Heregulin Induces Transcriptional Activation of the Progesterone Receptor by a Mechanism That Requires Functional ErbB-2 and Mitogen-Activated Protein Kinase Activation in Breast Cancer Cells

Abstract

The present study addresses the capacity of heregulin (HRG), a ligand of type I receptor tyrosine kinases, to transactivate the progesterone receptor (PR). For this purpose, we studied, on the one hand, an experimental model of hormonal carcinogenesis in which the synthetic progestin medroxyprogesterone acetate (MPA) induced mammary adenocarcinomas in female BALB/c mice and, on the other hand, the human breast cancer cell line T47D. HRG was able to exquisitely regulate biochemical attributes of PR in a way that mimicked PR activation by progestins. Thus, HRG treatment of primary cultures of epithelial cells of the progestindependent C4HD murine mammary tumor line and of T47D cells induced a decrease of protein levels of PRA and -B isoforms and the downregulation of progesterone-binding sites. HRG also promoted a significant increase in the percentage of PR localized in the nucleus in both cell types. DNA mobility shift assay revealed that HRG was able to induce PR binding to a progesterone response element (PRE) in C4HD and T47D cells. Transient transfections of C4HD and T47D cells with a plasmid containing a PRE upstream of a chloramphenicol acetyltransferase (CAT) gene demonstrated that HRG promoted a significant increase in CAT activity. In order to assess the molecular mechanisms underlying PR transactivation by HRG, we blocked ErbB-2 expression in C4HD and T47D cells by using antisense oligodeoxynucleotides to ErbB-2 mRNA, which resulted in the abolishment of HRG's capacity to induce PR binding to a PRE, as well as CAT activity in the transient-transfection assays. Although the inhibition of HRG binding to ErbB-3 by an anti-ErbB-3 monoclonal antibody suppressed HRG-induced PR activation, the abolishment of HRG binding to ErbB-4 had no effect on HRG activation of PR. To investigate the role of mitogen-activated protein kinases (MAPKs), we used the selective MEK1/MAPK inhibitor PD98059. Blockage of MAPK activation resulted in complete abrogation of HRG's capacity to induce PR binding to a PRE, as well as CAT activity. Finally, we demonstrate here for the first time that HRG-activated MAPK can phosphorylate both human and mouse PR in vitro.Recent evidence has clearly shown the presence of cross talks between steroid hormone and growth factor (GF) signaling pathways, which were previously thought to be distinctly separate processes. Members of the steroid receptor superfamily are heavily phosphorylated proteins that, upon ligand binding, act as nuclear transcription factors (for a review, see reference 72). Particularly, progesterone receptor (PR), the focus of the present work, is phosphorylated in the absence of hormone and undergoes an increase in phosphorylation upon hormonal stimulation (19,20,67). Although the functional role of PR phosphorylation remains elusive, increased evidence indicates that it plays a role in the regulation of PR transcriptional activity (3,7,14,22,40,66). On the other hand, most GFs bind to transmembrane receptors that carry an intrinsic activity of tyrosine kinase. Tyrosine resid...

Progestin-induced caveolin-1 expression mediates breast cancer cell proliferation

“…At this point, we cannot yet define the relationship between caveolin-1 and PR transcriptional activation, but we can hypothesize that caveolin-1 could facilitate PR phosphorylation/dephosphorylation in those sites required for transcriptional activity, by contacting the proper serine/threonine kinases or phosphatases. As PR is a phosphoprotein with active regulation of its phosphorylation status, and since many signal pathways implicated in PR transactivation such as MAPK (Labriola et al, 2003) and EGFR (Couet et al, 1997) are situated in caveolae, it is reasonable to predict that PR/caveolin-1 interaction may facilitate PR phosphorylation process.…”

“…Firstly, we performed a suppressive subtractive hybridization (SSH) (Diatchenko et al, 1996) assay to identify genes whose expressions were induced by in vivo progestin treatment in breast cancer. For that purpose, we used murine mammary C4HD tumor from an experimental model of hormonal carcinogenesis in which the synthetic progestin medroxyprogesterone acetate (MPA)-induced mammary adenocarcinomas in female BALB/c mice (Balana et al, 1999(Balana et al, , 2001Labriola et al, 2003;Salatino et al, 2004). C4HD tumor is from ductal origin, requires MPA administration for in vivo and in vitro growth, and expresses high PR levels.…”

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

“…Additionally, EGF and progestins synergistically activated transcription from the p21 and c-fos promoters that lack classical PRE sites, but presumably respond to liganded PR via the action of activated STATs (5,30) and/or PR interaction with SP-1 molecules (31). In contrast to studies with estrogen receptors, ligand-independent PR transcriptional activity in response to growth factors is seldom reported (17,18). To address whether EGF stimulation of breast cancer cells could similarly alter the effects of progestins on PRE-containing promoters, we transiently expressed a PRE-luciferase reporter gene in HeLa or T47D cells stably expressing the PR-B isoform ( Fig.…”

“…Further analysis of genes regulated by growth factors in a PR-dependent manner (independently of PR ligands) is required in order to more effectively target these pathways for breast cancer prevention and treatment. In addition to the activation of PRs via ligand-independent pathways (17,18), MAPK-dependent events may dramatically lower the EC 50 for gene activation by liganded PR (Fig. 4), ER (64), or AR (65).…”

“…We have previously defined functional roles for phosphorylation of human progesterone receptor (PR) Ser294 by mitogen-activated protein kinase (14)(15)(16) and PR Ser400 by cell-cycle-dependent protein kinase two (17). Phosphorylation of these sites is regulated in response to progestins or growth factor mitogens, including EGF and heregulin, and can potentiate ligand-dependent PR transcriptional activity, as well as induce ligand-independent PR actions (17,18). Thus, phosphorylation of liganded steroid hormone receptors allows for positive "feed-forward", perhaps insuring that appropriate (i.e.…”