LPA receptor 2 mediates LPA-induced endometrial cancer invasion Joanie Mayer Hope ⁎ , Feng-qiang Wang, Jill S. Whyte, Edgardo V. Ariztia, Walid Abdalla, Kara Long, David A. Fishman ⁎ Department of Obstetrics and Gynecology, New York University School of Medicine, 550 First Avenue, TH528 New York, NY 10016, USA Received 5 June 2008 Available online 18 November 2008 Abstract Objective. We have previously shown that lysophosphatidic acid (LPA) promotes the ovarian cancer metastatic cascade. In this study, we evaluated the role of LPA on endometrial cancer invasion. Methods. Transient mRNA knockdown was accomplished using pre-designed siRNA duplexes against LPA receptor 2 (LPA2) and human matrix metalloproteinase-7 (MMP-7). RT-PCR was used to characterize LPA receptor and MMP-7 expression. Analysis of in vitro invasion was performed with rat-tail collagen type I coated Boyden chambers. Gelatin zymography was used to evaluate the MMP activity in cell culture conditioned media. Cell–cell and cell–matrix attachment was also assessed upon LPA2 knockdown to further illuminate the LPA2 cascade. Results. LPA increases HEC1A cellular invasion at physiologic concentrations (0.1–1 μM). Of the four principle LPA receptors, LPA2 is predominantly expressed by HEC1A cells. Transient transfection of LPA2 siRNA reduced LPA2 mRNA expression in HEC1A cells by 93% (P b 0.01). Silencing LPA2 eliminated the LPA-stimulated increase in invasion (P b 0.05) and reduced LPA-induced MMP-7 secretion/activation, without significantly affecting cell–cell or cell–matrix adhesion. Silencing MMP-7 reduced overall invasion but did not eliminate LPA's pro- invasive effect on HEC1A cells, as compared to negative control (P b 0.05). Gelatin zymography confirmed that LPA2 and MMP-7 knockdown reduced MMP-7 activation in HEC1A conditioned media. Conclusion. LPA2 mediates LPA-stimulated HEC1A invasion and the subsequent activation of MMP-7. © 2008 Elsevier Inc. All rights reserved. Keywords: Lysophosphatidic acid (LPA); Matrix metalloproteinase-7 (MMP-7); LPA receptor 2 (LPA2); siRNA; HEC1A; Endometrial cancer invasion Introduction Endometrial cancer is one of the most common gynecologic malignancies with an estimated 40,100 new cases and 7470 deaths in 2008 [1]. When endometrial malignancies are detected early, the prognosis is excellent with a five-year survival approaching 90% [2–3]. For stage III and IV disease, however, the five-year survival decreases to 45% and 15%, respectively [1]. In 2006, the Gynecologic Oncology Group protocol 122 changed the standard of treatment for advanced stage disease from radiation alone to platinum-based chemotherapy [4]. While chemotherapy improved survival for stage III and IV disease, the associated toxicities are significant and in some cases intolerable. Thus the search for more effective, targeted, and less morbid therapies is critical. A potential novel locus for anti-cancer therapy is the lysophosphatidic acid (LPA) receptor family. LPA is a bioactive phospholipid with a potent regulatory role influencing cellular proliferation, migration, invasion and survival, as well as changes in morphology and differentiation [5]. LPA levels have been found to be elevated in the plasma and or ascites of ovarian, cervical, and endometrial cancer patients [6–8]. While LPA increases cellular proliferation and viability in some cell types, it induces apoptosis and necrosis in others [9–13]. LPA effects are mediated through five specific G protein-coupled receptors (LPA1–5), among which LPA1–3 are members of the endothelial differentiation gene (Edg) family. LPA receptors play a mechanistic role in cancer growth and metastasis [14–17]. LPA mediated signal transduction has been implicated in a number of oncogenic and physiologic processes including focal adhesion kinase phosphorylation, focal adhesion formation, Available online at www.sciencedirect.com Gynecologic Oncology 112 (2009) 215 – 223 www.elsevier.com/locate/ygyno ⁎ Corresponding authors. Fax: +1 212 263 5742. E-mail addresses: joanie.hope@nyumc.org (J.M. Hope), david.fishman@nyumc.org (D.A. Fishman). 0090-8258/$ - see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.ygyno.2008.09.019