Hypermethylation of the progesterone receptor A in constitutive antiprogestin-resistant mouse mammary carcinomas

Laboratory of Hormonal Carcinogenesis, Institute of Experimental Biology and Medicine (IBYME), National Research Council of Argentina (CONICET), Vuelta de Obligado 2490, C1428ADN, Buenos Aires, Argentina.
Breast Cancer Research and Treatment (Impact Factor: 3.94). 05/2010; 126(2):319-32. DOI: 10.1007/s10549-010-0908-x
Source: PubMed


Most breast carcinomas that are estrogen receptor (ER) and progesterone receptor (PR) positive respond initially to an endocrine therapy, but over time, they develop resistance (acquired hormone resistance). Others, however, fail to respond from the beginning (constitutive resistance). Overcoming hormone resistance is one of the major desirable aims in breast cancer treatment. Using the medroxyprogesterone acetate (MPA)-induced breast cancer mouse model, we have previously demonstrated that antiprogestin-responsive tumors show a higher expression level of PR isoform A (PRA) than PR isoform B (PRB), while tumors with constitutive or acquired resistance show a higher expression level of PRB. The aim of this study was to investigate whether PRA silencing in resistant tumors was due to PRA methylation. The CpG islands located in the PRA promoter and the first exon were studied by methylation-specific PCR (MSP) in six different tumors: two antiprogestin-responsive, two constitutive-resistant, and two with acquired resistance. Only in constitutive-resistant tumors, PRA expression was silenced by DNA methylation. Next, we evaluated the effect of a demethylating agent, 5-aza-2'-deoxycytidine, on PRA expression and antiprogestin responsiveness. In constitutive-resistant tumors, 5-aza-2'-deoxycytidine treatment in vitro and in vivo restored PRA expression and antiprogestin RU-486 responsiveness. Furthermore, high levels of DNA methyltransferase (Dnmts) 1 and 3b were detected in these tumors. In conclusion, our results suggest that methyltransferase inhibitors in combination with antiprogestins may be effective in the treatment of constitutive-resistant carcinomas with a high DNA methyltransferase level.

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    • "In vivo relationship between PRA/PRB ratio and antiprogestin responsiveness revealed that decreased PRA/PRB ratio is associated with antiprogestin resistance [18]. Furthermore, restoration of balanced PRA/PRB ratio using methyltransferase inhibitors re-established antiprogestin sensitivity in mouse mammary carcinomas [19] suggesting that PR isoforms differentially contribute towards cancer progression. "
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    ABSTRACT: Progesterone receptor isoforms (PRA and PRB) are expressed at equal levels in normal mammary cells. However, alteration in PRA/PRB expression is often observed in aggressive breast cancer suggesting differential contribution of PR isoforms in carcinogenesis. The mechanisms underlying such processes remain to be established mainly due to paucity of appropriate cellular models. To investigate the role of PR isoforms and the impact of imbalanced PRA/PRB ratio in transcriptional regulation, we have generated an original human breast cancer cell line conditionally expressing PRA and/or PRB in dose-dependence of non-steroid inducers. We first focused on PR-dependent transcriptional regulation of the paracrine growth factor gene amphiregulin (AREG) playing important role in cancer. Interestingly, unliganded PRA increases AREG expression, independently of estrogen receptor, yet inhibitable by antiprogestins. We show that functional outcome of epidermal growth factor (EGF) on such regulation is highly dependent on PRA/PRB ratio. Using this valuable model, genome-wide transcriptomic studies allowed us to determine the differential effects of PRA and PRB as a function of hormonal status. We identified a large number of novel PR-regulated genes notably implicated in breast cancer and metastasis and demonstrated that imbalanced PRA/PRB ratio strongly impact their expression predicting poor outcome in breast cancer. In sum, our unique cell-based system strongly suggests that PRA/PRB ratio is a critical determinant of PR target gene selectivity and responses to hormonal/growth factor stimuli. These findings provide molecular support for the aggressive phenotype of breast cancers with impaired expression of PRA or PRB.
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    ABSTRACT: ERα and PR levels are critical determinants for breast cancer prognosis and response to endocrine therapy. Although PR is known to be silenced by methylation of its promoter, few studies have correlated methylation with PR levels and outcome in breast cancer. There is only one previous small study comparing methylation of the two PR isoforms, PRA and PRB, which are expressed from different promoters, and finally, there is no prior knowledge of associations between isoform-specific methylation and outcome. We conducted a cohort-based study to test for associations between PRA and PRB methylation, expression, and clinical outcome in tamoxifen-treated patients (n = 500), and in patients who underwent surgery only (n = 500). Methylation and PR levels were measured by bisulfite pyrosequencing and ligand-binding assay, respectively. Low PR levels were significantly associated with worse outcome in all patients. PRA and PRB promoters were methylated in 9.6% and 14.1% of the breast tumors, respectively. The majority (74%) of PR-negative tumors were not methylated despite the significant inverse correlation of methylation and PR levels. PRA methylation was significantly associated with PRB methylation, although a subset of tumors had PRA only (3.9%) or PRB only (8.3%) methylated. Methylation of PRA, but not PRB was significantly associated with worse outcome in the tamoxifen-treated group. Mechanisms other than promoter methylation may be more dominant for loss of PR. Isoform-specific methylation events suggest independent regulation of PRA and PRB. Finally, this article shows for the first time that PRA methylation plays a unique role in tamoxifen-resistant breast cancer.
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