Abstract P3-04-10: Progesterone receptor (PR) blockade by antiprogestin, CDB4124 in hormone receptor positive breast cancer cells leads to significant inhibition of G2/M cell cycle genes

Abstract

Abstract Background: Several lines of evidence suggest that progesterone signaling is important in the breast cancer development, particularly in young women. Therefore, we sought to establish the effects of the antiprogestin CDB4124 (telapristone), and to identify PR related signature genes in hormone receptor positive (ER+ PR+) breast cancer cells. Methods: The PR expressing breast cancer cell line T47D was used to evaluate responses to PR ligands (P4, MPA and R5020, 10nM) alone or in combination with estradiol (E2, 1nM). The effects of the antiprogestin CDB4124 (0.1 or 1μM) were tested using varying hormonal conditions. The effect on (a) PRE promoter activity by dual luciferase assay; (b) cell proliferation using the MTT assay; (c) cell cycle by flow cytometry; (d) determination of gene expression signatures related to active PR responses. For the gene array experiment, cells were treated with either vehicle or R5020 (10nM) or R5020 (10nM) plus CDB4124 (1μM) in triplicate for 24hr. Total RNA was isolated and converted to cDNA and human Illumina chip microarray was performed. Data obtained from the microarray was further analyzed by Metacore Gene GO and Ingenuity Pathway Analysis. Real time PCR was performed in triplicate to confirm the expression of those genes related to the cell cycle and proliferation. ANOVA analysis and post-hoc Sidak test were used to determined the statistical significance of the data. Results: The PRE reporter activity resulting from P4, MPA and R5020 stimulation was inhibited by 80-90% in the presence of CDB4124 at 10 to 1000nM (p< 0.001). Cell proliferation was increased by PR ligands (P4, MPA and R5020) in the presence of E2; the addition of CDB4124 caused 50% inhibition of proliferation (p< 0.01) at 72 hours. Cell cycle analysis of T47D cells treated with P4, MPA and R5020 alone or in combination with E2 showed significant increases in S and G2/M phase and decreases in G0/G1. These were blocked by CDB4124 at 0.1 or 1.0μM (p<0.05 for all). Gene GO metacore analysis of genes identified in the microarray revealed significant enrichment of cell cycle pathways (FDR, p< 1.0X10-11 ) upon treatment with R5020. The addition of CDB4124 to R5020 treated samples showed inhibition of the same cell cycle pathways (FDR,p<1.0X10-14). A 16-gene panel related to G2/M phase of cell cycle was selected based on >1.5 fold upregulation (p<0.001) during treatment with R5020,10nM and blockade by CD4124. Real time PCR confirmed upregulation of this 16 gene panel ≥2.0 (p<0.05) in the presence of PR ligands alone or in combination with E2 which were significantly blocked by the addition of CDB4124. Conclusion: These data demonstrate that PR mediated cell proliferation occurs upon treatment with three different ligands of PR (P4, MPA and R5020); that PR actively engages key genes involved in the G2/M phase of the cell cycle to drive proliferation of ER+ and PR+ cells; and that the antiprogestin, CBD4124 is a potent transcriptional inhibitor for blockade of PR mediated cell proliferation in hormone receptor positive breast cancer cells. Citation Format: Akash Gupa, Mi Ran Choi, Susan E Clare, Jun Wang, Oukseub Lee, David Z Ivancic, J Julie Kim, Seema A Khan. Progesterone receptor (PR) blockade by antiprogestin, CDB4124 in hormone receptor positive breast cancer cells leads to significant inhibition of G2/M cell cycle genes [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-04-10.