The tumor-suppressor gene ARHI (DIRAS3) suppresses ovarian cancer cell migration through inhibition of the Stat3 and FAK/Rho signaling pathways.

Epithelial ovarian cancers have been thought to arise from cells that cover the ovarian surface or that line subserosal inclusion cysts. Recently, cancers that resemble ovarian primaries have been shown to arise from the fimbriae of the Fallopian tube, deposits of endometriosis and from the peritoneal surface. Whatever the site of origin, ovarian cancers spread through the abdominal cavity forming multiple implants on the peritoneal surface. Consequently, genetic and epigenetic changes that dysregulate motility are likely to be important for pathogenesis of ovarian cancer. Although ovarian cancer can be cured in up to 90% of cases while still confined to the ovary, approximately 70% are diagnosed after peritoneal dissemination has occurred, when the cure rate reduces to less than 30% ( Jemal et al ., 2011 ). Ovarian surface epithelial cells are generally quiescent with a low rate of proliferation. Following ovulation and rupture of the mature follicle, ovarian surface epithelial cells proliferate and migrate to the site of ovulation in order to repair the wound left by release of the ovum. Once integrity of the ovarian surface is restored, normal ovarian epithelial cells cease to migrate. Thus, the ability to proliferate and migrate is an intrinsic characteristic of normal ovarian cells that is under tight regulation ( Katabuchi and Okamura, 2003 ; Okamura and Katabuchi, 2005 ). During ovarian oncogenesis, ovarian cancer cells lose regulatory constraints on motility and invasion ( Katabuchi and Okamura, 2003 ; Liotta et al ., 1987 ). A number of factors can stimulate migration of ovarian cancer cells ( Bast et al ., 2009 ) including Stat3, EGF and fibronectin, but relatively little is known regarding the genetic or epigenetic factors that are dysregulated to increase motility and migration. Cell migration is a highly regulated process that involves continuous formation and turnover of protein complexes within focal adhesions that serve both as points of traction and as signaling centers ( Ridley et al ., 2003 ; Romer et al ., 2006 ). Regulation of cell migration during embryonic development, inflammation, and tumorigenesis is mediated by cytokines, growth factors, and integrins through activation of intracellular signaling molecules that include Stat3 and focal adhesion kinase (FAK) ( Naora and Montell, 2005 ; Jang et al ., 2007 ; Tomar and Schlaepfer, 2009 ). Stat3 is constitutively phosphorylated and activated in 70% of ovarian cancers ( Rosen, et al, 2004 ). Frequently, this is associated with autocrine stimulation of the IL-6 receptor recruiting JAK2 and phosphorylating Stat3. In addition to its nuclear role as 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 activation with chemical inhibitors or siRNA reduced the motility of ovarian cancer cells. Further, the Stat3-associated increase in cell motility was concomitant with upregulation of EMT-associated N-cadherin and vimentin expression ( Colomiere et al ., 2009 ). FAK is also activated in ovarian cancers. FAK is localized in focal adhesions and regulates the cycle of focal contact formation and disassembly required for efficient cell movement ( Geiger and Bershadsky, 2001 ). FAK activation correlates with, paxillin phosphorylation and subsequent actin stress fiber formation ( Parsons, 2003 ; Schaller, 2001 ). These events are often coupled to Rho-family GTPases that cycle between inactive GDP-bound and active GTP-bound forms. FAK can enhance the activation of RhoA GTPase ( Chikumi et al ., 2002 ; Zhai et al ., 2003 ), contributing to the regulation of actin cytoskeleton structure, focal adhesion complexes and cell polarity as well as cell-cell communication ( Etienne-Manneville and Hall, 2002 ; Kaibuchi et al ., 1999 ; Van Aelst and D’Souza-Schorey, 1997 ). Dysregulation of Stat3 and FAK could relate, in part, to loss of function in critical tumor suppressor genes. The maternally imprinted growth regulatory gene ARHI is a tumor suppressor gene whose function is downregulated or lost in >60% of ovarian cancers by several different mechanisms including loss of heterozygosity, hypermethylation, transcriptional regulation and shortened mRNA half-life ( Yu et al ., 2006 ). Loss of ARHI expression is associated with tumor progression in breast cancers and decreased disease-free survival in ovarian cancers ( Rosen et al ., 2004 ). ARHI encodes a 26 kDa protein with 55-62% homology to Ras. In contrast to Ras, ARHI inhibits, rather than stimulates cell growth. This growth inhibitory function has been attributed to a 34 amino-acid N-terminal extension that is unique to ARHI ( Luo et al ., 2003 ). Re-expression of ARHI in cancer cells inhibits signaling through Ras/MAPK and PI3 kinase, upregulates p21 WAF1/CIP1 , downregulates cyclin D1, induces JNK activation, and inhibits Stat3 signaling ( Luo et al ., 2003 ; Yu et al ., 1999 ). When ARHI is expressed at physiologic levels from a doxycycline (DOX)-inducible promoter, autophagy is induced, and cells undergo cell cycle arrest. Growth of ovarian and breast cancer xenografts is reversibly suppressed by ARHI and survival of dormant cells appears to depend, at least in part, on autophagy ( Lu et al ., 2008 ). The impact of ARHI on cell motility and migration has not previously been explored. In the present study, using conditional expression of physiological levels of ARHI in SKOv3 and Hey ovarian cancer cell lines, we demonstrate that re-expression of ARHI inhibits ovarian cancer cell motility by interfering with JAK/Stat signal transduction, and inhibiting the FAK signaling pathway, decreasing formation of focal adhesion complexes and stress fibers.

We have generated stable sublines of SKOv3 and Hey ovarian cancer cells with Tet-on inducible expression of ARHI (SKOv3-ARHI and Hey-ARHI). These two ovarian cancer cell lines were chosen because SKOv3 cells express low levels of endogenous ARHI while Hey cells have no detectable ARHI ( Lu et al ., 2008 ; Feng et al ., 2008 ). Incubation of each subline with 1 μg/ml of DOX induced moderate ARHI expression comparable to that observed in cultured normal surface epithelial cells ( Lu et al ., 2008 ) as well as to the normal ovarian epithelium tissue as determined by immunohistochemistry staining ( Fig. 1A ). To examine the effects of ARHI on cell motility, SKOv3-ARHI and the parental SKOv3 cell lines were treated with DOX and then inoculated (1×10 5 cells for chemotaxis and 2×10 4 for haptotaxis) in Boyden chambers and allowed to migrate toward 10% FBS or 5 μg/ml fibronectin for 16 hrs. Induction of ARHI expression significantly decreased FBS- and fibronectin-stimulated migration of SKOv3-ARHI cells when compared to uninduced cells ( Fig. 1B , middle panels). As expected, DOX treatment had no effect on the chemotactic or haptotactic migration of parental SKOv3 cells ( Fig. 1B , left panels). Similar inhibition in cell migration by ARHI was also observed in Hey-ARHI ovarian cancer cells after treatment with DOX ( Fig. 1B , right panels). To address whether expression of endogenous ARHI may also affect ovarian cancer cell migration, we knocked down ARHI in the parental SKOv3 and Hey cells and assessed their effects on cell migration. As shown in Figure 1C , knockdown of ARHI in ARHI-expressing SKOv3 cells resulted in an increase in cell migration while had no effect on Hey cells that do not express endogenous ARHI. To confirm that ARHI has an inhibitory effect on cell migration that is independent from its growth inhibitory effect, we performed live-cell time-lapse imaging of migrating cells in a scratch assay over a 4.5-hour interval. SKOv3-ARHI cells that express ARHI exhibited a 40% reduction in average migration speed when compared to those of uninduced cells ( Fig. 1D ). Many ovarian cancer cell lines have constitutively activated Stat3 ( Huang et al ., 2000 ) and >70% of ovarian cancers showed a higher nuclear localization of phosphorylated Stat3, which correlated with poor overall prognosis ( Rosen et al ., 2004 ). Furthermore, Stat3 has been reported to promote ovarian cancer cell motility ( Debidda et al ., 2005 ; Huang et al ., 2000 ; Silver et al ., 2004 ). We therefore sought to examine whether this Stat3-induced cell motility could be inhibited by ARHI. SKOv3-ARHI cells were stimulated with IL-6, and assayed for chemotaxis or haptotaxis. Treatment of SKOv3-ARHI cells with IL-6 induced both chemotactic and haptotactic cell migration by 2- to 3-fold ( Fig. 2A ). IL-6-induced cell migration was significantly reduced when ARHI was expressed. This ARHI-mediated inhibition of cell migration however is not due to inhibition of Stat3 tyrosine phosphorylation ( Fig. 2B ). As a control, SKOv3-ARHI cells were treated with AG490 to block IL-6-mediated Stat3 activation. Treatment with AG490 dramatically inhibited both basal and IL-6-induced cell migration. Interestingly, in the presence of AG490, ARHI expression can further reduce SKOv3-ARHI cell migration, suggesting that ARHI’s inhibitory effects on cell migration may not be limited to its inhibition of the JAK/Stat3 signaling pathway. To further evaluate the influence of Stat3 and ARHI on cell migration, SKOv3-ARHI cells were transfected with Stat3 siRNA to knockdown endogenous Stat3 and then treated with DOX to induce ARHI. Treatment of SKOv3-ARHI cells with different siStat3 effectively knocked down Stat3 ( Fig. 2C ) and reduced chemotactic cell migration by 25-45% when compared with siControl-transfected cells ( Fig. 2D ). Expression of ARHI in siStat3-transfected cells further reduced cell motility. Together, these data indicated that chemotaxis is IL-6-dependent while haptotaxis is not. Moreover, the expression of ARHI inhibited the basal and Stat3-mediated motility of ovarian cancer cells by Stat3-dependent and Stat3-independent mechanisms. Our earlier work demonstrated that Stat3 is an ARHI-interacting protein ( Nishimoto et al, 2005 ); however, these studies were done with adeno-ARHI infected breast cancer cells that produced supra-physiologic levels of ARHI. To document that ARHI expressed at physiological levels can interact with Stat3, we co-immunoprecipitated ARHI and Stat3 from cell lysates prepared from DOX-induced SKOv3-ARHI cells. Consistent with our previous results, Stat3 was co-immunoprecipitated with anti-ARHI antibody and anti-Stat3 co-immunoprecipitated ARHI ( Fig. 3A ), demonstrating that ARHI, expressed at physiological levels, can indeed interact with Stat3. As an integral component of cell motility, focal adhesion complexes form at the leading edge of moving cells and permit cells to spread in the forward direction ( Carragher and Frame, 2004 ). Silver and colleagues reported localization of activated Stat3 in focal adhesions and demonstrated a crucial role for Stat3 in the motility of ovarian cancer cells ( Silver et al ., 2004 ). Consistent with their observation, we also observed specific punctate Stat3 localization at the focal adhesion following stimulation with fibronectin ( Fig. 3B ). Since ARHI can interact with Stat3, we hypothesized that one mechanism by which ARHI may interfere with Stat3’s ability to promote cell motility is by sequestering Stat3 away from the focal adhesion complexes as well as from the nucleus. As such, ARHI may prevent Stat3 from affecting the function of focal adhesions or inducing genes that promote cell motility. To test this hypothesis, we performed immunofluorescent staining of ARHI, Stat3 and focal adhesion proteins to determine whether ARHI disrupts Stat3’s participation at the focal adhesion complexes and in the nucleus. SKOv3-ARHI cells were grown on fibronectin-coated slides with or without DOX and stained for Stat3, ARHI, and two focal adhesion markers, vinculin and paxillin. In the absence of ARHI, Stat3 staining was observed at the focal adhesions and colocalized with vinculin and paxillin ( Fig. 3B ). This co-localization was, however, disrupted when ARHI expression was induced. Our data thus demonstrate that expression of ARHI not only prevented Stat3 localization at the focal adhesions but also dramatically reduced the formation of focal adhesion complexes as reflected by the reduced vinculin and paxillin staining. To examine whether ARHI also affects Stat3 nuclear localization, SKOv3-ARHI cells were stimulated with IL-6 after DOX treatment. In control cells, IL-6 induced rapid Stat3 nuclear translocation within 30 min. However, expression of ARHI prevented Stat3 nuclear localization in response to IL-6, resulting in their co-localization in the cytoplasm ( Fig. 3C ). To confirm that the cytoplasmic sequestration of Stat3 by ARHI results in reduced Stat3-dependent transcription, we analyzed the expression of two Stat3 target genes, N-cadherin and vimentin, known to participate in motility of ovarian cancer cells ( Cheng et al ., 2008 ; Colomiere et al ., 2009 ; Vij et al ., 2008 ). Treatment of SKOv3-ARHI cells with IL-6 significantly increased N-cadherin and vimentin expression. This increase was inhibited in ARHI-expressing cells ( Fig. 3D ). However, in parental_SKOv3 cells incapable of expressing ARHI, DOX treatment did not change N-cadherin and vimentin expression (data not shown). Taken together, sequestration of Stat3 in the cytoplasm by ARHI reduces transcription of Stat3 target genes important for motility and also prevents translocation of Stat3 to focal adhesions where it is required for optimal cell migration. FAK is a major component of the focal adhesion complex that regulates cell motility. We therefore examined whether ARHI can also inhibit migration of SKOv3-ARHI cells through regulation of the FAK and Src signaling pathway. FAK activity is regulated by growth factor- and integrin-mediated cell adhesion. Activation of FAK by EGF or integrin clustering leads to autophosphorylation at Tyr397 (p-FAK Y397 ), a binding site for Src family kinases ( Long et al ., 2010 ; Schlaepfer and Mitra, 2004 ). We examined the effects of ARHI on FAK and Src activation by monitoring the phosphorylation of FAK Y397 and the phosphorylation of Src Y416 , respectively, in response to EGF or fibronectin. SKOv3-ARHI cells were treated with or without DOX in the presence or absence of fibronectin or EGF and then the levels of p-FAK Y397 and p-Src Y416 were analyzed. As shown in Figures 4A and 4B , expression of ARHI reduced p-FAK Y397 and p-Src Y416 levels induced by either fibronectin or EGF. Reduction in p-FAK Y397 but not total FAK levels by ARHI were further confirmed by immunofluorescent staining ( Fig. 4C ). Since ARHI interacts with and sequesters Stat3 in the cytoplasm, it raises the possibility that inhibition of FAK activation by ARHI might be due to reduced Stat3-FAK co-localization at the focal adhesion. Interestingly, knockdown of Stat3 did not reduce p-FAK Y397 levels ( Fig. 4D ). To further evaluate the independent roles of Stat3 and FAK in cell migration, SKOv3-ARHI cells were transfected with Stat3 siRNA and FAK siRNA to knockdown endogenous Stat3 and FAK ( Fig. 4E ) followed by DOX treatment to induce ARHI in the presence of IL-6 or EGF. Stimulation of SKOv3-ARHI cells with either IL-6 or EGF increased cell migration by 1.5- to 2-fold ( Fig. 4F ). Interestingly, the IL-6-stimulated increase was more dramatically affected by Stat3 knockdown (31% decrease; p <0.01) than by FAK knockdown. In contrast, EGF-mediated increase in cell migration was strongly affected by FAK knockdown (57% decrease; p <0.01) than by Stat3 knockdown ( Fig. 4F ). Finally, knockdown of both Stat3 and FAK resulted in most dramatic reduction in chemotactic cell migration. Additional expression of ARHI in these double knockdown SKOv3-ARHI cells did not further inhibit cell migration ( Fig. 4F ). Together, these data indicate that expression of ARHI inhibited the ovarian cancer cell motility through two distinct pathways, one dependent on Stat3 signaling and the other on FAK-mediated pathways. FAK activation is associated both with focal contact formation and the formation of actin stress fibers ( Orr and Wang, 2001 ; Orr et al ., 2000 ; Parsons, 2003 ). Furthermore, FAK signaling complex regulates fibronectin-associated RhoA activation and focal adhesion formation during cell migration ( Lim et al ., 2008 ). As EGF activates RhoA and stimulates motility ( Kakinuma et al ., 2008 ; Marcoux and Vuori, 2005 ), we asked whether inhibition of EGF-stimulated migration by ARHI may be mediated through inhibition of RhoA. Chemotaxis of SKOv3-ARHI cells was measured with or without EGF, the RhoA inhibitor C3 transferase, and a combination of the two agents. Addition of EGF increased motility of SKOv3-ARHI cells. Cell motility, however, was significantly reduced when ARHI was expressed ( Fig. 5A ). As expected, treatment with the RhoA inhibitor C3 transferase completely abrogated cell motility ( Fig. 5A ). Stimulation with EGF could only restore the migration slightly in both control and DOX-treated cells, suggesting that ARHI may regulate SKOv3-ARHI cell migration by inhibiting RhoA activation. We therefore tested directly whether ARHI can inhibit EGF-induced RhoA activation. A GFP-RhoA plasmid was transfected into cells treated with or without DOX and EGF. As predicted, the level of active RhoA was induced by EGF but was significantly reduced when ARHI was expressed ( Fig. 5B ). ARHI also significantly reduced stress fiber formation and cell migration stimulated by GFP-RhoA ( Fig. 5C and 5D ). Together, these data indicate that ARHI expression leads to decreased RhoA activity, and inhibition of stress fiber formation, both of which contribute to ARHI-mediated inhibition of SKOv3-ARHI cell migration.

Epithelial ovarian carcinoma is characterized by widespread intra-abdominal metastases mediated through surface shedding of tumor cells, migration and peritoneal implantation. Our current study demonstrates that re-expression of ARHI inhibited SKOv3 and Hey ovarian cancer cell migration and suggests that loss of ARHI expression may be one factor that contributes to increased motility and metastasis of ovarian cancer cells. Although ARHI has been implicated in tumor proliferation, dormancy, and autophagy, this is the first report that re-expression of ARHI inhibits motility, chemotaxis and haptotaxis, of human ovarian cancer cells. The underlying mechanism that accounts for the differential inhibitory effects between SKOv3 and Hey cells is unclear. However, factors such as the lower levels of ARHI expression in Hey-ARHI cells as well as differences in their genetic cell background are likely to influence the extent of inhibition. Interaction of ARHI and Stat3 is of particular interest in that Stat3 signaling is important both for proliferation and for motility. A significant fraction of ovarian cancers not only secrete IL-6 but also express IL-6 receptor, resulting in high cell proliferation and motility due to autocrine stimulation ( Lidor et al ., 1993 ; Martinez-Maza and Berek, 1991 ; Rustin et al ., 1993 ). Quiescent Stat3 exists mostly in the cytosol, whereas activated Stat3 translocates to the nucleus to induce gene transcription ( Zhong et al ., 1994 ). Nuclear Stat3 has been found in 70% of ovarian cancers, and was associated with poor prognosis. Using SKOv3-ARHI and Hey-ARHI inducible ovarian cancer cells, we have examined the effect of re-expression of ARHI at physiological levels on Stat3 signaling and motility. Although Stat3 tyrosine phosphorylation was unaffected by ARHI ( Fig. 2B ), ARHI expression was able to inhibit Stat3 translocation to the nucleus as well as to focal adhesion complexes ( Fig. 3B and C ). This inhibition is presumably due to physical interaction between ARHI and Stat3, resulting in sequestration of Stat3 in the cytoplasm. Stat3 has recently been shown to accumulate in the mitochondria and contribute to Ras-dependent cellular transformation ( Gough et al ., 2009 ). As such, ARHI may potentially regulate mitochondrial function by sequestering Stat3 in the cytoplasm. In addition, failure of Stat3 to translocate to the nucleus was associated with decreased expression of Stat3 target genes, N-cadherin and vimentin. Thus ARHI can inhibit both proliferation and motility by inhibiting a single critical mediator. Moreover, ovarian oncogenesis may require not only autocrine stimulation by IL-6, but also the loss of ARHI-mediated inhibition of Stat3 signaling. Our study confirms earlier reports that Stat3 plays a critical role in the motility of ovarian cancer cells ( Takeda et al ., 1997 ; Yamashita et al ., 2002 ). Localization of Stat3 at focal adhesions suggested a direct role in motility through protein-protein interaction. As such, Stat3 may serve as an adapter protein in integrin-mediated cell adhesion or could function as a sensor of adhesion, becoming activated in focal adhesion and translocating to the nucleus to alter gene expression in response to cell adhesion ( Roger et al ., 2006 ). Alternatively, nuclear translocation of activated Stat3 from focal adhesions may induce critical proteins needed for motility. In either case, sequestration by ARHI inhibits Stat3 function as it relates to motility. FAK is activated by a variety of growth factors receptors and integrins and transmits signals downstream to a variety of target molecules to regulate the cycle of focal contact formation and disassembly required for efficient cell movement. Thus, FAK acts as an integrator of cell motility-associated signaling events. Growth factor stimulates cell motility by inducing the phosphorylation of FAK; however FAK Y397 phosphorylation is inhibited when ARHI was induced ( Fig. 4 ). Given that Stat3 knockdown did not alter p-FAK Y397 levels, we reasoned that the effect of ARHI on FAK activity might relate to decreased association of paxillin and vinculin with focal adhesions ( Fig. 3B ). Indeed, correlation between FAK activation, paxillin and vinculin phosphorylation, and the subsequent actin stress fiber formation have been reported ( Parsons, 2003 ; Schaller, 2001 ). These events are often associated with Rho-family GTPases that act as switches between an inactive GDP-bound form or an active GTP-bound form. Importantly, FAK promotes p190RhoGEF tyrosine phosphorylation and enhances activation of RhoA ( Zhai et al ., 2003 ). ARHI decreased EGF-induced chemotaxis of SKOv3-ARHI cells. Chemotaxis in response to EGF was reduced by FAK knockdown ( Fig. 4F ), and also impaired by a RhoA inhibitor, C3 transferase; however, chemotaxis was, not further inhibited by ARHI ( Fig. 5A ), indicating that ARHI may regulate cell migration by reducing FAK-mediated RhoA activation. We confirmed that RhoA activity, which is critical for the formation of actin stress fiber and cell migration ( Kurokawa et al ., 2005 ), is largely abrogated upon ARHI induction ( Fig. 5B ). Additionally, cell migration requires integration of specific focal adhesion dynamics, including formation of actin stress fibers which is inhibited by ARHI ( Fig. 5C ). Together, our results suggest that ARHI regulates ovarian cell migration by interfering with the function of Stat3, by inhibiting FAK and RhoA activation, and by decreasing formation of stress fibers ( Fig. 6 ). Integrative functions that include the control of cell motility have been reported for other genes known to play a critical role in oncogenesis. They include the tumor suppressor proteins TP53 ( Roger et al ., 2006 ), PTEN ( Raftopoulou et al ., 2004 ) or LKB-1 ( Forcet et al ., 2005 ). Our data indicate that ARHI tumor suppressor function is not limited to its anti-proliferative activity but may also rely on its combined effects in cell migration of ovarian cancer cells. Considering that Stat3 and FAK are constitutively activated in many ovarian cancers, the loss of ARHI expression in the majority of ovarian cancers may result in up-regulation of Stat3 and FAK activity and thereby contribute to oncogenesis.

Antibodies against Stat3, p-Src Y416 , paxillin, FAK and β-actin were purchased from Cell Signaling Technology (Beverly, MA) and anti-p-FAK Y397 antibody was from Millipore (Worcester, MA). Antibodies against ARHI were generated in our laboratory. AG490 was purchased from Calbiochem (La Jolla, CA). Cell-permeable RhoA inhibitor C3 transferase was from Cytoskeleton (Denver, CO). IL-6, EGF, and anti-vinculin were purchased from Sigma (St. Louis, MO), and siRNAs and Dharmafect #4 transfection reagent were from Dharmacon Research (Lafayette, CO). Cells were maintained in McCoy’s 5A (SKOv3 and SKOv3-ARHI) or RPMI (Hey-ARHI) medium, supplemented with 10% Tet-system approved fetal bovine serum, 200 μg/ml G418, and 0.25 μg/ml puromycin, 100 mM L-glutamine, 100 μg/ml streptomycin and 100 U/ml penicillin and grown at 37°C in 95% humidified air and 5% CO 2 . Physiologic levels of ARHI were achieved 16-24 hrs after addition of 1 μg/ml DOX to the culture medium ( Lu et al ., 2008 ). Briefly, 1×10 5 cells (SKOv3 and SKOv3-ARHI) or 5×10 4 cells (Hey-ARHI) in 0.5 ml serum-free medium were introduced into the upper compartment of Boyden chambers (BD Discovery Labware, Franklin Lakes, NJ) fitted with membranes of 3.0 μm porosity separating the upper and lower compartments. The lower compartment was filled with normal culture medium, medium supplemented with IL-6 (chemotaxis) or serum-free media with 5 μg/ml fibronectin (haptotaxis). After 16 hrs of incubation, cells were wiped from the upper surface of each membrane. Cells on the lower surface were stained with Diff-Quick (Siemens, Deerfield, IL) and counted in ten representative fields. Each condition was assayed in triplicate and each experiment was repeated at least three times. ARHI expression was induced with DOX for 24 hrs before performing chemotaxis and hapotaxis assays and DOX treatment was maintained throughout the assay. For treatment with Stat3 inhibitors or activators, cells were incubated with 100 μM AG490, a JAK2 inhibitor, for 2 hrs followed by 30 min incubation with AG490, AG490 plus IL-6, IL-6 plus DMSO, or DMSO in serum-free medium. For knockdown experiments, cells were transfected with siRNA and treated with DOX for 48 hrs; 1×10 5 cells were inoculated into the upper chamber, and with complete medium in the bottom chamber. Cells migrated to the lower surface were counted 16 hrs later and migration was expressed as the mean number of cells in triplicate wells from three independent experiments. SKOv3-ARHI cells (4×10 5 ) were plated in glass bottom dishes (MatTek, Ashland, MA) and grown to confluence with or without DOX. Cells were scratched with a 10 μl pipette tip and placed onto the microscope stage in a 37°C chamber with 5% CO 2 . Scratch wound closure was monitored by DIC microscopy and an Olympus IX-81 inverted microscope (Olympus, Center Valley, PA). Images were captured every 15 min using the Slidebook software ( http://www.intelligent-imaging.com/home.php ) to manage the Hamamatus Orca II ER camera and microscope settings. Paraffin-embedded normal ovarian epithelium tissue and cell pellets from DOX-treated or untreated SKOv3-ARHI cells were sectioned. After initial deparaffinization, endogenous peroxidase activity was blocked using 0.3% hydrogen peroxide and steamed in 1X Diva Decloaker (Biocare medical DV2004LX) for 1 hr to restore latent epitopes. The slides were then incubated with anti-ARHI antibody (15E11) at final concentration of 15 μg/ml at 4°C overnight. The slides were washed and incubated 30 min each with biotin-labeled secondary antibody and then with streptavidin/peroxidase. The slides were then incubated with DAB substrate (Biocare medical BDB 2004L), counterstained with hematoxylin, and mounted in Permount. SKOv3-ARHI cells were plated onto slides with or without fibronectin and treated with DOX for 24 hrs, fixed in 4% paraformaldehyde, and permeabilized using 0.5% Triton X-100. Washed cells were blocked with 3% BSA and incubated with antibodies against Stat3, vinculin, paxillin, or p-FAK Y397 . After washing, cells were incubated with secondary antibodies conjugated with Alexa Fluor 488 or 594 (Molecular Probe, Invitrogen). Actin stress fibers were stained with rhodamin-labeled phalloidin (1/1000; Molecular Probes, Invitrogen). Finally, cells were mounted and examined using confocal microscopy (Olympus FluoView 500 or 1000, Olympus, Inc., Melville, NY). SKOv3-ARHI cells (1×10 6 ) were incubated with or without DOX for 24 hrs and then treated with lysis buffer (50 mM Hepes, pH7.0, 150 mM NaCl, 1.5 mM MgCl 2 , 1 mM EGTA, 100 mM NaF, 10 mM sodium pyrophosphate, 10% glycerol, 1% Triton X-100, 1 mM PMSF, 1 mM Na 3 VO 4 , 10 μg/ml leupeptin, and 10 μg/ml aprotinin). For immunoprecipitation, 3 μg of anti-Stat3 or anti-ARHI antibody was added to 2 mg of protein lysate. After overnight incubation at 4°C, protein G beads were added and incubated for 1.5 hrs at 4°C. After washing with wash buffer (10 mM Tris, pH7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM Na 3 VO 4 , 1 mM PMSF, 0.5% Triton X-100, and 0.2% NP-40), immunoprecipitated proteins were eluted for Western blotting. Proteins were separated on 12 or 15% SDS-PAGE, transferred to PVDF membranes, and subjected to Western blotting using an ECL chemiluminescence reagent (GE/Amersham, Piscataway, NJ). Quantification of protein band intensities was done using the Jave-based image processing program ImageJ developed at the NIH. A mixture of siRNA (100 nM final concentration) and Dharmafect #4 transfection reagents were incubated for 20 min at room temperature. This mixture was then added to cells and allowed to incubate for 48 hrs before cells were harvested for protein expression measurements or for motility assays. Expression of Stat3-responsive genes was measured using real-time quantitative reverse transcription-PCR (RQ RT-PCR). Total cDNA was synthesized using 2 μg of total RNA. The reverse transcriptase reaction was done according to the manufacturer’s instructions using oligo(dT)16 and SuperScript II reverse transcriptase (Invitrogen) followed by SYBR Green RT-PCR (ABI Prism 7000_Sequence Detection System, Applied Biosystems, Foster City, CA). The primers for N-cadherin were 5’-GCCTGCAGATTTTAAGGTGG-3’ and 5’-CTCTTGAGGAAAAGGTCCCC-3’ , and for vimentin were 5’-GAGAACTTTGCCGTTGAAGC-3’ and 5’-TTCAGGGAGGAAAAGTTTGG-3’. mRNA levels were normalized with a concurrent determination for GAPDH mRNA. SKOv3-ARHI cells (1×10 6 ) were transfected with 16 μg of GFP-RhoA plasmid (Addgene plasmid 12965, Cambridge, MA), and harvested 48 hrs later. RhoA activity was measured with a RhoA activation assay kit (Cell Biolabs, San Diego, CA). Levels of active RhoA were determined by Western analysis. Band intensity from Western blots were quantified by ImageJ ( Ferreira and Rasband, 2011 ). Fluorescence intensity was quantified by ImageJ ( Bongard, 2005 ). Briefly, cells in a region of interest were encircled using the program’s freehand selection tool. The total fluorescence corresponding to the staining for p-FAK Y397 or total FAK was quantified and the average fluorescence intensity calculated by dividing the total fluorescence intensity by the cell number. All experiments were repeated independently at least two times and the data expressed as mean ±SE. Statistical analysis was performed using Student’s t test (two-tailed). The criterion for statistical significance was taken as p <0.05 (two-sided).