Signal transducer and activator of transcription 3 (Stat3) enhances Progesterone Receptor (PR) activation of transcription in breast cancer cells

PROIETTI CJ ; BEGUELIN W ; ROSEMBLIT C ; CARNEVALE R ; RIVAS MA ; CHARREAU EH ; SCHILLACI R ; ELIZALDE PV

Los Angeles

Congreso; AACR Annual Meeting; 2007

American Associatiom of Cancer Research (AACR)

We have previously demonstrated that the synthetic progestin medroxyprogesterone acetate (MPA) is able to induce Stat3 transcriptional activation, which is in turn and absolute requirement for progestin stimulation of in vitro and in vivo breast cancer growth (Proc Amer Assoc Cancer Res 2005;46:5781).We here assessed whether the absolute requirement of Stat3 activation in progestin-stimulated breast cancer cell proliferation could be due to bi-directional cross-talks between progestins and Stat3, where Stat3 in turn regulates transcriptional activation of PR. Here, we studied whether Stat3, could modulate PR function acting as a coactivator, 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.We showed that MPA treatment of C4HD cells induced Stat3 and PR nuclear association, as detected by confocal microscopy.To study the effect of Stat3 on PR-mediated transcriptional activity, C4HD and T47D cells were transiently transfected with a luciferase reporter plasmid containing five copies of a progesterone-response element (PRE), together with a plasmid expressing a constitutively activated Stat3 mutant, Stat3-C. We found that Stat3-C enhanced progestin-induced PR transcriptional activity in a dose-dependent manner and this effect was abolished by the progestin antagonist, RU486.Assessment of the expression of the progestin-regulated bcl-X gene showed that MPA treatment of C4HD cells resulted in an increase of bcl-XL mRNA, as shown by Reverse Transcription-PCR assay. This effect was completely abolished by transfection with a dominant negative Stat3 expression vector, Stat3Y705-F, and in the presence of RU486. These results demonstrate that MPA-induced bcl-X gene expression is mediated by the classical PR and that this regulation depends on the presence of activated Stat3. Transfection of primary cultures of C4HD cells with a luciferase reporter plasmid under the control of the bcl-Xpromoter, resulted in an increase in luciferase activity, which was inhibited by RU486 and by cotransfection with Stat3Y705-F.We assessed the specific association of Stat3 and PR to the PRE region of the bcl-X gene in the context of living cells by performing Chromatin Immunoprecipitation Assays. We found that MPA treatment of primary cultures of C4HD cells induced PR and Stat3 recruitment to the bcl-X promoter.These results provide the first evidence that Stat3 acts as a coactivator in the molecular mechanism of progestin-induced transcriptional activation of PR.