Progesterone Receptor activation promotes GATA3 downregulation in breast cancer

FRANCO IZZO; LEANDRO VENTURUTTI; ROXANA SCHILLACI; ELIZALDE, PATRICIA; CECILIA J. PROIETTI

San Francisco

Congreso; 95th Annual Meeting of the Endocrine Society; 2013

Compelling evidence from the experimental and clinical fields points out to a critical role for the progesterone receptor (PR) in breast cancer. However, the molecular mechanisms of PR action remain elusive. On the other hand, the master transcription factor GATA3 has been shown to be involved in normal mammary gland development and differentiation. In breast cancer cells, the loss of GATA3 expression correlates with tumor progression and the onset of tumor dissemination. These studies prompted us to investigate the relationship between PR and GATA3 expression in breast cancer cells. For that purpose, we used the T47D human breast cancer cell line, and the C4HD model of mammary carcinogenesis induced by the synthetic progestin medroxyprogesterone acetate (MPA) in female BALB/c mice. We found that MPA treatment produces GATA3 protein and mRNA downregulation in both cellular types, as measured by western blot and quantitative RT-PCR, respectively. Preincubation with the antiprogestin RU486 prevented downregulation of both GATA3 protein and mRNA levels. The PR-null T47D-Y cell line, which derives from the T47D cell line, showed no GATA3 regulation upon MPA treatment. Restitution of PR expression restored MPA-mediated GATA3 downregulation. We next analyzed the phosphorylation of GATA3 at serine 309 (pGATA3). Confocal microscopy assays revealed that MPA increases pGATA3 levels. Noteworthily, cells displaying an increase in pGATA3 were undergoing mitosis. This observation was confirmed by flow cytometry analysis in MPA-treated cells, demonstrating an increase of pGATA3 in cells at the G2 phase of the cell cycle compared to those in G1. We next investigated whether GATA3 phosphorylation at serine 309 is required in order to achieve GATA3 downregulation. For that purpose, we generated a mutant form of GATA3 carrying a substitution of serine for alanine in position 309 (GATA3-S309A). The mutant form GATA3-S309A showed increased stability, and was unable to be downregulated by MPA treatment compared to wild type GATA3. Finally, we performed in vitro proliferation assays by [3H]-thymidine incorporation. When GATA3 was overexpressed, MPA failed to induce breast cancer cell proliferation. Overexpression of GATA3-S309A further reduced MPA-induced cell proliferation compared to wild type GATA3. To confirm the role of GATA3 as an inhibitor of cell proliferation in vivo, we transiently transfected C4HD cells with a GATA3 expression plasmid, inoculated them into BALB/c mice and measured tumor growth. Notably, there was a 50% reduction of tumor volume in the GATA3-transfected animals compared to their control counterparts. The presented results show for the first time PR activation as a stimulus that triggers GATA3 phosphorylation and downregulation in breast cancer cells, and underscore the importance of GATA3 downregulation for in vitro cell proliferation and in vivo tumor growth.