Molecular Pharmacology Fast Forward. Published on September 16, 2016 as DOI: 10.1124/mol.116.105338 This article has not been copyedited and formatted. The final version may differ from this version. MOL #105338 Title Page Gβγ pathways in cell polarity and migration linked to oncogenic GPCR signaling: potential relevance in tumor microenvironment Downloaded from José Vázquez-Prado, Ismael Bracho-Valdés, Rodolfo Daniel Cervantes-Villagrana, and Guadalupe Reyes-Cruz molpharm.aspetjournals.org Departments of Pharmacology (JVP, RDCV) and Cell Biology (GRC). CINVESTAV-IPN, Mexico City, MEXICO. Department of Pharmacology (IBV), School of Medicine, UABC, Mexicali, B.C., MEXICO. at ASPET Journals on September 27, 2021 1 Molecular Pharmacology Fast Forward. Published on September 16, 2016 as DOI: 10.1124/mol.116.105338 This article has not been copyedited and formatted. The final version may differ from this version. MOL #105338 Running Title Page Running title: Gβγ pathways in oncogenic GPCR signaling Address correspondence to: José Vázquez-Prado, Department of Pharmacology, CINVESTAV-IPN. Av. Instituto Politécnico Nacional 2508.Col. San Pedro Zacatenco, 14740 México, D.F., MEXICO. Tel: (52-55) 5747-3380; Fax: (52-55) 5747-3394; E-mail: [email protected] Downloaded from or Guadalupe Reyes-Cruz, Department of Cell Biology, CINVESTAV-IPN. Av. Instituto molpharm.aspetjournals.org Politécnico Nacional 2508.Col. San Pedro Zacatenco, 14740 México, D.F., MEXICO. E-mail: [email protected] Document statistics: at ASPET Journals on September 27, 2021 Text pages: 54 tables: 0 figures: 4 references:180 Number of words in: Abstract: 200 Body of manuscript: 6428 Abbreviations:bone marrow derived cells (BMDC), G protein coupled receptors (GPCRs), Calcium Sensing Receptor (CaSR) 2 Molecular Pharmacology Fast Forward. Published on September 16, 2016 as DOI: 10.1124/mol.116.105338 This article has not been copyedited and formatted. The final version may differ from this version. MOL #105338 Abstract Cancer cells and stroma cells in tumors secrete chemotactic agonists that exacerbate invasive behavior, promote tumor-induced angiogenesis, and recruit protumoral bone marrow- derived cells. In response to shallow gradients of chemotactic stimuli recognized by G protein coupled receptors (GPCRs), Gβγ-dependent signaling cascades contribute to specify the spatiotemporal assembly of cytoskeletal structures that can dynamically alter cell morphology. Downloaded from This sophisticated process is intrinsically linked to the activation of Rho GTPases and their cytoskeletal-remodeling effectors. Thus, Rho guanine nucleotide exchange factors, the activators molpharm.aspetjournals.org of these molecular switches, and their upstream signaling partners, are considered participants of tumor progression. Specifically, phosphoinositide-3 kinases (class-I PI3Ks, β and γ) and P-Rex1, a Rac-specific guanine nucleotide exchange factor, are fundamental Gβγ effectors in the pathways controlling directionally persistent motility. In addition, GPCR-dependent chemotactic at ASPET Journals on September 27, 2021 responses often involve endosomal trafficking of signaling proteins, and coincidently, endosomes serve as signaling platforms for Gβγ. In preclinical murine models of cancer, inhibition of Gβγ attenuates tumor growth, whereas in cancer patients, aberrant overexpression of chemotactic Gβγ-effectors and recently-identified mutations in Gβ correlate with poor clinical outcome. Here we discuss emerging paradigms of Gβγ signaling in cancer, which are essential for chemotactic cell migration and represent novel opportunities to develop pathway-specific pharmacological treatments. 3 Molecular Pharmacology Fast Forward. Published on September 16, 2016 as DOI: 10.1124/mol.116.105338 This article has not been copyedited and formatted. The final version may differ from this version. MOL #105338 Introduction In tumor microenvironments, cells adjust their shape and move in response to multiple factors secreted by cancer cells and stromal cells (Friedl and Alexander, 2011; Roussos et al., 2011). Inherent intracellular forces are generated by actin polymerization and ATPase activity of myosin motors, and these mechanical responses are tightly controlled by receptor-dependent signaling mechanisms that define the precise spatiotemporal reorganization of the actin Downloaded from cytoskeleton (Alexander and Friedl, 2012; Devreotes and Horwitz, 2015). In particular, chemotactic G protein-coupled receptors (GPCRs) elicit G protein-dependent signaling pathways molpharm.aspetjournals.org linked to metastatic dissemination of cancer cells, formation of new blood vessels, and recruitment of proangiogenic cells from the bone marrow, all of which are critical to maintain cancer progression (Figure 1) (Dorsam and Gutkind, 2007; Lappano and Maggiolini, 2011). Current models of GPCR-dependent chemotactic cell migration have evolved from at ASPET Journals on September 27, 2021 studies on fast-moving cells that migrate individually, such as Dictyostelium amoebae and neutrophils. These generic pathways mediating migration suggest the involvement of Gβγ- dependent effectors, particularly PI3Ks and phosphatidylinositol (3,4,5)-trisphosphate-dependent effectors, including guanine nucleotide exchange factors for Rho GTPases. Cytoskeletal reorganization is launched by actin nucleation sites generated by effectors of GTP-bound Rho GTPases; RhoA, Rac1 and Cdc42 are the best characterized members of this family of molecular switches (Figure 2). These Rho-GTPase-dependent downstream mechanisms will not be discussed here, but readers are encouraged to consult excellent reviews covering these topics (Burridge and Wennerberg, 2004; Charest and Firtel, 2007; Rickert et al., 2000; Ridley, 2015; Ridley et al., 2003; Van Haastert and Devreotes, 2004). General mechanisms of directional cell migration provide a basis to explore GPCR-dependent cell migration linked to cancer 4 Molecular Pharmacology Fast Forward. Published on September 16, 2016 as DOI: 10.1124/mol.116.105338 This article has not been copyedited and formatted. The final version may differ from this version. MOL #105338 progression. However, in cancer settings, this process is further complicated by the influence of a plethora of stimuli in the microenvironment. Moreover, oncogenic pathways participating in chemotactic migration are affected by mutational changes, overexpression and differential repertoire of signaling systems which, among many other factors, eventually are integrated to facilitate individual and collective migration of cancer and protumoral cells (Figure 1). βγ We focus this review on the role of G signaling in cancer. Emphasis is given to Downloaded from upstream effectors leading to the activation of Rho GTPases in the pathways that control chemotactic cell migration. In this window of molecular interactions, the participation of class-I molpharm.aspetjournals.org PI3Ks (β and γ) and P-Rex1, a Rac-specific guanine nucleotide exchange factor is considered critical. We highlight the properties of P-Rex1, known to be activated in concert by Gβγ and phosphatidylinositol (3,4,5)-trisphosphate (Welch, 2015; Welch et al., 2002), as a putative scaffold of chemotactic signaling. The proposed role of this multidomain effector is based on its at ASPET Journals on September 27, 2021 reported interactions with sphingosine-1-phosphate receptors and the serine/threonine kinases mTOR and type I PKA (Chavez-Vargas et al., 2016; Hernandez-Negrete et al., 2007; Ledezma- Sanchez et al., 2010). Overall, the proven relevance of multiple chemokines in oncogenic settings has pointed to GPCRs and their signaling effectors as possible pharmacological targets in cancer research. Protumoral chemotactic agonists Multiple agonists play a driving role in cancer progression. Oncogenic chemokines attract and consequently diversify the repertoire of cells associated with proliferating tumoral cells. Once incorporated as cellular components of the tumor stroma, fibroblasts, endothelial and immune cells also contribute to increase the variety of secreted chemokines that not only 5 Molecular Pharmacology Fast Forward. Published on September 16, 2016 as DOI: 10.1124/mol.116.105338 This article has not been copyedited and formatted. The final version may differ from this version. MOL #105338 stimulate the surrounding tissues, but also have a systemic effect: attracting cells from the bone marrow (Figure 1) (Kojima et al., 2010; Orimo et al., 2005; Scherz-Shouval et al., 2014; Wels et al., 2008). Tumor-associated bone marrow derived cells (BMDC) further support cancer progression via their ability to secrete chemokines, cytokines, growth factors, and proteins of the extracellular matrix (Gao et al., 2008; Nolan et al., 2007; Sekiguchi et al., 2011). Among BMDC’s protumoral effects, preparing the ground where metastatic niches are eventually Downloaded from established is particularly significant (Gao et al., 2008; Kaplan et al., 2005; Lyden et al., 2001; Peinado et al., 2012; Psaila and Lyden, 2009; Qian et al., 2011). molpharm.aspetjournals.org Examples of protumoral GPCR agonists with chemotactic properties include CCL2/5/22/25, CXCL1/2/5/8(IL-8)/12(SDF-1), and CX3CL1 (Bronte and Bria, 2016; Roussos et al., 2011; Ugel et al., 2015). In addition, bioactive lipids such as sphingosine-1-phosphate (Liang et al., 2013) and LPA (Jonkers and Moolenaar, 2009; Liu et al., 2009) also stimulate pro- at ASPET Journals on September 27, 2021 oncogenic migratory pathways. CXCL12