Stat3 modulates heregulin (HRG)-induced progesterone receptor (PR) transcriptional activation.

CECILIA J. PROIETTI, FRANCO IZZO, MARÍA CELESTE DÍAZ FLAQUÉ, ROCÍO VICARIO, MERCEDES TKACH, MARTÍN A. RIVAS, EDUARDO H. CHARREAU, ROXANA SCHILLACI, PATRICIA V. ELIZALDE

Orlando, Florida, USA.

Congreso; 102nd Annual Meeting of the American Association for Cancer Research; 2011

The signal transducer and activator of transcription (Stat) family of proteins was found to be involved in crosstalks with both steroid hormones and type I receptor tyrosine kinases (RTKs) signaling pathways. We have previously demonstrated that HRG, a ligand of RTKs, transactivates PR both in C4HD cells from an experimental model of hormonal carcinogenesis in which MPA induced mammary adenocarcinomas in Balb/c mice, and in the human breast cancer cell line T47D (Mol Cell Biol. 2003 Feb;23(3):1095-111). We have also shown that HRG induces Stat3 tyrosine phosphorylation and transcriptional activation by a mechanism that requires PR signaling (Mol Cell Biol. 2009 Mar;29(5):1249-65). In the present work we explored whether Stat3 acting as a coactivator, could modulate ligand-independent activation of PR by HRG. Assessment of the expression of the progestin-regulated gene bcl-X showed that HRG treatment of C4HD cells resulted in an increase in bcl-X protein levels. This effect was completely abolished when Stat3 expression was silenced using siRNAs. HRG treatment of cells transfected with a luciferase reporter plasmid under the control of the murine bcl-X promoter which contains two progesterone response elements (PREs), resulted in an increase in luciferase activity. HRG had no effect on PR transcriptional activation when cells were transfected with a mutant vector containing a deletion spanning both PREs in bcl-X promoter. We assessed the specific association of Stat3 and PR to the PRE region of bcl-X promoter in the context of living cells by performing Chromatin Immunoprecipitation (chIP) Assays. We found that HRG treatment of primary cultures of C4HD cells induced PR and Stat3 recruitment to the bcl-X promoter. HRG also induced PR and Stat3 occupancy of the stably integrated MMTV promoter in T47D-Cat0 breast cancer cells. By performing sequential chIP assays we showed that HRG induced simultaneous PR and Stat3 occupancy of the bcl-X promoter region. We explored Stat3 role in the non-classical mechanism of action of PR, where PR is recruited to p21 promoter indirectly through interaction with Sp1 transcription factor. Interestingly, when cells were treated with HRG, we detected Stat3 binding to the Sp1 binding sites of the PR-regulated p21 promoter, together with Sp1 and PR. These results provide the first evidence that Stat3 modulates ligand-independent activation of PR by HRG in breast cancer cells.