Active FOXO1 is a Key Determinant of Isoform-Specific Progesterone Receptor Transactivation and Senescence Programming

The progesterone receptor specific gene targets were investigated in ovarian and breast cancer cell lines where FOXO1 was found to be a primary factor that cooperates with PR to activate cellular senescence genes (including p21) specifically in ovarian cells. ABSTRACT: Progesterone promotes differentiation coupled to proliferation and pro-survival in the breast, but inhibits estrogen-driven growth in the reproductive tract and ovaries. Herein, it is demonstrated, using progesterone receptor (PR) isoform-specific ovarian cancer model systems, that PR-A and PR-B promote distinct gene expression profiles that differ from PR-driven genes in breast cancer cells. In ovarian cancer models, PR-A primarily regulates genes independently of progestin, while PR-B is the dominant ligand-dependent isoform. Notably, FOXO1 and the PR/FOXO1 target-gene p21 (CDKN1A) are repressed by PR-A, but induced by PR-B. In the presence of progestin, PR-B, but not PR-A, robustly induced cellular senescence via FOXO1-dependent induction of p21 and p15 (CDKN2B). Chromatin immunoprecipitation (ChIP) assays performed on PR-isoform specific cells demonstrated that while each isoform is recruited to the same PRE-containing region of the p21 promoter in response to progestin, only PR-B elicits active chromatin marks. Overexpression of constitutively active FOXO1 in PR-A-expressing cells conferred robust ligand-dependent upregulation of the PR-B target genes GZMA, IGFBP1, and p21, and induced cellular senescence. In the presence of endogenous active FOXO1, PR-A was phosphorylated on Ser294 and transactivated PR-B at PR-B target genes; these events were blocked by the FOXO1 inhibitor (AS1842856). PR isoform-specific regulation of the FOXO1/p21 axis recapitulated in human primary ovarian tumor explants treated with progestin; loss of progestin sensitivity correlated with high AKT activity. IMPLICATIONS: This study indicates FOXO1 as a critical component for progesterone signaling to promote cellular senescence and reveals a novel mechanism for transcription factor control of hormone sensitivity. The study contains 6 different sample groups measured in triplicate, for a total of 18 individual samples (18 arrays). From parental T47D-Y human breast cancer cell lines (that is a naturally occurring PR-null variant of T47D cells), we created two stable clones expressing either (1) the wild type progesterone receptor isoform A (pSG5-PR-A), or (2) the wild type progesterone receptor isoform B (pSG5-PR-B). These three cell lines were treated with either (1) vehicle control (ethanol) or (2) R5020 10e-8 M for 24 hours before total RNA harvest. Thus, the experiment contains three cell lines and two treatments (6 sample groups), treated and analyzed in triplicate (18 microarrays). Standard Illumina HumanHT-12 V4.0 chip controls were used during hybridization.