Molecular and Cellular Pathobiology TR 3 Modulates Platinum Resistance in Ovarian Cancer

In metastatic ovarian cancer, resistance to platinum chemotherapy is common. Although the orphan nuclear receptor TR3 (nur77/NR4A1) is implicated in mediating chemotherapy-induced apoptosis in cancer cells, its role in ovarian cancer has not been determined. In an ovarian cancer tissue microarray, TR3 protein expression was elevated in stage I tumors, but downregulated in a significant subset of metastatic tumors. Moreover, TR3 expression was significantly lower in platinum-resistant tumors in patients with metastatic disease, and low TR3 staining was associated with poorer overall and progression-free survival. We have identified a direct role for TR3 in cisplatin-induced apoptosis in ovarian cancer cells. Nucleus-to-cytoplasm translocation of TR3 was observed in cisplatin-sensitive (OVCAR8, OVCAR3, and A2780PAR) but not cisplatin-resistant (NCI/ADR-RES and A2780CP20) ovarian cancer cells. Immunofluorescent analyses showed clear overlap between TR3 and mitochondrial Hsp60 in cisplatin-treated cells, which was associated with cytochrome c release. Ovarian cancer cells with stable shRNAor transient siRNA-mediated TR3 downregulation displayed substantial reduction in cisplatin effects on apoptotic markers and cell growth in vitro and in vivo. Mechanistic studies showed that the cisplatin-induced cytoplasmic TR3 translocation required for apoptosis induction was regulated by JNK activation and inhibition of Akt. Finally, cisplatin resistance was partially overcome by ectopic TR3 overexpression and by treatment with the JNK activator anisomycin and Akt pathway inhibitor, wortmannin. Our results suggest that disruption of TR3 activity, via downregulation or nuclear sequestration, likely contributes to platinum resistance in ovarian cancer. Moreover, we have described a treatment strategy aimed at overcoming platinum resistance by targeting TR3. Cancer Res; 73(15); 4758–69. 2013 AACR. Introduction Ovarian cancer is the most lethal gynecologic malignancy and the vast majority of epithelial ovarian malignancies present as biologically aggressive, metastatic disease (1, 2). The high incidence of relapse following standard platinum-based therapy indicates that there is an urgent need for new treatment strategies andnovel insight intomechanisms of platinum resistance. As most high-grade ovarian cancers harbor mutations in TP53 (3), identifying antitumor effectors that act independently of p53 is an important goal. TR3 (also known as nur77 and NR4A1) has emerged as a major regulator of cancer cell survival and an attractive therapeutic target (4). TR3 is a member of the NR4A family of nuclear receptors, and mediates apoptosis in various cancer cell types in response to a wide range of chemotherapeutic agents. Knownmechanisms of TR3-induced apoptosis include p53-independent nuclear-cytoplasmic translocation, leading to cytochrome c release in response to various proapoptotic drugs (5–11), or upregulation of proapoptotic genes and/or downregulation of antiapoptotic genes (12–14). Nuclear export of TR3 is known to involve specific changes in its phosphorylation status, such as N-terminal serine phosphorylation by JNK and loss of Akt-mediated phosphorylation on serine 351 (15). At the mitochondria, TR3 binds Bcl-2, which induces a conformational change such that Bcl-2 assumes a proapoptotic function (9). Apoptosismediated at least partly throughTR3 activity has been reported in an ovarian-derived teratocarcinoma cell line, Pa-1 (5). However, it is unknown whether TR3 mediates apoptotic effects of established chemotherapeutic agents such as cisplatin in ovarian cancer cells of epithelial origin. Despite the clear proapoptotic role for TR3 identified in chemotherapy-treated cancer cells, accumulated evidence indicates that it may play a more complex role in tumorigenesis. TR3 expression is also induced bymitogenic factors in the Authors' Affiliations: Division of Gynecologic Oncology, Department of Obstetrics and Gynecology and Departments of Surgical Research, Biostatistics, and Pathology, Microbiology and Immunology, VanderbiltIngramCancer Center, Vanderbilt University School ofMedicine, Nashville, Tennessee Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Corresponding Author: Dineo Khabele, Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, Vanderbilt University School of Medicine, B1100 Medical Center North, Nashville, TN 37232. Phone: 615-322-8072; Fax: 615-343-8403; E-mail: dineo.khabele@vanderbilt.edu doi: 10.1158/0008-5472.CAN-12-456