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Cysteine-rich 61 (Cyr61/CCN1) is involved in human gastric cancer development and progression. Nonetheless, the role of Cyr61 as regards peritoneal dissemination of such cancers has not yet been completely characterized. We used liposome-mediated transfection to establish Cyr61, or antisense Cyr61, expression vectors into gastric cancer AGS or MKN45 cell lines. Transfectants were tested by means of a cancer-cell adhesion assay in vitro and ex vivo. Furthermore, a functional integrin fluorescence-activated cell sorting assay, reverse transcription-PCR, and an AP-1 reporter assay were performed to investigate the potential signaling pathway of Cyr61. It was shown that stable transfection of Cyr61 into the AGS cell line strongly enhanced its adhesion ability. The overexpression of Cyr61 within AGS cells significantly increased the functional expression of integrin alpha(2)beta(1). Function-neutralizing antibody to integrin alpha(2)beta(1) effectively suppressed the Cyr61-mediated enhanced adhesion of AGS cells to peritoneal tissue. Promoter assays of integrin alpha2 gene further revealed that the AP-1 pathway was evidently activated within Cyr61-expressing AGS cells. Animal studies have revealed that mice injected with Cyr61-overexpressed AGS cells featured a greater number of peritoneal seeding nodules and a lower survival rate than the Neo control cell lines, and when such cells were treated with functional blocking antibody to integrin alpha(2)beta(1), they were able to elicit a decline in the peritoneal dissemination. The data suggest that Cyr61 may contribute to the peritoneal dissemination of gastric cancer by promoting tumor-cell adhesion ability through the up-regulation of the functional integrin alpha(2)beta(1) via an AP-1-dependent pathway.

The aggressiveness of a tumor is partly attributed to its resistance to chemotherapeutic agent-induced apoptosis. Cysteine-rich 61 (Cyr61), from the CCN gene family, is a secreted and matrix-associated protein, which is involved in many cellular activities such as growth and differentiation. Here we established a cell model system to examine whether stable expression of Cyr61 in MCF-7 cells can confer resistance to apoptosis and identify possible participating mechanisms. We showed that stable cell lines overexpressing Cyr61 had acquired a remarkable resistance to apoptosis induced by paclitaxel, adriamycin, and beta-lapachone. Most interesting, gel shift and reporter assays showed that the Cyr61-overexpressing cells had significantly increased NF-kappaB activity compared with neo control cells. Blockage of NF-kappaB activity in Cyr61-expressing cells by transfecting with a dominant negative (DN)-IkappaB or with an NF-kappaB decoy rendered them more susceptible to anti-cancer drugs-induced apoptosis. In addition, several NF-kappaB-regulated anti-apoptotic genes were examined, and we found that only XIAP showed a significant 3-4-fold increase in mRNA and protein in Cyr61-overexpressing cells but not in neo control cells. Treatment with inhibitor of apoptosis protein (XIAP)-specific antisense, but not sense, oligonucleotides abolished the apoptosis resistance of the Cyr61-overexpressing cells. At the same time, transfection of these stable cells with DN-IkappaB to block NF-kappaB activity also effectively reduced the elevated XIAP level. Function-neutralizing antibodies to alpha(v)beta(3) and alpha(v)beta(5) could inhibit Cyr61-mediated NF-kappaB activation as well as XIAP expression. Taken together, our data suggested that Cyr61 plays an important role in resistance to chemotherapeutic agent-induced apoptosis in human breast cancer MCF-7 cells by a mechanism involving the activation of the integrins/NF-kappaB/XIAP signaling pathway.

Vascular endothelial growth factor C (VEGF-C) is a critical activator of tumor lymphangiogenesis that recently has been strongly implicated in the tumor metastasis process. In this study, we identified that HRG-beta 1 stimulated up-regulation of VEGF-C mRNA and protein of human breast cancer cells in a dosage- and time-dependent manner and that this up-regulation was de novo RNA synthesis-dependent. The HRG-beta 1-induced increase in VEGF-C expression was effectively reduced by treatment with Herceptin, an antibody specifically against HER2. Also, when HER2 was overexpressed in MCF-7 cells that resulted in an evident increase in the VEGF-C level, suggesting an essential role of HER2 in mediating VEGF-C up-regulation by HRG-beta 1. NF-kappa B has been shown to be probably involved in interleukin-1 beta- or tumor necrosis factor-alpha-induced VEGF-C mRNA expression in human fibroblasts. Here we found that HRG-beta 1 could stimulate NF-kappa B nuclear translocation and DNA-binding activity via the I kappa B alpha phosphorylation-degradation mechanism. Blockage of the NF-kappa B activation cascade caused a complete inhibition of the HRG-beta 1-induced elevation of VEGF-C. In promoter-reporter assay, the luciferase activities of the reporter constructs, including the putative NF-kappa B site deleted and mutated form were significantly reduced after HRG-beta 1 treatment as compared with the 1.5-kb VEGF-C promoter. Although investigating the upstream kinase pathway(s) involved in HRG-beta 1-elicited NF-kappa B activation and VEGF-C up-regulation, we found that HRG-beta1 could activate extracellular signal-regulated protein kinase 1/2, phosphatidylinositol 3'-kinase, and p38 mitogen-activated protein kinase (MAPK) in MCF-7. However, only SB203580 (a specific inhibitor of p38 MAPK), not PD98059 nor LY294002, blocked the up-regulation of VEGF-C by HRG-beta 1. A similar inhibition in VEGF-C expression was obtained by cell transfection with dominant-negative p38 (p38AF). Interestingly, the HRG-beta 1-induced NF-kappa B activation cascade was also effectively blocked by SB203580 treatment or p38AF transfection. Our data thus suggests that HRG-beta 1 stimulated a NF-kappa B-dependent up-regulation of VEGF-C through the p38 MAPK signaling pathway in human breast cancer cells.