Progestin-mediated GATA3 downregulation is required for breast cancer cell proliferation

IZZO, FRANCO, GALIGNIANA, NATALIA M, VENTURUTTI, LEANDRO, DÍAZFLAQUÉ, MARÍAC, TKACH, MERCEDES, ELIZALDE, PATRICIA V, PROIETTI, CECILIA J

Houston, Texas

Congreso; Endocrine Society 2012; 2012

Endocrine Society

The expression of the master transcription factor GATA3 positively correlates with disease-free survival and overall survival in breast cancer patients (Yoon et al, 2010). In addition, using an experimental model in mice, it has been shown that reduction of GATA3 expression correlates with tumor progression and marks the onset of tumor dissemination (Kouros-Mehr et al, 2008). On the other hand, compelling evidence points to a critical role of progesterone and the nuclear progesterone receptor (PR) in mammary tumorigenesis (Chlebowski et al, 2009). These studies prompted us to investigate the interaction between GATA3 and progestin, using the synthetic progestin medroxyprogesterone acetate (MPA) in the T47D human breast cancer cell line and in the murine mammary C4HD tumor model, which shows progestin-dependent growth. Our results for the first time showed that both GATA3 mRNA and protein are downregulated upon progestin treatment, as measured by western blot (WB) and quantitative PCR (qPCR) assays. The PR antagonist RU486 or siRNA-mediated PR knockdown prevented this regulation. Furthermore, T47D-Y cells which derive from T47D and lack PR expression, showed no regulation of GATA3. These findings are consistent with a PR-dependent regulation of GATA3 expression. To dissect the molecular mechanism involved, we used the translation inhibitor cycloheximide (CHX) and measured GATA3 protein half-life. Upon MPA addition there was a decrease in GATA3 half-life, and this effect was prevented by the use of the 26S proteasome inhibitor Bortezomib. Even more, pre-treatment of T47D and C4HD cells with the proteasome inhibitor induced GATA3 accumulation in both control and MPA-treated cells, proving that GATA3 downregulation occurs at a post-translational level in a proteasome-dependent fashion. In line with these results, co-immunoprecipitation (co-IP) experiments using an ubiquitin-HA expression plasmid showed that Bortezomib promotes accumulation of poly-ubiquitinated GATA3 species. Finally, to investigate the role of GATA3 on MPA biological effects, we performed in vitro proliferation assays by [H3]-thymidine incorporation. Upon GATA3 overexpression, MPA fails to induce breast cancer cell proliferation in both T47D and C4HD cells. This result underscores the importance of GATA3 downregulation for progestin-induced cell cycle progression. To further confirm the role of GATA3 in vivo, we transiently transfected C4HD cells with a GATA3 expression plasmid, and inoculated them into BALB/c mice. There was a significant reduction of tumor volume (p<0.05) in the GATA3-expressing animals compared to their control counterparts. The presented results account for a novel role of GATA3 in mediating progestin effects, and underscore the importance of GATA3 regulation for breast cancer cell proliferation.