CARCINOMA ASSOCIATED FIBROBLASTS (CAF) FROM HORMONE INDEPENDENT MAMMARY TUMORS INCREASE THE PROGESTERONE RECEPTOR AB HETERODIMER FORMATION AND THE PROGESTERONE RECEPTOR PHOSPHORYLATION STATUS OF EPITHELIAL TUMOR CELLS

CAROLINE A. LAMB; SEBASTIÁN GIULIANELLI; MARIA C. BOTTINO; MARIA A. GOROSTIAGA; CLAUDIA LANARI

Washington, DC. USA

Congreso; 97th AACR Annual Meeting; 2006

AACR

We have developed an experimental model of breast cancer in which metastatic ductal mammary carcinomas transit through different stages of hormone dependence and we have suggested that carcinoma associated fibroblasts (CAF) participate in the acquisition of the hormone independent (HI) phenotype. Hormone-independent tumors grow in vivo without exogenous progestin supply, although they retain high levels of estrogen and progesterone receptors (PR) and regress after antiprogestin treatment. In in vitro studies, we were able to demonstrate that 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 more efficiently than CAF-HD. We also demonstrated that FGF 2 was able to activate progesterone receptors (PR) inducing an increase in PR binding to DNA (EMSA assays). The aim of this study was to investigate if CAF-HI were also more efficient than CAF-HD inducing the activation of PR. Purified epithelial cells from HD tumors (EPI-HD) or from HI tumors (EPI-HI) were cultured alone or with equal number of CAF-HD or CAF-HI. Twenty four hours before harvesting, the cells were incubated in culture medium with 1% charcoalized fetal calf serum. In these experimental setting, only epithelial cells from mixed cultures proliferated as demonstrated by cell counting experiments (p<0.05). Nuclear extracts were normalized by total protein content or by cytosolic cadherin E protein expression that was used as a marker of epithelial cell content. In EMSA assays, an increase in PR binding to DNA was observed in mixed cultures as compared to epithelial purified cells. These data correlated with increased PR phosphorylation assessed by pSer190 and pSer294 expression by Western Blots. Again, the increased phosphorylation was higher for the CAF-HI co-cultures than for the purified EPI-HI. There was a prevalence of the band corresponding to the PRAB heterodimers in co-cultures as compared with the EPI-HI purified cells (Arbitrary units; EPI-HI: 15511±2870; EPI-HI2049µHI: 30804±6100, p<0.05).In addition, the ratio between the intensity of the band corresponding to PRAB heterodimers with respect to PRAA homodimers was different in epithelial purified cultures than in mixed cultures. The prevalence of PRAB heterdimers in proliferative epithelial tumor cells suggests their possible involvement in driving cell proliferation.