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

CECILIA J. PROIETTI; WENDY BEGUELIN; MARÍA C. DÍAZ FLAQUÉ; MARTÍN A. RIVAS; TKACH, MERCEDES; EDUARDO CHARREAU; ROXANA SCHILLACI; PATRICIA V. ELIZALDE

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

We have previously demonstrated that the synthetic progestinmedroxyprogesterone acetate (MPA) is able to induce Stat3transcriptional activation, which is in turn an absolute requirementfor progestin stimulation of in vitro and in vivo breast cancergrowth. We have performed our study both in C4HD cells from anexperimental model of hormonal carcinogenesis in which MPA inducedmammary adenocarcinomas in Balb/c mice, and in the human breast cancercell line T47D (Mol Cell Biol, 25: 4826, 2005).Therefore, we hereassessed whether the requirement of Stat3 activation inprogestin-stimulated breast cancer cell proliferation could be due tobi-directional cross-talks between progestins and Stat3, where Stat3in turn regulates PR transcriptional activation. We studied whetherStat3, acting as a coactivator, could modulate PR function, both whenPR binds to specific progesterone response elements (PRE) in thepromoter regions of target genes, such as bcl-X gene, and when PRregulates the transcription of  p21 gene, which lacks canonical PREsin its promoter region. Our present findings evidenced that MPAtreatment of C4HD cells induced an increase in bcl-XL mRNA levels.This effect was completely abolished by transfection with a dominantnegative Stat3 expression vector, Stat3Y705-F, and in the presence ofthe progestin antagonist RU486. In addition, we also found that MPAtreatment of both C4HD and T47D cells, induced an increase in thelevels of p21 protein expression, which was further enhanced in adose-dependent manner in the presence of a plasmid expressing aconstitutively activated Stat3 mutant, Stat3-C. To study the effect ofStat3 on PR-mediated transcriptional activity, C4HD and T47D cellswere transiently transfected with a luciferase reporter plasmid underthe control of the bcl-XP4 promoter, together with Stat3-C. MPAtreatment resulted in an increase in luciferase activity, inaccordance with previous results describing the presence of twohormone responsive elements present in the fourth promoter of bcl-Xgene. We found that Stat3-C enhanced progestin-induced PRtranscriptional activity in a dose-dependent manner and that thiseffect was abolished by RU486 and by cotransfection with Stat3Y705-F.Similar results were obtained with the well-characterizedprogesterone-responsive luciferase reporter plasmid MMTV-luc. We alsostudied the effect of Stat3 on PR capacity to regulate p21transcription by tethering to DNA-bound trans-acting factor Sp1. C4HDand T47D cells were transiently transfected with a p21 promoterreporter construct containing two Sp1 binding sites, together withincreasing amounts of Stat3-C. We found that Stat3-C enhancedprogestin-induced PR transcriptional activity in a dose-dependentmanner. This effect was completely abolished when Stat3 expression wassilenced using an siRNA strategy. Finally, we explored the specificassociation of Stat3 and PR to the PRE region of the bcl-X gene in thecontext of living cells, by performing Chromatin Immunoprecipitation(ChIP) and Sequential ChIP Assays. Our findings unraveled that MPAtreatment of primary cultures of C4HD cells induced PR and Stat3recruitment to the bcl-X promoter. We also evaluated the specificassociation of Stat3 and PR to the Sp-1 binding site in the p21promoter and found that MPA treatment of  T47D cells induced Sp-1, PRand Stat3 recruitment to the p21 promoter.These results provide the first evidence that Stat3 acts as acoactivator of PR when PR binds directly to target genes containingPREs in the promoter region, or when PR regulates transcription bytethering to Sp1.