Oncogene (2011) 30, 1566–1576 & 2011 Macmillan Publishers Limited All rights reserved 0950-9232/11 www.nature.com/onc ORIGINAL ARTICLE Ligand-independent activation of c-Met by fibronectin and a5b1-integrin regulates ovarian cancer invasion and metastasis AK Mitra1, K Sawada1, P Tiwari1, K Mui1, K Gwin2 and E Lengyel1 1Department of Obstetrics and Gynecology, Section of Gynecologic Oncology, Center for Integrative Science, University of Chicago, Chicago, IL, USA and 2Pathology, University of Chicago, Chicago, IL, USA The role of the fibronectin receptor, a5b1-integrin, as an route commonly observed in other cancers. The cancer adhesion receptor and in angiogenesis is well established. cells exfoliate from the surface of the ovary by shedding However, its role in cancer cell invasion and metastasis from the primary tumor or through hydroflotation is less clear. We describe a novel mechanism by which caused by circulating peritoneal fluid. They subse- fibronectin regulates ovarian cancer cell signaling and quently attach to the peritoneal surfaces and invade to promotes metastasis. Fibronectin binding to a5b1-integrin form metastatic tumors. This mode of metastasis places led to a direct association of a5-integrin with the receptor unique demands on the ovarian cancer cells and may tyrosine kinase, c-Met, activating it in a hepatocyte require a specific molecular mechanism different from growth factor/scatter factor (HGF/SF) independent manner. the intra- and extravasation involved in hematogenous Subsequently, c-Met associated with Src, and activated metastasis (Lengyel, 2010). Src and focal adhesion kinase (FAK). Inhibition of a5b1- Integrins are among the genes reported to be involved integrin decreased the phosphorylation of c-Met, FAK and in ovarian cancer metastasis. They are heterodimeric Src, both in vitro and in vivo. Independent activation of transmembrane proteins comprised of various combina- c-Met by its native ligand, HGF/SF, or overexpression tions of an a- and b-subunit, with each heterodimer of a constitutively active FAK in HeyA8 cells could possessing one or more ligands (Hynes, 2002). Integrins overcome the effect of a5b1-integrin inhibition on tumor anchor the cancer cells to the extracellular matrix cell invasion, indicating that a5b1-integrin is upstream of (ECM), which provides a contact point for invasion c-Met, Src and FAK. Inhibition of a5b1-integrin on cancer and metastasis. Signals from the ECM are transmitted cells in two xenograft models of ovarian cancer metastasis via integrins through interactions with multiple proteins resulted in a significant decrease of tumor burden, that eventually determine the cellular response to which was independent of the effect of a5b1-integrin on the ECM, including changes in cell shape, motility, angiogenesis. These data suggest that fibronectin pro- proliferation and so on (Giancotti and Tarone, 2003; motes ovarian cancer invasion and metastasis through an Mitra and Schlaepfer, 2006). The fibronectin receptor a5b1-integrin/c-Met/FAK/Src-dependent signaling path- (a5b1-integrin) primarily binds fibronectin to anchor way, transducing signals through c-Met in an HGF/ cells and subsequently activates the non-receptor tyrosine SF-independent manner. kinases (RTKs), focal adhesion kinase (FAK) and Src, Oncogene (2011) 30, 1566–1576; doi:10.1038/onc.2010.532; which play an important role in tumorigenesis by published online 29 November 2010 promoting the proliferation and invasion of cancer and endothelial cells (Mitra and Schlaepfer, 2006; Keywords: ovarian cancer; a5b1-integrin; c-Met; Src; Caswell et al., 2007). FAK; fibronectin Knocking out a5b1-integrin is associated with major vascular defects, such as a marked decrease in capillary branching and incompletely formed vessels in mouse Introduction embryos, leading to embryonic lethality (Yang et al., 1993; Francis et al., 2002). As a5b1-integrin is abun- Ovarian cancer is the fifth leading cause of cancer- dantly present on endothelial cells, research on this related death among women in the United States and integrin has primarily focused on its role in angiogenesis has the highest mortality rate of all gynecologic and elucidated its function in vascular endothelial cells malignancies (Jemal et al., 2009). It involves a unique (Kim et al., 2000; Taverna and Hynes, 2001; Magnussen metastatic process, which rarely takes the hematogenous et al., 2005). Endothelial cells in tumor vessels over- express a5b1-integrin, which helps to anchor them to the Correspondence: Dr E Lengyel, Department of Obstetrics and basement membrane. When antibodies against a5b1- Gynecology, Section of Gynecologic Oncology, Center for Integrative integrin are injected intravenously, they rapidly label Science, University of Chicago, 5841 South Maryland Avenue, MC tumor vessels (Magnussen et al., 2005), highlighting the 2050, Chicago, IL 60637, USA. E-mail: [email protected] importance of a5b1-integrin for tumor vasculature. Received 20 May 2010; revised 16 September 10; accepted 11 October However, the role of a5b1-integrin, when expressed on 2010; published online 29 November 2010 tumor cells, is less well understood. We have previously a5b1-integrin and c-Met in ovarian cancer AK Mitra et al 1567 shown that, during ovarian cancer progression, loss of Currently, the most completely characterized function E-cadherin-upregulated a5b1-integrin, which promoted of a5b1-integrin in cancer is its contribution to the adhesion and invasion of ovarian cancer cells. angiogenesis (Wingerter et al., 2005; Avraamides et al., Overexpression of a5b1-integrin indicated a very poor 2008). However, the human a5-integrin antibody used in prognosis in patients with epithelial ovarian cancer Figures 1a and b does not cross-react with murine a5b1- (Sawada et al., 2008). However, the molecular mechan- integrin (Bhaskar et al., 2007), indicating that the effects ism by which a5b1-integrin promotes cancer cell seen on metastasis and tumor growth are the result of a metastasis is currently unknown. Given that fibronectin direct effect of the antibody on the human tumor cells is one of the most abundant proteins in the ECM of rather than the blocking of host a5b1-integrin function. both the peritoneum and omentum (Kenny and Lengyel, To compare the contribution of a5b1-integrin in cancer 2009; Hafter et al., 1984; Franke et al., 2003), an cells with that in host stromal cells, we took advantage understanding of how fibronectin–a5b1-integrin interac- of an anti-murine a5Ab that inhibits angiogenesis by tions regulate metastasis in the ovarian cancer cell might blocking a5b1-integrin on mouse vascular endothelial provide additional insights into the biology of ovarian cells (Bhaskar et al., 2007). The specificity of the human cancer. and murine a5-integrin antibody was confirmed by In the present study, we show that upon binding to testing their effect on adhesion of the human ovarian fibronectin, a5b1-integrin interacts directly with the cancer cell line HeyA8 and the mouse ovarian cancer cell receptor tyrosine kinase c-Met, thereby, activating Src line ID-8 on fibronectin (Supplementary Figure S3). and FAK. This is the first report to demonstrate that the Treatment with the human or the murine a5-integrin fibronectin receptor transduces signals through c-Met, antibody showed a comparable reduction in tumor which is itself a major player in ovarian cancer weight (57 and 48%, respectively) and numbers (73 and metastasis. 64%, respectively) in a SKOV3ip xenograft model (Figure 1c). This reduction was similar to the effect of bevacizumab (Avastin, Genentech, South San Francisco, Results CA, USA), a humanized anti-VEGF antibody. Combin- ing the human and murine a5b1-integrin antibodies did Inhibition of a5b1-integrin inhibits ovarian cancer not significantly augment the antitumor effect of each metastasis individual antibody (Figure 1c). Immunohistochemical The ECM of the normal human omentum, the most staining of the tumors for CD31 confirmed an anti- common site of ovarian cancer metastasis, expresses angiogenic effect of murine a5Ab and bevacizumab, abundant fibronectin and vitronectin (Supplementary whereas, as expected, the human antibody had no effect Figure S1). We and others have previously shown that on blood vessel density (Supplementary Figure S4) or on the fibronectin receptor, a5b1-integrin, is upregulated in VEGF expression (data not shown). ovarian cancer cells during tumor progression (Yokoyama Next, we sought to determine whether a5b1-integrin et al., 2007; Sawada et al., 2008). Therefore, we decided plays a more important role in early or in late metastasis to inhibit a5b1-integrin to determine whether this would (Figure 1d). To study early metastasis, a single treat- result in a reduction of ovarian cancer dissemination. ment of the integrin antibody was given to the nude mice Two orthotopic xenograft models of ovarian cancer simultaneously with the intraperitoneal injection of metastasis were treated with a monoclonal antibody ovarian cancer cells to block the initial attachment and blocking human a5b1-integrin (Hu a5Ab) (Bhaskar invasion of tumor cells to the peritoneum (prevention et al., 2007). SKOV3ip1 or HeyA8 human ovarian study). Late metastasis was studied with the adminis- cancer cells, both of which have high expression levels of tration of repeated treatments beginning 8 days after the a5b1-integrin (Supplementary Figure S2), were injected tumors had established within the peritoneal cavity intraperitoneally into nude mice. Although SKOV3ip1 (intervention