Role of stromal fibroblasts in hormone-independent murine mammary tumor growth and in vivo effect of progesterone receptor antisense oligonucleotides (asPR).

LAMB, CAROLINE; HELGUERO, LUISA; VANZULLI, SILVIA; MOLINOLO, ALFREDO; LANARI, CLAUDIA

Orlando, EEUU

Congreso; American Association for Cancer Research; 2004

In previous studies, using the MPA-mouse mammary tumor model generated in our laboratory, we showed that bFGF stimulated the proliferation of hormone dependent (HD) tumor cells and suggested a cross-talk with the progesterone receptor (PR) pathway. As bFGF is mainly synthesized by stromal cells, we hypothesized that tumor stroma plays an active role in the acquisition of hormone independence. The aim of this study was a) to evaluate if stromal fibroblasts obtained from hormone independent (HI) tumors would increase hormone dependent cell proliferation as compared with fibroblasts from HD tumors, b) to compare bFGF expression in fibroblasts obtained from HD or HI tumors, c) to evaluate if bFGF activates PR and finally d) to evaluate the ability of PR phosphothiolated antisense oligonucleotides (asPR) to inhibit HI tumor growth in vivo. Epithelial cells or fibroblasts from HD or HI tumors were used in co-culture assays; cell proliferation was evaluated by 3H-thymidine uptake and cell counting. When co-cultured with HD or HI epithelial cells, HI fibroblasts induced a stimulation of cell proliferation higher than HD fibroblasts. This stronger synergistic effect was abolished in the presence of RU 486 10nM, suggesting that the responsive cells are the epithelial cells. HI epithelial cell proliferation significantly increased in response to 100ng bFGF (p<0.001). To investigate if bFGF could be a possible candidate involved in these stromal-parenchyma interactions, we evaluated bFGF expression in cell extracts obtained from PD or PI tumors and the ability of bFGF to activate PR.  Western blot analysis revealed an increased bFGF expression in HI fibroblasts (p<0.05) and Mobility Gel Shift assays revealed that bFGF activated PR of HD epithelial cells. Activated PRs were also observed in PI tumors. Having shown that stromal tissue from HI tumors may be driving epithelial cell proliferation through PR activation we decided to evaluate if the blockage of PR in a HI tumor would inhibit tumor growth. In in vivo experiments, a significant inhibition of tumor growth was observed in asPR-treated mice (0.8 mg/8 hs, ip for 5 days, n=4) (p<0.05), which correlated with histological signs of tumor regression, a large decrease in bromodeoxyuridine staining, and an increase in apoptosis (TUNEL). No differences were observed between scrambled antisense PR-treated or control mice. PR expression was significantly reduced in tumors from asPR-treated mice. In summary, our results suggest an active fibroblast participation in the hormone-independent phenotype in mammary tumors and point to bFGF as a stromal factor responsible for PR activation in hormone independent tumor growth. Our results provide evidence for a critical role of the PR pathway in mammary cancer, as well as substantiate its choice as an alternative therapeutic target.