A Dominant Negative Cx43 Mutant Differentially Affects Tumorigenic and Invasive Properties in Human Metastatic Melanoma Cells SHOSHANNA N. ZUCKER * , TARA A. BANCROFT, DAVID E. PLACE, BENJAMIN DES SOYE, ARCHIS BAGATI AND RONALD BEREZNEY Department of Biological Sciences, University at Buffalo, Buffalo, New York Previous reports have implicated connexin 43 (Cx43) as a tumor suppressor in early stages of tumorigenesis and in some cases as an enhancer of cell migration in later stages. To address the role of Cx43 in melanoma tumor progression, we utilized two melanoma cell lines derived from the same patient in pre-metastasis (WM793B) and following isolation from a lung metastasis in nude mice (1205Lu). Our results demonstrate a strikingly increased expression of Cx43 in both the pre-metastatic and metastatic melanoma cell lines that were actively migrating compared to non-migrating cells. To further investigate the role of Cx43 in these melanoma cells, we overexpressed wild type (wt) Cx43 as well as a mutant dominant negative Cx43 mutant that causes closed channels (T154A). The metastatic 1205Lu cells expressing Cx43-T154A showed a twofold decrease in colony formation on soft agar while the nonmetastatic WM793B cells showed no significant change. In invasion assays through a collagen matrix, the same Cx43-T154A 1205Lu cells demonstrated a three- to fourfold increase in the invasion index compared to either wt Cx43 or vector control cells. The increase in invasiveness was eliminated by migration towards media with charcoal-stripped serum, suggesting that migration may be directed towards a lipophilic compound(s). Our findings demonstrate that a dominant negative Cx43 mutant deficient in channel formation exhibits a dual pattern of regulation in metastatic melanoma cells with a decrease in anchorage-independent growth and an increase in invasive potential. J. Cell. Physiol. 228: 853–859, 2013. ß 2012 Wiley Periodicals, Inc. Gap junctions (GJ) mediate intercellular communication of ions and metabolites up to 1 kDa, affecting signaling processes that regulate proliferation, differentiation, and maintenance of homeostasis (Saez et al., 2003). Typically, GJ are clustered on the cell surface in large arrays termed gap junction plaques (Yeager, 1998). The protein constituent of gap junction plaques are connexins which are a family of 21 members (Sohl and Willecke, 2004). It is well established that connexins play a role in maintaining homeostasis and, therefore, mutations or alterations in expression of connexins have been associated with various disease phenotypes including cancer. For example, reduced expression of connexin protein and/or gap junctional intercellular communication (GJIC) has been implicated in numerous tumor types and transformed cell lines (Naus, 2002). Decreased GJIC was observed in glioblastoma (Huang et al., 1999), gastric carcinoma (Uchida et al., 1995), squamous cell carcinoma (Tada and Hashimoto, 1997) and tumors from the prostate (Tsai et al., 1996), lung (Jinn et al., 1998), liver (Krutovskikh et al., 1994), ovary (Hanna et al., 1997), colon (Friedman and Steinberg, 1982), and breast (Locke, 1998), as well as many chemically and virally transformed cells (Hanna et al., 1997; Cesen-Cummings et al., 1998). Furthermore, endogenously expressed connexins decreased the transformed phenotype of cell lines derived from hepatoma (Eghbali et al., 1991), mammary carcinoma (Hirschi et al., 1996), glioma (Huang et al., 1999), and melanoma (Su et al., 2000). In UACC903 melanoma cells, overexpression of Cx43 (connexin 43) was shown to suppress anchorage-independent growth and tumor formation in nude mice (Su et al., 2000). Due to these widely observed changes in expression patterns, connexins have often been termed, tumor suppressors (Naus, 2002). In addition to acting as mediators of growth suppression, another, seemingly contradictory role for connexins in cancer, is suggested by the findings that Cx43, which is the most prevalent connexin (expressed in at least 46 cell types and 34 tissues (Laird, 2006)), is up-regulated during metastasis in breast (Kanczuga-Koda et al., 2006), prostate (Miekus et al., 2005), and lung cancer (Elzarrad et al., 2008). While decreased GJIC is the widely accepted reason for reduced tumorigenic properties (Naus, 2002), some studies demonstrate that the inhibition of transformation may not depend on cell to cell coupling, but possibly the protein itself (Qin et al., 2002). Consistent with this hypothesis, the cytoplasmic tail of Cx43 can independently cause a reduction in growth in HeLa cells (Dang et al., 2003). The cytoplasmic tail of Cx43 is a binding site for several proteins including ZO-1 (Giepmans and Moolenaar, 1998) and b-tubulin (Giepmans et al., 2001), and also contains many phosphorylation sites, including MAPK, Src, PKC, PKA, CK1, and p34cdc2 (Lampe and Lau, 2004) Phosphorylation at specific residues can induce channel gating, either transiently or constitutively (Moreno and Lau, 2007). In addition, point mutations in Cx43 can alter its functionality. For instance, a dominant negative mutant that is Additional supporting information may be found in the online version of this article. Contract grant sponsor: National Institutes of Health; Contract grant number: GM 07431. *Correspondence to: Shoshanna N. Zucker, Senior Scientist, Roswell Park Cancer Institute, Department of Cell Stress Biology, Center for Genetics and Pharmacology, Elm & Carlton Streets, Buffalo, NY 14263. E-mail: shoshanna.zucker@roswellpark.org Manuscript Received: 12 September 2012 Manuscript Accepted: 20 September 2012 Accepted manuscript online in Wiley Online Library (wileyonlinelibrary.com): 5 October 2012. DOI: 10.1002/jcp.24235 ORIGINAL RESEARCH ARTICLE 853 Journal of Journal of Cellular Physiology Cellular Physiology ß 2012 WILEY PERIODICALS, INC.