Abstract A42: TR3/NR4A1 as a therapeutic target for ovarian cancer

Abstract

Abstract Background: A key clinical challenge in ovarian cancer is identifying new targets and strategies to treat recurrent and platinum-resistant disease. We and others have previously linked the nuclear orphan receptor NR4A1/Nur77 (TR3) to multiple malignancies; however, the role of TR3 in cancer is complex. In ovarian cancer cells, multiple chemotherapeutic agents stimulate translocation of TR3 to mitochondria to induce apoptosis, whereas nuclear TR3 is a transcription factor with progrowth and prosurvival effects. Moreover, the prognostic value of TR3 expression in ovarian tumors is not fully defined. Two prior reports had contradictory findings, with both positive and negative associations with ovarian cancer survival observed. Objective: We undertook this study to test the therapeutic potential of inhibiting TR3 in ovarian cancer cells and to clarify the prognostic value of TR3 expression in ovarian tumors. Methods: In a panel of established ovarian cancer cell lines, including cisplatin-resistant A2780CP20 and NCI/ADR-RES and their respective isogenic counterparts, A2780PAR and OVCAR-8, we inhibited TR3 function using established chemical antagonists, 1,1-bis(3′-indolyl)-1-(p-methoxyphenyl)methane, 3,3′-[(4-methoxyphenyl)methylene]bis-1H-indole, which is metabolized to an active p-hydroxyphenyl form in cells (C-DIM), and ethyl-2-(2,3,4-trimethoxy-6-(1-octanoyl)phenyl)acetate (TMPA), as well as two distinct siRNAs that targeted TR3. Cell growth was assessed in sulforhodamine B assays; apoptosis was measured by Western blot analysis of cleaved caspase-3 expression. A luciferase reporter plasmid with three consensus TR3 binding sites was used to measure TR3 transactivation and DNA binding activity. In ovarian tumors, TR3 was measured by immunohistochemistry (IHC) on 203 formalin-fixed, paraffin-embedded tissue samples from the Vanderbilt University Medical Center Tissue Repository for Ovarian Cancer (TROC) with linked clinical data abstracted from electronic medical records. TR3 staining intensity (1: weak; 2: moderate; 3: strong) and percent of positive nuclei (0-100) were multiplied to yield an H score. Associations with progression-free survival (PFS) and overall survival (OS) were quantified by hazards ratios (HR) and 95% confidence intervals (CI) from multivariable proportional hazards regression. Results: In our ovarian cancer cell line panel, C-DIM and TMPA induced concentration-dependent decreases in cell growth and TR3 transactivation and stimulated apoptosis. The magnitude of growth inhibition and apoptosis induction in cisplatin-resistant cells was strikingly similar to that seen in isogenic counterparts. Cells with high basal TR3 expression (OVCAR-3 and OVCAR-4) showed greater responses to TR3 inhibitors than those with low TR3 expression (SKOV-3). These effects were mimicked in cells transfected with TR3-targeting siRNA compared to non-targeting siRNA-transfected controls. In 203 TROC cases, TR3 expression lower than the median (<153.6) was more likely among later-stage, higher-grade, type II tumors, with suboptimal cytoreduction and platinum resistant disease. Higher TR3 was associated with better OS (HR: 0.52, 95% CI: 0.37-0.74) in unadjusted analyses. However, after adjustment for prognostic covariates, higher TR3 was associated with significantly shorter PFS (HR: 2.35, 95% CI: 1.29-4.28). Conclusions: Our current results reconcile the discrepancy between prior TR3 reports, as associations differed due to confounding by clinical covariates. Worse outcomes among cases with high TR3 expression are supported by new experimental evidence showing reduced ovarian cancer cell growth and survival following TR3 inhibition. Our findings support further development of TR3 inhibition as a novel therapeutic approach, which could benefit a large number of ovarian cancer cases with recurrent and platinum-resistant disease. Citation Format: Alicia Beeghly-Fadiel, Dajah Chase, Johnathan Cooks, Marta Crispens, Dineo Khabele, Andrew J. Wilson. TR3/NR4A1 as a therapeutic target for ovarian cancer. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A42.