Progestin-induced caveolin-1 expression mediates proliferation of breast cancer cells

MARIANA SALATINO, WENDY BEGUELIN, CECILIA J. PROIETTI, ROMINA CARNEVALE, CINTHIA ROSEMBLIT, MARTíN RIVAS, ROXANA SCHILLACI, EDUARDO H. CHARREAU, PATRICIA V. ELIZALDE

Washington DC, USA

Congreso; Annual Meeting of the American Association for Cancer Research; 2006

Caveolin-1 (cav-1) is the primary structural protein of caveolae and has been implicated in a variety of cellular processes including signal transduction. Even when cav-1 interaction with many of the signal transduction components is thought to have important consequences for cellular transformation, the role that cav-1 plays in cancer still remains controversial. We have previously demonstrated that cav-1 expression is induced both in vivo and in vitro by the synthetic progestin medroxiprogesterone acetate (MPA) in the murine mammary tumor line C4HD and that this regulation involves the classical progesterone receptor (PR) (Proc of the AACR 2003, vol 44, abs. 3587). C4HD tumor is a progestin dependent mammary adenocarcinoma which expresses high levels of PR. Here, we evaluated whether MPA was able to regulate cav-1 expression at transcriptional level in C4HD cells. For that purpose, we transiently transfected C4HD cells and human breast cancer T47D cells with a luciferase reporter vector under the transcriptional control of the mouse cav-1 promoter (PGL3-mcav-1-Luc) and we showed that MPA promoted strong luciferase activity clearly indicating that MPA induced cav-1 promoter transcriptional activation (C4HD: 2,5 fold, p<0,001, T47D: 5 fold, p<0,001). In both cases the effect was prevented by PR antagonist RU486. Furthermore, we showed that MPA regulation of cav-1 expression required at least activation of two signaling pathways: MAPK and PI-3K, since pretreatment of C4HD cells with U0126 (1, 5 and 10 ìM) or LY2940023 (1, 2 and 4 ìM) prevented MPA- induced cav-1 up-regulation. We next evaluated MPA capacity to induce cav-1 phosphorylation. MPA incubation of C4HD cells for 5 and 10 min led to tyrosine phosphorylation of cav-1 protein, detected with a specific antibody. This effect was prevented by PP2 (10 ìM), a specific inhibitor of Src activity, suggesting that Src was the kinase involved. We further confirmed that Src was able to directly phosphorylate cav-1 using a cold in vitro phosphorylation assay. Additionally, we showed that MPA induced the formation of a binary complex between cav-1 and PR, which was detected by coimmunoprecipitation assay and by confocal microscopy after 5 and 10 min treatment. Finally, we proved that MPA-induced proliferation of C4HD cells was significantly reduced by suppression of cav-1 expression with antisense oligodeoxynucleotides to cav-1 mRNA. Furthermore, we observed that inhibition of cav-1 expression inhibited PR capacity to induce luciferase activity from a progesterone response element-driven reporter plasmid. As a whole, our results demonstrated for the first time that cav-1 expression is regulated by progestin in breast cancer. We also demonstrated that cav-1 protein is a downstream effector of MPA that is responsible, al least in part, for the growth stimulation of C4HD breast cancer cells in these experimental conditions.