Published OnlineFirst September 5, 2013; DOI: 10.1158/0008-5472.CAN-13-1049 Cancer Molecular and Cellular Pathobiology Research GPR116, an Adhesion G-Protein–Coupled Receptor, Promotes Breast Cancer Metastasis via the Gaq-p63RhoGEF-Rho GTPase Pathway Xiaolong Tang1, Rongrong Jin1, Guojun Qu1, Xiu Wang1, Zhenxi Li1, Zengjin Yuan1, Chen Zhao1, Stefan Siwko2, Tieliu Shi1, Ping Wang1, Jianru Xiao1, Mingyao Liu1,2, and Jian Luo1 Abstract Adhesion G-protein–coupled receptors (GPCR), which contain adhesion domains in their extracellular region, have been found to play important roles in cell adhesion, motility, embryonic development, and immune response. Because most adhesion molecules with adhesion domains have vital roles in cancer metastasis, we speculated that adhesion GPCRs are potentially involved in cancer metastasis. In this study, we identified GPR116 as a novel regulator of breast cancer metastasis through expression and functional screening of the adhesion GPCR family. We found that knockdown of GPR116 in highly metastatic (MDA-MB- 231) breast cancer cells suppressed cell migration and invasion. Conversely, ectopic GPR116 expression in poorly metastatic (MCF-7 and Hs578T) cells promoted cell invasion. We further showed that knockdown of GPR116 inhibited breast cancer cell metastasis in two mammary tumor metastasis mouse models. Moreover, GPR116 modulated the formation of lamellipodia and actin stress fibers in cells in a RhoA- and Rac1-dependent manner. At a molecular level, GPR116 regulated cell motility and morphology through the Gaq-p63RhoGEF-RhoA/Rac1 pathway. The biologic significance of GPR116 in breast cancer is substantiated in human patient samples, where GPR116 expression is significantly correlated with breast tumor progres- sion, recurrence, and poor prognosis. These findings show that GPR116 is crucial for the metastasis of breast cancer and support GPR116 as a potential prognostic marker and drug target against metastatic human breast cancer. Cancer Res; 73(20); 1–13. Ó2013 AACR. Introduction leading to metastasis (2). The Rho family GTPases, including Breast cancer is the leading cause of cancer-related mor- RhoA, Rac1, and Cdc42, are essential for cytoskeletal dynam- tality in females worldwide (1). Death caused by breast ics, especially cancer cell migration (3, 4). Among them, fi cancer primarily results from cancer cells metastasizing to RhoAregulatestheactincytoskeletoninstress ber forma- fi distal organs such as lung, bone, liver, or brain. Cell motility tion (5). Rac1 modulates a meshwork of actin laments at is regulated by the cytoskeleton, and disruptions in cyto- the cell periphery to produce lamellipodia and membrane fl skeletal regulation are a means by which cancer cells devel- ruf es (5). Activated Rho GTPases induce multiple down- op inappropriate migratory and invasive characteristics, stream signaling pathways during cancer cell migration, such as ROCK1/2 signaling (4). All Rho GTPases cycle between an inactive guanosine diphosphate (GDP)-bound Authors' Affiliations: 1East China Normal University and Shanghai and an active guanosine triphosphate (GTP)-bound state. Changzheng Hospital Joint Research Center for Orthopedic Oncology, The process is accelerated by a large family of Rho guanine Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, nucleotide exchange factors (Rho GEF; ref. 6). One such Rho Shanghai, China; and 2Center for Cancer and Stem Cell Biology, Alkek GEF is p63RhoGEF (also called GEFT), which is known as an Institute of Biosciences and Technology, Texas A&M University Health – Science Center, Houston, Texas effector of the heterotrimeric guanine nucleotide binding protein GaqandtherebylinksGaq-coupled receptors Note: Supplementary data for this article are available at Cancer Research a Online (http://cancerres.aacrjournals.org/). (GPCR) to the activation of the Rho GTPases (6). G q- p63RhoGEF-Rho GTPase has been reported as a protein X. Tang and R. Jin contributed equally to this work. complex (6), which is involved in multiple physiologic func- J. Xiao and M. Liu are co-senior authors of this article. tions such as vascular smooth muscle contractility (7) and cell movement (8). Corresponding Author: Jian Luo, The Institute of Biomedical Sciences, – School of Life Sciences, East China Normal University, 500 Dongchuan G-protein coupled receptors (GPCR or GPR) are integral Road, Shanghai 200241, China. Phone: 86-212420-6947; Fax: 86-215434- membrane proteins participating in the transmission of 4922; E-mail: [email protected] signals from the extracellular environment to the cytoplasm. doi: 10.1158/0008-5472.CAN-13-1049 A variety of external stimuli, including neurotransmitters, Ó2013 American Association for Cancer Research. hormones, phospholipids, growth factors, and proteases, can www.aacrjournals.org OF1 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2013 American Association for Cancer Research. Published OnlineFirst September 5, 2013; DOI: 10.1158/0008-5472.CAN-13-1049 Tang et al. activate GPCRs (9, 10). Activated GPCRs typically transduce were purchased from Santa Cruz Biotechnology; and mono- signals to effector proteins through their heterotrimeric G clonal anti-Rac1 (23A8) antibody was obtained from Millipore, proteins. These signaling pathways represent important and GPR116 (Ab111169) antibody used to conduct immuno- specific targets for a variety of physiologic functions and histochemistry on human breast cancer tissue was purchased therapeutic approaches, ranging from the control of blood from Abcam. pressure, allergic response, kidney function, and hormonal disorders to neurologic diseases (11). As a result, approxi- GPR116 mouse monoclonal antibody production mately 50% of marketed pharmaceuticals target human The fragment of 27 amino acid residues from the N terminal GPCRs or their signaling pathways (12). However, there are region of human GPR116, with the sequence of "WNYES- few GPCRs that are cancer therapeutic targets. The adhesion TIHPLSLHEHEPAGEEALRQKR," was synthesized and used as GPCRs are the second largest GPCR family with 33 members, immunogen. BALB/c mice were immunized by intraperitoneal most of which are orphan receptors (13). This family is very injection with 100 mg of immunogen (dissolved in PBS) in a 50% special because of the so-called adhesion domains in their emulsion with complete Freunds adjuvant on day 1 and long N-terminal extracellular regions. Adhesion domains, boosted by intraperitoneal injection on days 21 and 36 with which are thought to have adhesive properties, mainly exist immunogen in incomplete Freunds adjuvant. After 60 days, in some adhesion molecules such as integrins, cadherins, freshly harvested spleen cells obtained from the immunized and selectins. However, limited studies have shown that mice were prepared for cell fusion to generate hybridoma lines, adhesion GPCRs are involved in the regulation of cell which were subsequently screened by ELISA and immuno- adhesion, motility, embryonic development, and immune blotting. The specificity of the antibody for immunoblotting response (12–14). GPR116, named Ig-hepta in the rat, is a was examined using a competition strategy with the antigen member of the adhesion GPCR family. Previous studies peptide. showed that rat Gpr116 forms a dimer and is cleaved at multiple sites in the N-terminal region yielding several Immunofluorescence staining fragments with unknown functions (15–17). Recent evidence After plating on coverslips and culturing in 24-well plates, from an adipocyte-specific GPR116-knockout mouse model cells were fixed in 4% paraformaldehyde for 15 minutes, shows that GPR116 plays a critical role in adipocyte biology permeabilized with 0.1% Triton/PBS for 5 minutes at room and systemic energy homeostasis (18). Further research on temperature, and blocked with 1% bovine serum albumin for lung development indicates that GPR116 regulates pulmo- 30 minutes. Then the cells were incubated with primary nary surfactant pool size and is important for lung surfac- antibody for 1 hour, followed by incubation with the appro- tant homeostasis (19, 20). These discoveries implicate the priate secondary antibody or Alexa Fluor 594 phalloidin for 30 physiologic and pathologic functions of GPR116. minutes at room temperature. Finally, cells were photographed As the adhesion molecules (integrins, cadherins, and selec- with a confocal laser scanning microscope (Leica TCS SP5). tins, etc.) have an important function in cancer metastasis (12), The concentrations of antibodies or dyes were: anti-paxillin it is reasonable to speculate that adhesion GPCRs also have (BD, 610051), 1 mg/mL; Alexa Fluor 488–conjugated anti-rabbit vital functions in cancer progression and metastasis. In this IgG (Invitrogen), 1 mg/mL; and Alexa Fluor 594 phalloidin study, we screened the adhesion GPCR family and identified (Invitrogen), 1:500 dilutions. GPR116 as a novel regulator of breast cancer cell migration and invasion in vitro and metastasis in vivo by modulation of the GTPase activity assay Gaq-p63RhoGEF-Rho GTPases signaling pathway. Moreover, RhoA activity in cell lysates was measured using the fusion GPR116 is strongly correlated with breast cancer progression, protein of glutathione S-transferase (GST) and the RhoA- metastasis and poor prognosis by analysis of human breast binding domain of Rhotekin (RBD). For Rac1 and Cdc42 cancer clinical samples. Our results suggest that GPR116 is a activities,