GATA3 and progestin interaction in mammary cancer.

FRANCO IZZO, MARÍA C. DÍAZ FLAQUÉ, ROCÍO VICARIO, MARTÍN A. RIVAS, MERCEDES TKACH, EDUARDO H. CHARREAU, ROXANA SCHILLACI, PATRICIA V. ELIZALDE, CECILIA PROIETTI.

Orlando, Florida, USA.

Congreso; 102nd Annual Meeting of the American Association for Cancer Research; 2011

GATA3 is a Master Transcription Factor crucial in mammary gland development and differentiation. Due to the role of progesterone in mammary gland development and in the etiology and progression of breast cancer, we explored the regulation of GATA3 by the synthetic progestin medroxyprogesterone acetate (MPA) in breast cancer cells. The experiments 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. Our results indicate that MPA produces GATA3 downregulation, as measured by Western Blot (WB) experiments, and this effect is abolished by the progestin antagonist RU486. In order to assess MPA regulation of GATA3 mRNA levels we performed Real Time PCR, and observed that MPA induced GATA3 transcriptional downregulation. By performing in silico analysis, we detected that GATA3 aminoacidic sequence contains a PEST sequence, recognized as a site for protein degradation. This finding lead us to explore whether MPA affects GATA3 post-translational regulation. For that purpose we treated T47D cells with cicloheximide, and a reduction of GATA3 half-life was observed with MPA treatment. To evaluate whether the B isoform of Progesterone Receptor (PR-B) was sufficient to maintain GATA3 downregulation, we used T47D - YB cell line, which expresses only PR-B. As assessed by WB analysis, PR-B proved to be sufficient for MPA-induced GATA3 downregulation. Remarkably, MPA treatment of T47D-Y- DBD cells, which express a mutant form of PR that is unable to bind to DNA or to tether with other transcription factors, was able to cause GATA3 downregulation. This result suggests that GATA3 downregulation occurs via PR citoplasmatic signaling. In order to dissect the signaling pathway involved in MPA regulation of GATA3 expression, cells were incubated with U0126 + MPA. We found that the addition of U0126 abrogated GATA3 dowregulation by MPA. This result suggests that ERK 1/2 phosphorylation is necessary to maintain this regulation. However, when Src phosphorylation was prevented, together with Src downstream target ERK 1/2 phosphorylation, GATA3 downregulation persisted. This result points to an off-target effect of U0126 inhibitor, probably preventing ERK 5 phosphorylation, as it was previously reported. Finally, we performed in silico analysis of cyclin D1 gene proximal promoter and found three potential GATA3 binding sites. To assess the effect of GATA3 on cyclin D1 expression, we overexpressed human GATA3 in T47D cells, and measured cyclin D1 levels by WB. Overexpression of GATA3 resulted in lower cyclin D1 levels. This study shows for the first time GATA3 downregulation by MPA in breast cancer cells, both at transcriptional and post-translational levels. It also shades light over the PR mechanism involved in this process and the signaling pathway potentially implicated. Finally, we demonstrate that GATA3 overexpression affects cyclin D1 levels.