Membranous Β-catenin Research Articles

β-catenin expression in in situ and infiltrative ductal carcinomas of the breast

Cascades that include β-catenin that has a function in adhesion and interaction with tumor suppressor genes such as APC have important roles in many neoplasms. The aim of the current study was to confirm the effect of the β-catenin pathway in breast tumor carcinogenesis and invasion. Polyclonal rabbit β-catenin antibody was applied to 52 cases of infiltrative ductal carcinoma and 28 cases of ductal carcinoma in situ using the Avidin Biotin complex immune peroxidase method. The intensity and cellular localization of immunostaining were evaluated and compared. β-catenin immunoreactivity similar to that of normal epithelium was observed in 7 (8.75%) cases and weak or absent β-catenin expression was noted in 45 (56.25%) infiltrative ductal carcinoma cases. β-catenin expression was strong in 5 (6.25%) cases of ductal carcinoma in situ but weak or absent immunostaining was observed in 23 (28.75%) cases. Membranous β-catenin immunoreactivity was observed in 18 (22.5%) cases of infiltrative and 14 (%17.5) cases of ductal carcinoma in situ. Cytoplasmic immunostaining or complete absence of staining was noted in 34 (42.5%) cases of infiltrative and 14 (17.5%) cases of ductal carcinoma in situ. Similar quantitative and qualitative changes in β-catenin expression were detected in a considerable proportion of in situ and infiltrative ductal carcinomas in the current study. These findings suggest that β-catenin plays a role in the carcinogenesis of infiltrative ductal carcinoma but similar expression patterns of β-catenin in infiltrative and in situ ductal carcinomas indicates that changes in β-catenin expression occur early in carcinogenesis.

Expression of Cell Adhesion Molecules and Doublecortin in Canine Anaplastic Meningiomas

Tumor cell invasion into the surrounding nervous tissue is one of the histologic hallmarks of anaplastic meningiomas. To identify other possible markers for aggression in canine meningiomas, the relationship between histologic features and the expression of molecules involved in cell adhesion, cell proliferation, and invasion was examined. Immunohistochemistry for epithelial cadherin (E-cadherin), neural cadherin (N-cadherin), β-catenin, doublecortin (DCX), and Ki-67 was performed for 55 cases of canine meningioma. DCX was preferentially expressed in tumor cells invading the brain parenchyma (12 of 14 cases), suggesting its involvement in the invasion process. Regardless of the histologic type, E-cadherin and N-cadherin expression was observed in 31 of 55 and 44 of 55 cases, respectively. There was a significant positive correlation between DCX and N-cadherin expression and a significant negative correlation between E-cadherin and N-cadherin expression, suggesting that decreased E-cadherin and increased N-cadherin expression induce DCX expression. Typical membranous β-catenin expression was observed in 10 of 55 cases, whereas nuclear translocation was observed in 33 cases. Nuclear β-catenin expression was frequently found in anaplastic meningiomas (12 of 14 cases). The Ki-67 labeling indices were significantly higher in anaplastic meningiomas than in other types. These findings indicate that the expression of N-cadherin and DCX and the nuclear translocation of β-catenin are closely associated with the presence of invasion and anaplasia in canine meningiomas. Notably, granular cell meningiomas were negative for almost all the molecules examined, suggesting that they have a different tumor biology than other meningiomas.

β-catenin/Wnt signalling pathway in fibromatosis, metaplastic carcinomas and phyllodes tumours of the breast

Wnt signalling pathway is known to have a critical role in carcinogenesis and in epithelial-to-mesenchymal transition. Upon Wnt activation, β-catenin is translocated from the membrane to the cytoplasm and nucleus, where it interacts with transcriptional activators. It has been suggested that various spindle cell lesions of the breast may harbour Wnt pathway activation. Given that β-catenin nuclear localization constitutes a good surrogate marker of Wnt canonical pathway activation, we have investigated the distribution of β-catenin in spindle cell lesions of the breast and whether it could be employed in the differential diagnosis of these lesions. A total of 52 metaplastic breast carcinomas, eight fibromatoses and 23 phyllodes tumours were retrieved from our institutions' archives. We performed immunohistochemistry using two anti-β-catenin antibodies. In all, three fibromatoses and 21 metaplastic breast carcinomas were subjected to CTNNB1 (β-catenin encoding gene) mutation analysis by direct gene sequencing. A good correlation between the two antibodies was observed (Spearman's r>0.82, P<0.001). All fibromatoses and 23% of metaplastic breast carcinomas expressed nuclear β-catenin. In fibromatosis, β-catenin was more often diffusely expressed, whereas in metaplastic breast carcinomas, expression was more frequently focal. Membranous β-catenin expression was significantly lower in spindle cell carcinomas than in other subtypes of metaplastic breast carcinomas. In phyllodes tumours, stromal cells of benign and malignant subtypes displayed nuclear β-catenin expression in 94 and 57% of cases, respectively. No CTNNB1 mutation was identified in any of the 21 metaplastic carcinomas analysed, whereas the mutations 45S>S/P and 41T>T/A were found in samples of fibromatosis. In conclusion, β-catenin nuclear expression is a common feature in fibromatoses and in the stromal component of phyllodes tumours, but may also be observed in metaplastic breast carcinomas. β-catenin nuclear expression should not be used as a single marker to differentiate fibromatosis from other spindle cell tumours of the breast.

PF-03732010: A Fully Human Monoclonal Antibody against P-Cadherin with Antitumor and Antimetastatic Activity

P-cadherin is a membrane glycoprotein that functionally mediates tumor cell adhesion, proliferation, and invasiveness. We characterized the biological properties of PF-03732010, a human monoclonal antibody against P-cadherin, in cell-based assays and tumor models. The affinity, selectivity, and cellular inhibitory activity of PF-03732010 were tested in vitro. Multiple orthotopic and metastatic tumor models were used for assessing the antitumor and antimetastatic activities of PF-03732010. Treatment-associated pharmacodynamic changes were also investigated. PF-03732010 selectively inhibits P-cadherin-mediated cell adhesion and aggregation in vitro. In the P-cadherin-overexpressing tumor models, including MDA-MB-231-CDH3, 4T1-CDH3, MDA-MB-435HAL-CDH3, HCT116, H1650, PC3M-CDH3, and DU145, PF-03732010 inhibited the growth of primary tumors and metastatic progression, as determined by bioluminescence imaging. Computed tomography imaging, H&E stain, and quantitative PCR analysis confirmed the antimetastatic activity of PF-03732010. In contrast, PF-03732010 did not show antitumor and antimetastatic efficacy in the counterpart tumor models exhibiting low P-cadherin expression. Mechanistic studies via immunofluorescence, immunohistochemical analyses, and 3'-[(18)F]fluoro-3'-deoxythymidine-positron emission tomography imaging revealed that PF-03732010 suppressed P-cadherin levels, caused degradation of membrane β-catenin, and concurrently suppressed cytoplasmic vimentin, resulting in diminished metastatic capacity. Changes in the levels of Ki67, caspase-3, and 3'-[(18)F]fluoro-3'-deoxythymidine tracer uptake also indicated antiproliferative activity and increased apoptosis in the tested xenografts. These findings suggest that interrupting the P-cadherin signaling pathway may be a novel therapeutic approach for cancer therapy. PF-03732010 is presently undergoing evaluation in Phase 1 clinical trials.

Glycogen synthase kinase-3β does not correlate with the expression and activity of β-catenin in gastric cancer

The regulation of β-catenin activation by glycogen synthase kinase-3β (GSK-3β) in cancer has been shown to be cell type-specific. This study was performed to investigate the relationship between activated GSK-3β (phosphorylated at Tyr216) and β-catenin in gastric cancer. Immunohistochemical tissue array analysis of 278 human gastric carcinoma specimens showed positive immunoreactivity for activated GSK-3β in 44% of the samples, whereas membranous β-catenin and nuclear β-catenin were observed in 19% and 20% of the samples, respectively. However, GSK-3β activation was not correlated with the expression of either membranous β-catenin or nuclear β-catenin. Moreover, SNU gastric cancer cell lines over-expressing kinase dead GSK-3β and the same cells treated with a GSK-3β inhibitor showed that GSK-3β inhibition did not alter either the protein expression or transcriptional activity of β-catenin. In addition, GSK-3β activation was positively correlated with the expressions of anti-adenomatous polyposis coli (p = 0.002), p16 (p < 0.001), p21 (p < 0.001), p27 (p = 0.001), and p53 (p = 0.013). On the other hand, the nuclear expression of β-catenin was positively correlated with those of Bcl-2 (p = 0.025) and cyclin D1 (p = 0.043), but these expressions were not correlated with GSK-3β activation. Thus, the GSK-3β pathway seems to function in gastric cancer cells without involving the β-catenin pathway.

GSK3β and β-Catenin Modulate Radiation Cytotoxicity in Pancreatic Cancer

BACKGROUND: Knowledge of factors and mechanisms contributing to the inherent radioresistance of pancreatic cancer may improve cancer treatment. Irradiation inhibits glycogen synthase kinase 3β (GSK3β) by phosphorylation at serine 9. In turn, release of cytosolic membrane β-catenin with subsequent nuclear translocation promotes survival. Both GSK3β and β-catenin have been implicated in cancer cell proliferation and resistance to death. METHODS: We investigated pancreatic cancer cell survival after radiation in vitro and in vivo, with a particular focus on the role of the function of the GSK3β/β-catenin axis. RESULTS: Lithium chloride, RNAi-medicated silencing of GSK3β, or the expression of a kinase dead mutant GSK3β resulted in radioresistance of Panc1 and BxPC3 pancreatic cancer cells. Conversely, ectopic expression of a constitutively active form of GSK3β resulted in radiosensitization of Panc1 cells. GSK3β silencing increased radiation-induced β-catenin target gene expression asmeasured by studies of AXIN2 and LEF1 transcript levels. Western blot analysis of total and phosphorylated levels of GSK3β and β-catenin showed that GSK3β inhibition resulted in stabilization of β-catenin. Xenografts of both BxPC3 and Panc1 with targeted silencing of GSK3β exhibited radioresistance in vivo. Silencing of β-catenin resulted in radiosensitization, whereas a nondegradable β-catenin construct induced radioresistance. CONCLUSIONS: These data support the hypothesis that GSK3β modulates the cellular response to radiation in a β-catenin-dependent mechanism. Further understanding of this pathway may enhance the development of clinical trials combining drugs inhibiting β-catenin activation with radiation and chemotherapy in locally advanced pancreatic cancer.

Abstract 3180: Altered β-catenin phosphorylation pattern in human prostate cancer

Abstract Protein kinase D1 (PKD1) phosphorylates β-catenin at Thr112 and Thr120. The phosphorylation is required for β-catenin membrane trafficking via trans Golgi network (TGN). Data presented in this study indicate that activation of PKD1 shrinks the cytoplasmic pool of β-catenin which mainly is the non-phosphorylation (S33/S37/T41/S45) isoform of β-catenin, suggesting that PKD1 interacts with Wnt/GSK3β signaling pathway. The phosphorylated Thr120 β-catenin (pT120) isoform predominately locates at membrane fraction in cultured prostate cancer cell line C4-2. A newly developed phospho-threonine antibody specifically targeting the pT120 of β-catenin shows accumulation of pT120 β-catenin in TGN in normal prostate tissues. Our study demonstrates that distribution of Thr120 phosphorylated β-catenin is distinct in normal and malignant prostate tissues. In 16/22 (72%) of normal prostate tissues, the pT120 β-catenin is enriched at TGN. In contrast, only 15/200 (7.5%; p &amp;lt; 0.001) of prostate cancer samples had accumulation of pT120 β-catenin in TGN. A vast majority of cancer samples (92.5%) lacking of TGN staining shows the pT120 β-catenin distributed diffusely in cytoplasm, nucleus and plasma membrane. can be more discriminatory compared to conventional β-catenin antibody staining. We propose that β-catenin T120 phosphospecific antibody can be used to study alteration in subcellular distribution of β-catenin and possibly as biomarker in prostate and other tissues, which may facilitate risk stratification or predict disease outcomes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3180.

Proteins from the Wnt pathway are involved in the pathogenesis and progression of mammary phyllodes tumours

Background: The Wnt pathway is important in cell signalling transduction and is involved in the pathogenesis of multiple tumour types. A comprehensive analysis of the expression of Wnt signalling pathway...

The influence of fasudil on the epithelial-mesenchymal transdifferentiation of renal tubular epithelial cells from diabetic rats

To investigate the influence of fasudil on the epithelial-mesenchymal transdifferentiation of renal tubular epithelial cells from diabetic rats and explore the mechanisms of this effect. Wistar rats were randomly divided into the following three groups: control, diabetes and fasudil-treatment. All rats were sacrificed after three months of feeding with or without fasudil treatment. Pathological changes to the glomeruli and renal interstitium were studied using Periodic acid-Schiff's staining and Masson staining, respectively. Expression of ROCK1, α-SMA, E-cadherin and the distribution of β-catenin in rat renal cortex were revealed by immunohistochemistry. Changes in the MYPT1 phosphorylation profile and α-SMA, E-cadherin and membrane β-catenin expression were revealed by western blot. Changes in the levels of ROCK1, E-cadherin and total β-catenin mRNA expression were analyzed by real-time PCR. Fasudil treatment notably attenuates renal interstitial fibrosis in diabetic rats. Compared to the control rats, diabetic rats showed elevated phosphorylation of MYPT1, increased expression of ROCK1 and α-SMA, decreased expression of E-cadherin and membrane β-catenin, and increased expression of ROCK1 and total β-catenin mRNA, decreased expression of E-cadherin mRNA. Fasudil treatment of diabetic rats resulted in attenuated MYPT1 phosphorylation, decreased ROCK1 and α-SMA expression, increased E-cadherin and membrane β-catenin expression, and reduced ROCK1 and total β-catenin mRNA expression, increased expression of E-cadherin mRNA. In conclusion, fasudil may reduce the epithelial-mesenchymal transdifferentiation and renal interstitial fibrosis in diabetic rats through a mechanism by which ROCK activity is inhibited, which further facilitates the recovery of the cell-cell adhesions among renal tubular epithelial cells and adhesion complex formation.

Cyclophilin C-associated protein (CyCAP) knock-out mice spontaneously develop colonic mucosal hyperplasia and exaggerated tumorigenesis after treatment with carcinogen azoxymethane1

BackgroundThe discovery of a "serrated neoplasia pathway" has highlighted the role of hyperplastic lesions of the colon as the significant precursor of colorectal adenocarcinoma. In mice, hyperplasia of the colonic mucosa is a regular phenomenon after a challenge with colonic carcinogens indicating that mucosal hyperproliferation and thickening, even without cytological dysplasia, represents an early pre-malignant change. Cyclophilin C-associated protein (CyCAP) has been described to down-modulate endotoxin signaling in colorectal murine mucosa and is a murine orthologue of the tumor-associated antigen 90 K (TAA90K)/mac-2-binding protein.MethodsFemale Balb/c wild-type (WT) and CyCAP knock-out (KO) mice (6–8 weeks old) were administered 2 or 6 weekly subcutaneous injections of azoxymethane. The animals were evaluated post-injection at six weeks for aberrant crypt foci (ACF) study and at five months for colon tumor measurement. The thickness of the colon crypts was measured in microns and the number of colonocytes per crypt was also determined in well-oriented crypts. Morphometric analyses of the colon mucosa were also performed in untreated 6–8 weeks old KO and WT animals. Formalin-fixed/paraffin-embedded colon sections were also studied by immunohistochemistry to determine the Ki-67 proliferation fraction of the colon mucosa, β-catenin cellular localization, cyclin D1, c-myc, and lysozyme in Paneth cells.ResultsCyclophilin C-associated protein (CyCAP)-/- mice, spontaneously developed colonic mucosal hyperplasia early in life compared to wild-type mice (WT) (p < 0.0001, T-test) and crypts of colonic mucosa of the (CyCAP)-/- mice show higher proliferation rate (p = 0.039, Mann-Whitney Test) and larger number of cyclin D1-positive cells (p < 0.0001, Mann-Whitney Test). Proliferation fraction and cyclin D1 expression showed positive linear association (p = 0.019, Linear-by-Linear Association). The hyperplasia was even more pronounced in CyCAP-/- mice than in WT after challenge with azoxymethane (p = 0.005, T-test). The length of the crypts (r = 0.723, p = 0.018, Spearman Correlation) and the number of colonocytes per crypt (r = 0.863, p = 0.001, Spearman Correlation) in non-tumorous areas were positively associated with azoxymethane-induced number of tumors. CyCAP-/- developed larger numbers of tumors than WT animals (p = 0.003, T-Test) as well as overall larger tumor mass (p = 0.016, T-Test). Membranous β-catenin was focally overexpressed in KO mice including proliferative zone of the crypts.ConclusionCyCAP-/- represent the first described model of spontaneous colonic mucosal hyperplasia. We conclude that CyCAP-deficient mice spontaneously and after challenge with carcinogen develop significantly more colorectal mucosal hyperplasia, an early stage in murine colonic carcinogenesis.

β-Catenin expression in uterine sarcomas and its relation to clinicopathological parameters

Aberrations in the Wnt/β-catenin signalling pathway are suggested as mediators of chromosomal instability and carcinogenesis. β-catenin acts both as a component of the membranous adhesion system, and as a transcription activator in the nucleus. β-Catenin immunoreactivity was evaluated in 353 uterine sarcomas (US) including 231 leiomyosarcomas (LMS), 82 endometrial stromal sarcomas (ESS), 22 adenosarcomas (AS) and 18 undifferentiated uterine sarcomas (UUS). Up-regulated membranous β-catenin was observed in 25% of the LMS ( p = 0.039), 21% of the ESS ( p = 0.072) and 39% of the UUS ( p = 0.025). Cytoplasmic β-catenin was up-regulated in 36% of the LMS ( p = 0.008) and 33% of the UUS ( p = 0.028). Nuclear β-catenin expression was observed in 23% of the LMS ( p = 0.051), 61% of ESS ( p = 0.628) and in the sarcoma component of 68% of the AS. In patients with LMS, membranous β-catenin was associated with poor crude survival in univariate ( p = 0.045), but not in multivariate analyses. In patients with ESS, nuclear β-catenin expression was related to spread of tumour ( p = 0.033), but not to survival. The observation of up-regulated β-catenin expression in US might suggest a so far undocumented role for the Wnt/β-catenin pathway in these malignancies.

Expression of Notch-1 and alteration of the E-cadherin/β-catenin cell adhesion complex are observed in primary cutaneous neuroendocrine carcinoma (Merkel cell carcinoma)

Increasing evidence indicates that Notch signaling contributes to physiological processes, including development and differentiation, as well as tumorigenesis, either as a tumor promoter or suppressor, depending on cellular context, expression levels and cross talk with other signaling systems. Recent studies reported absent or minimal Notch-1 expression in neuroendocrine tumors of the lung and gastrointestinal tract, suggesting a tumor-suppressor function of Notch-1. Merkel cell carcinoma is a rare and highly aggressive primary cutaneous neuroendocrine carcinoma. Because no information is available on Notch-1 expression in this tumor, we have investigated a series of 31 Merkel cell carcinoma for Notch-1 immunoreactivity. Immunoreactivities for E-cadherin and β-catenin were also analyzed. All but 1 Merkel cell carcinoma (30 of 31) retained cytoplasmic and membrane Notch-1 expression in more than 50% of cells. β-Catenin displayed a prevalent membrane-associated staining in 30 of 31 cases, and 22 cases showed more than 50% of immunoreactive cells whereas nuclear β-catenin was seen only in 2 of 31 cases. E-cadherin membranous expression was remarkably low, as only 1 of 26 cases was found positive in more than 50% of cells. In contrast with neuroendocrine tumors in other tissues, evident Notch-1 expression was found in Merkel cell carcinoma. This finding does not support a tumor-suppressor function of Notch-1 in Merkel cell carcinoma. Downregulation of E-cadherin and diffuse membranous β-catenin expression suggest a dysregulation of the E-cadherin/β-catenin complex in Merkel cell carcinoma. This may contribute to local invasion and distant metastasis.

Expression of cell cycle regulatory proteins in endometrial adenocarcinoma: variations in conventional tumor areas and in microcystic, elongated and fragmented glands

Endometrial adenocarcinomas may show a distinctive pattern of invasion characterized by the presence of microcystic, elongated and fragmented glands, often most evident along the advancing tumor margin. Earlier, we have shown that these changes appear restricted to low-grade endometrioid carcinomas, many of which show focal mucinous differentiation and lymphovascular space invasion. However, the molecular alterations associated with this morphological alteration are not known. In this study, we have examined immunoreactivity for the cell cycle regulatory proteins cyclin D1, p16 and β-catenin in 22 endometrial carcinomas, specifically comparing the results in conventional tumor areas and in foci in which the glands exhibited microcystic, elongated and fragmented appearances. The conventional neoplastic glands exhibited cyclin D1 and p16 expression in most cases, with >50% tumor cells positive in 8 cases and 11 tumors, respectively. Membranous expression of β-catenin was usually preserved, with variable cytoplasmic and nuclear staining. Cyclin D1 and β-catenin predominantly stained cells at the peripheral or basal aspect of the conventional glands, whereas p16 was more uniformly expressed centrally. Tumor foci composed of microcystic, fragmented and elongated glands showed strong expression of cyclin D1 and p16, sometimes in contrast to unstained contiguous or adjacent conventional neoplastic elements, and there was also loss or fragmentation of membranous β-catenin staining. Intravascular tumor cells also expressed cyclin D1 and p16 and therefore the immunostains often highlighted subtle foci of lymphovascular invasion. The heterogenous expression of cell cycle regulatory proteins within endometrial adenocarcinoma illustrates the importance of assessing microanatomical variations in immunoreactivity, particularly at the advancing margin of tumors. The upregulation of cyclin D1 and p16, together with loss of membranous β-catenin expression in microcystic, fragmented and elongated glands, is similar to epithelial–mesenchymal transitions observed in other malignancies and suggests that this pattern of invasion represents an active rather than a degenerative cellular process.

β-catenin expression:the role in carcinogenesis and development of human breast cancer

Objective To explore the role of β-catenin expression in breast carcinogenesis. Methods β-cateninin expression of the breast diseases was detected by immunohistochcmical staining. A-mong them, there were cases with intraductal papilloma n=25, DBCH (disease of the breast cystic hyper-plasia) n = 27, DBCH with ADH(atypical ductal hyperplasia) n=26, DCIS(ductal carcnoma in situ) n=29 and IDC(invasive ductal cancinoma)n=58. Results The positive rates of β-catcnin expression in in-traductal papilloma,DBCH, DBCH with ADH, DCIS, and IDC were 4.0%, 3.7%, 23.1%, 72.4% and 79.3%, respectively. The expression of simple membranous β-catenin was statistically related with tumor size, lymph node metastasis and TNM staging (P<0.05), the expression rate was higher in patients with smaller size, no lymph node metastasis and stage Ⅰ or Ⅱ, but not with tumor grading and age of the pa-tient. The co-expression of membranous and cytoplasmic β-catenin was significantly correlated with lymph node metastasis, tbe rate rised higherly only in patients with lymph node metastasis (P<0.05). Further-more, the possibility of lymph node metastasis was higher in membranous and cytoplasmic β-catenin co-ex-pression group than in membranous group. Conclusion Positive expression of β-catcnin occurred early and continued to exist during neoplastic transformation of mammary cells;the co-expression of membranous and cytoplasmic β-catenin was significantly related with lymph node metastasis, which implied they might be a risk factor of poor prognosis, whereas the expression of membranous β-catenin only was negtively cor-related with tumor size,lymph node metastasis, and TNM staging,it might be an indicator of good progno-sis. Key words: β-catenin; Breast cancer

Lingual cyst with respiratory epithelium: a histopathological and immunohistochemical analysis of two cases

Cysts of the tongue are rare, usually derived from epithelia of the embryonic gastrointestinal and respiratory tracts, and classified according to the predominant epithelium lining. These cysts are usually discovered during infancy, more frequently in males, but they may not appear until well into adulthood. The authors report two lingual cysts lined mainly with respiratory, and focally by squamous, epithelium. Periodic acid-Schiff and mucicarmine staining revealed focal positivity in intracystic mucoid material and goblet cells. Immunohistochemical analysis with vimentin, cytokeratins (AE1/AE3, 34βE12, CK1, CK5, CK6, CK7, CK8, CK10, CK13, CK14, CK16, CK18, and CK19), E-cadherin, β-catenin, and epithelial membrane antigen showed a similar profile of normal respiratory epithelium, suggesting well-differentiated states. Owing to their controversial origin, these cysts should be named descriptively, as suggested by Manor et al., as lingual cysts with respiratory epithelium.

Proteins from the wnt pathway are involved in the pathogenesis and progression of mammary phyllodes tumours

Background Alterations in the Wnt pathway may be involved in the pathogenesis of mammary phyllodes tumours (PTs) and be associated with prognosis. Methods 65 PTs (34 benign, 23 borderline and 8 malignant) from 62 patients diagnosed at a single institution between 1990 and 2006 were analysed. The PTs were alternatively subdivided into two grades: all benign PTs were classified as low grade, and borderline and malignant tumours were combined into a high grade group. Immunohistochemical stains were performed on tissue microarrays for β-catenin, Wnt1, Wnt5a, SFRP4 and E-cadherin. Stroma and epithelium were scored separately. Results Stromal cytoplasmic Wnt5a and SFRP4 expression showed significant progressive increases in expression with increasing grade on both the two and three tiered grading schemes (Wnt 5a p = 0.002 and 0.002;SFRP4 p = 0.02 and 0.03, respectively). Epithelial membranous and stromal nuclear β-catenin, epithelial cytoplasmic Wnt1 and epithelial E-cadherin all had a similar pattern of expression but the differences were not significant. Disease-free survival was significantly decreased (p = 0.0017) with positive epithelial E-cadherin staining. Conclusion The results suggest that alterations in the Wnt pathway are important in the progression and in the epithelial and stromal interactions in PTs. Alterations in the Wnt pathway may be involved in the pathogenesis of mammary phyllodes tumours (PTs) and be associated with prognosis. 65 PTs (34 benign, 23 borderline and 8 malignant) from 62 patients diagnosed at a single institution between 1990 and 2006 were analysed. The PTs were alternatively subdivided into two grades: all benign PTs were classified as low grade, and borderline and malignant tumours were combined into a high grade group. Immunohistochemical stains were performed on tissue microarrays for β-catenin, Wnt1, Wnt5a, SFRP4 and E-cadherin. Stroma and epithelium were scored separately. Stromal cytoplasmic Wnt5a and SFRP4 expression showed significant progressive increases in expression with increasing grade on both the two and three tiered grading schemes (Wnt 5a p = 0.002 and 0.002;SFRP4 p = 0.02 and 0.03, respectively). Epithelial membranous and stromal nuclear β-catenin, epithelial cytoplasmic Wnt1 and epithelial E-cadherin all had a similar pattern of expression but the differences were not significant. Disease-free survival was significantly decreased (p = 0.0017) with positive epithelial E-cadherin staining. The results suggest that alterations in the Wnt pathway are important in the progression and in the epithelial and stromal interactions in PTs.

β-catenin is involved in N-cadherin–dependent adhesion, but not in canonical Wnt signaling in E2A-PBX1–positive B acute lymphoblastic leukemia cells

The t(1;19)(q23;13) translocation, resulting in the production of the E2A-PBX1 chimeric protein, is a common nonrandom translocation in pediatric B-lineage acute lymphoblastic leukemia (B-ALL). The E2A-PBX1 chimeric protein activates expression of several genes, including Wnt16. In the present study, we explored the role of Wnt16 and beta-catenin in t(1;19) B-ALL cells. Canonical Wnt signaling was measured by TOPflash activity. Localization of beta-catenin in the cell membrane and its involvement in leukemia-stroma interaction were studied by confocal microscopy. Adhesion to N-cadherin was analyzed by adding (3)H-thymidin-labeled cells to N-cadherin-coated wells. In contrast to previous reports, we detected no effects on cell viability or proliferation upon modulation of the Wnt16 levels. Moreover, despite high levels of Wnt16 and beta-catenin, the cells had very low levels of canonical Wnt signaling. Instead, beta-catenin was located in the cell membrane along with N-cadherin. E2A-PBX1-positive leukemia cells adhered strongly to bone marrow stroma cells, and we showed that adherence junctions stained strongly for both proteins. Moreover, knockdown of beta-catenin reduced the adhesion of E2A-PBX1-positive leukemia cells to N-cadherin, suggesting that beta-catenin and N-cadherin play a central role in homotypic cell-to-cell adhesion and in leukemia-stroma adhesion. Interestingly, knockdown of Wnt16 by small interfering RNA reduced the level of N-cadherin. Wnt16 does not activate canonical Wnt signaling in E2A-PBX1-positive cells. Instead, beta-catenin is involved in N-cadherin-dependent adherence junctions, suggesting for the first time that leukemia-stroma interactions may be mediated via an N-cadherin-dependent mechanism.

Overexpression of fatty acid synthase is associated with palmitoylation of Wnt1 and cytoplasmic stabilization of β-catenin in prostate cancer

Fatty acid synthase (FASN), a key metabolic enzyme for liponeogenesis highly expressed in several human cancers, displays oncogenic properties such as resistance to apoptosis and induction of proliferation when overexpressed. To date, no mechanism has been identified to explain the oncogenicity of FASN in prostate cancer. We generated immortalized prostate epithelial cells (iPrECs) overexpressing FASN, and found that 14C-acetate incorporation into palmitate synthesized de novo by FASN was significantly elevated in immunoprecipitated Wnt-1 when compared to isogenic cells not overexpressing FASN. Overexpression of FASN caused membranous and cytoplasmic β-catenin protein accumulation and activation, whereas FASN knockdown by short-hairpin RNA resulted in a reduction in the extent of β-catenin activation. Orthotopic transplantation of iPrECs overexpressing FASN in nude mice resulted in invasive tumors that overexpressed β-catenin. A strong significant association between FASN and cytoplasmic (stabilized) β-catenin immunostaining was found in 862 cases of human prostate cancer after computerized subtraction of the membranous β-catenin signal (P<0.001, Spearman's ρ=0.33). We propose that cytoplasmic stabilization of β-catenin through palmitoylation of Wnt-1 and subsequent activation of the pathway is a potential mechanism of FASN oncogenicity in prostate cancer.

Regulation of β-catenin trafficking to the membrane in living cells

β-catenin is a key mediator of the Wnt signaling process and accumulates in the nucleus and at the membrane in response to Wnt-mediated inhibition of GSK-3β. In this study we used live cell photobleaching experiments to determine the dynamics and rate of recruitment of β-catenin at membrane adherens junctions (cell adhesion) and membrane ruffles (cell migration). First, we confirmed the nuclear-cytoplasmic shuttling of GFP-tagged β-catenin, and found that a small mobile pool of β-catenin can move from the nucleus to membrane ruffles in NIH 3T3 fibroblasts with a t 0.5 of ~ 30 s. Thus, β-catenin can shuttle between the nucleus and plasma membrane. The localized recruitment of β-catenin-GFP to membrane ruffles was more rapid, and the strong recovery observed after bleaching (mobile fraction 53%, t 0. 5 ~5 s) is indicative of high turnover and transient association. In contrast, β-catenin-GFP displayed poor recovery at adherens junctions in MDCK epithelial cells (mobile fraction 10%, t 0. 5 ~8 s), indicating stable retention at these membrane structures. We previously identified IQGAP1 as an upstream regulator of β-catenin at the membrane, and this is supported by photobleaching assays which now reveal IQGAP1 to be more stably anchored at membrane ruffles than β-catenin. Further analysis showed that LiCl-mediated inactivation of the kinase GSK-3β increased β-catenin membrane ruffle staining; this correlated with a faster rate of recruitment and not increased membrane retention of β-catenin. In summary, β-catenin displays a high turnover rate at membrane ruffles consistent with its dynamic internalization and recycling at these sites by macropinocytosis.

Calpastatin overexpression attenuates amyloid-β-peptide toxicity in differentiated PC12 cells

Amyloid β peptide (Aβ) plays a major role in the pathogenesis of Alzheimer's disease (AD). Aβ is toxic to neurons, possibly through causing initial synaptic dysfunction and neuronal membrane dystrophy, promoted by increased cellular Ca 2+. Calpain (Ca 2+-dependent protease) and caspase have been implicated in AD. Previously, we used calpain and caspase pharmacological inhibitors to study effects of Aβ25–35 (sAβ) on neuronal-like differentiated PC12 cells. We reported that sAβ-treated cells exhibited calpain activation and protein degradation (due to both calpain and caspase-8). We have now found that overexpression of the calpain specific inhibitor calpastatin in differentiated PC12 cells significantly inhibited the sAβ-induced calpain activation and decreased the protease activity. Calpastatin overexpression inhibited the sAβ-promoted degradation of fodrin, protein kinase Cε, β-catenin (membrane structural proteins and proteins involved in signal transduction pathways), and prevented the sAβ-induced alteration of neurite structure (manifested by varicosities). Overexpression of calpastatin also inhibited Ca 2+-promoted calpain activation and protein degradation; this is consistent with the notion that the Aβ-induced increase in calpain activity results from a rise in cellular Ca 2+, provided the calpastatin level is not so high as to strongly inhibit calpain. Carrying out transfection without selection allowed the comparison in the same culture of calpastatin-overexpressing with non-overexpressing cells. In cultures transfected with green fluorescent protein (GFP)–calpastatin plasmid, calpastatin overexpression (indicated by GFP-labeling) led to inhibition in sAβ-induced membrane propidium iodide (PI) permeability, whereas non-transfected, GFP-unlabeled cells exhibited PI permeability. Overall, the results demonstrate that the effects of Aβ-toxicity studied here were attenuated to a large extent by calpastatin overexpression, indicating that the protease calpain is involved in Aβ-toxicity (obviating a primary, direct role for caspases). Increased expression of calpastatin and/or decrease in calpain may serve as one of the means for ameliorating some of the early symptoms of AD.