Progesterone receptor A (PRA) down regulation in constitutive resistant mouse mammary carcinomas is due to promoter methylation

WARGON VICTORIA; FERNANDEZ SANDRA; NOVARO VIRGINIA; GOIN MERCEDES; RUSSO JOSE; LANARI CLAUDIA

Denver, Colorado, USA

Congreso; American Association for Cancer Research; 2009

AACR

Mouse mammary carcinomas induced by medroxyprogesterone acetate (MPA) are maintained by syngeneic transplantation and tumor variants with different hormone responsiveness have been generated. Tumors which grow without the supply of MPA, were defined as progestin or hormone independent (HI). Most of these tumors regress after antiprogestin (mifepristone, MIF) administration (responsive). Eventually, some of them became resistant to antiprogestin therapy and they were refered as acquired resistant tumors. Other variants show constitutive resistance to hormone therapy. We have previously demonstrated that responsive tumors present higher levels of progesterone receptor A (PRA) than PRB, while the opposite occurs in both types of hormone resistant tumors, indicating that PR isoform ratio might be a predictor of antiprogestin responsiveness.In addition we have demonstrated that acquired resistance was a reversible phenomenon suggesting the involvement of epigenetic mechanisms regulating PRA expression. The aim of this study was to investigate whether PRA silencing in resistant tumors was regulated by methylation of the CpG islands in its promoter and first exon. We also evaluated if a demethylating agent could restore PRA expression and antiprogestin sensitivity in tumor epithelial cells. Isolated DNA from responsive, constitutive and acquired resistant tumors was treated with bisulphite and methylation specific PCR (MSP) was carried on with specific primers for the CpG islands included into PRA promoter and the first exon. In addition, the fragments obtained by MSP were cloned and sequenced (Macrogen Inc). As it was expected, the PRA promoter and first exon was unmethylated in responsive tumors. Interestingly, only in constitutive resistant tumors, but not in acquired resistant ones, PRA expression was found to be regulated by DNA methylation. PRA re-expression, as determined by immunofluorescence and western blot, was achieved in primary cell cultures from constitutive resistant tumors incubated with the demethylating agent 5-aza-2´-deoxycytidine (5x10-6M, 48 hs). Neither MIF nor 5´aza dC independently reduced cell (3H)-thymidine uptake, whereas the combination of both agents inhibited constitutive resistant cell proliferation. In conclusion, we report for the first time that constitutive resistance to antiprogestins in PR positive tumors may be associated with PRA silencing by DNA methylation. In addition, our results indicate that the pharmacological induction of PRA in these tumors may induce their responsiveness to antiprogestins.