ABSTRACT: INTRODUCTION: Progesterone receptors (PR) are emerging as important breast cancer drivers. Phosphorylation events common to breast cancer cells impact PR transcriptional activity, in part by direct phosphorylation. PR-B but not PR-A isoforms are phosphorylated on Ser294 by mitogen activated protein kinase (MAPK) and cyclin dependent kinase 2 (CDK2). Phospho-Ser294 PRs are resistant to ligand-dependent Lys388 SUMOylation (i.e. a repressive modification). Antagonism of PR small ubiquitin-like modifier protein (SUMO)ylation by mitogenic protein kinases suggests a mechanism for derepression (i.e. transcriptional activation) of target genes. As a broad range of PR protein expression is observed clinically, a PR gene signature would provide a valuable marker of PR contribution to early breast cancer progression. METHODS: Global gene expression patterns were measured in T47D and MCF-7 breast cancer cells expressing either wild-type (SUMOylation-capable) or K388R (SUMOylation-deficient) PRs and subjected to pathway analysis. Gene sets were validated by RT-qPCR. Recruitment of coregulators and histone methylation levels were determined by chromatin immunoprecipitation. Changes in cell proliferation and survival were determined by MTT and western blotting. Finally, human breast tumor cohort datasets were probed to identify PR-associated gene signatures; metagene analysis was employed to define survival rates in patients whose tumors express a PR gene signature. RESULTS: "SUMO-sensitive" PR target genes primarily include genes required for proliferative and pro-survival signaling. DeSUMOylated K388R receptors are preferentially recruited to enhancer regions of derepressed genes (i.e. MSX2, RGS2, MAP1A, and PDK4) with the steroid receptor coactivator, CREB-(cAMP-response element-binding protein)-binding protein (CBP), and mixed lineage leukemia 2 (MLL2), a histone methyltransferase mediator of nucleosome remodeling. PR SUMOylation blocks these events, suggesting that SUMO modification of PR prevents interactions with mediators of early chromatin remodeling at "closed" enhancer regions. SUMO-deficient (phospho-Ser294) PR gene signatures are significantly associated with human epidermal growth factor 2 (ERBB2)-positive luminal breast tumors and predictive of early metastasis and shortened survival. Treatment with antiprogestin or MEK inhibitor abrogated expression of SUMO-sensitive PR target-genes and inhibited proliferation in BT-474 (estrogen receptor (ER)+/PR+/ERBB2+) breast cancer cells. CONCLUSIONS: We conclude that reversible PR SUMOylation/deSUMOylation profoundly alters target gene selection in breast cancer cells. Phosphorylation-induced PR deSUMOylation favors a permissive chromatin environment via recruitment of CBP and MLL2. Patients whose ER+/PR+ tumors are driven by hyperactive (i.e. derepressed) phospho-PRs may benefit from endocrine (antiestrogen) therapies that contain an antiprogestin.