosarcoma.

BackgroundUterine carcinosarcoma (UCS) represents a true example of cancer associated with epithelial-mesenchymal transition (EMT), which exhibits cancer stem cell (CSC)-like traits. Both Sox and β-catenin signal transductions play key roles in the regulation of EMT/CSC properties, but little is known about their involvement in UCS tumorigenesis. Herein, we focused on the functional roles of the Sox/β-catenin pathway in UCSs.MethodsEMT/CSC tests and transfection experiments were carried out using three endometrial carcinoma (Em Ca) cell lines. Immunohistochemical investigation was also applied for a total of 32 UCSs.ResultsEm Ca cells cultured in STK2, a serum-free medium for mesenchymal stem cells, underwent changes in morphology toward an EMT appearance through downregulation of E-cadherin, along with upregulation of Slug, known as a target gene of β-catenin. The cells also showed CSC properties with an increase in the aldehyde dehydrogenase (ALDH) 1high activity population and spheroid formation, as well as upregulation of Sox4, Sox7, and Sox9. Of these Sox factors, overexpression of Sox4 dramatically led to transactivation of the Slug promoter, and the effects were further enhanced by cotransfection of Sox7 or Sox9. Sox4 was also able to promote β-catenin-mediated transcription of the Slug gene through formation of transcriptional complexes with β-catenin and p300, independent of TCF4 status. In clinical samples, both nuclear β-catenin and Slug scores were significantly higher in the sarcomatous elements as compared to carcinomatous components in UCSs, and were positively correlated with Sox4, Sox7, and Sox9 scores.ConclusionsThese findings suggested that Sox4, as well as Sox7 and Sox9, may contribute to regulation of EMT/CSC properties to promote development of sarcomatous components in UCSs through transcriptional regulation of the Slug gene by cooperating with the β-catenin/p300 signal pathway.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2090-y) contains supplementary material, which is available to authorized users.

Bifunctional roles of survivin-ΔEx3 and survivin-2B for susceptibility to apoptosis in endometrial carcinomas.

Alternative splicing variants of survivin have different biological roles on cell kinetics. Here, we focused on the effects of different variants, including wild type (wt), survivin-ΔEx3, and survivin-2B, on apoptosis and cell proliferation in endometrial carcinomas (Em Cas). Expression of survivin-wt, survivin-ΔEx3, and survivin-2B with reference to cell death and proliferation was investigated, using Em Ca cell lines and its clinical tissues. Ishikawa cells stably overexpressing either survivin-ΔEx3 (Surv-ΔEx3#34) or survivin-2B (Surv-2B#17) demonstrated considerably lower proliferative activity, along with up-regulation of p21waf1. After TNF-α treatment, Surv-ΔEx3#34 cells showed an increase in apoptotic cells, while the effects were relatively minor in Surv-2B#17 cells. In contrast, doxorubicin treatment resulted in increased apoptotic cells in Surv-2B#17 but not Surv-ΔEx3#34 cells, along with decreased expression of bcl-2 relative to bax. Control Ishikawa cells also showed relatively higher endogenous mRNA expression of survivin-ΔEx3 and survivin-2B during treatment of TNF-α and doxorubicin, respectively. In addition, exogenous overexpression of each survivin variant resulted in inhibition of other endogenous isoforms, indicating that the relative proportion may contribute to regulation of the splicing machinery. In clinical samples, level of survivin-ΔEx3 relative to either survivin-wt or survivin-2B was significantly higher in Em Cas than non-neoplastic lesions. Moreover, survivin-ΔEx3 and survivin-wt were positively correlated with apoptosis and cell proliferation, respectively, in Em Cas. These findings provided evidence that the balance among expression level of survivin variants may contribute to modulation of cell kinetics in Em Ca cells.

Sox4 functions as a positive regulator of β-catenin signaling through upregulation of TCF4 during morular differentiation of endometrial carcinomas

Sox factors function as either activators or repressors of β-catenin/TCF transcription depending on the cellular context and associated interacting proteins. Our previous study provided evidence that alteration in β-catenin signaling is an essential event during transdifferentiation toward the morular phenotype of endometrial carcinomas (Em Cas). Here, we focused on related functional roles of Sox factors. Of eight Sox factors investigated, Sox4 could enhance β-catenin/TCF4 transcription, through upregulation of TCF4 at the transcription level, without any direct β-catenin association. Cells stably overexpressing Sox4 showed significant decreases in proliferation rate, along with increases in expression of p21WAF1, as well as TCF4, in contrast to increased cell growth observed with knockdown. Of these factors, only Sox7 could transcriptionally upregulate Sox4 expression, but it also resulted in not only inhibition of Sox4-meditated activation of β-catenin/TCF4-driven transcription, but also repression of its own promoter activity, indicating the existence of very complex feedback loop for Sox-mediated signal cascades. Finally, Sox4 immunoreactivity was frequently pronounced in morular lesions of Em Cas, the expression being positively correlated with status of β-catenin, TCF4, and Sox7, and inversely with cell proliferation. These data therefore suggest that Sox4 may serve as a positive regulator of β-catenin signaling through alteration in TCF4 expression during morular differentiation of Em Ca cells, leading to inhibition of cell proliferation. In addition, Sox7 may also participate in the process, having complex roles in modulation of signaling.

Pin1 acts as a modulator of cell proliferation through alteration in NF‐κB but not β‐catenin/TCF4 signalling in a subset of endometrial carcinoma cells

Prolyl isomerase Pin1 is frequently up-regulated in a variety of human malignancies, modulating signalling in several oncogenic pathways, including those involving NF-κB and β-catenin. Our previous study provided evidence that alterations in these signal pathways are essential events during trans-differentiation of endometrial carcinoma (Em Ca) cells. Here we focused on the functional roles of Pin1. In normal endometrium, Pin1 expression showed a stepwise decrease from proliferative to secretory phases during the menstrual cycle, correlating positively with cell proliferation and expression of several cell cycle-related molecules including E2F1 and pRb. Transfection of E2F1 caused transactivation of Pin1, indicating control by E2F1/Rb pathways. In Em Cas with morules, Pin1 expression was found to be significantly increased in glandular but not in morular components, correlating inversely with nuclear accumulation of β-catenin. Overexpression also caused an increase in the stability of nuclear p65, leading to enhancement of NF-κB-mediated transactivation of the cyclin D1 gene, in contrast to minimal inhibition of β-catenin/TCF4 transcription activity. These findings indicate that Pin1 may play an important role in preserving cell proliferative activity in glandular carcinoma components through enhancement of NF-κB signalling, but its down-regulation may be a key signal for induction of trans-differentiation of Em Ca cells, contributing to a shift from NF-κB to β-catenin/TCF signalling pathways.

Stromal reaction of adenomyosis protects from uterine endometrial cancer invasion to myometrium

16064 Background: It is unclear that stromal reaction plays role in endometrial cancer (Em Ca) invasion to uterine muscle with adenomyosis. Methods: One hundred and five patients with Em Ca (stage 0=38, 1a=18, 1b=26, 1c=10, 2b=3, 3a=4, 3c=5, 4a=1) received operative therapy with hysterectomy. Evaluation of stromal reaction using immunohistochemical method with alpha-smooth muscle actin and h-caldesmon antibody. In case of alpha-smooth muscle actin had immunoreactivity and h-caldesmon had not immunoreactivity, there was stromal reaction with myofibroblast. Evaluation of cell proliferation, using immunohistochemical method with epidermal growth factor receptor (EGFR) by global score with six degrees and MIB-1 by labeling Index (LI). Results: All cases had stromal reaction with myofibloblast in myometrial invasion area in forty nine cases with myometrial invasion equal or more than stage 1b. Em Ca invaded near to and in adenomyosis lesion in eleven cases. In stroma of adenomyosis without cancer invasion, EGFR was positive in one case (9%) and global score was two (average 0.18). In contrast, EGFR was positive in eight cases (73%) with Em Ca invasion to adenomyosis stroma and global score was from two to three (average 1.6). LI of MIB-1 was from 0 to 0.2 (average 0.05) in adenomyosis stroma without Em Ca invasion and 0.2 to 0.75 (average 0.41) with Em Ca invasion. Conclusions: When Em Ca invades to adenomyosis, myofibloblast cell proliferation in stromal reaction increases more than in adenomyosis without Em Ca invasion. Stromal reaction of adenomyosis plays role in protecting from Em Ca invasion to uterine muscle and it may contribute to being good prognosis compared with Em Ca without adenomyosis. No significant financial relationships to disclose.

A functional role of Cdx2 in β-catenin signaling during transdifferentiation in endometrial carcinomas

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.

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 beta-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 beta-catenin could inhibit cell proliferation through activation of p14(ARF)/p53/p21(WAF1) pathway during trans-differentiation toward morular phenotype of endometrial carcinoma (Em Ca) cells. Here, we focused on associations with alterations in p16(INK4A) and pRb expression during this process. In clinical cases, p16(INK4A) immunoreactivity was found to frequently overlap with nuclear beta-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 beta-catenin and p21(WAF1) 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 p16(INK) (4A) promoter by active form beta-catenin, as well as p21(WAF1), 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 p16(INK4A) expression, while its inhibition by serum starvation caused decreased expression of total pRb but not p16(INK4A), resulting in high relative amounts of the latter. These findings indicate that induction of p16(INK4A) mediated by nuclear beta-catenin and p21(WAF1), 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.

The role of adenomyosis in uterine endometrial cancer (Em Ca) invasion to myometrium

5179 Background: It is unclear what adenomyosis play roles in Em Ca invasion. Methods: Eighty five patients with Em Ca (stage 0=31, 1a=15, 1b=21, 1c=8, 2b=2, 3a=3, 3c=4, 4a=1, Age 22–82 years old, mean 54.1) received operative therapy with hysterectomy between January 2001 and November 2004. Evaluation of stromal reaction using immunohistochemical method with alpha-smooth muscle actin and h-caldesmon antibody. In case of alpha-smooth muscle actin had immunoreactivity and h-caldesmon had not immunoreactivity, there was stromal reaction with myofibroblast. Immunoreactivity scored from zero to four degrees. Results: Forty seven patients had adenomyosis in 85 Em Ca patients. In nine cases, Em Ca invaded near to adenomyosis lesion. In thirty nine cases with myometrial invasion (stage 1b-), all cases had stromal reaction with myofibloblast in myometrial invasion area (scored 1–10 cases, 2–15 cases, 3–10 cases, 4–4 cases). In contrast, nine cases with Em Ca invasion near to adenomyosis, stromal reaction with myofibloblast score were more high (scored 1–0 cases, 1–0 case, 2–3 cases, 3–3 cases, 4–3 cases). Conclusions: When Em Ca invaded around the adenomyosis in myometrium, stromal rection with myofibloblasts appeared stronger than in myometrium without adenomyosis. This may be the adenomyosis reaction of protection from Em Ca invasion. No significant financial relationships to disclose.

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.

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 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.

THE MEAN-VARIANCE APPROACH TO PORTFOLIO OPTIMIZATION SUBJECT TO TRANSACTION COSTS

Tra,nsact>ion costss are a. source of concern for port,folio managers. Due to nonlinearity of the cost function, the ordinary quadratic programming solution technique cannot be applied. This paper addresses the portfolio optinlization problem subject to transaction costs. The transaction cost is assumed to be a V-sha,ped function of difference between an existing and new portfolio. A nonlinear programming solution technique is used to solve t,he proposed problem. The port,folio optimiza,t,ion syst,em ca,lled POSTRAC (Portfolio Optirniza,tion System with TRAnsaction Costs) is proposed. The experimental analysis indicates that ignoring the transa,ction costss results in inefficient portfolios. It is also shown tlmt there does not exist statistica,lly significant difference in portfolio performance with different methods to estimate the expected return of se~urit~ies, when considering the tra,nsact,ion costs int,o the p~rt~folio return.