CARCINOMA ASSOCIATED FIBROBLASTS IN MAMMARY CARCINOMAS: KEY PLAYERS IN THE ACQUISITION OF HORMONE INDEPENDENCE

CAROLINE A. LAMB; SEBASTIÁN GIULIANELLI; JUAN P. CERLIANI; MARÍA C. BOTTINO; ROCÍO SOLDATI; MARÍA A. GOROSTIAGA; VICTORIA WARGON; VICTORIA FABRIS; ALFREDO MOLINOLO; CLAUDIA LANARI

La Jolla, California. USA

Congreso; AARC. Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; 2005

AACR

Background: Hormone-independent (HI) medroxyprogesterone acetate-induced mouse mammary carcinomas grow in vivo without exogenous progestin supply, although they retain high levels of estrogen and progesterone receptors (PR) and regress after antiprogestin treatment. Interestingly, their purified epithelial cells, the malignant compartment, grow very slowly when cultured in vitro, unless they are co-cultured with carcinoma associated fibroblasts (CAF). In previous experiments we have demonstrated that MPA and FGF-2 exerted the same proliferative effect in primary cultures of epithelial hormone-dependent (HD) cells and that this effect was blocked by antiprogestins or asPR. Our working hypothesis is that CAF from HI tumors participate in the acquisition of the hormone independent phenotype secreting paracrine factors such as FGF 2 which favor PR activation. Objectives: The goal of this study was a) to investigate if FGF 2 was also able to stimulate in vitro HI epithelial growth b) evaluate differences in FGF expression between HD and HI CAF and differences in FGF receptors in the HD and HI epithelial cells and c) evaluate if  CAF-HI when co-cultered with epithelial cells were able to induce an increase in  PR binding to DNA as compared with CAF-HD and thus participate in the induction of cell proliferation.  Results:  FGF 2 increased cell proliferation as MPA in primary cultures of HI epithelial (Proliferation index. Control: 1±0.2; MPA: 3.62±1; FGF-2: 3.11±0.09; p<0.001) indicating no differences between HD and HI epithelial cells growing in vitro. FGF 2 was more expressed  in CAF-HI than CAF-HD as evaluated by Western blots using CAF-HI or CAF-HD whole cell extracts (20 fold, p<0.001) and FGFR 2 and 3 were highly expressed in parenchyma of HI tumors showing nuclear staining (immunohistochemistry). These results suggest a paracrine loop between the stromal and epithelial compartments and that FGF 2 may be one stromal candidate involved in HI tumor growth. Interestingly exogenous addition of FGF 2 to HD epithelial cells increased PR-DNA binding as revealed by Electrophoretic mobility shift assay (EMSA) indicating a crosstalk between the FGF and the PR pathways. To further challenge our hypothesis we co-cultured epithelial HD or HI tumor cells with purified CAF-HD or HI and evaluated proliferative effects using  3H-thymidine-uptake assays. An increase in cell proliferation was observed when epithelial HD were co-cultered with CAF-HI as compared with CAF-HD (Epithelial HD CAF-HD: 649.6±162.6 cpm; Epithelial HD CAF-HI: 1891±99.56 cpm; p<0.001). Results were confirmed by cell counting in which it could also be concluded that the epithelial cells (cytokeratin positive) are the ones which take profit of this interaction. EMSA assays also confirmed the ability of CAF-HI to induce PR binding to DNA in the epithelial cells. Conclusion: Our results strongly suggest a key role for stromal fibroblasts promoting hormone-independent tumor growth under the modulatory control of the progesterone receptor.