Abstract NT-088: PROGNOSTIC SIGNIFICANCE OF NR4A1/TR3 EXPRESSION IN OVARIAN CANCER
Authors: Alicia Beeghly-Fadiel, Johnathan Cooks, Dajah Chase, Marta Crispens, Dineo Khabele, Andrew J Wilson
Journal: Clinical Cancer Research (2019)

Abstract BACKGROUND: A key clinical challenge in ovarian cancer is identifying new strategies to treat patients who do not respond to poly ADP-ribose polymerase (PARP) inhibitor (PARPi) therapy. We previously linked the nuclear orphan receptor NR4A1/TR3 to pro-growth and pro-survival effects in ovarian cancer cells. However, it is unknown whether inhibiting NR4A1 function has therapeutic effects alone or in combination with PARPi in vitro. Moreover, the prognostic value of NR4A1 expression in patient tumors remains ill-defined, as two prior reports had contradictory findings for associations with ovarian cancer survival. OBJECTIVE: We undertook this study to test the therapeutic potential of inhibiting NR4A1 in ovarian cancer cells, and to clarify the prognostic value of NR4A1 expression in patient tumors. METHODS: In a panel of established ovarian cancer cell lines (OVCAR-3, OVCAR-4, SKOV-3), we inhibited NR4A1 using the chemical antagonist, 1,1-Bis(3'-indolyl)-1-(p-hydroxyphenyl) methane (C-DIM) and siRNA targeting NR4A1 (siNR4A1). Effects of C-DIM on cell growth, alone and in combination with the PARPi, olaparib and rucaparib, were assessed in sulforhodamine B (SRB) in vitro assays. Markers of apoptosis (cleaved PARP, cleaved caspase-3) and proliferation (Ki67, PCNA, p21) were measured by western blot or immunohistochemistry (IHC). In ovarian tumors, NR4A1 was measured by IHC in 203 clinically annotated formalin-fixed paraffin-embedded (FFPE) tissue samples from the Vanderbilt University Medical Center (VUMC) Tissue Repository for Ovarian Cancer (TROC). Staining intensity (1: weak; 2: moderate; 3: strong) and percent of positive nuclei (0-100) were multiplied to yield an H score for NR4A1 expression. Associations with progression-free survival (PFS) and overall survival (OS) were quantified by Hazards Ratios (HR) and 95% Confidence Intervals (CI) from proportional hazards regression. RESULTS: In ovarian cancer cell lines, C-DIM induced concentration-dependent decreases in cell growth and markers of proliferation, and stimulated apoptosis. These effects were mimicked in cells transfected with siNR4A1 compared to a non-targeting siRNA-transfected control. In combination with PARPi, C-DIM induced synergistic growth inhibition and apoptosis in vitro. In tumors, NR4A1 expression lower than the median (H score &amp;lt;153.6) was more common among later stage, higher grade, serous tumors with suboptimal cytoreduction or platinum resistant disease. Higher NR4A1 expression was associated with better OS (HR: 0.52, 95% CI: 0.37–0.74) in unadjusted analyses. However, after adjustment for important prognostic covariates, including age, stage, grade, and histologic subtype, higher NR4A1 was associated with significantly shorter PFS (HR: 2.35, 95% CI: 1.29–4.28). CONCLUSIONS: Our current results reconcile the discrepancy between prior NR4A1 reports, as associations differed due to confounding by clinical covariates. Shorter survival among cases with higher NR4A1 expression is supported by experimental evidence showing reduced ovarian cancer cell growth and increased apoptosis following NR4A1 inhibition, both alone and when combined with a PARPi. Together, our findings support further development of NR4A1 inhibition as a novel therapeutic approach that could improve response to PARPi therapy among ovarian cancer patients with chemoresistant disease. Citation Format: Alicia Beeghly-Fadiel, Johnathan Cooks, Dajah Chase, Marta Crispens, Dineo Khabele, and Andrew J Wilson. PROGNOSTIC SIGNIFICANCE OF NR4A1/TR3 EXPRESSION IN OVARIAN CANCER [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr NT-088.