Phosphorylated and sumoylation-deficient progesterone receptors drive proliferative gene signatures during breast cancer progression.
BACKGROUND
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 (that is, a repressive modification). Antagonism of PR small ubiquitin-like modifier (SUMO)ylation by mitogenic protein kinases suggests a mechanism for derepression (that is, 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 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays 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 (that is, 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 (that is, derepressed) phospho-PRs may benefit from endocrine (antiestrogen) therapies that contain an antiprogestin.
Departments of Medicine (Division of Hematology, Oncology, and Transplantation) and Pharmacology, Masonic Cancer Center, University of Minnesota, 420 Delaware Street SE, Minneapolis, MN 55455 USA.
Todd P
TP
Knutson
Andrea R
AR
Daniel
Danhua
D
Fan
Kevin At
KA
Silverstein
Kyle R
KR
Covington
Suzanne Aw
SA
Fuqua
Carol A
CA
Lange
D017209:Apoptosis; D001943:Breast Neoplasms / Q000235:genetics* / Q000378:metabolism*; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002470:Cell Survival; D017362:Cyclic AMP Response Element-Binding Protein / Q000235:genetics; D051357:Cyclin-Dependent Kinase 2 / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics; D018450:Disease Progression; D005260:Female; D020869:Gene Expression Profiling; D006801:Humans; D061986:MCF-7 Cells; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009363:Neoplasm Proteins / Q000235:genetics; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000378:metabolism; D011980:Receptors, Progesterone / Q000235:genetics* / Q000378:metabolism*; D015398:Signal Transduction; D025841:Small Ubiquitin-Related Modifier Proteins / Q000037:antagonists & inhibitors; D058207:Sumoylation; D015533:Transcriptional Activation; D059467:Transcriptome
D017362:Cyclic AMP Response Element-Binding Protein; D051357:Cyclin-Dependent Kinase 2; D004268:DNA-Binding Proteins; D020928:Mitogen-Activated Protein Kinases; D009363:Neoplasm Proteins; D018719:Erb-b2 Receptor Tyrosine Kinases; D011980:Receptors, Progesterone; D025841:Small Ubiquitin-Related Modifier Proteins; C115163:CREB1 protein, human; C120898:KMT2D protein, human; C508053:ERBB2 protein, human; C495894:CDK2 protein, human