Supplementary MaterialsTable S1: lists primer sequences. 1 (ADORA1) and AKT signaling. Accordingly, pharmacological inhibition of ADORA1 or AKT signaling caused leader cell collapse and halted 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. This identifies autocrine purinergic signaling, through Cx43 hemichannels, as a critical pathway in leader cell function and collective invasion. Graphical Abstract Open in a separate window Introduction Collective invasion of multicellular groups with intact cellCcell junctions is an important mechanism to local invasion and metastasis in epithelial cancers (Gaggioli et al., 2007; Friedl and Gilmour, 2009; Cheung and Ewald, 2016). Collective invasion is initiated and maintained by leader cells that extend actin-rich protrusions, engage with the ECM to exert traction and ECM remodeling, and maintain cellCcell coupling with follower cells (Cheung et al., 2013; Westcott et al., 2015). Mechanical connection and cell-to-cell signaling between moving cells are provided by adherens junctions, through E-, N-, and other cadherins (Theveneau and Mayor, 2012). Cadherins coordinate the actomyosin cytoskeleton through catenins and vinculin (Hartsock and Nelson, 2008; Han and de Rooij, 2016) and secure supracellular force balance, frontCrear and apicobasal polarity, and multicellular branching Aminothiazole (Tambe et al., 2011). 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). Gap junctions consist of connexins (Cxs) oligomerized into hemichannels that engage across cell membranes (Goodenough and Paul, 2009). The resulting transmembrane connections mediate gap-junctional transfer of ions and small molecules ( 1 kD), including Ca2+, phosphoinosites, and nucleotides (Goodenough and Paul, 2009). 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 Ntrk2 al., 2009). The mechanisms by which Cxs support coordinated cytoskeletal contractility and multicellular dynamics vary. Direct cell-to-cell signaling occurs through gap-junctional intercellular transfer of second messengers, including IP3 and cAMP, energy equivalents (ATP and glucose), and Ca2+ wave propagation (Boitano et al., 1992; Goldberg et al., 2004; Howe, 2004). 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). In morphogenesis, Cxs are indispensable for coordinated tissue growth (Sinyuk et al., 2018). By regulating cell differentiation, Cxs further counteract Aminothiazole 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). 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). 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). 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). Whereas a role of Cxs in collective processes in morphogenesis has been established, the contribution of gap-junctional communication in collective tumor cell invasion Aminothiazole remains unclear (Friedl and Gilmour, 2009). We here revisited Cx43 expression and function in breast cancer models of collective invasion. Using pharmacological inhibition, molecular interference, and rescue strategies, we show that Cx43 mediates gap-junctional intercellular communication (GJIC) between collectively invading cells and, via hemichannels, the release of purine derivatives into the extracellular space. Cx43 hemichannel-mediated release of adenosine (ADO) nucleotides initiate an autocrine loop inducing leader cell function and collective invasion, but not single-cell motility, Aminothiazole through ADO receptor 1 (ADORA1) and AKT/protein kinase B signaling. Results Breast cancer cells express Cx43 during collective invasion To address whether Cxs are expressed.