Connexin26 Regulates the Expression of Angiogenesis-Related Genes in Human Breast Tumor Cells by Both GJIC-Dependent and -Independent Mechanisms

Abstract

We previously reported that over-expression of connexins in mammary tumor cells retarded tumor growth in vivo in the absence of appreciable gap junction formation, highlighting a possible connexin-linked, but gap junctional intercellular communication (GJIC)-independent mechanism. In the current study, we engineered GJIC-deficient MDA-MB-435 human breast tumor cells to express a chimeric Cx26 where the green fluorescent protein was fused to the amino-terminal of Cx26 (GFP-Cx26). Characterization of this chimeric protein revealed that GFP-Cx26 assembled into non-functional gap junction-like clusters that were impermeable to Lucifer Yellow. In contrast, expression of wild-type Cx26 or Cx26 tagged at the carboxy terminal with yellow fluorescent protein, efficiently rescued GJIC in these tumor cells. Interestingly, by screening 96 tumor-related genes, we observed that the expression of Cx26 or GFP-Cx26 in the tumor cells up-regulated both the transcription and the translation of thrombospondin-1 (TSP-1), an anti-angiogenic molecule. Affymetrix array analysis extended the list of Cx26 or GFP-Cx26 regulated genes by ten candidates including connective tissue growth factor (CTGF), another angiogenesis-related gene. CTGF mRNA and protein levels were found to be down-regulated by both Cx26 and GFP-Cx26. Thus, our data indicates that Cx26 regulates angiogenesis-related molecules by mechanisms that are both GJIC-dependent and -independent.

Down-regulation of Cx43 by Retroviral Delivery of Small Interfering RNA Promotes an Aggressive Breast Cancer Cell Phenotype

“…In order to determine the mechanism(s) by which Cx43 regulates tumor growth and progression, we examined potential Cx43-regulated molecules. Our previous study using overexpression models revealed that one connexin-regulated mechanism involved in tumor growth may be related to key molecules involved in angiogenesis (32). In the present study, Western blot analysis revealed that TSP-1, an antiangiogenesis molecule, was dramatically down-regulated by >60% in siRNA-1 and siRNA-3 expressing cells (Fig.…”

“…Our earlier results revealed that Cx43 suppressed breast tumor growth in vivo (3) raising possibilities that Cx43 may be regulating factors that are important in the vascularization of the developing tumor (3,32). Previously, Huang et al (43) identified that the tumor-promoting cytokine, monocyte chemotactic protein-1, was down-regulated in Cx43 overexpressing cells and this cytokine regulated glioblastoma cell growth.…”

“…Consistently, the regulation of both of these key angiogenic factors was tightly correlated with the degree of Cx43 silencing in the various Hs578T cell lines. Previously, we showed that TSP-1 was up-regulated in MDA-MB-435 as a consequence of Cx26 overexpression (32), suggesting that several connexin family members regulate TSP-1 expression. Often human breast tumors overexpress the oncogene ErbB2 which has been shown to decrease TSP-1 expression levels further supporting the key role of TSP-1 in tumor progression (44).…”

Connexins: a junctional crossroad to breast cancer

“…The same group investigated the role of Cx26 as a tumor suppressor gene in two independent studies. In the first one, they showed that expression of Cx26 and its GJIC-incompetent variants in MDA-MB-435 cells upregulated anti-angiogenic molecules by both GJIC-dependent and -independent mechanisms (Qin et al, 2003). In their second study, only GJIC-incompetent variants inhibited MDA-MB-435 cell growth, migration and invasion and regulated the expression of several genes involved in adhesion (b-integrin) and invasion (MMP-9 and TIMP-1), suggesting a GJIC-independent role of Cx26 in breast cancer suppression (Kalra et al, 2006).…”

Hopes and Disillusions in Therapeutic Targeting of Intercellular Communication in Cancer.

“…Cx43 affect angiogenesis in vitro and in vivo (McLachlan et al 2006) and improves the resistance to the chemotherapeutic agent cisplatin (CDDP) (Sato et al 2009) in a GJIC-independent fashion. Cx26 regulates angiogenesis-related molecules by mechanisms that are both GJIC-dependent andindependent (Kalra et al 2006;Qin et al 2003b). GJIC-independent functions of Cx32 in blocking proliferation, invasion and metastasis in human renal cell carcinoma RCC cells, have also been reported (Sato et al 2007b).…”