FGF receptors (FGFR). stromal-parenchymal interactions and acquisition of hormone independence in the MPA breast cancer model

CERLIANI, JUAN PABLO; LAMB, CAROLINE; BOTTINO, MARÍA CECILIA; GIULIANELLI, SEBASTIÁN; HEWITT, SM; MOLINOLO, ALFREDO; LANARI, CLAUDIA

Los Angeles, EEUU

Congreso; American Association for Cancer Research: Annual Meeting; 2007

We have developed a murine model of breast cancer in which mammary carcinomas transit through different stages of hormone dependence. Hormone independent (HI) tumors do not need the exogenous administration of medroxyprogesterone acetate (MPA) to grow. Both tumor types retain high levels of estrogen and progesterone receptors (PR). In vitro, however, there are no differences in hormone responsiveness between both types, suggesting the involvement of host factors regulating in vivo tumor growth. In in vitro studies, we demonstrated that carcinoma associated fibroblasts (CAF) obtained from HI (CAF-HI) tumors expressed high levels of FGFs and in co-cultures they increase epithelial cell (EPI) proliferation and PR activation more efficiently as compared with CAF-HD. The aim of this study was to evaluate a) FGFR expression in hormone-dependent (HD) and HI tumors, b) MPA regulation of FGFR and c) the role of FGFR as possible mediators of CAF-induced EPI proliferation, d) FGFR and PR interactions. Samples of C4-HD and C4-HI were processed for western blotting. An increase in FGFR 1-4 expression (p<0.05) was observed in C4-HI tumors and in MPA-treated C4-HD tumors as compared with untreated tumors. Co-immunoprecipitation studies with p-tyr or FGFR antibodies and further blotting with the respective antibodies indicated that MPA induced FGFR activation. Immunohistochemical and immunofluorescence studies using paraffin embedded tissues or frozen tissues respectively, showed FGFR expression mainly in the parenchymal cells. FGFR nuclear staining was a common finding. Incubation of HI cells with MPA, induced nuclear co-localization of FGFR2 and PR. The interaction of both proteins was confirmed by co-immunoprecipitation assays. Having shown that a) HI fibroblasts express high levels of FGFs and b) that the EPI express mainly FGFR2 and FGFR3, we decided to evaluate if the blockage of these receptors with siRNA would inhibit CAF-induced EPI proliferation in co-cultures. EPI C4-HI were transfected  with siRNA of FGFR2 or FGFR3 (Santa Cruz Biotech). The blockage of the corresponding FGFR was confirmed by Western blots. Co-cultures of CAF-HI togther with wild type EPI-HI induced a significant increase in EPI proliferation (3H-thymidone uptake) which was not observed when siRNA FGFR2, or siRNA FGFR3 EPI transfected cells were used. BrdU labeling together with cytokertin staining confirmed that the decrease in cell proliferation was specific of the EPI. The incubation of HI cells with a specific FGFR inhibitor (PD 173074) inhibited cell proliferation in cell culture (2D) and in matrigel (3D). In summary, our results confirm an active CAF participation in the HI phenotype in mammary tumors pointing to FGFR 2 and 3 as key players mediating CAF-induced HI tumor growth and suggest that novel interactions between FGFR and PR may be involved in the regulation of cell proliferation in these tumors.