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.

Focal squamous differentiation is a common feature of endometrioid endometrial and ovarian carcinomas (E-Em and E-Ova Cas). A close association between mutated beta-catenin accumulation and alteration in cellular morphology has recently been demonstrated in murine L cell lines. To clarify the possible role of beta-catenin abnormalities and changes in tumour morphology, 60 grade (G) 1 or G2 E-Em Cas with areas of squamous differentiation (SqD), including morules and squamous metaplastic (SqM) foci, as well as 32 G1 or G2 tumours without such lesions, were investigated and the results compared with findings for c-jun and wnt-1 expression. Twenty-three E-Ova Cas, with and without SqD lesions, were also examined. In E-Em Cas, frequent nuclear beta-catenin accumulation was observed in 22 (84.6%) of 26 tumours with morules and 15 (45.5%) of 33 with SqM foci, in contrast to 4 (12.5%) of 32 without such lesions. Similar findings were also noted for mutations in exon 3 of the beta-catenin gene, involving codons 32, 33, 34, 37, 41, and 45, the single nucleotide substitutions being identical between SqD and the surrounding carcinoma tissue in most informative cases. The mutations were positively related to nuclear immunopositivity, but inversely to membrane expression, while there was no association with the status of c-jun or wnt-1. These E-Ova Cas, nuclear beta-catenin accumulation and mutations were limited to tumours with SqD features, independent of c-jun and wnt-1 status. These data indicate that beta-catenin abnormalities are relatively common in E-Em and E-Ova Cas with SqD features, implying a role in the squamous differentiation of tumour cells, not necessarily related to c-jun and/or wnt-1 status.

The current histologic classifications of gastric cancers define only carcinoids and small cell carcinomas in the neuroendocrine (NE) category. This study aimed to characterize the histologic and clinical features of high-grade gastric NE carcinomas of nonsmall cell type, tentatively named large cell neuroendocrine carcinoma (LCNEC). Tumors with histologic features suspicious of NE differentiation were selected by a histologic review of 2835 resected gastric cancers, and those with a NE phenotype in > 50% and 1% to approximately 50% tumor cells assessed by expressing chromogranin A and/or synaptophysin were defined as LCNEC and adenocarcinoma with neuroendocrine differentiation (ACNED), respectively. One hundred ninety-nine tumors were selected and of the 109 positive for chromogranin A and/or synaptophysin, 42 and 44 met the criteria for LCNEC and ACNED, respectively. Generally, LCNECs demonstrated less predominant NE morphology than carcinoids, and could be roughly divided into solid (30 cases), tubular (7 cases), and scirrhous (5 cases) subtypes with reference to their main growth pattern. The prognosis of LCNECs was significantly worse than that of conventional adenocarcinomas (P < 0.0001). Thus, this study shows that the spectrum of gastric NE tumors is broader than has previously been recognized and LCNEC is not only a distinct histopathologic entity, but also a distinct clinical entity. Furthermore, the prognosis of ACNEDs was also significantly worse than that of adenocarcinomas (P < 0.0001), and some ACNEDs might actually have been LCNECs, and survival analysis showed that > 20% positivity of NE markers could be enough to characterize LCNEC, as long as light microscopic NE morphology was present in the tumor.

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

Summary To clarify the relation between bcl-2 and bax protein (Bcl-2 and Bax) expression with regard to apoptosis and cell proliferation, 82 gastric carcinomas were immunohistochemically investigated. The significance of apoptosis for biological behaviour of the tumours was also examined. The apoptotic indices (Als) were significantly lower in early-stage than in advanced-stage lesions (P<0.05), being positively correlated with the mitotic indices (MIs) (r=0.447, P<0.001). No association between either Als or Mls and tumour size (diameter of intramural spreading) was noted. The Als in the high Bcl-2-immunoreactive score group were significantly smaller than in either the low or the negative categories, whereas they were relatively elevated in the high Bax score group. In addition, an inverse correlation between Bcl-2 and Bax expression was revealed for both Als and MIs. Although depth of tumour invasion and lymph node status were clearly associated with favourable outcome, no relation between survival rates and average values of either Als or MIs, or immunoreactive scores for Bcl-2 and Bax was observed. These results indicate that in gastric carcinomas, apoptosis is closely associated with cell proliferation and expression of Bcl-2 and Bax, but appears likely to have no particular biological significance as a prognostic factor.

BACKGROUND:The impact of somatostatin receptor type 2 (SSTR-2a) expression levels on outcomes in patients with pancreatic neuroendocrine tumors (PNETs) has not been evaluated. METHODS: Correlations between clinicopathologic characteristics, including SSTR-2a expression and outcomes, were retrospectively studied in 79 patients with pancreatic neuroendocrine tumors (PNETs). RESULTS: The SSTR-2a score was 0 in 27% of patients, 1 in 24% of patients, 3 in 30% of patients, and 4 in 18% of patients. The overall survival rate was 87% at 1 year, 77% at 3 years, and 71% at 5 years. On univariate analysis, a pancreatic tumor that measured 20 mm in greatest dimension, stage IV disease, vascular invasion, neuroendocrine carcinoma (NEC), and an SSTR-2a score of 0 were associated significantly with poor outcomes. On multivariate analysis, NEC (P 5.000; hazard ratio, 28.8; 95% confidence interval, 7.502-111.240) and an SSTR-2a score of 0 (P 5.001; hazard ratio, 3.611; 95% confidence interval, 1.344-9.702) were related independently to poor outcomes. CONCLUSIONS: The current analysis of prognostic factors in patients with PNETs demonstrated that NEC and an SSTR-2a score of 0 both were significant independent predictors of poor outcomes. The results suggest that the assessment of SSTR-2a may facilitate the selection of treatment regimens and the prediction of outcomes. Because a considerable proportion of patients with NEC have SSTR-2a-positive tumors, further analyses of the usefulness of somatostatin analogues are warranted in patients who have SSTR-2a-positive NEC. Cancer 2013;119:4094-102.