GEP Oncogene Promotes Cell Proliferation Through YAP Activation in Ovarian Cancer

ORIGINAL ARTICLE GEP oncogene promotes cell proliferation through YAP activation in ovarian cancer

H Yagi, K Asanoma, T Ohgami, A Ichinoe, K Sonoda and K Kato

G-protein-coupled receptors (GPCRs) and their ligands function in the progression of human malignancies. Gα12 and Gα13, encoded by GNA12 and GNA13, respectively, are referred to as the GEP oncogene and are implicated in tumor progression. However, the molecular mechanisms by which Gα12/13 activation promotes cancer progression are not fully elucidated. Here, we demonstrate elevated expression of Gα12/13 in human ovarian cancer tissues. Gα12/13 activation did not promote cellular migration in the ovarian cancer cell lines examined. Rather, Gα12/13 activation promoted cell growth. We used a synthetic biology approach using chimeric G proteins and GPCRs activated solely by artificial ligands to selectively trigger signaling pathways downstream of specific G proteins. We found that Gα12/13 promotes proliferation of ovarian cancer cells by activating the transcriptional coactivator YAP, a critical component of the Hippo signaling pathway. Furthermore, we reveal that inhibition of YAP by short hairpin RNA or a specific inhibitor prevented the growth of ovarian cancer cells. Therefore, YAP may be a suitable therapeutic target in ovarian cancer.

Oncogene (2016) 35, 4471–4480; doi:10.1038/onc.2015.505; published online 25 January 2016

INTRODUCTION residue.11–15 Phosphorylation of YAP represses its activity through Ovarian cancer generally has a poor prognosis because of spread creation of a 14-3-3 binding site, which promotes cytoplasmic 15 of cancer cells to the peritoneal cavity. Furthermore, approxi- accumulation and ubiquitin-mediated proteolysis. mately 70% of ovarian cancer patients have late-stage disease at YAP promotes tissue growth and cell viability by regulating the the time of diagnosis.1 Although chemotherapeutic protocols activity of multiple transcription factors, including TEA domain 11,12 have improved over time, ovarian cancer mortality rates remain family (TEADs) and Sma- and Mad- related family (SMADs). 1 However, the precise mechanisms by which YAP drives tissue unchanged. Therefore, new therapeutic strategies for advanced 16 ovarian cancer are urgently required. growth are incompletely understood. Studies using mice harbor- fi G-protein-coupled receptors (GPCRs) are seven transmembrane- ing tissue-speci c deletions of Hippo pathway genes have revealed the oncogenic activities of this signaling cascade in colorectal, domain cell surface receptors, and represent the largest family of 17 cell surface receptors. Ligand binding induces a conformational ovarian and liver cancers. Furthermore, Hippo pathway dysregula- change within GPCRs, which activates intracellular heterotrimeric tion occurs at a high frequency in human lung, colorectal, ovarian, liver and prostate cancers.16–19 This Hippo pathway dysregulation G proteins, enabling downstream signaling via various second 18–21 messengers. GPCRs regulate diverse biological functions including correlates with poor patient prognosis. cell proliferation, chemotaxis and angiogenesis, all of which are The core Hippo pathway genes, including the core kinase important aspects of cancer progression.2 Moreover, cancer cells cassette and downstream transcriptional regulators, are infre- quently mutated. Rather, indirect disruption of the Hippo cascade frequently overexpress GPCRs to exploit the growth and survival- 17 enhancing functions conferred by these receptors.2 by other signaling pathways appears critical. The Hippo fl Recent genome-wide analyses have revealed that overexpres- signaling cascade is regulated by various upstream in uences, – sion of or activating mutations to heterotrimeric G proteins— including cell cell contact, organ size sensing machinery, and without a concurrent increase in the receptor component—can other signaling pathways regulated by WNT, transforming growth β α contribute to cancer progression.3 Among G proteins, Gα and factor beta (TGF- ) and several GPCRs, especially G 12/13-linked 12 GPCRs.17,22 Furthermore, recent data indicate that gain-of-function Gα13, encoded by GNA12 and GNA13, respectively, are known as 4,5 mutations in GNAQ and GNA11 oncogenes promote uveal GEP oncogene. GEP oncogene is expressed at a high level in 23 some cancers and is involved in the metastatic spread of breast, melanoma growth through YAP activation. This study reveals 6–9 that Gα12/13 signaling contributes to YAP-dependent growth in prostate and oral cancers. Gα12/13 activates Rho by directly binding Rho guanine nucleotide exchange factors (RhoGEF), and ovarian cancer. Therefore, YAP is a potential therapeutic target for ovarian cancer treatment. the Gα12/13-Rho signaling axis has a critical role in cancer progression.6–8,10 However, the downstream events underlying Gα -driven malignancy remain ill-defined. 12/13 RESULTS The transcriptional coactivator Yes-associated protein (YAP; α encoded by YAP1) is a critical component of the Hippo signaling G 12/13 is highly expressed in human ovarian cancer tissues pathway. This pathway influences organ size in mammals and Human ovarian cancer tissues were subjected to immunohisto- involves activation of large tumor-suppressor homolog (LATS) chemistry to determine protein levels of Gα12 and Gα13. Both Gα12 1 and 2. LATS1 and LATS2 phosphorylate YAP on the Ser127 and Gα13 were overexpressed in ovarian cancer tissues, but not in

Department of Obstetrics and Gynecology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan. Correspondence: Dr H Yagi, Department of Obstetrics and Gynecology, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. E-mail: [email protected] Received 9 June 2015; revised 7 December 2015; accepted 11 December 2015; published online 25 January 2016 Gα12/13 drives ovarian cancer growth through YAP H Yagi et al 4472

normal ovarian tissues (Figure 1). Gα12/13 overexpression was Gα13QL inhibited colony formation by SHIN3 and OVAS cells observed in various histological subtypes of ovarian cancers, but (Figure 3d and Supplementary Figure S2b). Taken together, these ﬁ no speci c pattern for any histological subtype was observed results suggest that Gα13 expression affects the proliferation of (Table 1). ovarian cancer cells.

α G 12/13 is involved in cell proliferation of ovarian cancer Gα12/13 activation induces YAP nuclear translocation and We examined whether upregulation of Gα12/13 was involved in the transcriptional upregulation of YAP-regulated genes progression of ovarian cancer using RGS-GFP, which binds to the YAP localized to the cytoplasm of OVAS cells after serum activated form of Gα12/13 and thus behaves as a dominant- starvation, whereas addition of FBS promoted YAP nuclear 24 negative mutant of Gα12/13. Expression of RGS-GFP in ES2 translocation (Figures 4a and b). YAP was highly phosphorylated ovarian cancer cells inhibited Rho activation (Figures 2a–c). The following serum starvation, and addition of FBS induced a rapid Gα12/13-Rho signaling axis can promote cellular motility and decrease in YAP phosphorylation at Ser127 (Figure 4c). We next invasion in multiple cancer cells. However, lysophosphatidic acid- used RGS-GFP to assess whether Gα12/13 had a role in the induced or fetal bovine serum (FBS)-induced migration of ES2 or localization and dephosphorylation of YAP in response to FBS. RMG1 cells was not blocked by RGS-GFP expression (Figure 2d and RGS-GFP blocked FBS-induced nuclear translocation and depho- Supplementary Figure S1a). Meanwhile, FBS-induced cell prolif- sphorylation of YAP and reduced expression of the YAP-targeted eration and colony formation in ES2 or OVAS cells was blocked by gene connective tissue growth factor (CTGF) in OVAS cells RGS-GFP-induced Gα inhibition (Figures 2e and f, and 12/13 (Figures 4d–g). These results suggest a role for Gα12/13 in the Supplementary Figure S1b). We next investigated the role of nuclear translocation of YAP and YAP-regulated gene α G 12/13 in ovarian cancer cell proliferation by stably expressing transcription. short hairpin RNAs (shRNAs) targeting Gα12 and Gα13 in ES2, RMG1 and MCAS cells (Figure 2g and Supplementary Figure S1c). Colony A synthetic biology approach reveals a key role for Gα in the formation by ES2, RMG1 and MCAS cells was blocked by Gα 13 12/13 nuclear translocation of YAP in ovarian cancer cells knockdown (Figure 2h, Supplementary Figures S1d and e). ﬁ α Furthermore, Gα knockdown markedly inhibited the tumor We con rmed the role of G 13 in YAP activation by engineering a 12/13 α growth of RMG1 or MCAS cells in vivo (Figure 2i). Together, these GPCR-G 13-coupled system that was activated solely by an inert artiﬁcial ligand, as previously described.8 We created Gα ,a data suggest that Gα12/13 has a role in ovarian cancer cell 13i5 proliferation.

α Expression of G 13 promotes the proliferation of ovarian cancer Table 1. Expression of Ga12/13 in human ovarian cancer tissues cells Histological subtype n Number of positive expression We then confirmed the biological significance of Gα13 in ovarian cancer by establishing ovarian cancer cells stably expressing Gα13 or a constitutively active mutant of Gα13 (Gα13QL) by lentiviral Ga12 Ga13 infection (Figures 3a and c). Cellular morphology was altered by Serous 21 21 21 forced expression of Gα13QL, but not Gα13 (Figure 3d and Supplementary Figure S2a). Gα QL-expressing cells had an Clear cell 29 29 29 13 Endometrioid 11 11 11 expanded and flattened cytoplasm, similar to the morphology of 25 α Mucinous 8 8 7 senescent cells. Expression of G 13 promoted ovarian cancer cell Others 24 24 24 colony formation in vitro and tumor formation in vivo (Figures 3d–f 93 93 93 and Supplementary Figure S2b). Meanwhile, forced expression of

Figure 1. Gα12/13 protein levels are upregulated in human ovarian cancer tissues. Sections of formalin-ﬁxed parafﬁn-embedded ovarian tissues were stained for Gα12 (a–d) and Gα13 (e–h), respectively. (a, e) normal ovary (b, f) serous adenocarcinoma (c, g) clear cell adenocarcinoma (d, h) mucinous adenocarcinoma.

Figure 2. Gα12/13 activation is involved in proliferation of ovarian cancer cells. (a, b) Expression of GFP and RGS-GFP—a chimeric protein encoding the RGS domain of PDZ-RhoGEF fused to GFP—in ES2 cells. (c–f) Effect of Gα12/13 inhibition by RGS-GFP on the activation of Rho (c), cell migration (d), cell proliferation as measured by WST-1 assay (e) and colony formation (f) in ES2 cells. Error bars, s.e.m., n = 3, *Po0.05 with respect to the corresponding cells infected with GFP lentivirus. (g) Knockdown of Gα12/13 by lentiviruses encoding Gα12 shRNA and Gα13 shRNA in ES2 cells. (h) Effect of Gα12/13 knockdown on colony formation in ES2 cells. Error bars, s.e.m., n = 3, *Po0.05 with respect to ES2 cells infected with lentivirus-shRNA control. (i) Effect of Gα12/13 knockdown on RMG1 (left panel) or MCAS (right panel) cell growth in vivo. Tumor size was measured every week after the injection of each cell line. Error bars, s.e.m., n = 3, *Po0.05 with respect to the corresponding cells infected with lentivirus-shRNA control. chimeric molecule, in which the C-terminal ﬁve amino acids of and enhanced CTGF expression (Figures 5e–h). Taken together, ﬁ α Gα13 were replaced with those of Gαi (Figure 3a). This molecule these ndings suggest that G 13 activation may stimulate YAP could be coupled to and activated by GiRASSL—a GPCR activated nuclear translocation and YAP-dependent transcription. solely by synthetic ligand, Clozapine N-oxide (CNO) (Figure 5a).

The functional activity of this reconstituted system was examined Gα13-induced phosphorylation of LATS1 at Ser909 is involved in by assessing its ability to activate extracellular signal-regulated LATS1 degradation kinase (ERK)-1 and -2 in HEK-293A cells. CNO activated ERK1/2 in We next determined whether Gα13-regulated signals act through HEK-293A cells transfected with expression constructs encoding LATS1 kinase—a core component of the Hippo pathway—to α GiRASSL, but co-transfection of G 13i5 and GiRASSL constructs regulate YAP phosphorylation. We examined the effect of Gα13 enhanced CNO-induced ERK1/2 activation (Figure 5b). CNO also activation on LATS1 kinase activity, which was determined by induced dephosphorylation and nuclear translocation of YAP in assessing phosphorylation of LATS1 at the activation loop (Ser909) α – 26 HEK-293A cells expressing both GiRASSL and G 13i5 (Figures 5b d). and the hydrophobic motif (Thr1079). Activation of Gα13 in We established ES2 ovarian cancer cell lines, which stably HEK-293A cells induced rapid phosphorylation of LATS1 at Ser909, expressed GiRASSL and Gα13i5. CNO-induced Gα13 activation whereas phosphorylation at Thr1079 was decreased following promoted nuclear translocation and dephosphorylation of YAP, CNO treatment (Supplementary Figure S3a). In addition,

Figure 3. Gα13 expression promotes cell proliferation of ovarian cancer. (a) Expression of Gα13, Gα13QL and Gα13i5 in HEK-293T cells. (b, c) Expression of Gα13 in control and Gα13-expressing SHIN3 cells examined in real-time PCR (b) and western blot (c). (d) Morphological changes of SHIN3 or RMG1 ovarian cancer cells stably expressing Gα13 and Gα13QL. (e) Effect of Gα13 and Gα13QL expression on colony forming properties of SHIN3 cells. Error bars, s.e.m., n = 3, *Po0.05 with respect to SHIN3 cells infected with control lentivirus. (f) Effect of Gα13 expression on RMG1 (upper panel) or MCAS (lower panel) cell growth in vivo. Tumor size was measured every week after the injection of each cell line. Error bars, s.e.m., n = 3, *Po0.05 with respect to the corresponding cells infected with control lentivirus.

phosphorylation of LATS1 at Ser909 was observed after 5 min of (Figures 7a–e). The extent and pattern of YAP staining in each CNO stimulation in ES2, OVAS and TYK-nu ovarian cancer cells tissue was scored using the scoring system deﬁned in Table 2. (Figures 6a and b, and Supplementary Figure S3b). Phosphoryla- Cytoplasmic staining of YAP was not present in surface epithelial tion of LATS1 at Thr1079 was not detected in OVAS and TYK-nu cells (Figure 7a). Ovarian cancer tissues exhibited generally strong cells (Figure 6b and Supplementary Figure S3b). Furthermore, cytoplasmic and nuclear staining (Figures 7b–e, Table 3). These LATS1 protein expression gradually decreased following addition data suggest that YAP may have a role in human ovarian cancer of CNO (Figures 6a and b, Supplementary Figures S3a and b). progression. However, LATS1 mRNA levels were unchanged (Figure 6c). Finally, α the potent proteasome inhibitor MG-132 inhibited G 13-mediated YAP represents a therapeutic target in ovarian cancer LATS1 protein degradation (Figure 6d). Together, these data α We next explored the role of YAP activation in ovarian cancer cell suggest that G 13-induced LATS1 phosphorylation at Ser909 may proliferation. Lentiviral-delivered shRNAs were used to impair YAP result in proteasome-dependent LATS1 protein degradation, expression in OVAS ovarian cancer cells (Figures 8a and b). thereby promoting YAP function. Knockdown of YAP led to a reduced colony formation by OVAS cells (Figures 8c and d). Furthermore, FBS-induced CTGF expres- YAP is highly expressed in human ovarian cancer tissues sion was blocked following knockdown of YAP in OVAS cells Immunohistochemical staining for YAP was performed on human (Figure 8e). We then conﬁrmed the role of the Gα13-YAP signaling normal and cancerous ovarian tissues. Cancers of serous, clear axis in colony formation in ovarian cancer cells by silencing YAP cell and mucinous adenocarcinoma types were examined expression using shRNA in OVAS cells stably expressing Gα13.

Figure 4. Gα12/13 signaling activates YAP by dephosphorylation. (a, b) Immunoﬂuorescence detection of YAP in OVAS ovarian cancer cells in the presence and absence of FBS. Endogenous YAP is indicated in green. 4′,6-Diamidino-2-phenylindole (DAPI) staining (blue) indicates nuclear DNA. Levels of nuclear YAP were quantiﬁed using cell count software (BZ-9000, Keyence, Osaka, Japan). Error bars: s.e.m., n = 3, *Po0.05. (c) OVAS cells were treated with FBS for the indicated times. Cell lysates were subjected to immunoblotting with the indicated antibodies. (d–f) Effect of Gα12/13 inhibition by RGS-GFP on YAP nuclear translocation (d, e), dephosphorylation (f), and CTGF expression (g) induced by FBS in OVAS cells. Error bars: s.e.m., n = 3, *Po0.05.

3 Overexpression of Gα13 promoted colony formation, whereas protein α-subunits. Among them, gain-of-function mutations of knockdown of YAP inhibited Gα13-induced colony formation the GNAQ and GNA11 oncogenes represent the initial genetic (Figure 8f). Taken together, these results suggest that YAP alteration promoting development of uveal melanoma and a activation represents a molecular event important for subset of melanomas arising in the skin.23 Consistently, recent Gα13-mediated growth progression in ovarian cancer. meta-analysis of expression signatures from ~ 18 000 human Verteporfin (VP) is a potent inhibitor of the YAP–TEAD4 tumors with overall survival outcomes across 39 malignancies interaction.27 Therefore, we investigated whether VP exerts an revealed associations between gene expression of GNA12 and anti-proliferative effect on ovarian cancer cells. Treatment with GNA13 and poor prognosis in patients with glioblastoma, oral, 29 1 μM VP significantly reduced colony formation by OVAS or SHIN3 breast, lung, kidney and ovarian cancers. A meta-analysis of cells (Figure 8g and Supplementary Figure S4a). In addition, cancer patient databases using Oncomine (http://www.oncomine. Gα13-induced expression of the YAP-target gene CTGF was blocked org.) revealed a significant increase in the DNA copy number of by VP treatment in OVAS or SHIN3 ovarian cancer cells coexpressing GEP oncogene in renal cell, breast and bladder carcinoma, and GiRASSL and Gα13i5 (Figures 8h and i, and Supplementary malignant glioma compared with corresponding normal tissues. Figure S4b). Together, these findings suggest that YAP is a No alteration to the DNA copy numbers of GEP oncogene potential therapeutic target for the treatment of ovarian cancer. components was observed in ovarian cancer, but this may be because of the difficulty involved in obtaining pure normal ovarian surface epithelium. Contrasting with the oncogenic role of DISCUSSION Gα12/13, loss-of-function mutations in GEP oncogene promote Cancer cells commonly exploit the normal physiological functions proliferation and dissemination of B-cell-derived lymphoma.30 The of GPCRs leading to enhanced tumor growth and metastasis. underlying mechanisms by which increased signaling through Although activating mutations of GPCRs have been known to be Gα12/13 regulates cancer progression remain to be fully elucidated. involved in tumorigenesis for some time, more recent studies Much of the work surrounding the role of Gα12/13 in cancer has have revealed the importance of heterotrimeric G proteins in concentrated on Gα12/13 promotion of cell migration through Rho 8,9,23,28 tumor progression. Deep-sequencing efforts have revealed GTPase activation. However, Gα12/13 was originally identified as an a high frequency of functional mutations in heterotrimeric G oncogene with the potential for neoplastic transformation.4,5,31

Figure 5. A synthetic biology approach reveals that Gα13 signaling induces activation of YAP by dephosphorylation. (a) Schematic of cells coexpressing GiRASSL and Gα13i5. CNO promotes activation of Gα13 in cells coexpressing GiRASSL and Gα13i5.(b) Activation of ERK and YAP induced by CNO in HEK-293A cells expressing indicated genes. (c, d) YAP nuclear translocation in HEK-293A cells coexpressing GiRASSL and Gα13i5; representative images (b) and quantiﬁcation (c). Error bars: s.e.m., n = 3, *Po0.05 compared with cells without stimulation. (e–h) Nuclear localization (e, f) and dephosphorylation of YAP, and protein (g) and mRNA expression of CTGF (h) induced by CNO in ES2 cells coexpressing GiRASSL and Gα13i5. Error bars: s.e.m., n = 3, *Po0.05 compared with cells without stimulation.

Gα12/13 activation can also promote tumorigenesis and cell growth pathway component genes. Recent reports suggest that GPCR in ovarian and hepatocellular cancer cells—but not in breast and signaling can regulate the Hippo pathway.22 GPCRs linked to 7,8,32,33 prostate cancer cells. This differential effect of Gα12/13 Gα12/13 can inhibit LATS activation—which is determined by suggests that it exerts a complex regulatory influence on cancer phosphorylation at both the activation loop (Ser909) and cell proliferation and growth. We used a synthetic biology hydrophobic motif (Thr1079)—thereby releasing YAP from inhibi- approach to begin to unravel the effects of Gα12/13, and have tion by LATS-dependent inhibitory phosphorylation of 16,35–37 revealed that YAP activation is a key molecular event in GEP Ser127. Our present findings suggest that Gα13-mediated oncogene-regulated tumorigenesis in ovarian cancer. phosphorylation of LATS1 at Ser909 may induce proteasome- YAP is a tumor promoter and transcriptional coactivator of the dependent degradation of LATS1, thereby inhibiting its activity. Hippo signaling pathway. Activation of YAP is a frequent event in However, the precise mechanisms by which LATS1 protein 15–21 many human cancers. However, the mechanisms by which degradation is induced by Gα13 remain unknown. YAP activation promote tumor progression remain incompletely Our study has revealed that YAP activation is involved in understood. YAP and its Drosophila homolog Yorkie promote Gα12/13-regulated proliferation of ovarian cancer cells. YAP may be tissue growth and cell viability by regulating the activity of a suitable therapeutic target for cancer treatment, and several different transcription factors, including TEADs and SMADs.11,12,34 small molecule compounds that inhibit YAP transcriptional activity Although distinct tumor phenotypes emerge when core compo- in vitro have been identified.38 Among them, the benzoporphyrin nents of the Hippo pathway are perturbed in mice, relatively few derivative VP is used clinically as a photosensitizer in photo- upstream components of the cascade have been studied. coagulation therapy of wet age-related macular degeneration.27 To date, few somatic or germline mutations in Hippo pathway We found that both YAP knockdown and VP treatment inhibited components have been discovered in common human cancers ovarian cancer cell growth in vitro. when compared with other well-defined signaling pathways that Structural studies of GPCRs suggest that activated GPCRs can are disrupted in cancer.17 This suggests that the frequent adopt multiple conformations, which engage distinct downstream perturbation of Hippo pathway activity in human cancers results signaling pathways.39 Different ligands can preferentially activate from molecular events other than the somatic mutation of or inhibit subsets of the G protein-linked signaling pathways

Figure 6. Gα13-induced LATS1 phosphorylation at Ser909 is involved in LATS1 protein degradation. (a, b) Phosphorylation of LATS1 at Ser909 and Thr1079 in ES2 (a) and OVAS (b) ovarian cancer cells coexpressing GiRASSL and Gα13i5 induced by CNO. (c) LATS1 mRNA expression after CNO addition in OVAS cells coexpressing GiRASSL and Gα13i5.(d) Effect of MG-132 on LATS1 protein degradation induced by CNO in OVAS cells coexpressing GiRASSL and Gα13i5.

Figure 7. Immunohistochemical analysis of YAP in ovarian cancer tissues. Sections of formalin-ﬁxed parafﬁn-embedded ovarian tissues were stained using and anti-YAP antibody. (a) Normal ovary. (b) Serous adenocarcinoma. (c) Clear cell adenocarcinoma. (d) Mucinous adenocarcinoma. (e) Endometrioid adenocarcinoma.

Table 2. YAP immunohistochemical scoring system Table 3. YAP expression in human ovarian tissues Score Grading Tissue type Histological Cytoplasmic Nuclear Complete absence of reactivity Negative subtype expression expression

Cytoplasmic expression Negative Low High Negative Low High Weak reactivity Low Strong reactivity in o50% of cells Low Normal ovary 3/3 0/3 0/3 3/3 0/3 0/3 Strong reactivity in 450% of cells High Serous 0/10 2/10 8/10 0/5 3/10 7/10 Clear cell 0/9 1/9 8/9 1/9 2/9 6/9 Nuclear expression Ovarian cancer Expression in o10% of cells Low Endometrioid 0/4 0/4 4/4 0/4 1/4 3/4 Expression in 410% of cells High Mucinous 1/4 1/4 2/4 1/4 0/4 3/4 Abbreviation: YAP, Yes-associated protein. Abbreviation: YAP, Yes-associated protein.

Figure 8. YAP represents a therapeutic target in ovarian cancer. (a, b) qPCR and western blot analysis of YAP mRNA and protein levels following knockdown of YAP expression by shRNA (YAP#1 and YAP#2) in OVAS ovarian cancer cells. (c, d) Colony formation in OVAS cells following YAP knockdown by shRNA. Representative images (c) and quantiﬁcation (d). Error bars: s.e.m., n = 3, *Po0.05 compared with OVAS cells infected with lentivirus encoding control shRNA. (e) Effect of shRNA-mediated YAP knockdown on FBS-induced CTGF mRNA expression in OVAS cells. (f) Effect of YAP knockdown on colony forming properties of control and Gα13-expressing OVAS cells. Error bars: s.e.m., n = 3, *Po0.05. (g) Colony formation in OVAS cells following treatment with VP. Error bars: s.e.m., n = 3, *Po0.05. (h, i) Effect of VP on CNO-induced CTGF protein (h), and mRNA (i) expression in OVAS cells coexpressing GiRASSL and Gα13i5. Ovarian cancer cells coexpressing GiRASSL and Gα13i5.

engaged by a given GPCR.40 Furthermore, recent reports reveal a unique opportunity to control the pathological functions that the expression level of a G protein itself may regulate of GPCRs. downstream signaling of GPCRs. In breast cancer cells, elevated expression of Gα is required for CXCR4-mediated metastatic 13 MATERIALS AND METHODS spread, whereas most of the physiological functions of CXCR4 8 involve the activation of Gαi. In addition, high levels of Gα12/13 Cell culture promotes cell motility and invasion in prostate and oral cancer.7,9 SKOV3 human ovarian cancer and human epithelial kidney 293T and 293A These findings highlight the potential clinical benefits of devel- cell lines were purchased from the American Type Culture Collection oping GPCR antagonists or negative allosteric regulators that (Manassas, VA, USA). OVK18 cells were obtained from RIKEN Cell Bank fi α (Ibaraki, Japan). ES2 cells were purchased from Summit Pharmaceuticals speci cally prevent the activation of G 12/13. Indeed, GPCRs are International Corporation (Tokyo, Japan). RMG1, RMG2 and OVISE cells the direct or indirect target of approximately one in four of the were purchased from the Health Science Research Resources Bank (Osaka, 41 current therapeutic agents currently available. Therefore, the Japan). MCAS cells were purchased from the Japanese Collection of development of a class of signaling-selective GPCRs may represent Research Biosources (Osaka, Japan). KOC-7C cells were provided by Dr Toru

Oncogene (2016) 4471 – 4480 © 2016 Macmillan Publishers Limited, part of Springer Nature. Gα12/13 drives ovarian cancer growth through YAP H Yagi et al 4479 Sugiyama, Kurume University, Japan, and OVAS cells by Professor Hiroshi Immunocytochemistry Minaguchi, Yokohama City University, Japan. TU-OC1, OVMG, SHIN3 and Ovarian cancer and HEK-293A cells were plated on coverslips coated with TYK-nu human ovarian cancer cells were derived as described fibronectin. After 24-h incubation, coverslips were washed with PBS and 42–44 previously. Cells lines were cultured according to the manufacturer's cells fixed with 4% paraformaldehyde in PBS. After three washes with PBS, instructions. cells were permeabilized with 0.05% Triton X in PBS for 15 min at room temperature. Cells were washed three times with PBS and then incubated Patients and tissue samples with 3% FBS in PBS for 30 min at room temperature. Fixed cells were Ninety-three ovarian cancer patients who underwent surgery at the incubated with primary antibody (anti-YAP, 1:50) for 1 h at room Department of Obstetrics and Gynecology of Kyushu University Hospital temperature, followed by 45-min incubation with secondary antibody between 2005 and 2010 were recruited for this study. Normal control (goat anti-rabbit Alexa Fluor 488 or 647). Coverslips were then mounted ovarian tissues were obtained from 10 patients who underwent surgery for with Prolong gold antifade reagent with 4′,6-diamidino-2-phenylindole benign reasons such as uterine myoma. All patients involved in this study (Life Technologies, Waltham, MA, USA) onto glass slides. provided their written informed consent. This study was approved by the Ethical Committee of Kyushu University. Immunoblot analysis Cells were lysed at 4 °C in lysis buffer (20 mM HEPES (pH 7.5), 100 mM NaCl, Reagents and antibodies 200 mM MgCl2,10mM EGTA, 40 mM β-glycerol phosphate and 1% Rabbit polyclonal anti-phospho-ERK1/2 (#9101), anti-phospho-LATS1 Triton X-100) supplemented with protease inhibitors (0.5 mM (Ser909, #9157) and anti-glyceraldehyde-3-phosphate dehydrogenase phenylmethyl-sulfonyl fluoride, 10 μg/ml aprotinin and 10 μg/ml leupep- and rabbit monoclonal anti-phospho-YAP (Ser127, #13008), anti- tin). Equal amounts of proteins were subjected to sodium dodecyl phospho-LATS1 (Thr1079, #8654) and LATS1 (#3477) antibodies and sulfate–polyacrylamide gel electrophoresis and transferred onto a poly- MG-132 were purchased from Cell Signaling Technology (Beverly, MA, vinylidene difluoride membrane (Immobilon P, Millipore, Billerica, MA, USA). Rabbit polyclonal anti-ERK2 (sc-154), anti-Gα12 (sc-409), ant-Rho (sc- USA). Membranes were then incubated with appropriate antibodies. 179) and anti-YAP (sc-15407) and goat polyclonal anti-CTGF (sc-14939) antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, RNA extraction, reverse transcription and real-time PCR USA). Rabbit monoclonal anti-Gα13 antibody (LS-C152439) was purchased from LifeSpan BioSciences (Seattle, WA, USA). CNO and VP were purchased RNA samples were prepared using the RNeasy Plus mini kit (Qiagen, from Sigma-Aldrich (St Louis, MO, USA). Hilden, Germany). Reverse transcription was performed using ReverTra Ace-α (Toyobo, Osaka, Japan). Real-time PCR was performed using SYBR Premix ExTaqII (Takara, Kusatsu, Japan). Transfection Cells were transfected with plasmid DNA using TurboFect Transfection Reagent (Thermo Scientific, Waltham, MA, USA) according to the Cell viability assay 3 manufacturer’s instructions. Approximately 3 × 10 cells were seeded into each well of a 96-well plate. WST-1 was added after 48 h of culture, according to the manufacturer’s instructions (Takara). Incubation continued for 1 h at 37 °C, and then Plasmid constructs absorbance at 450 nm was measured via a microtiter plate reader (Bio-Rad Expression vectors for GFP and GFP fused to the RGS domain of Laboratories, Hercules, CA, USA). Each assay was performed in triplicate. PDZ-RhoGEF (RGS-GFP) have been described previously.45 Lentiviral vectors 8 for Gα13QL, Gi RASSL and Gα13i5 have been described previously. Lentiviral Colony formation assay vectors for Gα12,Gα13 and YAP were purchased from GE Healthcare (Buckinghamshire, UK). Approximately 1 × 103 cells were seeded onto each well of a six-well plate and incubated at 37 °C for 10 days. Colonies were washed once with PBS fi Lentivirus production and infection and xed with 4% paraformaldehyde in PBS for 20 min. Cells were then stained with crystal violet, and the number of colonies per well counted. Lentiviral stocks were prepared and titrated using HEK-293T cells as packaging cells as described previously.46 Ovarian cancer and HEK-239 A cells were incubated with virus-containing HEK-293T-cell supernatant for Tumor growth in nude mice 24 h. After incubation, this supernatant was replaced with normal growth A total volume of 250 μl of serum-free RPMI1640 containing 5 × 106 cells medium. Infected cells were selected with puromycin (1 μg/ml). was injected subcutaneously into female BALB/c nu/nu mice at 5 weeks of age (Charles River Laboratories, Yokohama, Japan). Injected mice were Immunohistochemistry examined every week for tumor apparition. Tumor volume was calculated according to the following formula: tumor volume (mm3) = 0.5 × (major The following antibodies were used for tissue immunohistochemistry: diameter) x (minor diameter)2. All experimental use of animals complied rabbit polyclonal anti-Gα (1:100, Santa Cruz Biotechnology); rabbit 12 with the guidelines of Animal Care of Kyushu University. polyclonal anti-YAP (1:100, Santa Cruz Biotechnology); and rabbit monoclonal anti-Gα13 (1:100, LifeSpan BioSciences). Unstained 5 μm paraffin sections were dewaxed, hydrated through graded alcohol and Statistical analysis distilled water solutions, and washed three times with phosphate-buffered All experiments were repeated at least three times with similar results in saline (PBS). Antigens were retrieved by incubation with citric acid (10 mM) each instance. Statistical analysis of real-time PCR, cell viability assay, in a microwave for 20 min. Slides were then rinsed twice with PBS, and colony formation assay, YAP nuclear localization and tumor formation immersed in 3% hydrogen peroxide in PBS for 10 min to quench the in vivo was performed using GraphPad Prism version 6 for Windows endogenous peroxidase. Sections were washed in distilled water and PBS (GraphPad Software, San Diego, CA, USA). The data were analyzed by sequentially, and then incubated in blocking solution (2.5% bovine serum analysis of variance test or t-test (NS, not significant, P40.05; *Po0.05). albumin in PBS) for 30 min at room temperature. Primary antibodies diluted in blocking solution were applied to slides, followed by incubation at 4 °C overnight. After three washes with PBS, sections were incubated CONFLICT OF INTEREST with biotinylated secondary antibody (1:400, Vector Laboratories, fl Burlingame, CA, USA) for 30 min at room temperature. After three The authors declare no con ict of interest. more washes with PBS, sections were incubated with avidin solution (Vector Laboratories) for 30 min at room temperature, then with ACKNOWLEDGEMENTS diaminobenzidine solution (Sigma) under microscopic control. After three final washes in distilled water, sections were counterstained We are grateful to Ms Emiko Hori, Ms Yoko Miyanari and Research Support Center, with hematoxylin. Slides were then mounted with Vectashield (Vector Graduate School of Medical Science, Kyushu University for technical supports. This Laboratories). study was supported in part by a Grant-in-Aid for Young Scientists (B) from the

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