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Interactions between steroid hormone receptors and Stats-mediated signaling pathways have already been described. We have previously demonstrated that the synthetic progestin medroxyprogesterone acetate (MPA) was able to induce Stat3 nuclear translocation, binding to DNA and transcriptional activation in C4HD cells from an experimental model of hormonal carcinogenesis in which MPA induced mammary adenocarcinomas in female Balb/c mice (Proc Am Assoc Cancer Res 45:105, 2004). Here, we explored the molecular mechanisms involved in progestin-induced Stat3 activation in C4HD cells and in human breast cancer cell line T47D. MPA was able to induce rapid, nongenomic Jak1 and Jak2 tyrosine phosphorylation. MPA treatment of C4HD for 2 min also resulted in c-Src tyrosine phosphorylation in C4HD cells. These effects were completely abolished by progestin antagonist RU486. Transient transfections of C4HD and T47D cells with a luciferase reporter plasmid containing four copies of the m67 high-affinity Stat3 binding site gene, demonstrated that MPA promoted a significant increase in luciferase activity. Blockage of Jak1 and Jak2 activity, by the use of dominant negative (DN) Jak1 or Jak2 expression vectors, and of Src activity with the selective Src family kinase inhibitor, PP2, abolished MPA capacity to induce Stat3 transcriptional activation. To investigate the correlation between MPA-induced Stat3 activation and cell growth, C4HD cells were transiently transfected with a DN Stat3 expression vector, Stat3Y705-F, or with a constitutively activated Stat3 mutant, Stat3-C. While expression of Stat3Y705-F had an inhibitory effect on MPA-induced growth of C4HD cells, transfection with the constitutively activated Stat3-C vector resulted in MPA-independent proliferation. Furthermore, the level of apoptosis increased significantly in Stat3Y705-F-transfected cells. Assessment of the expression of Bcl-xL gene, member of the Bcl-2 family of anti-apoptotic regulatory proteins, showed that MPA treatment of C4HD cells resulted in up-regulation of Bcl-xL protein expression, which was completely abolished by transfection with Stat3Y705-F. On the other hand, the Stat3-C mutant induced Bcl-xL protein levels to increase. Finally, we here addressed the effect of targeting Stat3 in in vivo growth of C4HD breast tumors. Blockage of Stat3 activation by transfection of C4HD cells with the DN Stat3Y705-F expression vector significantly inhibited these cells’ ability to form tumors in syngeneic mice. Our findings have for the first time demonstrated that progestins are able to induce Stat3 transcriptional activation by a mechanism requiring Jaks and c-Src activity in both mouse and human mammary tumor cells. We also found activated Stat3 to be a requisite for progestin stimulation of in vitro and in vivo breast cancer growth.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]