Dysfunction of cadherin cell adhesion system in cancer invasion and metastasis

Dysfunction of the cadherin-mediated cell adhesion system involved in cancer metastasis occurs by several mechanisms: alterations of E-cadherin, alpha- and beta-catenin genes, CpG methylation of the promoter region of E-cadherin, and aberrant tyrosine phosphorylation of beta-catenin. In addition to the cell adhesion function, beta-catenin, which is an intracytoplasmic cadherin binding molecule and thought to be a regulator of cadherin-mediated cell adhesion function, has been proven to associate with both the growth factor receptors, including c-erbB-2, EGF receptor and k-sam and APC tumor suppressor gene product. These data indicate that the cadherin-mediated cell adhesion system plays important roles not only in cancer metastasis but in carcinogenesis.     

连环蛋白p120的转位与肝癌细胞恶性行为的关系

目的 探讨p120^ctn磷酸化转位与肝癌细胞恶性行为的关系,以及p120^ctn和β-连环蛋白在上皮钙黏蛋白介导细胞信号转导中的相互影响。方法用DNA转染技术、免疫印迹、免疫荧光显微镜、免疫沉淀等方法检测p1210^ctn的表达、转位以及酩氨酸磷酸化情况;同时检测细胞黏附和迁移能力以及增殖能力的改变。结果肝癌细胞经转染p120^ctn同功蛋白3A后,p120^ctn的表达增强,且主要表达于细胞与细胞连接处;当细胞经表皮生长因子(EGF)刺激后,p120^ctn酩氨酸发生磷酸化,其在细胞内的分布发生了改变,表现为胞膜表达减弱,胞浆表达增强,出现核表达(即转位);细胞黏附能力降低,而迁移能力增强。此外,过表达p120^ctn后,β-连环蛋白在细胞内的分布也发生了改变,表现为向核外转移,细胞增殖能力减弱。EGF刺激使细胞增殖能力增加。结论p121^ctn在肝癌细胞黏附、迁移和增殖等细胞生物学行为中起重要作用,其磷酸化转位可能与这些生物学行为改变有关;p120^ctn与β-连环蛋白在一定条件下存在某种竞争关系。     

Anti-sense suppression of epidermal growth factor receptor expression alters cellular proliferation, cell-adhesion and tumorigenicity in ovarian cancer cells

Over-expression of epidermal growth factor receptor (EGFR) in ovarian cancer has been well documented. Human NIH:OVCAR-8 ovarian carcinoma cells were transfected with an expression vector containing the anti-sense orientation of truncated human EGFR cDNA. EGFR anti-sense over-expression resulted in decreased EGFR protein and mRNA expression, cell proliferation and tumor formation in nude mice. In accordance with the reduced levels of EGFR in EGFR anti-sense-expressing cells, tyrosine phosphorylation of EGFR was decreased compared to untransfected parental cells treated with EGF. In EGFR anti-sense-transfected cells, expression of erbB-3, but not erbB-2, was increased. In addition, basal and heregulin-beta 1-stimulated tyrosine phosphorylation of erbB-3 was higher in EGFR anti-sense vector-transfected cells. A morphological alteration in EGFR anti-sense gene-expressing cells was correlated with a decrease in the expression of E-cadherin, alpha-catenin and, to a lesser extent, beta-catenin. Changes in the expression of these proteins were associated with a reduction in complex formation among E-cadherin, beta-catenin and alpha-catenin and between beta-catenin and EGFR in EGFR anti-sense-expressing cells compared to sense-transfected control cells. These results demonstrate that EGFR expression in ovarian carcinoma cells regulates expression of cell adhesion proteins that may enhance cell growth and invasiveness.     

Expression of e-cadherin, alpha-catenins and Beta-catenins in human gastric carcinomas - correlation with histology and tumor progression

The expression of cell-cell adhesion molecule, E-cadherin and its associated proteins, alpha- and beta-catenins in human gastric carcinomas was examined by Western blotting. All the seven gastric carcinoma cell lines expressed E-cadherin except KATOIII, which was derived from pleural effusion of a scirrhous type stomach cancer or Borrmann's type-4 carcinoma. The expression of alpha-catenin was not detected in HSC43 derived from scirrhous carcinoma, while HSC39 expressed abnormal beta-catenin caused by genetic alteration. In gastric carcinoma cases, the levels of E-cadherin and alpha-catenin were significantly lower in poorly differentiated adenocarcinomas and scirrhous carcinomas when compared to other types of gastric carcinomas. Deeply invasive carcinomas expressed E-cadherin and alpha-catenin at lower levels. However, the expression level of alpha-catenin was not necessarily consistent with that of E-cadherin. One of 10 gastric carcinomas examined showed complete deletion of alpha-catenin gene in Southern blotting. beta-catenin was expressed at lower level in poorly differentiated adenocarcinomas than in well-differentiated adenocarcinomas. These findings suggest that reduction in the expression of E-cadherin and its associated molecules, catenins, is involved in the development and infiltrative growth of scirrhous type gastric carcinomas.     

Carnosol inhibits beta-catenin tyrosine phosphorylation and prevents adenoma formation in the C57BL/6J/Min/+ (Min/+) mouse

Carnosol, a constituent of the herb, rosemary, has shown beneficial medicinal and antitumor effects. Using the C57BL/6J/Min/+ (Min/+) mouse, a model of colonic tumorigenesis, we found that dietary administration of 0.1% carnosol decreased intestinal tumor multiplicity by 46%. Previous studies showed that tumor formation in the Min/+ mouse was associated with alterations in the adherens junctions, including an increased expression of tyrosine-phosphorylated beta-catenin, dissociation of beta-catenin from E-cadherin, and strongly reduced amounts of E-cadherin located at lateral plasma membranes of histologically normal enterocytes. Here, we confirm these findings and show that treatment of Min/+ intestinal tissue with carnosol restored both E-cadherin and beta-catenin to these enterocyte membranes, yielding a phenotype similar to that of the Apc(+/+) wild-type (WT) littermate. Moreover, treatment of WT intestine with the phosphatase inhibitor, pervanadate, removed E-cadherin and beta-catenin from the lateral membranes of enterocytes, mimicking the appearance of the Min/+ tissue. Pretreatment of WT tissue with carnosol inhibited the pervanadate-inducible expression of tyrosine-phosphorylated beta-catenin. Thus, the Apc(Min) allele produces adhesion defects that involve up-regulated expression of tyrosine-phosphorylated proteins, including beta-catenin. Moreover, these data suggest that carnosol prevents Apc-associated intestinal tumorigenesis, potentially via its ability to enhance E-cadherin-mediated adhesion and suppress beta-catenin tyrosine phosphorylation.     

Csk defines the ability of integrin-mediated cell adhesion and migration in human colon cancer cells: implication for a potential role in cancer metastasis

Progression of human colon cancer is often associated with elevated expression and activity of the Src family tyrosine kinase (SFK). SFK is ordinarily in equilibrium between inactive and primed states by a balance of negative regulatory kinase Csk and its counteracting tyrosine phosphatase(s), both of which act on the regulatory C-terminal tyrosine of SFK. To evaluate the contribution of the regulatory system of SFK in cancer progression, we here modulated the equilibrium status of SFK by introducing wild-type or dominant-negative Csk in human epithelial colon cancer cells, HCT15 and HT29. Overexpression of wild-type Csk induced decreased SFK activation, increased cell-cell contacts mediated by E-cadherin, decreased the number of focal contacts and decreased cell adhesion/migration and in vitro invasiveness. Conversely, expression of a dominant-negative Csk resulted in elevated SFK activation, enhanced phosphorylation of FAK and paxilllin, enhanced cell scattering, an increased number of focal contacts, dramatic rearrangement of actin cytoskeleton and increased cell adhesion/migration and in vitro invasiveness. In these scattered cells, however, localization, expression and phosphorylation of either E-cadherin or beta-catenin were not significantly affected, suggesting that the E-cadherin-mediated cell-cell contact is indirectly regulated by SFK. Furthermore, all these events occurred absolutely dependent on integrin-mediated cell adhesion. These findings demonstrate that Csk defines the ability of integrin-SFK-mediated cell adhesion signaling that influences the metastatic potential of cancer cells.     

Collagen type I induces disruption of E-cadherin-mediated cell-cell contacts and promotes proliferation of pancreatic carcinoma cells

Pancreatic cancer is characterized by its invasiveness, early metastasis, and the production of large amounts of extracellular matrix (ECM). We analyzed the influence of type I collagen and fibronectin on the regulation of cellular adhesion in pancreatic cancer cell lines to characterize the role of ECM proteins in the development of pancreatic cancer. We show that collagen type I is able to initiate a disruption of the E-cadherin adhesion complex in pancreatic carcinoma cells. This is due to the increased tyrosine phosphorylation of the complex protein beta-catenin, which correlates with collagen type I-dependent activation of the focal adhesion kinase and its association with the E-cadherin complex. The activation and recruitment of focal adhesion kinase to the E-cadherin complex depends on the interaction of type I collagen with beta1-containing integrins and an integrin-mediated activation of the cellular kinase Src. The disassembly of the E-cadherin adhesion complex correlates with the nuclear translocation of beta-catenin, which leads to an increasing expression of the beta-catenin-Lef/Tcf target genes, cyclin D1 and c-myc. In addition to that, cells grown on collagen type I show enhanced cell proliferation. We show that components of the ECM, produced by the tumor, contribute to invasiveness and metastasis by reducing E-cadherin-mediated cell-cell adhesion and enhance proliferation in pancreatic tumor cells.     

Aberrant expression and phosphorylation of beta-catenin in human colorectal cancer.

The cytoplasmic domain of cadherins is known to associate with the intracellular proteins, catenins, which link cadherins to the actin-based cytoskeleton. In this study, we immunohistochemically investigated the expression of beta-catenin as well as E-cadherin and alpha-catenin in 86 human colorectal cancers, and we analysed their coexpression pattern and relationship to clinicopathological factors. In cancerous tissues, the frequency of reduced expression of beta-catenin (28 of 86, 33%) was similar to that of E-cadherin (19 of 86, 22%), but less than that of alpha-catenin (47 of 86, 55%). All three molecules were expressed strongly, as was the normal epithelium, in 36 cases (42%), whereas the rest (50 cases, 58%) showed reduction in one of the molecules. The reduction of beta-catenin expression was significantly correlated with dedifferentiation, Duke''s stage, lymph node metastasis and liver metastasis. Next, we examined tyrosine phosphorylation in the protein complex immunoprecipitated with E-cadherin, as E-cadherin function is down-regulated by receptor-type tyrosine kinase in vitro. It was of interest that up-regulation of tyrosine phosphorylation of beta-catenin was more frequently observed in cancerous tissues than in the matching normal mucosa. These results suggest that beta-catenin may have important regulatory roles within an E-cadherin-mediated adhesion system in human colorectal cancers.     

A novel function of CD82/KAI-1 on E-cadherin-mediated homophilic cellular adhesion of cancer cells

In this study, we analyzed the effect of the metastasis suppressor CD82/KAI-1, a member of the tetraspanin superfamily, on intercellular adhesion on cancer cells. The newly established invasion assay and the cell aggregation assay revealed that CD82 strengthens E-cadherin-mediated intercellular adhesion. Interestingly, ectopic expression of CD82 stabilized E-cadherin/beta-catenin complex formation. Furthermore, CD82 reduced tyrosine phosphorylation of beta-catenin on HGF stimulation. Taken together, CD82 may stabilize or strengthen E-cadherin-dependent intercellular adhesion by regulating beta-catenin-mediated signal transduction on cancer cells, and consequently, prevent cancer cells from seceding from the primary tumor site.     

Integrins and E-cadherin cooperate with IGF-I to induce migration of epithelial colonic cells.

Although the detailed mechanisms of cell migration remain largely unknown, it is now clear that growth factors and cell adhesion molecules are crucial for this process. We have shown that type I insulin-like growth factor (IGF-I) promotes migration of human colonic tumour cells. Since morphological analysis suggested an involvement of adhesion molecules, we have now examined the role of integrins (cell-matrix adhesion molecules) and E-cadherin/catenins complex (cell-cell adhesion molecules) in the IGF-I-induced migration. Using a monolayer wounding assay, we have determined that, except for alpha2beta1, all of the integrins expressed in HT29-D4 cells are involved in the induced cell migration. Immunofluorescence studies revealed that upon IGF-I stimulation the integrins reorganized at the leading edge of migrating cells. We also demonstrate that E-cadherin is involved in cell migration. A rapid tyrosine phosphorylation of E-cadherin and beta-catenin was detected upon IGF-I stimulation. Tyrosine phosphorylation was associated with reduced membranous expression of E-cadherin and promotion of cell motility, suggesting a regulation of the E-cadherin/catenins complex. This effect can be reversed by incubating cells with tyrosine kinase inhibitors. Taken together, our results suggest that IGF-I promotes colonic cell migration through reorganization of integrin receptors and through modulation of E-cadherin/catenins complex function.     

Endostatin regulates endothelial cell adhesion and cytoskeletal organization

Endostatin, an endogenous angiogenesis inhibitor, attenuates endothelial cell migration through an unknown mechanism. We show that endostatin induced tyrosine phosphorylation of focal adhesion kinase and paxillin, and promoted formation of focal adhesions and actin stress fibers, similar to fibroblast growth factor-2 (FGF-2). In cells cotreated with endostatin and FGF-2, focal adhesions and actin stress fibers were decreased, indicating that endostatin disturbs cell-matrix adhesion. Reduced tyrosine phosphorylation and cytoplasmic relocalization of beta-catenin in cells treated with FGF-2 and endostatin indicates that loosening of cell-cell adhesion is also disturbed by endostatin. These data provide a molecular basis both for the lack of effect of endostatin on the normal, quiescent vasculature, and its antagonistic effects on stimulated tumor vessels.     

FGF-1 and FGF-2 regulate the expression of E-cadherin and catenins in pancreatic adenocarcinoma.

E-cadherin is a transmembrane protein that mediates Ca2+-dependent cell-cell adhesion and is implicated in a number of biologic processes, including cell growth and differentiation, cell recognition and cell sorting during development. We have previously demonstrated that both cell-cell adhesion and invasion are modulated by fibroblast growth factor (FGF)-1 and FGF-2 in a panel of pancreatic adenocarcinoma cell lines (BxPc3, T3M4 and HPAF). Here, we examine further the role of FGFs in the expression and activation of the E-cadherin/catenin system. We demonstrate that both FGF-1 and FGF-2 upregulate E-cadherin and beta-catenin at the protein level in the BxPc3 and HPAF cell lines and modestly in T3M4 cells. FGF-1 and FGF-2 facilitate the association of E-cadherin and alpha-catenin with the cytoskeleton, as demonstrated by the increase in the detergent-insoluble fraction of E-cadherin in BxPc3 and HPAF cells. Since the correct function of the E-cadherin/catenin complex requires its association with the cytoskeleton, our data suggest that FGF-1 and FGF-2 contribute to the integrity and thus the function of the complex. Furthermore, FGFs facilitate the assembly of the E-cadherin/catenin axis. The effect is associated with elevation of tyrosine phosphorylation of E-cadherin, alpha-catenin, beta-4051 mu-catenin and gamma-catenin, but not p120ctn. These findings indicate that the E-cadherin/catenin system is a target of the FGF/FGFR system and that coordinated signals from both systems may determine the ultimate biologic responses.     

FGF-1 and FGF-2 modulate the E-cadherin/catenin system in pancreatic adenocarcinoma cell lines

Fibroblast growth factors (FGFs) and fibroblast growth factor receptors (FGFRs) have been increasingly recognized to play an important role in the pathobiology of pancreatic malignancy. We have investigated the effects of FGF-1 and FGF-2 on the behaviour and adhesion properties of human pancreatic adenocarcinoma cell lines (BxPc3, T3M4 and HPAF) that were previously characterised for the expression of FGFRs. Here we show that exposure to FGF-1 and FGF-2 leads to significant and dose-dependent increase in E-cadherin-dependent cell-cell adhesion, tubular differentiation, and a reduced capacity to invade collagen gels. FGF stimulation produces phosphorylation of E-cadherin and beta-catenin on tyrosine residues, as well as increased E-cadherin localisation to the cytoplasmic membrane and association with FGFR1 demonstrable by coimmunoprecipitation. These results demonstrate that FGF-1 and FGF-2 may be involved in the regulation of cell adhesion, differentiation and invasion of pancreatic cancer. Copyright Cancer Research Campaign.     

Abnormal expression and function of the E-cadherin-catenin complex in gastric carcinoma cell lines.

Dysfunction of the cadherin-catenin complex, a key component of adherens junctions, is thought to confer invasive potential to cells. The aim of this study is to examine the expression and function of the E-cadherin/catenin complex in gastric carcinoma cell lines. Expression of E-cadherin, alpha, beta and gamma-catenin and p120ctn, and of the adenomatous polyposis coli protein (APC), together with function of the cadherin-catenin complex was examined in a panel of gastric carcinoma cell lines, using immunocytochemistry, Western blotting and a cell-cell aggregation assay. Protein interactions were examined by sequential immunoprecipitation and immunoblotting with antibodies to E-cadherin, alpha, beta and gamma-catenin, p120ctn and APC. Abnormalities of E-cadherin, alpha- and beta-catenin expression, were associated with disturbance of E-cadherin-catenin complex composition, loss of membranous localization and loss of calcium-dependent aggregation in six gastric carcinoma cell lines. APC protein expression and interaction with beta-catenin was preserved in five cell lines. We demonstrate frequent abnormalities of expression and function of E-cadherin and catenins, and associated disturbance of E-cadherin-mediated intercellular adhesion in gastric carcinoma cell lines. These findings support the tumour suppressor role of the E-cadherin and its contribution to the development and progression of the neoplastic phenotype in gastric carcinoma.     

Ribonucleotide reductase small subunit p53R2 promotes oral cancer invasion via the E-cadherin/beta-catenin pathway

The p53-inducible p53R2 gene has been isolated and shown to play a crucial role in DNA repair and synthesis after DNA damage. Moreover, the expression and activity of p53R2 has been reported to be associated with the anticancer agent resistance of human cancer cells. Previously, we reported that the presence of p53R2 expression was a predictive factor for regional lymph node metastasis in oral squamous cell carcinoma; however, the mechanism of cancer metastasis by p53R2 expression is still unclear. In the present study, we analyzed the correlation of p53R2 expression with cancer invasion in vitro. Three human oral cancer cell lines (SAS, HSC-3 and Ca9-22) were cultured, and the invasive potential of these cancer cells was evaluated using Matrigel invasion assay. To investigate the effect of p53R2 on cancer invasion, the down-regulation of p53R2 was examined by small interfering RNA (siRNA). Moreover, we examined the intracellular localization of cell adhesion molecules (E-cadherin and beta-catenin) in subcellular extractions of cancer cells by immunoblotting. The proteolytic activity of matrix metalloproteinases (MMPs) was assessed by gelatin zymography. Down-regulation of p53R2 significantly enhanced the invasion potential (p<0.01), and enhanced nuclear translocation of beta-catenin with loss of total cellular E-cadherin expression in p53 mutant cancer cells, but not in p53 wild-type cancer cells. These changes in the invasion index by p53R2 siRNA transfection were not accompanied by alterations in MMP activity and expression. These results suggested that the expression of p53R2 could be associated with the invasion of cancer cells, and indicated that p53R2 might promote cancer invasion via the E-cadherin/beta-catenin pathway without the alteration of MMP activity.