© 2017. Published by The Company of Biologists Ltd | Journal of Cell Science (2017) 130, 1064-1077 doi:10.1242/jcs.195552 RESEARCH ARTICLE A RhoG-mediated signaling pathway that modulates invadopodia dynamics in breast cancer cells Silvia M. Goicoechea, Ashtyn Zinn, Sahezeel S. Awadia, Kyle Snyder and Rafael Garcia-Mata* ABSTRACT micropinocytosis, bacterial uptake, phagocytosis and leukocyte One of the hallmarks of cancer is the ability of tumor cells to invade trans-endothelial migration (deBakker et al., 2004; Ellerbroek et al., surrounding tissues and metastasize. During metastasis, cancer cells 2004; Jackson et al., 2015; Katoh et al., 2006, 2000; van Buul et al., degrade the extracellular matrix, which acts as a physical barrier, by 2007). Recent studies have revealed that RhoG plays a role in tumor developing specialized actin-rich membrane protrusion structures cell invasion and may contribute to the formation of invadopodia called invadopodia. The formation of invadopodia is regulated by Rho (Hiramoto-Yamaki et al., 2010; Kwiatkowska et al., 2012). GTPases, a family of proteins that regulates the actin cytoskeleton. Invadopodia are actin-rich adhesive structures that form in the Here, we describe a novel role for RhoG in the regulation of ventral surface of cancer cells and allow them to degrade the invadopodia disassembly in human breast cancer cells. Our results extracellular matrix (ECM) (Gimona et al., 2008). Formation of show that RhoG and Rac1 have independent and opposite roles invadopodia involves a series of steps that include the disassembly in the regulation of invadopodia dynamics. We also show that SGEF of focal adhesions and stress fibers, and the relocalization of several (also known as ARHGEF26) is the exchange factor responsible of their components into the newly formed invadopodia (Hoshino for the activation of RhoG during invadopodia disassembly. When et al., 2012; Oikawa et al., 2008). Invadopodia assembly starts with the expression of either RhoG or SGEF is silenced, invadopodia the formation of actin- and cortactin-rich puncta, followed by the are more stable and have a longer lifetime than in control cells. recruitment of adhesion proteins, such as vinculin and paxillin, and Our findings also demonstrate that RhoG and SGEF modulate the proteinases that allow ECM degradation (Hoshino et al., 2013). phosphorylation of paxillin, which plays a key role during invadopodia Even though many of the molecular components required for disassembly. In summary, we have identified a novel signaling invadopodia formation have been identified, the signaling pathways pathway involving SGEF, RhoG and paxillin phosphorylation, which that regulate these events are still poorly understood (Linder et al., functions in the regulation of invadopodia disassembly in breast 2011). Invadopodia formation is controlled by the integrated cancer cells. activity of several GTPases, including Cdc42, RhoA, RhoC and Rac1 (Spuul et al., 2014). Cdc42 promotes invadopodia formation KEY WORDS: RhoG, Invadopodia, SGEF, Guanine-nucleotide in almost every system tested (Ayala et al., 2009; Di Martino et al., exchange factors, Src, Paxillin, Rac1 2014; Moreau et al., 2006, 2003; Nakahara et al., 2003; Tatin et al., 2006). However, generalized conclusions cannot be drawn from INTRODUCTION studies of other Rho GTPases, since their activities can both inhibit Rho GTPases control many aspects of cell behavior ranging from or promote invadopodia formation depending on the experimental the regulation of cytoskeletal organization, cell motility and cell conditions or cell type used (Spuul et al., 2014). polarity, to nuclear gene expression and control of cell growth In this study, we describe a novel function for RhoG in the (Hodge and Ridley, 2016). Rho proteins cycle between an active regulation of invadopodia dynamics in human breast cancer cells. (GTP-bound) and an inactive (GDP-bound) state. The activation of Our findings describe a signaling pathway involving SGEF and Rho proteins involves the exchange of GDP for GTP, which is RhoG that regulates invadopodia disassembly independently of catalyzed by specific guanine-nucleotide-exchange factors (GEFs). Rac1 through regulation of paxillin phosphorylation. Once activated, Rho GTPases interact with a wide variety of downstream effectors to modulate their activity and/or localization. RESULTS The hydrolysis of GTP to GDP, a reaction that is stimulated by RhoG is a negative regulator of invadopodia formation GTPase-activating proteins (GAPs), inactivates the GTPases and We have previously shown that the breast cancer cell line SUM159 terminates the signal. With more than 80 Rho GEFs, 70 Rho GAPs forms invadopodia when treated with phorbol esters such phorbol and over 100 effectors, cells regulate the activity of Rho proteins 12,13-dibutyrate (PDBu) and 12-O-tetradecanoylphorbol-13- through multiple pathways, thus acting as key nodes for signal acetate (PMA), and that the formation of these structures integration and dissemination (Bustelo et al., 2007; Rossman et al., correlates with their metastatic potential (Goicoechea et al., 2009). 2005; Tcherkezian and Lamarche-Vane, 2007). To determine the requirement of RhoG in invadopodia formation, RhoG, a Rho protein related to Rac, has been associated we generated stable SUM159 cell lines in which RhoG expression with cell migration, neurite outgrowth, microtubule dynamics, was silenced using lentivirally encoded shRNA. We used two different RhoG-specific shRNAs (shRNA#1 and shRNA#4) to rule Department of Biological Sciences, University of Toledo, Toledo, OH 43606, USA. out off-target effects. A stable cell line expressing a non-targeting shRNA was used as control (CTRL). Fig. 1A shows that RhoG *Author for correspondence ([email protected]) silencing was efficient, especially for shRNA #4. SUM159 cells are S.M.G., 0000-0003-1107-389X; R.G.-M., 0000-0002-7116-4411 able to form invadopodia spontaneously, so we first looked at the effect of RhoG depletion in untreated cells. To identify invadopodia Received 22 July 2016; Accepted 14 January 2017 structures, we stained the cells with the invadopodia markers Journal of Cell Science 1064 RESEARCH ARTICLE Journal of Cell Science (2017) 130, 1064-1077 doi:10.1242/jcs.195552 Fig. 1. RhoG negatively regulates invadopodia formation in SUM159 cells. (A) Cell lysates from SUM159 cells stably expressing non-targeting (CTRL) or RhoG-specific shRNAs (shRNA#1 and shRNA #4) were analyzed by western blotting and probed for RhoG and Rac1, and for tubulin as a loading control. (B) Quantification of cortactin- and actin-containing invadopodia in CTRL and RhoG KD cells. Results are expressed as the percentage of cells with invadopodia. Data are mean±s.e.m. for at least three independent experiments. Cells were either untreated, or treated with PDBu or PMA. (C) CTRL or RhoG KD SUM159 cells were treated with PDBu for 30 min and stained with anti-cortactin antibody (red), Alexa-Fluor-488–phalloidin (green) and Hoechst 33342 (blue). Arrowheads indicate representative invadopodia. (D) Cell lysates from CTRL, RhoG KD and RhoG KD cells expressing shRNA resistant Myc– RhoG (rescue) were immunoblotted with anti-RhoG and -Myc antibodies. Tubulin was used as a loading control. (E) Quantification of cortactin- and actin-containing invadopodia in CTRL, RhoG KD and rescue cells. Data are mean±s.e.m. of three independent experiments. (F) Cell lysates from CTRL, Myc–RhoG wt (RhoG wt) or Myc–RhoG Q61L (RhoG Q61L) cells were immunoblotted with anti-RhoG and -Myc antibodies. Tubulin was used as a loading control. (G) SUM159 cells transiently transfected with Myc- tagged wild-type (RhoG wt) or constitutively active RhoG (RhoG Q61L) were treated with PDBu for 30 min and stained with anti-cortactin antibody (red), anti-Myc antibody (green), Alexa-Fluor-647–phalloidin (magenta) and Hoechst 33342 (blue). Arrowheads indicate transfected cells and arrows indicate representative invadopodia. (H) Invadopodia were quantified in CTRL (non-transfected cells), and RhoG wt and RhoG Q61L transfected cells. Results are representative of three independent experiments in which at least 100 cells per experiment were counted. Data are mean±s.e.m. (error bars). Scale bars: 10 µm. *P<0.05; **P<0.01; ***P<0.001; ns, not significant. cortactin and actin. We found that silencing RhoG induced a depletion in cells treated with phorbol esters. In PDBu-treated cells, significant increase in the number of spontaneous invadopodia the number of cells with invadopodia increased significantly in when compared to CTRL cells, from 7% in CTRL cells to 14% and RhoG-knockdown (KD) cells, from 35% in CTRL cells to 64% 23% in RhoG shRNA#1 and shRNA#4 cells, respectively (Fig. 1B, and 81% in RhoG shRNA#1 and shRNA#4 cells respectively untreated; Fig. S1A). We next looked at the effect of RhoG (Fig. 1B,C). Similarly, the percentage of cells with invadopodia Journal of Cell Science 1065 RESEARCH ARTICLE Journal of Cell Science (2017) 130, 1064-1077 doi:10.1242/jcs.195552 increased from 31% in CTRL cells to 79% of cells in PMA-treated SUM159 cells, using specific shRNAs. We tested two independent RhoG KD cells (Fig. 1B; Fig. S1B). Based on these results, and shRNAs for each of the GEFs and compared them to cells unless otherwise indicated, we used shRNA#4 for the rest of these expressing a non-targeting shRNA (CTRL). The KD efficiency was studies (referred to as RhoG KD). We next performed a rescue verified either by quantitative real-time PCR (qRT-PCR; Fig. 3A; experiment