Adherence to osteopontin via alphavbeta3 suppresses phorbol ester-mediated apoptosis in MCF-7 breast cancer cells that overexpress protein kinase C-alpha

Overexpression of protein kinase C-alpha in MCF-7 breast cancer cells (MCF-7-PKC-alpha cells) results in anchorage-independent growth and increased tumorigenicity of these cells in nude mice. MCF-7-PKC-alpha cells, unlike their parental MCF-7 cells, are sensitized to apoptosis by phorbol esters. When adhered to osteopontin, a bone matrix protein, MCF-7-PKC-alpha cells were resistant to phorbol ester mediated apoptosis. Fluorescence-activated cell sorting revealed that osteopontin receptors, alphavbeta3 and alphavbeta5, are expressed on MCF-7-PKC-alpha cells and that both are used to adhere to osteopontin. Addition of an RGD-containing peptide inhibited survival of MCF-7-PKC-alpha cells exposed to phorbol ester and adhered to osteopontin. This indicated that an integrin was involved in the cell death suppression signal. Whereas, anti-alphavbeta5 antibody did not reduce survival of MCF-7-PKC-alpha cells adhered to osteopontin, anti-alphavbeta3 antibody could efficiently block suppression of apoptosis. Phorbol ester also induced increased expression of alphavbeta3 on MCF-7-PKC-alpha cells by upregulating expression of a second species of beta3 mRNA. This study suggests that breast cancer cells that have metastasized to bone may have a survival advantage resulting from interaction of alphavbeta3 on these cells with the bone protein osteopontin.     

Integrin alpha 5 beta 1 suppresses apoptosis triggered by serum starvation but not phorbol ester in MCF-7 breast cancer cells that overexpress protein kinase C-alpha

MCF-7 breast cancer cells grow as adherent cells, but following overexpression of protein kinase C-alpha these cells (MCF-7-PKC-alpha cells) become anchorage-independent and exhibit increased tumorigenicity in nude mice. MCF-7-PKC-alpha cells are also sensitized to apoptosis in response to phorbol ester but not serum starvation. Flourescence-activated cell sorting revealed that several integrin subunits were down-regulated in MCF-7-PKC-alpha cells, however, the fibronectin receptor alpha 5 beta 1 was upregulated. MCF-7-PKC-alpha cells growing under non-adherent conditions underwent cell death when antibodies to alpha 5 beta 1 were added to growth media lacking serum but not when serum was present. Addition of soluble fibronectin to cells incubated without serum suppressed apoptosis triggered by anti-alpha 5 beta 1 antibodies but not by phorbol esters. MCF-7-PKC-alpha cells also were shown to express more fibronectin on their cell surface than MCF-7V cells (MCF-7 cells transfected with pSV(2)M(2)6 vector only). This study indicates that the survival of MCF-7-PKC-alpha cells under non-adherent conditions in the absence of serum results from the ligation of alpha 5 beta 1 with surface-bound fibronectin, which may account, in part, for the increased aggressiveness of these cells.     

Adriamycin activates E-cadherin-mediated cell-cell adhesion in human breast cancer cells

E-cadherin-mediated cell-cell adhesion plays a crucial role in intercellular communication, which is related to the regulation of cell proliferation, differentiation, and apoptosis. Our previous study showed that decreased expression of MUC1 can induce E-cadherin-mediated cell-cell adhesion in human breast cancer cell lines proliferating in suspension without aggregation. Using such a cell line (YMB-S), we observed the effects of an anticancer agent, adriamycin, on cell-cell adhesion and expression of E-cadherin-catenin complex and MUC1. The cells showed E-cadherin-mediated cell-cell adhesion after 48 h exposure to 0.4 micromol/l adriamycin. And in these cells, expression of E-cadherin and beta-catenin mRNA obviously began to increase, while expression of MUC1 mRNA decreased, as demonstrated by Northern blot analysis. Such change in mRNA levels were followed by increases in E-cadherin and beta-catenin protein levels and a decrease in MUC1 protein level. Though expression of alpha-catenin mRNA began to increase on day 2, its protein level did not change. In immunohistochemical analysis, beta-catenin protein in untreated cells showed diffuse cytoplasmic localization, whereas beta-catenin in treated cells was present in cytoplasm with a clear submembranous localization, indicating that increased beta-catenin mainly bound with E-cadherin, participating in cell-cell adhesion. These findings show for the first time that adriamycin can induce E-cadherin-mediated cell-cell adhesion by increasing expression of E-cadherin and beta-catenin and decreasing expression of MUC1 during breast cancer cell apoptosis induced by this drug.     

Wnt-5a has tumor suppressor activity in thyroid carcinoma

Stabilization of beta-catenin by inhibition of its phosphorylation is characteristic of an activation of the canonical Wnt/beta-catenin signaling pathway and is associated with various human carcinomas. It contrasts to an as yet incompletely characterized action of an alternative noncanonical Wnt signaling pathway on neoplastic transformation. The aim of the present study was to test the effects of a member of the noncanonical Wnt signaling pathway, Wnt-5a, in primary thyroid carcinomas and in thyroid carcinoma cell lines. Compared to normal tissue Wnt-5a mRNA expression was clearly increased in thyroid carcinomas. Immunohistochemically, a bell-shaped response was observed with low to undetectable levels in normal tissue and in anaplastic tumors whereas differentiated thyroid carcinomas showed strong positive immunostaining for Wnt-5a. Transfection of Wnt-5a in a thyroid tumor cell line FTC-133 was able to reduce proliferation, migration, invasiveness and clonogenicity in these cells. These effects of Wnt-5a are associated with membranous beta-catenin translocation and c-myc oncogene suppression and are mediated through an increase in intracellular Ca(2+) release, which via CaMKII pathways promotes beta-catenin phosphorylation. Specific inhibition of beta-catenin phosphorylation by W-7, a calmodulin inhibitor, or by KN-93, a CaMKII inhibitor, supports these findings whereas PKC inhibitors were without effect. This interaction occurs downstream of GSK-3 beta as no Wnt-5a effect was seen on the Ser(9) phosphorylation of GSK-3 beta. Our data are compatible with the hypothesis that Wnt-5a serves as an antagonist to the canonical Wnt-signaling pathway with tumor suppressor activity in differentiated thyroid carcinomas.     

Restoration of E-cadherin-based cell-cell adhesion by overexpression of nectin in HSC-39 cells,a human signet ring cell gastric cancer cell line

Nectin is an immunoglobulin-like adhesion molecule that comprises a family consisting of four members, nectin-1, -2, -3, and -4. Nectin is associated with the actin cytoskeleton through afadin, a nectin- and actin filament-binding protein. The nectin-afadin and cadherin-catenin systems are associated with each other and cooperatively form cell-cell adherens junctions in intact epithelial cells. HSC-39 cells, a human signet ring cell gastric cancer cell line, express E-cadherin but do not form cell-cell adhesion. The beta-catenin gene has been shown to be truncated at the N-terminal region including the alpha-catenin-binding domain in HSC-39 cells, but overexpression of normal beta-catenin failed to form cell-cell adhesion. HSC-39 cells expressed nectin-1, -2, and afadin, but not nectin-3. Overexpression of nectin-3 or -2 formed cell-cell adhesion and accumulation of E-cadherin, but not actin filaments, at the cell-cell adhesion sites. Overexpression of a truncated form of nectin-2 incapable of interacting with afadin failed to form cell-cell adhesion. However, the nectin-formed cell-cell adhesion was not so strong as that observed in epithelial cells, such as CaCo-2 cells. Co-expression of nectin-2 and normal beta-catenin did not form strong cell-cell adhesion. These results suggest that an unidentified mechanism, by which nectin and E-cadherin form the actin cytoskeleton-associated adherens junctions to form strong cell-cell adhesion, is impaired in HSC-39 cells.     

Expression of endothelial nitric oxide synthase is induced by estrogen with glycogen synthase 3beta phosphorylation in MCF-7 cells

The endothelial nitric oxide synthase (eNOS) gene is induced by a variety of extracellular signals and NOS plays a key role in many physiological as well as pathological processes, including tumorgenesis. Some studies showed a positive correlation between the level of NOS protein and progression of malignancy in human breast cancer. In this study, we examined eNOS mRNA expression in human breast cancer cell lines. MCF-7 cells, which showed an estrogen receptor positive phenotype, were treated with estradiol or LiCl, a selective inhibitor of glycogen synthase kinase (GSK)-3beta. Both estradiol and LiCl enhanced the expression of eNOS mRNA with the phosphorylation of GSK-3beta, but not Akt. The induction was completely suppressed by the phosphatidylinositol 3-kinase (PI3-kinase) inhibitor LY294002, but not by PD98059, MEK-1 inhibitor nor rapamycin, p70S6 kinase inhibitor. We conclude that the estradiol-induced eNOS expression is modulated by PI3-kinase-dependent GSK-3beta pathway.     

Dominant-negative E-cadherin alters adhesion and reverses contact inhibition of growth in breast carcinoma cells

Cadherins play a crucial role in epithelial morphogenesis and mediate intercellular adhesion. These receptors bind catenins and are involved in signal transduction pathways that regulate cell growth and apoptosis, and are frequently down-regulated in invasive and metastatic carcinomas. In order to assess the role of E-cadherin in cell adhesion and growth, we transfected MCF-7 cells, a human breast cancer cell line, with a dominant-negative construct of E-cadherin (H-2kd-E-cad). The dominant-negative form of E-cadherin disrupted cell-cell adhesion of monolayer cells and induced an epithelial-to-fibroblastic conversion without any significant change in integrin profiles. Whereas control cells rapidly formed multicellular aggregates that tightly compacted into spheroids, dominant-negative transfected cells failed to compact and remained as loosely-associated cells. The transfectants exhibited down-regulation and redistribution of endogenous E-cadherin as well as increased levels of alpha- and beta-catenin. Importantly, the H-2kd-E-cad-transfected cells, when grown as multicellular aggregates, showed an increase in cell proliferation rate, compared to control cells. Overall, these observations suggest that in breast carcinoma, disruption of E-cadherin and catenin function modulates both cell-cell adhesion and permits escape from cell-cell contact-involved inhibition of cell growth.     

Evidences that leptin up-regulates E-cadherin expression in breast cancer: effects on tumor growth and progression

Leptin, a cytokine mainly produced by adipocytes, seems to play a crucial role in mammary carcinogenesis. In the present study, we explored the mechanism of leptin-mediated promotion of breast tumor growth using xenograft MCF-7 in 45-day-old female nude mice, and an in vitro model represented by MCF-7 three-dimensional cultures. Xenograft tumors, obtained only in animals with estradiol (E(2)) pellet implants, doubled control value after 13 weeks of leptin exposure. In three-dimensional cultures, leptin and/or E(2) enhanced cell-cell adhesion. This increased aggregation seems to be dependent on E-cadherin because it was completely abrogated in the presence of function-blocking E-cadherin antibody or EGTA, a calcium-chelating agent. In three-dimensional cultures, leptin and/or E(2) treatment significantly increased cell growth, which was abrogated when E-cadherin function was blocked. These findings well correlated with an increase of mRNA and protein content of E-cadherin in three-dimensional cultures and in xenografts. In MCF-7 cells both hormones were able to activate E-cadherin promoter. Mutagenesis studies, electrophoretic mobility shift assay, and chromatin immunoprecipitation assays revealed that cyclic AMP-responsive element binding protein and Sp1 motifs, present on E-cadherin promoter, were important for the up-regulatory effects induced by both hormones on E-cadherin expression in breast cancer MCF-7 cells. In conclusion, the present study shows how leptin is able to promote tumor cell proliferation and homotypic tumor cell adhesion via an increase of E-cadherin expression. This combined effect may give reasonable emphasis to the important role of this cytokine in stimulating primary breast tumor cell growth and progression, particularly in obese women.     

The interaction between beta-catenin, GSK3beta and APC after motogen induced cell-cell dissociation, and their involvement in signal transduction pathways in prostate cancer

The effect of HGF/SF was examined on the interactions between APC, GSK3beta and beta-catenin in prostate cancer cells LNCapFGC (E-cadherin positive) and PC-3 (E-cadherin negative). Using immunoprecipitation, APC was found to be co-precipitated with either GSK3beta or beta-catenin in both cell lines. Stimulation with HGF/SF showed no change in the co-precipitation status of these protein molecules. In contrast, co-precipitation between GSK3beta and beta-catenin was only observed in LNCapFGC cells, and increased upon continued exposure to the motogen HGF/SF. Furthermore, using immunofluorescence, stimulation with HGF/SF was found to increase the level of co-localised cytoplasmic staining between beta-catenin and GSK3beta, in prostate cancer cells. RT-PCR revealed that there were no mutations within the binding regions between beta-catenin and GSK3beta. It is concluded, that uncomplexed cytoplasmic pools of beta-catenin associate more readily with the Axin complex in the absence of E-cadherin. Whereas, in the presence of E-cadherin, beta-catenin is stabilised by forming tight cell-cell contacts which may influence the invasive potential of cancer cells.     

Role of Wnt pathway in medulloblastoma oncogenesis

To clarify the roles of Wnt pathway in medulloblastoma oncogenesis, immunohistochemical staining of beta-catenin and Wnt-1 and genomic analyses of CTNNB1 (beta-catenin) and AXIN1 (axin 1) were examined in 23 sporadic cases. Accumulation of beta-catenin in tumor cells was immunohistochemically proven in 5 cases; 2 cases showed positive immunoreactivity for Wnt-1 and another 2 showed mutation of either CTNNB1 or AXIN1. AXIN1 mutation was in exon 3, corresponding to GSK-3beta binding site and CTNNB1 mutation was in exon 3, corresponding to its phosphorylation site. Disruption of these proteins could result in upregulation of the Wnt signaling and accumulation of beta-catenin, followed by cell proliferation and medulloblastoma oncogenesis.     

Alteration of beta-catenin expression in esophageal squamous-cell carcinoma

beta-catenin regulates cadherin-mediated cell-cell adhesion and also functions as a signaling molecule. In this study, we examined the expression pattern of E-cadherin, alpha-catenin and beta-catenin in 22 cases of esophageal squamous-cell carcinoma by Western-blot analysis. Expression of E-cadherin, alpha-catenin and beta-catenin was lower in carcinomas than in normal esophageal mucosa in 4 cases (18.2%) for E-cadherin, 6 cases (27.3%) for alpha-catenin and 9 cases (40.9%) for beta-catenin. Expression of beta-catenin was not always correlated with that of E-cadherin. Over-expression of beta-catenin was observed in 3 cases (13.6%). Of 3 cases that presented with over-expression of beta-catenin, 2 showed cytoplasmic staining by immunohistochemistry. Nuclear localization of beta-catenin was observed in one case that had higher beta-catenin level in tumor tissue (1.4-fold higher than normal mucosa). The genomic DNA sequences of the beta-catenin and the APC gene were analyzed. No mutation of the beta-catenin gene was observed in any cases. Silent mutation of the APC gene was found in all the cases that showed over-expression or nuclear localization of the beta-catenin protein. These results indicate that alterations of the cadherin-catenin complex may play an important role in a sub-set of esophageal carcinogenesis. Furthermore, it is suggested that beta-catenin over-expression is not caused by genetic alteration of either the beta-catenin or the APC gene.