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

PROIETTI CJ ; ROSEMBLIT C ; BEGUELIN W ; DIAZ FLAQUE MC ; RIVAS MA ; TKACH M ; CHARREAU EH ; SCHILLACI R ; ELIZALDE PV

Carefree

Congreso; Extra-Nuclear Steroid Receptors: Integration with Multiple Signaling Pathways; 2008

FASEB

Signal transducer and activator of transcription 3 (Stat3) enhances Progesterone Receptor (PR) activation of transcription in breast cancer cells. We have previously demonstrated that the synthetic progestin medroxyprogesterone acetate (MPA) is able to induce Stat3 transcriptional activation, which is in turn an absolute requirement for progestin stimulation of in vitro and in vivo breast cancer growth. We have performed our study 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, 25: 4826, 2005).Therefore, we here assessed whether the 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 PR transcriptional activation. We studied whether Stat3, acting as a coactivator, could modulate PR function, both when PR binds to specific progesterone response elements (PRE) in the promoter regions of target genes, such as bcl-X gene, and when PR regulates the transcription of  p21 gene, which lacks canonical PREs in its promoter region. Our present findings evidenced that MPA treatment of C4HD cells induced an increase in bcl-XL mRNA levels. This effect was completely abolished by transfection with a dominant negative Stat3 expression vector, Stat3Y705-F, and in the presence of the progestin antagonist RU486. In addition, we also found that MPA treatment of both C4HD and T47D cells, induced an increase in the levels of p21 protein expression, which was further enhanced in a dose-dependent manner in the presence of a plasmid expressing a constitutively activated Stat3 mutant, Stat3-C. To study the effect of Stat3 on PR-mediated transcriptional activity, C4HD and T47D cells were transiently transfected with a luciferase reporter plasmid under the control of the bcl-XP4 promoter, together with Stat3-C. MPA treatment resulted in an increase in luciferase activity, in accordance with previous results describing the presence of two hormone responsive elements present in the fourth promoter of bcl-X gene. We found that Stat3-C enhanced progestin-induced PR transcriptional activity in a dose-dependent manner and that this effect was abolished by RU486 and by cotransfection with Stat3Y705-F. Similar results were obtained with the well-characterized progesterone-responsive luciferase reporter plasmid MMTV-luc. We also studied the effect of Stat3 on PR capacity to regulate p21 transcription by tethering to DNA-bound trans-acting factor Sp1. C4HD and T47D cells were transiently transfected with a p21 promoter reporter construct containing two Sp1 binding sites, together with increasing amounts of Stat3-C. We found that Stat3-C enhanced progestin-induced PR transcriptional activity in a dose- dependent manner. This effect was completely abolished when Stat3 expression was silenced using an siRNA strategy. Finally, we explored 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 (ChIP) and Sequential ChIP Assays. Our findings unraveled that MPA treatment of primary cultures of C4HD cells induced PR and Stat3 recruitment to the bcl-X promoter. We also evaluated the specific association of Stat3 and PR to the Sp-1 binding site in the p21 promoter and found that MPA treatment of  T47D cells induced Sp-1, PR and Stat3 recruitment to the p21 promoter. These results provide the first evidence that Stat3 acts as a coactivator of PR when PR binds directly to target genes containing PREs in the promoter region, or when PR regulates transcription by tethering to Sp1.