ARF 1 regulates the Rho / MLC pathway to control EGF-dependent breast cancer cell invasion

Invasion of tumor cells is a key step in metastasis that depends largely on the ability of these cells to degrade the extracellular matrix. Although we have showed that the GTPase ADP-ribosylation factor 1 (ARF1) is overexpressed in highly invasive breast cancer cell lines and that epidermal growth factor stimulation can activate this ARF isoform to regulate migration as well as proliferation, the role of this small GTP-binding protein has not been addressed in the context of invasiveness. Here we report that modulation of ARF1 expression and activity markedly impaired the ability of M.D. Anderson-metastatic breast-231 cells, a prototypical highly invasive breast cancer cell line, to degrade the extracellular matrix by controlling metalloproteinase-9 activity. In addition, we demonstrate that this occurs through inhibition of invadopodia maturation and shedding of membrane-derived microvesicles, the two key structures involved in invasion. To further define the molecular mechanisms by which ARF1 controls invasiveness, we show that ARF1 acts to modulate RhoA and RhoC activity, which in turn affects myosin light-chain (MLC) phosphorylation. Together our findings underscore for the first time a key role for ARF1 in invasion of breast cancer cells and suggest that targeting the ARF/Rho/MLC signaling axis might be a promising strategy to inhibit invasiveness and metastasis. INTRODUCTION Cell invasion is a process tightly regulated by multiple signaling proteins, such as plasma membrane receptors, adaptors, and small GTP-binding proteins. During cancer progression, transformed cells migrate and invade surrounding tissues to form metastasis. Expression of small GTPases and their regulatory factors is frequently modulated in human cancers (Karnoub and Weinberg, 2008; Vigil et al., 2010; Mardilovich et al., 2012). The ADP-ribosylation factor (ARF) family of proteins has been associated with cancer progression (Sabe et al., 2009). We showed that the GTPase ARF1 is overexpressed in highly invasive cancer cells and that epidermal growth factor (EGF) stimulation can activate this ARF isoform to regulate migration (Boulay et al., 2008; Lewis-Saravalli et al., 2013). Like all GTPases, ARFs cycle between their inactive (GDP bound) and active (GTP bound) form. This process is controlled by guanine nucleotide exchange factors (GEFs), which catalyze the exchange of GDP to GTP, and GTPase-activating proteins (GAPs), which accelerate the hydrolysis of bound GTP. Six ARF genes have been identified, and ARF1 and ARF6 are the best characterized. In invasive breast cancer cells, we reported that stimulation of the epidermal growth factor receptor (EGFR) activates ARF1 in order to regulate cell proliferation and migration by affecting the phosphoinositide 3-kinase (PI3K) pathway (Boulay et al., 2008). In contrast, ARF6 regulates migration and invasion by activating the mitogen-activated protein kinase pathway (Hashimoto et al., 2004; D’Souza-Schorey and Chavrier, 2006; Boulay et al., 2008). GTPases of the Rho family, RhoA and RhoC, play a major role in several key steps of cancer progression, such as invasion (Simpson et al., 2004). Depletion of RhoA or RhoC inhibits the invasiveness of M.D. Anderson-metastatic breast-231 (MDA-MB-231) cells (Pille et al., 2005; Wu et al., 2010). Positive correlation was reported between the expression level of RhoA and disease progression in breast cancer tumors (Fritz et al., 2002). RhoC was also reported as a Monitoring Editor J. Silvio Gutkind National Institutes of Health Received: Jun 20, 2013 Revised: Oct 4, 2013 Accepted: Oct 30, 2013 This article was published online ahead of print in MBoC in Press (http://www .molbiolcell.org/cgi/doi/10.1091/mbc.E13-06-0335) on November 6, 2013. Address correspondence to: Audrey Claing (audrey.claing@umontreal.ca). © 2014 Schlienger et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0). “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society of Cell Biology. Abbreviations used: ARF, ADP-ribosylation factor; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; MDA-MB-231, M.D. Anderson-metastatic breast-231; MLC, myosin light chain; MMP, metalloproteinase; PI3K, phosphoinositide 3-kinase; siRNA, small interfering RNA. http://www.molbiolcell.org/content/suppl/2013/11/04/mbc.E13-06-0335v1.DC1 Supplemental Material can be found at: