Mechanisms of epigenetic silencing of the Rassf1a gene during estrogen-induced breast carcinogenesis in ACI rats

Abstract

Breast cancer, the most common malignancy in women, emerges through a multistep process, encompassing the progressive sequential evolution of morphologically distinct stages from a normal cell to hyperplasia (with and without atypia), carcinoma in situ, invasive carcinoma and metastasis. The success of treatment of breast cancer could be greatly improved by the detection at early stages of cancer. In the present study, we investigated the underlying molecular mechanisms involved in breast carcinogenesis in Augustus and Copenhagen-Irish female rats, a cross between the ACI strains, induced by continuous exposure to 17beta-estradiol. The results of our study demonstrate that early stages of estrogen-induced breast carcinogenesis are characterized by altered global DNA methylation, aberrant expression of proteins responsible for the proper maintenance of DNA methylation pattern and epigenetic silencing of the critical Rassf1a (Ras-association domain family 1, isoform A) tumor suppressor gene. Interestingly, transcriptional repression of the Rassf1a gene in mammary glands during early stages of breast carcinogenesis was associated with an increase in trimethylation of histones H3 lysine 9 and H3 lysine 27 and de novo CpG island methylation and at the Rassf1a promoter and first exon. In conclusion, we demonstrate that epigenetic alterations precede formation of preneoplastic lesions indicating the significance of epigenetic events in induction of oncogenic pathways in early stages of carcinogenesis.

Fig. 1.

Histomorphological changes in mammary gland of female ACI rats exposed to 17β-estradiol for 6 and 12 weeks. (A) Mammary gland from untreated (control) ACI female rats (original magnification ×100); (B) Typical alveolar hyperplasia after 6 weeks of continuous E₂ treatment (original magnification ×100); (C) Typical hyperplasia showing alveolar lobule (AL) and a normal appearing intralobular duct (D) after 6 weeks of continuous E₂ treatment (original magnification ×400); (D) Typical hyperplasia after 12 weeks of continuous E₂ treatment (original magnification ×100; (E) Mammary gland hyperplasia with atypical (arrows) and typical (arrowheads) hyperplastic alveoli after 12 weeks of continuous E₂ treatment (original magnification ×100) and (F) Mammary gland hyperplasia with atypical hyperplastic ducts (arrows) line with variably sized epithelial cells (arrowheads). Duct lumen (L) contains intensely eosinophilic secretion; compare with normal appearing duct in panel C. Original magnification ×400.

Fig. 2.

Western blot analysis of DNMT1, DNMT3A, DNMT3B and MeCP2 proteins in mammary glands of control rats and rats continuously exposed to E₂ for 6 and 12 weeks. Mammary gland tissue lysates were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and subjected to western immunoblotting using specific antibodies against DNMT1, DNMT3A, DNMT3B and MeCP2. Equal sample loading was confirmed by immunostaining against β-actin. These results were reproduced in two independent experiments. (A) Representative western immunoblot images. (B) Quantitative analysis of DNMT1, DNMT3A, DNMT3B and MeCP2 protein levels. Data are presented as relative to age-matched control rats. Control values at each time point were considered as 100%. The histograms in each of the panels are the mean ± SD. *Significantly different from the control at the same time point.

Fig. 3.

Rassf1a promoter methylation in mammary glands of control rats and rats continuously exposed to E₂ for 6 and 12 weeks. (A) MSP analysis of the Rassf1a first exon methylation. Bisulfite-modified DNA was PCR amplified with two sets of primers specific to unmethylated (U) and methylated (M) cytosine residues in the promoter region of Rassf1a gene. The PCR consisted of initial denaturation at 95°C for 7 min followed by 37 cycles of denaturation at 95°C for 30 s, annealing at 60°C for 60 s and extension at 72°C for 60 s. Presence of methylated PCR products after amplification with methylation-specific primers indicates appearance of de novo methylation of the first exon of Rassf1a gene in mammary glands of E₂-exposed rats. (B) Bisulfite sequencing analysis of Rassf1a promoter methylation. Bisulfite-modified DNA from control (n = 4) and E₂-treated rats (n = 4) was PCR amplified and PCR products were cloned into pCR2.1-TOPO vector (Invitrogen). Ten independent clones from each DNA sample were sequenced. Each row represents an individual clone and each column represents an individual CpG site. Open and closed circles represent unmethylated and methylated CpG sites, respectively. Open circles indicate unmethylated in four of four samples; half filled circles indicate methylated in two of four samples; three quarter-filled circles indicate methylated in three of four samples and closed circles indicate methylated in four of four samples.

Fig. 4.

Western blot analysis of Rassf1a, Cdkn2a, Socs1, Cx26 and Cdh1 proteins in the mammary glands of control rats and rats exposed to 17β-estradiol for 6 and 12 weeks. Mammary gland tissue lysates (n = 5) from control rats and rats treated with E₂ were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and subjected to western immunoblotting using specific antibodies against Rassf1a, Cdkn2a, Socs1, Cx26 and Cdh1 proteins. Equal sample loading was confirmed by immunostaining against β-actin. These results were reproduced in two independent experiments. Representative western immunoblot images are shown.