THE MPA MURINE BREAST CANCER MODEL: ROLE OF FGF2 AS A STROMAL FACTOR MEDIATING PROGESTERONE RECEPTOR ACTIVATION IN HORMONE INDEPENDENT MAMMARY TUMOR GROWTH

SEBASTIÁN GIULIANELLI; JUAN PABLO CERLIANI; CAROLINE A LAMB; MARÍA ALICIA GOROSTIAGA; ADRIÁN GÓNGORA; ALBERTO BALDI; CLAUDIA LANARI

Hyatt Regency Cambridge, Cambridge, MA. USA

Congreso; AACR Mouse Models of Cancer; 2006

We have developed an experimental model of breast cancer in BALB/c female mice in which metastatic ductal mammary carcinomas transit through different stages of hormone dependence. HD tumors need the exogenous administration of progestins to grow while hormone-independent (HI) carcinomas grow in vivo without exogenous progestin supply, although they retain high levels of estrogen and progesterone receptors (PR). In vitro, however, there are no differences in hormone responsiveness between both tumor types, thus suggesting the involvement of host factors regulating in vivo tumor growth. In in vitro studies, we were able to demonstrate that carcinoma associated fibroblasts (CAF) obtained from HI (CAF-HI) tumors are different from those from hormone dependent tumors (CAF-HD), they express higher levels of FGF 2 and in co-cultures they increase epithelial cell proliferation and PR activation more efficiently as compared with CAF-HD. The aim of these experiments was to assess that FGF 2 was able to activate PR from HI epithelial cells and to confirm FGF 2 involvement in CAF-HI increased PR activation.  Epithelial purified (EPI-HI) cells from C4-HI were cultured in the presence of 10% fetal calf serum (FCS) and with 1% charcoalized FCS (chFCS) for the last 48 hs. FGF-2 (50ng/ml) or MPA 10nM were added for 5 minutes or one hour. Cells were harvested and total extracts processed for western blotting. An increase in pSer190 PR was observed in both, MPA or FGF 2 treated cells, after one h of incubation. This correlated with increased binding to DNA (labeled PRE) as confirmed in electrophoretic mobility shift assays (EMSA). On the other hand, EPI-HI or EPI obtained from C4-HD (EPI-HD), were co-cultured with either CAF-HI or CAF-HD and after 48 h of incubation with 1%chFCS they were processed for EMSA studies. CAF-HI induced a higher PR-DNA binding as compared with CAF-HD in both cases (p<0.05). Incubation of the co-cultures with a neutralizing antibody to FGF 2 (developed by A. Baldi), inhibited PR binding to DNA. Control of EMSA assays included the use of T47D cells overexpressing PRA or PRB, exclusive displacement with excess of unlabeled PRE and supershift with the C-19 PR antibody.  These results indicate that FGF 2 is a paracrine factor involved in ligand independent PR activation leading to hormone independent tumor growth.