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

Abstract

Ovarian cancers migrate and metastasize over the surface of the peritoneal cavity. Consequently, dysregulation of 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 >60% of ovarian cancers, and its loss is associated with decreased progression-free survival. ARHI encodes a 26-kDa GTPase with homology to Ras. In contrast to Ras, ARHI inhibits cell growth, but whether it also regulates cell motility has not been studied previously. Here we report that re-expression of ARHI decreases the motility of IL-6- and epidermal growth factor (EGF)-stimulated SKOv3 and Hey ovarian cancer cells, inhibiting both chemotaxis and haptotaxis. ARHI binds to and sequesters Stat3 in the cytoplasm, preventing its translocation to the nucleus and localization in focal adhesion complexes. Stat3 siRNA or the JAK2 inhibitor AG490 produced similar inhibition of motility. However, the combination of ARHI expression with Stat3 knockdown or inhibition produced greatest inhibition in ovarian cancer cell migration, consistent with Stat3-dependent and Stat3-independent mechanisms. Consistent with two distinct signaling pathways, knockdown of Stat3 selectively inhibited IL-6-stimulated migration, whereas knockdown of focal adhesion kinase (FAK) preferentially inhibited EGF-stimulated migration. In EGF-stimulated ovarian cancer cells, re-expression of ARHI inhibited FAKY397 and SrcY416 phosphorylation, disrupted focal adhesions, and blocked FAK-mediated RhoA signaling, resulting in decreased levels of GTP-RhoA. Re-expression of ARHI also disrupted the formation of actin stress fibers in a FAK- and RhoA-dependent manner. Thus, ARHI has a critical and previously uncharacterized role in the regulation of ovarian cancer cell migration, exerting inhibitory effects on two distinct signaling pathways.

Introduction

Epithelial ovarian cancers have been thought to arise from cells that cover the ovarian surface or that line sub-serosal 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 the 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 the occurrence of peritoneal dissemination, 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 (Liotta et al., 1987; Katabuchi 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 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 interleukin-6 (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 small interfering RNA (siRNA) reduced the motility of ovarian cancer cells. Further, the Stat3-associated increase in cell motility was concomitant with upregulation of epithelial-mesenchymal transition-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 (Schaller, 2001; Parsons, 2003). 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 the actin cytoskeletal structure, focal adhesion complexes and cell polarity, as well as cell–cell communication (Van Aelst and D’Souza-Schorey, 1997; Kaibuchi et al., 1999; Etienne-Manneville and Hall, 2002).

Dysregulation of Stat3 and FAK could relate, in part, to loss of function of 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 phosphatidylinositol-3-kinase, upregulates p21WAF1/CIP1, downregulates cyclin-D1, induces JNK activation and inhibits Stat3 signaling (Yu et al., 1999; Luo et al., 2003). When ARHI is expressed at physiological 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 been explored previously.

In the present study, using conditional expression of physiological levels of ARHI in SKOv3 and Hey ovarian cancer cell lines, we show that re-expression of ARHI inhibits ovarian cancer cell motility by interfering with JAK/Stat signal transduction, and inhibiting the FAK signaling pathway, decreasing the formation of focal adhesion complexes and stress fibers.

Results

ARHI reduces chemotactic and haptotactic responses, and decreases the speed of cell migration

We have generated stable sub-lines of SKOv3 and Hey ovarian cancer cells by 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, whereas Hey cells have no detectable ARHI (Feng et al., 2008; Lu et al., 2008). Incubation of each sub-line with 1 μg/ml 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 immunohistochemical staining (Figure 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 × 105 cells for chemotaxis and 2 × 104 for haptotaxis) in Boyden chambers and allowed to migrate toward 10% fetal bovine serum or 5 μg/ml fibronectin for 16 h. Induction of ARHI expression significantly decreased the fetal bovine serum- and fibronectin-stimulated migration of SKOv3-ARHI cells when compared with un-induced cells (Figure 1b, middle panels). As expected, DOX treatment had no effect on the chemotactic or haptotactic migration of parental SKOv3 cells (Figure 1b, left panels). Similar inhibition in cell migration by ARHI was also observed in Hey-ARHI ovarian cancer cells after treatment with DOX (Figure 1b, right panels). To address whether the 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, whereas 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-h interval. SKOv3-ARHI cells that express ARHI showed a 40% reduction in average migration speed when compared with those of un-induced cells (Figure 1d).

Figure 1
figure1

ARHI expression inhibits the chemotaxis, haptotaxis and migration speed of ovarian cancer cells. (a) Comparable ARHI expression in DOX-induced SKOv3-ARHI cells and the normal ovarian epithelium (NOE) tissue. Immunohistochemical staining for ARHI expression in NOE and SKOv3-ARHI cells with and without DOX. (b) Chemotaxis and haptotaxis. Parental SKOv3, SKOv3-ARHI and Hey-ARHI cells were treated with or without 1 μg/ml DOX for 24 h before addition to the upper compartment of Boyden chambers. The cells were allowed to migrate for 16 h toward the lower chambers, which contained either 10% fetal bovine serum (chemotaxis) or 5 μg/ml fibronectin (haptotaxis). The cells that had migrated through the membrane were counted and expressed as the mean number of cells in triplicates from three independent experiments. The columns indicate the mean, and the bars indicate the s.e. (*P<0.05; **P<0.01). (c) Knockdown of endogenous ARHI increases cell motility. Left panel: Parental SKOv3 and Hey cells were transfected with siARHI and assayed for cell motility (**P<0.01). Right panel: Endogenous ARHI expression was determined by real-time quantitative reverse transcription–PCR. (d) Migration speed in scratch assays. SKOv3-ARHI cells were grown to confluence with or without DOX. Following a scratch wound, wound closure was recorded over a 4.5-h time period by live-cell time-lapse microscopy. Representative images at the indicated time points are shown. The speeds of migration are calculated as distance over time and expressed as the mean of cell migration speed (n=6 in each group; **P<0.01). DOX, doxycycline.

ARHI inhibits cell migration induced by the JAK/Stat3 signaling pathway

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 (Huang et al., 2000; Silver et al., 2004; Debidda et al., 2005). 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 (Figure 2a). IL-6-induced cell migration was significantly reduced when ARHI was expressed. This ARHI-mediated inhibition of cell migration, however, is not because of inhibition of Stat3 tyrosine phosphorylation (Figure 2b). As 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.

Figure 2
figure2

ARHI inhibits cell migration by interfering with JAK/Stat signaling. (a) Stat3 activation enhances and Stat3 inhibition decreases ovarian cancer cell migration. SKOv3-ARHI cells in Boyden chambers were treated with IL-6 and AG490, individually or together, and allowed to migrate toward the lower chambers containing either 10% fetal bovine serum (chemotaxis) or 5 μg/ml fibronectin (haptotaxis). The columns indicate the mean, and the bars indicate the s.e. (**P<0.01). (b) ARHI expression does not affect Stat3 phosphorylation at tyrosine-705. (c) Knockdown of Stat3 in SKOv3-ARHI cells with siStat3 as indicated by western blotting. Two individual Stat3 siRNAs (1 and 2) as well as a pooled Stat3 siRNA (P) were used in the knockdown. (d) Knockdown of Stat3 and induction of ARHI produce additive inhibition of ovarian cancer cell migration. Migration assays of cells transfected with three different Stat3 siRNAs or a control siRNA with or without pretreatment with DOX for 48 h. The columns indicate the mean, and the bars indicate the s.e. (*P<0.05; **P<0.01). DOX, doxycycline; IL, interleukin; siRNA, small interfering RNA.

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 (Figure 2c) and reduced chemotactic cell migration by 25–45% when compared with that in siControl-transfected cells (Figure 2d). Expression of ARHI in siStat3-transfected cells further reduced cell motility. Together, these data indicated that chemotaxis is IL-6-dependent whereas haptotaxis is not. Moreover, expression of ARHI inhibited the basal and Stat3-mediated motility of ovarian cancer cells by Stat3-dependent and Stat3-independent mechanisms.

ARHI complexes with Stat3 and prevents its localization in the focal adhesions and in the nucleus

Our earlier work showed that Stat3 is an ARHI-interacting protein (Nishimoto et al, 2005); however, these studies were performed with adeno-ARHI-infected breast cancer cells that produced supra-physiological 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 an anti-ARHI antibody and anti-Stat3 co-immunoprecipitated ARHI (Figure 3a), showing that ARHI, expressed at physiological levels, can indeed interact with Stat3.

Figure 3
figure3

ARHI interacts with Stat3 and prevents its localization to the focal adhesion complex and to the nucleus. (a) Stat3 and ARHI co-immunoprecipitation. SKOv3 cells were incubated with or without DOX for 48 h. Cell lysates were incubated with anti-ARHI or anti-Stat3 antibody, and analyzed by western blotting. IgG was included as a negative control. (b) ARHI expression disrupts the colocalization of Stat3, vinculin and paxillin on immunofluorescence staining. SKOv3-ARHI cells were treated with fibronectin and stained with an anti-Stat3 antibody in the absence or presence of a Stat3-blocking peptide (upper panels 1–3). DOX-induced SKOv3-ARHI cells were stained with anti-vinculin and anti-Stat3 (middle panels), or anti-paxillin and anti-Stat3 antibodies (lower panels), and examined by fluorescence confocal microscopy. (c) ARHI and Stat3 colocalize in the cytoplasm. SKOv3-ARHI cells were pretreated with DOX to induce ARHI expression and then with IL-6 for 30 min to activate Stat3. Treated cells were stained using anti-ARHI and anti-Stat3, and the subcellular localization of Stat3 was examined by confocal microscopy. (d) ARHI reduces N-cadherin and vimentin expression. SKOv3-ARHI cells were treated with DOX for 24 h before addition of IL-6 (10 ng/ml). mRNA levels were normalized with GAPDH mRNA and the fold changes are relative to the untreated controls to which a value of 1.0 was assigned (*P<0.05; **P<0.01). DOX, doxycycline; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IL, interleukin.

As an integral component of cell motility, focal adhesion complexes form at the leading edge of moving cells and permit the cells to spread in the forward direction (Carragher and Frame, 2004). Silver et al. (2004) reported localization of activated Stat3 in focal adhesions and showed a crucial role for Stat3 in the motility of ovarian cancer cells. Consistent with their observation, we also observed specific punctate Stat3 localization at the focal adhesion following stimulation with fibronectin (Figure 3b). As 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 immunofluorescence 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 (Figure 3b). This colocalization was, however, disrupted when ARHI expression was induced. Our data thus show that expression of ARHI not only prevented Stat3 localization at the focal adhesions, but also reduced dramatically 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 colocalization in the cytoplasm (Figure 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 the motility of ovarian cancer cells (Cheng et al., 2008; Vij et al., 2008; Colomiere et al., 2009). Treatment of SKOv3-ARHI cells with IL-6 significantly increased N-cadherin and vimentin expression. This increase was inhibited in ARHI-expressing cells (Figure 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 the 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.

ARHI regulates cell migration by inhibiting FAK activation

FAK is a major component of the focal adhesion complex that regulates cell motility. We therefore examined whether ARHI can also inhibit the 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-FAKY397), a binding site for Src family kinases (Schlaepfer and Mitra, 2004; Long et al., 2010). We examined the effects of ARHI on FAK and Src activation by monitoring the phosphorylation of FAKY397 and the phosphorylation of SrcY416, 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-FAKY397 and p-SrcY416 were analyzed. As shown in Figures 4a and b, expression of ARHI reduced p-FAKY397 and p-SrcY416 levels induced by either fibronectin or EGF. Reduction in p-FAKY397 but not total FAK levels by ARHI was further confirmed by immunofluorescence staining (Figure 4c). As ARHI interacts with and sequesters Stat3 in the cytoplasm, it raises the possibility that inhibition of FAK activation by ARHI might be because of reduced Stat3-FAK colocalization at the focal adhesion. Interestingly, knockdown of Stat3 did not reduce p-FAKY397 levels (Figure 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 (Figure 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 (Figure 4f). Interestingly, the IL-6-stimulated increase was more dramatically affected by Stat3 knockdown (31% decrease; P<0.01) than by FAK knockdown. By contrast, the EGF-mediated increase in cell migration was strongly affected by FAK knockdown (57% decrease; P<0.01) than by Stat3 knockdown (Figure 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 inhibit cell migration any further (Figure 4f). Together, these data indicate that expression of ARHI inhibited ovarian cancer cell motility through two distinct pathways, one dependent on Stat3 signaling and the other on FAK-mediated pathways.

Figure 4
figure4

ARHI regulates cell migration by inhibiting FAK activation. (a) ARHI inhibits FAKY397 phosphorylation. SKOv3-ARHI cells were treated with DOX for 24 h and then stimulated with fibronectin (FN) (1 μg/ml for 1 h) or EGF (10 ng/ml for 30 min). The levels of FAKY397 phosphorylation are expressed as the ratio of p-FAKY397 to total FAK, to which the ratio for control cells is assigned a value of 1.0. (b) ARHI inhibits Src phosphorylation. SKOv3-ARHI cells were treated with DOX for 24 h and then stimulated with FN (1 μg/ml for 1 h) or EGF (10 ng/ml for 30 min). The levels of Src phosphorylation are expressed as the ratio of p-Src to total Src, to which the ratio for control cells is assigned a value of 1.0. (c) ARHI inhibits p-FAKY397 at focal adhesions. Cells were plated on fibronectin-coated cover slips, treated with or without DOX for 48 h, and stained with anti-p-FAKY397, anti-FAK and anti-ARHI antibodies. Fluorescence intensities were quantified by ImageJ. The columns indicate the mean, and the bars indicate the s.e. (**P<0.01). (d) Stat3 knockdown does not reduce p-FAKY397 levels. (e) Knockdown of Stat3 and FAK in SKOv3-ARHI cells by siRNAs. (f) Migration assays of cells transfected with Stat3 and FAK siRNAs, or control siRNA, with or without pretreatment with DOX for 48 h, and with or without IL-6 or EGF stimulation for 16 h. The columns indicate the mean, and the bars indicate the s.e. (*P<0.05; **P<0.01). In IL-6-stimulated cells, siStat3 significantly inhibited migration (P<0.01), whereas siFAK did not (P>0.11). Similarly, in EGF-stimulated cells, siFAK significantly inhibited migration (P<0.01), whereas siStat3 did not (P>0.08). DOX, doxycycline; EGF, epidermal growth factor; FAK, focal adhesion kinase; IL, interleukin; siRNA, small interfering RNA.

ARHI inhibits FAK-mediated focal contact formation

FAK activation is associated with both focal contact formation and the formation of actin stress fibers (Orr et al., 2000; Orr and Wang, 2001; Parsons, 2003). Furthermore, the 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 (Marcoux and Vuori, 2005; Kakinuma et al., 2008), 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 the motility of SKOv3-ARHI cells. Cell motility, however, was reduced significantly when ARHI was expressed (Figure 5a). As expected, treatment with the RhoA inhibitor C3 transferase completely abrogated cell motility (Figure 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 green fluorescent protein (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 (Figure 5b). ARHI also reduced stress fiber formation and GFP-RhoA-stimulated cell migration significantly (Figures 5c and d). 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.

Figure 5
figure5

ARHI decreases RhoA activity and inhibits actin stress fiber formation. (a) SKOv3-ARHI cells were treated with DOX for 24 h before they were treated with EGF and C3 transferase, individually or in combination (n=9 in each group; **P<0.01). (b) ARHI inhibits RhoA GTPase activity. SKOv3-ARHI cells were induced with DOX and then transfected with the GFP-RhoA plasmid. The cells were treated with DOX for an additional 24 h before stimulation with EGF (30 min). (c) ARHI reduces actin stress fiber formation. SKOv3-ARHI cells were treated with DOX for 24 h before they were transfected with the GFP-RhoA plasmid for 24 h. Actin stress fibers were stained with rhodamine-labeled phalloidin. The yellow arrowheads indicate the GFP-RhoA-transfected cells and the blue arrowheads indicate the non-transfected cells. (d) ARHI inhibits RhoA-stimulated cell migration. SKOv3-ARHI cells were treated with DOX and transfected with GFP-RhoA before the migration assay. The columns indicate the mean, and the bars indicate the s.e. (*P<0.05). DOX, doxycycline; EGF, epidermal growth factor; GFP, green fluorescent protein.

Discussion

Epithelial ovarian carcinoma is characterized by widespread intra-abdominal metastases mediated through surface shedding of tumor cells, migration and peritoneal implantation. Our current study shows 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 the 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 the 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 for both proliferation and motility. A significant fraction of ovarian cancers not only secrete IL-6 but also express the IL-6 receptor, resulting in high cell proliferation and motility owing to autocrine stimulation (Martinez-Maza and Berek, 1991; Lidor et al., 1993; 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 (Figure 2b), ARHI expression was able to inhibit Stat3 translocation to the nucleus as well as to focal adhesion complexes (Figures 3b and c). This inhibition is presumably because of physical interaction between ARHI and Stat3, resulting in the sequestration of Stat3 in the cytoplasm. Stat3 has been shown recently to accumulate in 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 loss of ARHI-mediated inhibition of Stat3 signaling.

Our study confirms earlier reports that Stat3 has 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, the 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, FAKY397 phosphorylation is inhibited when ARHI was induced (Figure 4). Given that Stat3 knockdown did not alter p-FAKY397 levels, we reasoned that the effect of ARHI on FAK activity might relate to decreased association of paxillin and vinculin with focal adhesions (Figure 3b). Indeed, correlation between FAK activation, paxillin and vinculin phosphorylation, and the subsequent actin stress fiber formation has been reported (Schaller, 2001; Parsons, 2003). These events are often associated with Rho-family GTPases that act as switches between an inactive, GDP-bound form and an active, GTP-bound form. Importantly, FAK promotes p190RhoGEF tyrosine phosphorylation and enhances the activation of RhoA (Zhai et al., 2003). ARHI decreased the EGF-induced chemotaxis of SKOv3-ARHI cells. Chemotaxis in response to EGF was reduced by FAK knockdown (Figure 4f) and also impaired by the RhoA inhibitor, C3 transferase; however, chemotaxis was not inhibited further by ARHI (Figure 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 (Figure 5b). Additionally, cell migration requires the integration of specific focal adhesion dynamics, including formation of actin stress fibers, which is inhibited by ARHI (Figure 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 the formation of stress fibers (Figure 6).

Figure 6
figure6

A molecular model. The tumor-suppressor gene ARHI functions as a regulatory molecule in controlling ovarian cancer cell migration through modulation of Stat3- and FAK-mediated signaling pathways. FAK, focal adhesion kinase.

Integrative functions that include control of cell motility have been reported for other genes known to have 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's tumor-suppressor function is not limited to its antiproliferative activity, but may also rely on its combined effects in the 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 the upregulation of Stat3 and FAK activity, and thereby contribute to oncogenesis.

Materials and methods

Antibodies and reagents

Antibodies against Stat3, p-SrcY416, paxillin, FAK and β-actin were purchased from Cell Signaling Technology (Beverly, MA, USA), and the anti-p-FAKY397 antibody was purchased from Millipore (Worcester, MA, USA). Antibodies against ARHI were generated in our laboratory. AG490 was purchased from Calbiochem (La Jolla, CA, USA). The cell-permeable RhoA inhibitor C3 transferase was purchased from Cytoskeleton (Denver, CO, USA). IL-6, EGF and anti-vinculin were purchased from Sigma (St Louis, MO, USA), and siRNAs and the Dharmafect #4 transfection reagent were from Dharmacon Research (Lafayette, CO, USA).

Cell culture

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% CO2. Physiological levels of ARHI were achieved 16–24 h after addition of 1 μg/ml DOX to the culture medium (Lu et al., 2008).

Chemotaxis and haptotaxis

Briefly, 1 × 105 cells (SKOv3 and SKOv3-ARHI) or 5 × 104 cells (Hey-ARHI) in 0.5 ml of serum-free medium were introduced into the upper compartment of Boyden chambers (BD Discovery Labware, Franklin Lakes, NJ, USA) 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 containing 5 μg/ml fibronectin (haptotaxis). After 16 h of incubation, cells were wiped from the upper surface of each membrane. The cells on the lower surface were stained with Diff-Quick (Siemens, Deerfield, IL, USA) and counted in 10 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 h 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 h followed by 30-min incubation with AG490, AG490 plus IL-6, IL-6 plus dimethylsulfoxide or dimethylsulfoxide in serum-free medium. For knockdown experiments, cells were transfected with siRNA and treated with DOX for 48 h; 1 × 105 cells were inoculated into the upper chamber and with complete medium in the bottom chamber. Cells migrated to the lower surface were counted 16 h later and migration was expressed as the mean number of cells in triplicate wells from three independent experiments.

Live-cell time-lapse microscopy

SKOv3-ARHI cells (4 × 105) were plated in glass-bottom dishes (MatTek, Ashland, MA, USA) and grown to confluence with or without DOX. The cells were scratched with a 10-μl pipette tip and placed onto a microscope stage in a 37 °C chamber with 5% CO2. Scratch-wound closure was monitored by DIC microscopy and an Olympus IX-81 inverted microscope (Olympus, Center Valley, PA, USA). Images were captured every 15 min using the Slidebook software (http://www.intelligent-imaging.com/home.php) to manage the Hamamatsu Orca II ER camera and microscope settings.

Immunohistochemical staining of ARHI

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 1 × Diva Decloaker (Biocare Medical, Concord, CA, USA) for 1 h to restore latent epitopes. The slides were then incubated with an anti-ARHI antibody (15E11) at a final concentration of 15 μg/ml at 4 °C overnight. The slides were washed and incubated for 30 min with each with biotin-labeled secondary antibody, and then with streptavidin/peroxidase. The slides were then incubated with the diaminobenzidine substrate (Biocare Medical), counterstained with hematoxylin and mounted in Permount.

Immunofluorescence staining

SKOv3-ARHI cells were plated onto slides with or without fibronectin and treated with DOX for 24 h, fixed in 4% paraformaldehyde and permeabilized using 0.5% Triton X-100. Washed cells were blocked with 3% bovine serum albumin and incubated with antibodies against Stat3, vinculin, paxillin or p-FAKY397. After washing, the cells were incubated with secondary antibodies conjugated with Alexa Fluor-488 or Alexa Fluor-594 (Molecular Probes, Invitrogen, Eugene, OR, USA). Actin stress fibers were stained with rhodamine-labeled phalloidin (1/1000; Molecular Probes, Invitrogen). Finally, cells were mounted and examined by confocal microscopy (Olympus FluoView 500 or 1000; Olympus Inc., Melville, NY, USA).

Immunoprecipitation

SKOv3-ARHI cells (1 × 106) were incubated with or without DOX for 24 h and then treated with lysis buffer (50 mM HEPES (pH 7.0), 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 100 mM NaF, 10 mM sodium pyrophosphate, 10% glycerol, 1% Triton X-100, 1 mM phenylmethylsulphonyl fluoride, 1 mM Na3VO4, 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 h at 4 °C. After washing with wash buffer (10 mM Tris (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM Na3VO4, 1 mM phenylmethylsulphonyl fluoride, 0.5% Triton X-100 and 0.2% NP-40), the immunoprecipitated proteins were eluted for western blotting.

Western blot analysis

Proteins were separated on 12 or 15% sodium dodecyl sulfate–PAGE, transferred to polyvinylidene difluoride membranes and subjected to western blotting using an ECL chemiluminescence reagent (GE/Amersham, Piscataway, NJ, USA). Protein band intensities were quantified using the Java-based image-processing program ImageJ developed at the NIH.

siRNA transfection

A mixture of siRNA (100 nM final concentration) and the Dharmafect #4 transfection reagent was incubated for 20 min at room temperature. This mixture was then added to cells and allowed to incubate for 48 h before cells were harvested for protein expression measurements or for motility assays.

Real-time quantitative reverse transcription–PCR

Expression of Stat3-responsive genes was measured using real-time quantitative reverse transcription–PCR. Total cDNA was synthesized using 2 μg of total RNA. The reverse transcriptase reaction was performed 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, USA). The primers for N-cadherin were 5′-IndexTermGCCTGCAGATTTTAAGGTGG-3′ and 5′-IndexTermCTCTTGAGGAAAAGGTCCCC-3′, and for vimentin were 5′-IndexTermGAGAACTTTGCCGTTGAAGC-3′ and 5′-IndexTermTTCAGGGAGGAAAAGTTTGG-3′. mRNA levels were normalized with a concurrent determination for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA.

Transient transfection and RhoA GTPase activation assay

SKOv3-ARHI cells (1 × 106) were transfected with 16 μg of GFP-RhoA plasmid (Addgene plasmid 12 965; Cambridge, MA, USA) and harvested 48 h later. RhoA activity was measured with a RhoA activation assay kit (Cell Biolabs, San Diego, CA, USA). Levels of active RhoA were determined by western blot analysis.

Quantitation of band intensity and average fluorescence intensity

Band intensity from western blots was 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 staining for p-FAKY397 or total FAK was quantified and the average fluorescence intensity was calculated by dividing the total fluorescence intensity by the cell number.

Statistical analysis

All experiments were repeated independently at least two times and the data have been expressed as the mean±s.e. Statistical analysis was performed using Student's t-test (two-tailed). The criterion for statistical significance was taken as P<0.05 (two-sided).

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Acknowledgements

This work was supported by Grants NCI P01 CA064602 and 1RO1 CA135354-01 from the National Institutes of Health. We also acknowledge the support of the MD Anderson SPORE in Ovarian Cancer 5P50 CA83639 and the CCSG shared resources funded, in part, by CA 5 P30 CA016672. Support was also provided by the Ovarian Cancer Research Fund through a Program Project Award. DBB was supported by an Excellence Award from the Ovarian Cancer Research Fund. AAA was supported by a Cancer Research UK Clinician Scientist fellowship. We thank Jodie Polan for excellent technical support with confocal microscopy and Jared Burks from the Flow Cytometry and Cell Imaging core laboratory for the live-cell time-lapse microscopy partially supported by the MD Anderson Cancer Center CCSG NCI P30 CA16672.

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Correspondence to R C Bast Jr.

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Keywords

  • tumor-suppressor gene ARHI
  • migration suppression
  • Stat3
  • RhoA GTPase
  • cytoskeleton

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