Phosphorylated and sumoylation-deficient progesterone receptors drive proliferative gene signatures during breast cancer progression

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. .
Breast cancer research: BCR (Impact Factor: 5.49). 06/2012; 14(3):R95. DOI: 10.1186/bcr3211
Source: PubMed


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.
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.
'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.
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.

Download full-text


Available from: Kyle Covington, Jul 20, 2015
  • Source
    • "These differences are perfectly in line with the altered androgen regulation of several genes influencing cell growth. Fittingly, SUMOylation modulates the target gene selection of progesterone receptor (PR) as well as glucocorticoid receptor (GR) such that their SUMO sensitive targets include genes associated with cell growth and proliferation (15,34). This is also the case with SUMOylation-defective microphthalmia-associated transcription factor (MITF) mutant associated with the predisposition to melanoma and renal carcinoma (39). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Androgen receptor (AR) plays an important regulatory role in prostate cancer. AR's transcriptional activity is regulated by androgenic ligands, but also by post-translational modifications, such as SUMOylation. To study the role of AR SUMOylation in genuine chromatin environment, we compared androgen-regulated gene expression and AR chromatin occupancy in PC-3 prostate cancer cell lines stably expressing wild-type (wt) or doubly SUMOylation site-mutated AR (AR-K386R,K520R). Our genome-wide gene expression analyses reveal that the SUMOylation modulates the AR function in a target gene and pathway selective manner. The transcripts that are differentially regulated by androgen and SUMOylation are linked to cellular movement, cell death, cellular proliferation, cellular development and cell cycle. Fittingly, SUMOylation mutant AR cells proliferate faster and are more sensitive to apoptosis. Moreover, ChIP-seq analyses show that the SUMOylation can modulate the chromatin occupancy of AR on many loci in a fashion that parallels their differential androgen-regulated expression. De novo motif analyses reveal that FOXA1, C/EBP and AP-1 motifs are differentially enriched at the wtAR- and the AR-K386R,K520R-preferred genomic binding positions. Taken together, our data indicate that SUMOylation does not simply repress the AR activity, but it regulates AR's interaction with the chromatin and the receptor's target gene selection.
    Nucleic Acids Research 06/2014; 42(13). DOI:10.1093/nar/gku543 · 9.11 Impact Factor
  • Source
    • "We also compared our dataset with several other previously published datasets of PR-regulated genes generated by microarray expression experiments in T47D cells [10], [11], [12], [13], [14], [15]. We found that both stringency groups contained previously reported PR-regulated genes (350 out of the 1287) (Table S1). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Despite the pleiotropic effects of the progesterone receptor in breast cancer, the molecular mechanisms in play remain largely unknown. To gain a global view of the PR-orchestrated networks, we used next-generation sequencing to determine the progestin-regulated transcriptome in T47D breast cancer cells. We identify a large number of PR target genes involved in critical cellular programs, such as regulation of transcription, apoptosis, cell motion and angiogenesis. Integration of the transcriptomic data with the PR-binding profiling of hormonally treated cells identifies numerous components of the small-GTPases signaling pathways as direct PR targets. Progestin-induced deregulation of the small GTPases may contribute to the PR's role in mammary tumorigenesis. Transcript expression analysis reveals significant expression changes of specific transcript variants in response to the extracellular hormonal stimulus. Using the NET1 gene as an example, we show that the PR can dictate alternative promoter usage leading to the upregulation of an isoform that may play a role in metastatic breast cancer. Future studies should aim to characterize these selectively regulated variants and evaluate their clinical utility in prognosis and targeted therapy of hormonally responsive breast tumors.
    PLoS ONE 06/2014; 9(6):e98404. DOI:10.1371/journal.pone.0098404 · 3.23 Impact Factor
  • Source
    • "The PR is a member of the steroid hormone receptor subgroup of ligand-activated transcription factors within the large nuclear receptor superfamily, and is an important down-stream effector of estrogen-receptor (ER) signaling; in most circumstances, estrogen is required for robust PR expression. PR binding to DNA, either directly through progesterone response elements or indirectly through tethering interactions with other transcription factors, activates transcriptional profiles associated with mammary gland proliferation and breast cancer [5-9]. Additionally, PR binding interactions with transcriptional co-activators and repressors are critical to PR transcription factor function [10]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The ovarian steroid hormone, progesterone, and its nuclear receptor, the progesterone receptor, are implicated in the progression of breast cancer. Clinical trial data on the effects of hormone replacement therapy underscore the importance of understanding how progestins influence breast cancer growth. The progesterone receptor regulation of distinct target genes is mediated by complex interactions between the progesterone receptor and other regulatory factors that determine the context-dependent transcriptional action of the progesterone receptor. These interactions often lead to post-translational modifications to the progesterone receptor that can dramatically alter receptor function, both in the normal mammary gland and in breast cancer. This review highlights the molecular components that regulate progesterone receptor transcriptional action and describes how a better understanding of the complex interactions between the progesterone receptor and other regulatory factors may be critical to enhancing the clinical efficacy of anti-progestins for use in the treatment of breast cancer.
    BMC Medicine 02/2014; 12(1):32. DOI:10.1186/1741-7015-12-32 · 7.25 Impact Factor
Show more