Abstract P5-03-02: Progestin Activation of p42/p44 MAPK Induces Stat3 Phosphorylation at Serine 727 Promoting Breast Cancer Growth
Authors: M Tkach, C Rosemblit, W Béguelin, C Proietti, MA Rivas, MC Diaz Flaqué, F Cayrol, EH Charreau, PV Elizalde, R. Schillaci
Journal: Cancer Research (2010)

Abstract We have previously demonstrated that the synthetic progestin medroxyprogesterone acetate (MPA) induces Stat3 phosphorylation at Tyr705 leading to Stat3 transcriptional activation, which is an absolute requirement for progestin stimulation of in vitro and in vivo breast cancer growth. These studies were performed 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. Besides tyrosine phosphorylation, Stat3 can also be phosphorylated on a single serine residue (Ser727) leading to a full transcriptional activation of Stat3. Here, we explored the effect of progestins on Stat3 Ser727 phosphorylation and its biological significance. MPA treatment of C4HD and T47D cells for 5 to 10 min induced phosphorylation of Stat3 on Ser727. The 727 Ser residue in Stat3 is situated in a conserved PMSP motive which resembles the consensus PxS/TP motive for mitogen-activated protein kinase (MAPK) targets. To address whether MPA activation of p42/p44 MAPK signaling pathway is directly involved in Ser727 Stat3 phosphorylation, we pretreated C4HD cells with U0126, a p42/p44 MAPK inhibitor, which suppressed phosphorylation of Stat3 on Ser727. To strengthen the finding that Ser phosphorylation of Stat3 proceeds in a p42/p44 MAPK-dependent manner, we performed a cold in vitro phosphorylation assay. We observed that immunoprecipitated p42/p44MAPK from C4HD cells activated with MPA was able to phosphorylate Stat3 at Ser727 obtained from non treated cells. Neither p42/p44 MAPK obtained from control cells nor p42/p44 MAPK inactivated by U0126 increased Stat3 Ser727 phosphorylation. Next, we explored the relevance of MPA-induced Stat3 Ser727 phosphorylation on cyclin D1 promoter activation, as it contains Stat3 response elements, named GAS sites. C4HD and T47D cells transiently co-transfected with a cyclin D1 promoter luciferase construct and a Stat3 expression vector, showed an enhanced transcriptional activity upon MPA treatment. Co-transfection of Ser727 to alanine-mutated Stat3 (Stat3S727-A) expression vector resulted in abrogation of MPA-induced cyclin D1 promoter activation. Moreover, MPA-induced cyclin D1 protein expression in C4HD and T47D cells was inhibited in cells transfected with Stat3S727-A. To further investigate the correlation between MPA-induced Stat3 Ser727 phosphorylation and cell growth, C4HD and T47D cells were transiently transfected with Stat3S727-A expression vector. Expression of Stat3S727-A had an inhibitory effect on in vitro MPA-induced growth of C4HD and T47D cells. Finally we explored the requirement of Stat3 Ser727 for in vivo progestin-driven breast cancer growth. Transfection of C4HD cells with Stat3S727-A significantly inhibited the ability of these cells to grow in syngeneic mice. Our findings have for the first time demonstrated that progestins are able to induce Stat3 Ser727 phosphorylation through a mechanism requiring p42/p44 MAPK activity in both mouse and human mammary tumor cells. We also found Stat3 phosphorylated at Ser727 to be a requisite for progestin-induced cyclin D1 promoter activation and protein up-regulation, as well as for stimulation of breast cancer growth in vitro and in vivo Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P5-03-02.