EGFR-dependent pancreatic carcinoma cell metastasis through Rap1 activation

Oncogene (2012) 31, 2783–2793 & 2012 Macmillan Publishers Limited All rights reserved 0950-9232/12 www.nature.com/onc ORIGINAL ARTICLE EGFR-dependent pancreatic carcinoma cell metastasis through Rap1 activation M Huang1, S Anand1, EA Murphy1, JS Desgrosellier1, DG Stupack1, SJ Shattil2, DD Schlaepfer3 and DA Cheresh1 1 Department of Pathology, Moores University of California San Diego Cancer Center, La Jolla, CA, USA; 2Department of Medicine, University of California San Diego, La Jolla, CA, USA and 3Reproductive Medicine, Moores University of California San Diego Cancer Center, La Jolla, CA, USA Tyrosine kinase receptors have an essential role in various aspects of tumor progression. In particular, epidermal growth factor receptor (EGFR) and its ligands have been implicated in the growth and dissemination of a wide array of human carcinomas. Here, we describe an EGFRmediated signaling pathway that regulates human pancreatic carcinoma cell invasion and metastasis, yet does not influence the growth of primary tumors. In fact, ligation/activation of EGFR induces Src-dependent phosphorylation of two critical tyrosine residues of p130CAS, leading to the assembly of a Crk-associated substrate (CAS)/Nck1 complex that promotes Ras-associated protein-1 (Rap1) signaling. Importantly, GTP loading of Rap1 is specifically required for pancreatic carcinoma cell migration on vitronectin but not on collagen. Furthermore, Rap1 activation is required for EGFR-mediated metastasis in vivo without impacting primary tumor growth. These findings identify a molecular pathway that promotes the invasive/metastatic properties of human pancreatic carcinomas driven by EGFR. Oncogene (2012) 31, 2783–2793; doi:10.1038/onc.2011.450; published online 3 October 2011 Keywords: pancreatic cancer; Rap1; CAS; EGFR; metastasis; tyrosine phosphorylation Introduction Although the significance of metastatic burden on mortality from pancreatic carcinoma is well appreciated, the molecular mechanisms that govern such aggressive invasive behavior remain poorly understood. Current efforts have focused on targeted therapies, including inhibitors of epidermal growth factor receptor (EGFR) signaling, a pathway, which is often dysregulated in pancreatic adenocarcinoma (Rivera et al., 2009). HowCorrespondence: Dr DA Cheresh, Department of Pathology, Moores University of California San Diego Cancer Center, Room 2344, 3855 Health Sciences Drive, MC 0803, La Jolla, CA 92093-1503, USA. E-mail: Received 29 April 2011; revised 5 August 2011; accepted 30 August 2011; published online 3 October 2011 ever, resistance to anti-EGFR therapies frequently occurs through mechanisms that activate downstream mediators independent of EGFR activation (Faller and Burtness, 2009). This could include signaling through other growth factor receptors, such as recepteur d’Origine nantais (RON) and insulin-like growth factor (IGF)-1 receptor (Guix et al., 2008). Both receptors have been shown to cross-talk with EGFR (Peace et al., 2003; Morgillo et al., 2006) and their expression correlates with disease progression (Thomas et al., 2007; Wolpin et al., 2007). The small GTPase Ras-associated protein-1 (Rap1) is activated downstream from EGFR and is a regulator of integrin activation, cell adhesion and migration (Shimonaka et al., 2003; Bos, 2005; Jenei et al., 2005; Shattil et al., 2010). Rap1 acts as a signaling switch that cycles between an inactive GDP-bound form and an active GTP-bound form with the assistance of guanine exchange factors (GEFs) and GTPase-activating proteins (GAPs). Because of its low intrinsic GTPase activity, Rap1 relies on GTP hydrolysis by GAPs such as Rap1GAP, which has been identified as a putative tumor suppressor deficient in pancreatic carcinoma. Ectopic expression of Rap1GAP inhibits migration in pancreatic carcinoma cells and serves as a metastasis suppressor, which suggests that Rap1 activity is a critical determinant of tumor cell invasiveness (Zhang et al., 2006). Previously, we described two distinct pathways of tumor cell migration: one requiring a cross-talk between growth factor receptors and integrin-avb5, whereas the other independent of growth factor receptors and using one or more b1-integrins (Klemke et al., 1994; Brooks et al., 1997). We have also reported that growth factor induced carcinoma cell migration on the avb5 substrate vitronectin correlates with the cell’s metastatic properties (Klemke et al., 1998). In fact, EGFR-induced activation of Src-family kinases is necessary and sufficient for induction of migration on vitronectin in vitro and metastasis in vivo. Subsequent to Src-family kinase activation, the adaptor protein Crk-associated substrate (CAS) is phosphorylated on specific tyrosine residues in its substrate domain, which contains 15 YxxP motifs (Ricono et al., 2009). Deletion of the CAS substrate domain or mutation of all 15 YxxP motifs to FxxP has been shown to EGFR-mediated metastasis through Rap1 activation M Huang et al 2784 block tumor cell migration, invasion and metastasis (Klemke et al., 1998; Brabek et al., 2005). Tyrosine phosphorylation of CAS on the 15 YxxP motifs in its substrate domain creates docking sites for proteins that contain SH2 domains, including other pro-migratory signaling molecules such as Crk and Nck (Schlaepfer et al., 1997; Klemke et al., 1998; Rivera et al., 2006). In this study, we characterize a novel mechanism of Rap1 activation downstream from EGFR that requires the formation of a CAS/Nck1 complex. Activation of Rap1 is required to promote migration on vitronectin but not on collagen. Furthermore, Rap1-GTP loading is critical for EGFR-induced metastasis, without enhancing primary tumor growth. Our results reveal new insights into the mechanism of EGFR-mediated metastasis and establish a previously unrecognized connection between the CAS/Nck1 signaling module and Rap1 activation. Results Rap1 activation is required for EGFR-mediated metastasis Hyper-activation of EGFR induces the metastasis of a wide range of carcinoma cells (Kim and Muller, 1999; Papouchado et al., 2005; Matsumoto et al., 2006; Ueno et al., 2008). EGF stimulation also results in the selective induction of migration of these cells on a vitronectin substrate in vitro, suggesting that EGFR-induced migration on vitronectin might recapitulate specific aspects of metastatic invasion in the context of EGFR stimulation in vivo (Klemke et al., 1994; Brooks et al., 1997; Ricono et al., 2009). Previously, we implicated a role for Rap1 specifically in EGFR-dependent migration on vitronectin in the fast growing variant of COLO 357 (FG) human pancreatic tumor cells (Ricono et al., 2009). To determine whether Rap1 is also required for EGFR-mediated metastasis, FG cells stably expressing either a non-silencing short-hairpin RNA (shRNA) or a Rap1 shRNA were treated with or without EGF ex vivo and subsequently implanted on the chorioallantoic membrane (CAM) of 10-day-old chick embryos. Primary tumor g (...truncated)