Linkage of progestin and epidermal growth factor signaling: Phosphorylation of progesterone receptors mediates transcriptional hypersensitivity and increased ligand-independent breast cancer cell growth

Department of Medicine, Division of Hematology, Oncology, and Transplantation and Pharmacology, University of Minnesota Cancer Center, Minneapolis, MN 55455, United States.
Steroids (Impact Factor: 2.64). 03/2007; 72(2):188-201. DOI: 10.1016/j.steroids.2006.11.009
Source: PubMed


Progesterone receptor (PR) action is linked to epidermal growth factor (EGF) initiated signaling pathways at multiple levels; mitogen-activated protein kinases (MAPKs) are key mediators of this important cross-talk. Herein, we probed the effects of EGF on PR function and regulation of breast cancer cell growth. EGF stimulated rapid and transient phosphorylation of PR-B Ser294 relative to persistent phosphorylation of this site induced by the synthetic progestin, R5020. EGF induced nuclear translocation and DNA binding of unliganded wild-type, but not mutant PRs containing an Ala at position 294 (S294A). However, EGF alone induced little to no PR-B transcriptional activity; S294A PR-B was transcriptionally impaired. In contrast, pretreatment of cells with EGF (30min) significantly increased the potency and efficacy of wild-type, but not S294A PR transcriptional activity in response to progestin, and enhanced ligand-dependent downregulation of wild-type but not S294A PR. Replacement of Ser294 with aspartic acid (S294D) to mimic phosphorylation at this site decreased receptor stability and, as predicted, heightened progestin-induced transcription relative to wild-type PR-B. RT-PCR demonstrated the Ser294 phosphorylation-dependence of selected PR target genes (TGFalpha and HB-EGF). Surprisingly, PR-B expressing cells growing in soft agar were highly responsive to EGF or progestin, and this was further stimulated by the combination of both hormones. Cells expressing S294A PR exhibited reduced soft agar growth, and were also sensitive to R5020 alone, but failed to respond to EGF. These results suggest that PR Ser294 is an important "sensor" for growth factor inputs that affects PR function and breast cancer cell growth in the absence of progestin or in the presence of low or "sub-threshold" progestin concentrations. PR function likely contributes to breast cancer progression when EGFR family members or their ligands are overexpressed, a condition that predicts low abundance, but highly active and nuclear PR.

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    • "The fact, that the reduction in ERK-1/-2 activity was limited to day 21 only, might point to a change in endocrine milieu at day 28, which might be reflected by a significant increase in serum progesterone in our IUGR rats at this time-point. Growing evidence points to a cross-talk of progesterone and ERK-1/-2 action [68], [69]. Interestingly, mammary glands of IUGR animals fed a high-fat diet after puberty, show an increased phosphorylation of ERK-1/-2 and an induction of progesterone receptor at 4 months of age, which was associated with an increase in mammary tumor risk [17]. "
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    ABSTRACT: Background Intrauterine growth restriction (IUGR) is thought to lead to fetal programming that in turn contributes to developmental changes of many organs postnatally. There is evidence that IUGR is a risk factor for the development of metabolic and cardiovascular disease later in life. A higher incidence of breast cancer was also observed after IUGR. This could be due to changes in mammary gland developmental pathways. We sought to characterise IUGR-induced alterations of the complex pathways of mammary development at the level of the transcriptome in a rat model of IUGR, using pathways analysis bioinformatics. Methodology/Principal Findings We analysed the mammary glands of Wistar rats with IUGR induced by maternal low protein (LP) diet at the beginning (d21) and the end (d28) of pubertal ductal morphogenesis. Mammary glands of the LP group were smaller in size at d28, however did not show morphologic changes. We identified multiple differentially expressed genes in the mammary gland using Agilent SurePrint arrays at d21 and d28. In silico analysis was carried out using Ingenuity Pathways Analysis. In mammary gland tissue of LP rats at d21 of life a prominent upregulation of WT1 and CDKN1A (p21) expression was observed. Differentially regulated genes were associated with the extracellular regulated kinase (ERK)-1/-2 pathway. Western Blot analysis showed reduced levels of phosphorylated ERK-1/-2 in the mammary glands of the LP group at d21. To identify possible changes in circulating steroid levels, serum LC-Tandem mass-spectrometry was performed. LP rats showed higher serum progesterone levels and an increased corticosterone/dehydrocorticosterone-ratio at d28. Conclusions/Significance Our data obtained from gene array analysis support the hypothesis that IUGR influences pubertal development of the rat mammary gland. We identified prominent differential regulation of genes and pathways for factors regulating cell cycle and growth. Moreover, we detected new pathways which appear to be programmed by IUGR.
    PLoS ONE 06/2014; 9(6):e100504. DOI:10.1371/journal.pone.0100504 · 3.23 Impact Factor
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    • "We show that AREG expression is regulated not only by liganded-PRB but interestingly also by unliganded PRA. PR transcriptional activity is known to be highly influenced by EGF stimulation [46], [70]. The differential roles of PR isoforms in regulating HBEGF and AREG expression under P4 and EGF stimulation have not been described. "
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    ABSTRACT: Progesterone receptor isoforms (PRA and PRB) are expressed at equal levels in normal mammary cells. However, alteration in PRA/PRB expression is often observed in aggressive breast cancer suggesting differential contribution of PR isoforms in carcinogenesis. The mechanisms underlying such processes remain to be established mainly due to paucity of appropriate cellular models. To investigate the role of PR isoforms and the impact of imbalanced PRA/PRB ratio in transcriptional regulation, we have generated an original human breast cancer cell line conditionally expressing PRA and/or PRB in dose-dependence of non-steroid inducers. We first focused on PR-dependent transcriptional regulation of the paracrine growth factor gene amphiregulin (AREG) playing important role in cancer. Interestingly, unliganded PRA increases AREG expression, independently of estrogen receptor, yet inhibitable by antiprogestins. We show that functional outcome of epidermal growth factor (EGF) on such regulation is highly dependent on PRA/PRB ratio. Using this valuable model, genome-wide transcriptomic studies allowed us to determine the differential effects of PRA and PRB as a function of hormonal status. We identified a large number of novel PR-regulated genes notably implicated in breast cancer and metastasis and demonstrated that imbalanced PRA/PRB ratio strongly impact their expression predicting poor outcome in breast cancer. In sum, our unique cell-based system strongly suggests that PRA/PRB ratio is a critical determinant of PR target gene selectivity and responses to hormonal/growth factor stimuli. These findings provide molecular support for the aggressive phenotype of breast cancers with impaired expression of PRA or PRB.
    PLoS ONE 09/2012; 7(9):e45993. DOI:10.1371/journal.pone.0045993 · 3.23 Impact Factor
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    • "We first engineered multiple clones of vector-matched PR-null T47D breast cancer cells expressing either WT PR-B or mutant K388R (KR) PR-B that is unable to undergo SUMO modification at Lys388; this SUMO-deficient receptor is a functional mimic for PR-B that is persistently phosphorylated on Ser294 [13,41]. Phospho-Ser294 and S294D receptors are hyperactive transcription factors that undergo rapid ligand-dependent (ubiquitin-mediated) downregulation relative to WT PRs [39]. Cells expressing either WT or KR PR-B were then treated with the synthetic progestin, R5020 (10-8 M), for six hours (Figure 1B). "
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    ABSTRACT: 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.
    Breast cancer research: BCR 06/2012; 14(3):R95. DOI:10.1186/bcr3211 · 5.49 Impact Factor
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