Abstract B46: Preclinical testing of TR3 activators in cisplatin-resistant ovarian cancer

Introduction: The vast majority of epithelial ovarian malignancies present as biologically aggressive, metastatic tumors. High incidence of recurrence following standard platinum-based therapy indicates an urgent need for new treatment strategies in advanced disease. We and others have shown that the immediate-early gene NR4A1/Nur77 (TR3) is linked with malignant tumors and is a potential novel target for therapy. Nucleus-to-mitochondria translocation of TR3, leading to cytochrome C release, is an important mechanism of pro-apoptotic chemotherapeutic agents, including cisplatin, in ovarian cancer cells. Furthermore, two distinct chemical classes of compound that activate TR3 by direct binding induce anti-tumor effects in certain cancer cell lines: C-DIM (an analog of diindoylmethane, a metabolite of the indole-3-carbinol chemopreventive agent found in cruciferous vegetables) and the fungal metabolite, cytosporone B (CsnB). Both classes are known to activate TR3-dependent transcription (transactivation) of pro-apoptotic genes, while CsnB also exclusively stimulates TR3 expression and cytoplasmic translocation. Objective: We hypothesized that targeted activation of TR3 will be an effective therapeutic strategy in ovarian cancer cells resistant to established cisplatin chemotherapy. To test this hypothesis, we determined the anti-tumor effects of C-DIM and CsnB in preclinical models of cisplatin-resistant ovarian cancer. Methods: The isogenic cell line pairs, OVCAR-8 and cisplatin-resistant NCI/ADR-Res, and A2780 parental (A2780PAR) and cisplatin-resistant A2780CP20 cells, were treated with C-DIM, CsnB or 0.1% DMSO as vehicle control. Sulforhodamine B colorimetric assays were used to assess cell growth and cytotoxicity in vitro. Western Blot and immunofluorescence assays were used to measure apoptotic markers (cleaved PARP, cleaved caspase 3 and cytochrome C release). TR3 DNA binding was measured by a luciferase reporter plasmid containing 3 copies of its consensus response element (NurRE). Results: C-DIM and CsnB reduced cell growth in all cell lines tested in a concentration-dependent manner. CsnB induced approximately 2-fold more potent effects than C-DIM (IC50 concentrations ranging from 20-25μM to 40-50μM, respectively). Strikingly, there were no statistically significant differences in IC50 concentrations for either drug in cisplatin-resistant cells compared to their respective isogenic counterpart. Consistent with their growth-inhibitory effects, both compounds induced p21 expression. C-DIM and CsnB treatment induced apoptosis, although different mechanisms likely underlie these effects. While both compounds stimulated TR3 transactivation, only CsnB also induced nucleus-to-mitochondrial translocation of TR3, accompanied by cytochrome C release. Conclusions: These studies highlight the potential of targeted activation of TR3, particularly by CsnB-like drugs, as a novel therapeutic strategy in cisplatin-resistant ovarian cancer. Citation Format: Andrew J. Wilson, Jeanette Saskowski, Dineo Khabele. Preclinical testing of TR3 activators in cisplatin-resistant ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr B46.