Because Cxs exert a range of functions, and these may vary between tumor types and experimental conditions, an integrating concept on how Cxs either suppress or enhance neoplastic progression is lacking ( Aasen et al., 2019 Naus and Laird, 2010). Interference with Cx43 expression or channel function inhibits cancer cell migration in vitro ( Ogawa et al., 2012) and reduces metastatic seeding by reducing binding of circulating tumor cells to vascular endothelial cells ( el-Sabban and Pauli, 1991 Elzarrad et al., 2008 Stoletov et al., 2013). On the other hand, Cxs remain expressed in several solid tumors, and their expression increases in metastases ( Bos et al., 2009 Elzarrad et al., 2008 Kanczuga-Koda et al., 2006 Stoletov et al., 2013). By regulating cell differentiation, Cxs further counteract neoplastic transformation ( Bazzoun et al., 2019 Fostok et al., 2019 Saunders et al., 2001 Zhang et al., 2003), and forced expression of Cxs in transformed cells reduces tumor cell growth and inhibits invasion by reverting the epithelial-to-mesenchymal transition ( Kazan et al., 2019 McLachlan et al., 2006). In morphogenesis, Cxs are indispensable for coordinated tissue growth ( Sinyuk et al., 2018). Cxs further contribute to gene expression of cadherins ( Kotini et al., 2018) and/or the release of chemotactic factors to induce cell polarity and migration ( Barletta et al., 2012 Haynes et al., 2006 Kaczmarek et al., 2005). Direct cell-to-cell signaling occurs through gap-junctional intercellular transfer of second messengers, including IP 3 and cAMP, energy equivalents (ATP and glucose), and Ca 2+ wave propagation ( Boitano et al., 1992 Goldberg et al., 2004 Howe, 2004). The mechanisms by which Cxs support coordinated cytoskeletal contractility and multicellular dynamics vary. Cxs mediate multicellular contractility of cardiomyocytes and the myoepithelial layer of mammary ducts ( Kumai et al., 2000 Mroue et al., 2015) and, by unknown mechanisms, collective movement of neural and endothelial cells during morphogenesis ( Ashton et al., 1999 Huang et al., 1998 Marins et al., 2009). The resulting transmembrane connections mediate gap-junctional transfer of ions and small molecules (<1 kD), including Ca 2+, phosphoinosites, and nucleotides ( Goodenough and Paul, 2009).
Gap junctions consist of connexins (Cxs) oligomerized into hemichannels that engage across cell membranes ( Goodenough and Paul, 2009). In nonneoplastic collective processes during morphogenesis and regeneration, moving cell sheets are further connected by gap junctions ( Ashton et al., 1999 Huang et al., 1998 Kotini et al., 2018 Marins et al., 2009). This identifies autocrine purinergic signaling, through Cx43 hemichannels, as a critical pathway in leader cell function and collective invasion. ADORA1 inhibition further reduced local invasion of orthotopic mammary tumors in vivo, and joint up-regulation of Cx43 and ADORA1 in breast cancer patients correlated with decreased relapse-free survival. Accordingly, pharmacological inhibition of ADORA1 or AKT signaling caused leader cell collapse and halted collective invasion. Using molecular interference and rescue strategies, we identify that Cx43 hemichannel function, but not intercellular communication, induces leader cell activity and collective migration through the engagement of the adenosine receptor 1 (ADORA1) and AKT signaling. We here show that Cx43 mediates gap-junctional coupling between collectively invading breast cancer cells and, via hemichannels, adenosine nucleotide/nucleoside release into the extracellular space. Collectively invading breast cancer cells express the gap junction protein connexin-43 (Cx43), yet whether Cx43 regulates collective invasion remains unclear. Progression of epithelial cancers predominantly proceeds by collective invasion of cell groups with coordinated cell–cell junctions and multicellular cytoskeletal activity.
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