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

Medical School, University of Minnesota Duluth, Duluth, Minnesota, 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: 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.
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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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    • "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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