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Integrity of SOS1/EPS8/ABI1 Tri-Complex Determines Ovarian Cancer Metastasis

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Integrity of SOS1/EPS8/ABI1 Tri-Complex Determines Ovarian Cancer Metastasis Published OnlineFirst November 30, 2010; DOI: 10.1158/0008-5472.CAN-10-2394 Cancer Tumor and Stem Cell Biology Research Integrity of SOS1/EPS8/ABI1 Tri-Complex Determines Ovarian Cancer Metastasis Huijun Chen1,2, Xufeng Wu3, Zhixing K. Pan4, and Shuang Huang2 Abstract Ovarian cancer is mainly confined in peritoneal cavity and its metastasis is often associated with the formation of malignant ascites. As lysophosphatidic acid (LPA) is present at high levels in ascites of ovarian cancer patients and potently stimulates cell migration, we reason that LPA-stimulated cell migration may play an important role in ovarian cancer metastasis. Here, we show that only ovarian cancer cell lines with LPA migratory response undergo peritoneal metastatic colonization. LPA-stimulated cell migration is required for metastatic colonization because knockdown of LPA receptor subtype 1 (LPAR1) abolishes this event. However, the difference in metastatic potentials is not caused by the absence of LPAR1 because both metastatic and nonmetastatic lines express similar levels of LPAR1. Instead, we find that LPA can activate Rac only in metastatic cells and that metastatic colonization of ovarian cancer cells necessitates Rac activity. These results thus suggest that LPA-induced Rac activation is a prerequisite for ovarian cancer metastasis. In metastatic cells, Rac activation is facilitated by SOS1/EPS8/ABI1 tri-complex and the integrity of this tri-complex is essential for LPA- stimulated cell migration and metastatic colonization. We show that at least 1 member of SOS1/EPS8/ABI1 tri- complex is absent in nonmetastatic ovarian cancer cells and reexpressing the missing one conferred them with metastatic capability. Importantly, coexpression of SOS1, EPS8, and ABI1, but not of any individual member of SOS1/EPS8/ABI1 tri-complex, correlates with advanced stages and shorter survival of ovarian cancer patients. Our study implicates that the integrity of SOS1/EPS8/ABI1 tri-complex is a determinant of ovarian cancer metastasis. Cancer Res; 70(23); 9979–90. Ó2010 AACR. Introduction (3, 4). Apparently, the ability of ovarian cancer cells to migrate is essential for ovarian cancer metastasis. Ovarian cancer has the highest mortality rate among gyne- Cell migration is a complex process involving the reorga- cologic cancers mainly due to complication of metastasis (1). nization of actin cytoskeleton that is facilitated by the mem- Unlike other solid tumors that rely on the vasculature for bers of the Rho GTPases including Rac, Cdc42, and Rho (5–7). metastasis, ovarian cancer is predominantly confined within In the process of cell migration, Rac facilitates the formation the abdominal cavity and spread by direct extension to of actin-rich membrane ruffle, called lamellipodia, at the adjacent organs and/or disseminate throughout the perito- leading edge of migrating cells. Cdc42 regulates cell polarity neal cavity (2, 3). A widely recognized behavior of ovarian and filopodia formation, thus controlling the direction of cell cancer is its ability to seed the abdominal cavity with tumor movement. Rho controls the formation of stress fibers and implants, subsequent migration of tumor cells into perito- maintains focal adhesions at the rear of the cells (6). Ras has neum and underlying organs, and the formation of ascites also been implicated to play an important role in cell migra- tion because microinjection of Ras-neutralizing antibody blocks cell migration (7) and forced expression of constitu- Authors' Affiliations: 1Department of Gynecological Oncology, Zhongnan tively active Ras mutant enhances ovarian cancer cell migra- Hospital of Wuhan University, Wuhan, Hubei, China; 2Department of tion (8). As Ras can activate Rac through Tiam1 (9), b-PIX (10), Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, Georgia; 3Department of Gynecology, Hubei Maternity and Child or SOS1/EPS8/ABI1 tri-complex (11, 12), it is likely that Ras Care Hospital, Wuhan, Hubei, China; and 4Department of Medical Immu- may facilitate cell migration by regulating Rac activity. nology and Microbiology, University of Toledo Medical Center, Toledo, Lysophosphatidic acid (LPA) is a growth factor–like phos- Ohio pholipid and has been uniquely linked to ovarian malignan- Note: Supplementary material for this article is available at Cancer cies. For example, LPA triggers protease production/ Research Online (http://cancerres.aacrjournals.org/). activation (13, 14) and Cox-2 expression (15), thus facilitating Corresponding Author: Shuang Huang, Department of Biochemistry and Molecular Biology, Medical College of Georgia, 1459 Laney Walker ovarian cancer cell invasion. It also promotes angiogenesis by Blvd, Augusta, GA 30907. Phone: 706-721-1637; Fax: 706-721-6608; inducing the expression of various proangiogenic factors E-mail: [email protected]. including VEGF (16), IL8 (17), and Gro-a (18). Moreover, doi: 10.1158/0008-5472.CAN-10-2394 LPA can be produced and secreted into peritoneal cavity Ó2010 American Association for Cancer Research. by both ovarian cancer cells (19, 20) and mesothelial cells www.aacrjournals.org 9979 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2010 American Association for Cancer Research. Published OnlineFirst November 30, 2010; DOI: 10.1158/0008-5472.CAN-10-2394 Chen et al. nu (21). Importantly, LPA is present at high levels in ascites of female nude mice (Hsd: athymic Nude-Foxn1 , Harlan Spra- ovarian cancer patients (22–25) and potently stimulates ovar- gue Dawley) were intraperitoneally injected with 107 cells per ian cancer cell migration (8, 26, 27). Therefore, ascite-borne mouse. To determine the role of LPAR1, SOS1, EPS8, and ABI1 LPA is likely to play a critical role in ovarian cancer metastasis in metastatic colonization, SK-OV3 and HEY cells expressing by facilitating cell migration. shRNAs against these genes were intraperitoneally injected In this study, we demonstrated an excellent correlation into mice. To determine the importance of Rac activity, cells between LPA migratory responses and metastatic potentials expressing constitutively active Rac1 (Rac1G12V) or dominant- in a panel of ovarian cancer cell lines. Preventing LPA-stimu- negative Rac1 (Rac1T17N) were injected into mice. Five weeks lated cell migration by silencing LPA receptor subtype 1 after injection, the mice were sacrificed and autopsied. Visible (LPAR1) diminished peritoneal metastatic colonization of metastatic implants were also collected and weighed. All ovarian cancer cells, implicating the importance of LPA- procedures were approved by the Institution Animal Care stimulated cell migration in ovarian cancer metastasis. LPA Committee at Medical College of Georgia. activates Rac only in metastatic cells, and a signaling pathway consisting of Ras-SOS1/EPS8/ABI1 tri-complex mediates LPA- Real-time quantitative PCR induced Rac activation. Interestingly, 1 or more members of Total RNA was extracted from cells using Trizol (Invitro- SOS1/EPS8/ABI1 tri-complex are absent in nonmetastatic gen), treated by DNase I and reverse transcribed with Super- ovarian cancer cells; however, reexpressing the missing mem- Scriptase II (Invitrogen). Generated cDNA was subjected to ber converts the nonmetastatic lines to metastatic ones. real-time PCR to measure LPAR1, LPAR2, LPAR3, and GAPDH Finally, we show that SOS1/EPS8/ABI1 coexpression, but levels with the respective TaqMan probes (Applied Biosys- not any one alone, correlates with advanced clinical stage tem). The expression levels were standardized by comparing and shorter survival of ovarian cancer patients. the Ct values of target with that of GAPDH. Materials and Methods Rac, Cdc42, and Rho activity assays Activation of Rac/Cdc42, and Rho was measured with the Cells, shRNAs, and other reagents Rac/Cdc42 and the Rho activity assay kits (Cellbio Labs). To All cells were maintained in DMEM containing 10% fetal determine the effect of LPA on Rac, Cdc42, or RhoA activity, calf serum at 37C in a humidified incubator supplied with 5% cells(2 Â 106 cellsper10-cm dish)wereserum-starved for2 days CO2. The shRNA sequences for each target genes were and then stimulated with 10 mmol/L of LPA for various times. designed using web-based Block-iT program (Invitrogen) The cells were lysed and analyzed for Rac, Cdc42, or RhoA and subcloned into pLV-shRNA vector (Biosettia). Informa- activity. To determine the effect of constitutively active Ras on tion for shRNA sequences, antibodies, and other reagents are Rac activity, HEY, IGROV1, OVCAR3, and SK-OV3 cells were described in Supplementary Data. infected with empty or H-RasG12V-containing retrovirus for 2 days followed by 2-day puromycin selection. The cells were Transwell cell migration assay lysed and analyzed for Rac activity. To determine how silencing Transwell cell migration was assayed as previously Tiam1, SOS1, EPS8, and ABI1 affected LPA-induced Rac activa- described (28). Briefly, the lower phase of Transwell was tion, HEY and SK-OV3 cells were incubated with lentiviral coated with 10 mg/mL of collagen I or laminin, and serum- vectors containing the respective shRNAs for 2 days and starved free medium with or without 10 mmol/L of LPA was added to for another 2 days. The cells were stimulated with 10 mmol/L of lower chambers. Serum-starved cells (105 per well) were added LPA for 2 or 5 minutes followed by the analysis of Rac activity. to Transwells and allowed to migrate for 4 hours. Cells that To determine the effect of forced expression of SOS1, EPS8, and remained
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