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The Tumor-Suppressor Gene ARHI (DIRAS3) Suppresses Ovarian Cancer Cell Migration Through Inhibition of the Stat3 and FAK/Rho Signaling Pathways

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The Tumor-Suppressor Gene ARHI (DIRAS3) Suppresses Ovarian Cancer Cell Migration Through Inhibition of the Stat3 and FAK/Rho Signaling Pathways Oncogene (2012) 31, 68–79 & 2012 Macmillan Publishers Limited All rights reserved 0950-9232/12 www.nature.com/onc ORIGINAL ARTICLE The tumor-suppressor gene ARHI (DIRAS3) suppresses ovarian cancer cell migration through inhibition of the Stat3 and FAK/Rho signaling pathways DB Badgwell1,3,ZLu1,3,KLe1, F Gao1, M Yang1, GK Suh1, J-J Bao1, P Das1, M Andreeff 2, W Chen1,YYu1, AA Ahmed1, W S-L Liao1 and RC Bast Jr1 1Department of Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, TX, USA and 2Department of Blood and Marrow Transplantation, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Ovarian cancers migrate and metastasize over the surface Keywords: tumor-suppressor gene ARHI; migration of the peritoneal cavity. Consequently, dysregulation of suppression; Stat3; RhoA GTPase; cytoskeleton mechanisms that limit cell migration may be particularly important in the pathogenesis of the disease. ARHI is an imprinted tumor-suppressor gene that is downregulated in 460% of ovarian cancers, and its loss is associated Introduction with decreased progression-free survival. ARHI encodes a 26-kDa GTPase with homology to Ras. In contrast Epithelial ovarian cancers have been thought to arise to Ras, ARHI inhibits cell growth, but whether it also from cells that cover the ovarian surface or that line sub- regulates cell motility has not been studied previously. serosal inclusion cysts. Recently, cancers that resemble Here we report that re-expression of ARHI decreases the ovarian primaries have been shown to arise from the motility of IL-6- and epidermal growth factor (EGF)- fimbriae of the Fallopian tube, deposits of endometriosis stimulated SKOv3 and Hey ovarian cancer cells, inhibit- and from the peritoneal surface. Whatever the site of ing both chemotaxis and haptotaxis. ARHI binds to and origin, ovarian cancers spread through the abdominal sequesters Stat3 in the cytoplasm, preventing its trans- cavity, forming multiple implants on the peritoneal location to the nucleus and localization in focal adhesion surface. Consequently, genetic and epigenetic changes complexes. Stat3 siRNA or the JAK2 inhibitor AG490 that dysregulate motility are likely to be important for produced similar inhibition of motility. However, the the pathogenesis of ovarian cancer. Although ovarian combination of ARHI expression with Stat3 knockdown cancer can be cured in up to 90% of cases while still or inhibition produced greatest inhibition in ovarian confined to the ovary, approximately 70% are diag- cancer cell migration, consistent with Stat3-dependent nosed after the occurrence of peritoneal dissemination, and Stat3-independent mechanisms. Consistent with when the cure rate reduces to less than 30% (Jemal two distinct signaling pathways, knockdown of Stat3 et al., 2011). selectively inhibited IL-6-stimulated migration, whereas Ovarian surface epithelial cells are generally quies- knockdown of focal adhesion kinase (FAK) preferentially cent, with a low rate of proliferation. Following inhibited EGF-stimulated migration. In EGF-stimulated ovulation and rupture of the mature follicle, ovarian ovarian cancer cells, re-expression of ARHI inhibited surface epithelial cells proliferate and migrate to the site FAKY397 and SrcY416 phosphorylation, disrupted focal of ovulation in order to repair the wound left by release adhesions, and blocked FAK-mediated RhoA signaling, of the ovum. Once integrity of the ovarian surface is resulting in decreased levels of GTP-RhoA. Re-expression restored, normal ovarian epithelial cells cease to of ARHI also disrupted the formation of actin stress fibers migrate. Thus, the ability to proliferate and migrate is in a FAK- and RhoA-dependent manner. Thus, ARHI has an intrinsic characteristic of normal ovarian cells that is a critical and previously uncharacterized role in the regu- under tight regulation (Katabuchi and Okamura, 2003; lation of ovarian cancer cell migration, exerting inhibitory Okamura and Katabuchi, 2005). During ovarian onco- effects on two distinct signaling pathways. genesis, ovarian cancer cells lose regulatory constraints Oncogene (2012) 31, 68–79; doi:10.1038/onc.2011.213; on motility and invasion (Liotta et al., 1987; Katabuchi published online 6 June 2011 and Okamura, 2003). A number of factors can stimulate the migration of ovarian cancer cells (Bast et al., 2009), including Stat3, epidermal growth factor (EGF) and Correspondence: Dr RC Bast Jr, Department of Experimental fibronectin, but relatively little is known regarding the Therapeutics, The University of Texas MD Anderson Cancer Center, genetic or epigenetic factors that are dysregulated to 1515 Holcombe Boulevard, Box 355, Houston, TX 77030, USA. increase motility and migration. E-mail: [email protected] 3These authors contributed equally to this work. Cell migration is a highly regulated process that Received 23 September 2010; revised 13 March 2011; accepted 11 April involves continuous formation and turnover of protein 2011; published online 6 June 2011 complexes within focal adhesions that serve both as ARHI suppresses ovarian cancer cell migration DB Badgwell et al 69 points of traction and as signaling centers (Ridley et al., cycle arrest. Growth of ovarian and breast cancer 2003; Romer et al., 2006). Regulation of cell migration xenografts is reversibly suppressed by ARHI and during embryonic development, inflammation and survival of dormant cells appears to depend, at least in tumorigenesis is mediated by cytokines, growth factors part, on autophagy (Lu et al., 2008). The impact of and integrins through activation of intracellular signal- ARHI on cell motility and migration has not been ing molecules that include Stat3 and focal adhesion explored previously. kinase (FAK) (Naora and Montell, 2005; Jang et al., In the present study, using conditional expression of 2007; Tomar and Schlaepfer, 2009). physiological levels of ARHI in SKOv3 and Hey Stat3 is constitutively phosphorylated and activated ovarian cancer cell lines, we show that re-expression of in 70% of ovarian cancers (Rosen et al., 2004). ARHI inhibits ovarian cancer cell motility by interfering Frequently, this is associated with autocrine stimulation with JAK/Stat signal transduction, and inhibiting the of the interleukin-6 (IL-6) receptor recruiting JAK2 and FAK signaling pathway, decreasing the formation of phosphorylating Stat3. In addition to its nuclear role as focal adhesion complexes and stress fibers. a transcription factor, phosphorylated Stat3 has also been found to localize to focal adhesions, interact with focal adhesion proteins and contribute to ovarian cancer cell motility (Silver et al., 2004). Inhibition of Stat3 Results activation with chemical inhibitors or small interfering RNA (siRNA) reduced the motility of ovarian cancer ARHI reduces chemotactic and haptotactic responses, and cells. Further, the Stat3-associated increase in cell decreases the speed of cell migration motility was concomitant with upregulation of epithe- We have generated stable sub-lines of SKOv3 and Hey lial-mesenchymal transition-associated N-cadherin and ovarian cancer cells by Tet-on-inducible expression of vimentin expression (Colomiere et al., 2009). ARHI (SKOv3-ARHI and Hey-ARHI). These two FAK is also activated in ovarian cancers. FAK is ovarian cancer cell lines were chosen because SKOv3 localized in focal adhesions and regulates the cycle of cells express low levels of endogenous ARHI, whereas focal contact formation and disassembly required for Hey cells have no detectable ARHI (Feng et al., 2008; efficient cell movement (Geiger and Bershadsky, 2001). Lu et al., 2008). Incubation of each sub-line with 1 mg/ml FAK activation correlates with paxillin phosphorylation DOX induced moderate ARHI expression comparable and subsequent actin stress fiber formation (Schaller, to that observed in cultured normal surface epithelial 2001; Parsons, 2003). These events are often coupled to cells (Lu et al., 2008), as well as to the normal ovarian Rho-family GTPases that cycle between inactive, GDP- epithelium tissue as determined by immunohistochem- bound and active, GTP-bound forms. FAK can enhance ical staining (Figure 1a). To examine the effects of the activation of RhoA GTPase (Chikumi et al., 2002; ARHI on cell motility, SKOv3-ARHI and the parental Zhai et al., 2003), contributing to the regulation of the SKOv3 cell lines were treated with DOX and then actin cytoskeletal structure, focal adhesion complexes inoculated (1 Â 105 cells for chemotaxis and 2 Â 104 for and cell polarity, as well as cell–cell communication haptotaxis) in Boyden chambers and allowed to migrate (Van Aelst and D’Souza-Schorey, 1997; Kaibuchi et al., toward 10% fetal bovine serum or 5 mg/ml fibronectin 1999; Etienne-Manneville and Hall, 2002). for 16 h. Induction of ARHI expression significantly Dysregulation of Stat3 and FAK could relate, in part, decreased the fetal bovine serum- and fibronectin- to loss of function of critical tumor-suppressor genes. stimulated migration of SKOv3-ARHI cells when The maternally imprinted growth-regulatory gene ARHI compared with un-induced cells (Figure 1b, middle is a tumor-suppressor gene whose function is down- panels). As expected, DOX treatment had no effect on regulated or lost in 460% of ovarian cancers by several the chemotactic or haptotactic migration of parental different mechanisms, including loss of heterozygosity, SKOv3 cells (Figure 1b, left panels). Similar inhibition hypermethylation, transcriptional regulation and shor- in
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