Abstract 1317: Antiprogestin drug development: in vitro validation of a potential clinical biomarker.

Abstract

Abstract Background: Antihormonal agents (AH) are some of the most clinically useful anticancer drugs. AH resistance and the availability of alternative treatments have made patient selection increasingly important for AH drug development. We hypothesized that the progesterone receptor (PR) may be constitutively active because of the aberrant biological pathways and that selecting patients with baseline tumor PR activation would predict for antiprogestin activity. Activated PR (APR) can be visualized in nuclei as fluorescent aggregates via fluorescent green protein engineered PR cell lines in vitro and by immunofluorescence (IF) in tissues. The detection of PR foci has been associated with transcription and gene activation in vitro. PR phosphorylation (phos) is induced by a variety of pathways. We have developed a IHC method that allows APR determination in tumor biopsies on a routine basis (SABCS 2012). The predictive nature of the observation of APR by IHC in vitro has been tested with onapristone (ONA) for cell survival and proliferation. Methods: 8 human breast cancer (BT-474, CAMA-1, EVSA-T, HCC-1954, MCF-7, MDA-MB-231, T-47D, ZR-75-1) and 2 human endometrial cancer (Ishikawa, HEC-1-A) cell lines were studied. Cytoblocks for APR analysis were made for each time point and experimental condition, baseline ER/PR expression was determined by IHC and PR phos was analyzed with specific antibodies (pSER162, 190, 294, 400, 554) by Western Blotting and IHC. Culture conditions: normal and stripped FBS, +/- ONA and growth factor/hormone exposure (EGF, FGF2, E2 and P4) in triplicate. Analysis was performed at baseline (BL), 6h, 96h and 7 days. Cell viability was assessed by MTS assay, proliferation was studied by Ki67 analysis. APR foci were determined by IHC and read by a pathologist blinded to the experimental conditions. Results: At BL only two cell lines were PR pos/APR pos (T47D, CAMA-1) and all other cell lines where PR pos/APR neg (BT-474, EVSA-T, HCC-1954, MCF-7) or PR neg/APR neg (Ishikawa, HEC-1-A, MDA-MB-231, ZR-75-1). ONA exerted inhibitory effect only in APR pos cell lines. In stripped FBS at 6h, T47D APR pos status was converted to APR neg by ONA except with E2 stimulation which required longer treatment. The CAMA-1 data are not yet available. No APR neg cell lines were converted into APR pos except in one experiment after protracted exposure to EGF. ONA generally decreased phos; phos decrease measured by WB did not correlate with growth inhibition. Phos status determined by IHC and Ki67 proliferation assessment are currently in process and will be presented with the confirmatory data. Conclusions: Baseline APR positivity is predictive of ONA activity in vitro. ONA converts APR pos cells to APR neg within 6h in most cases, a time frame consistent with PR biology. The growth inhibitory effects of ONA were not correlated to PR phos in the cell lines and conditions tested. The APR IHC methodology may be applicable in the clinical setting. Citation Format: Erard Gilles, Laura Caplier, Alexander Valent, Guillaume Serin, Anne Gompel, Jacques Bosq, Alexander Zukiwski. Antiprogestin drug development: in vitro validation of a potential clinical biomarker. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1317. doi:10.1158/1538-7445.AM2013-1317