Frequent RASSF1A gene promoter hypermethylation in breast cancer

Abstract

RASSF1A identified at 3p21.3 was suggested as the major target tumor suppressor on the basis of its frequent epigenetic silencing in human cancers, including breast cancer. We performed a retrospective survey of RASSF1A methylation among 181 breast cancer patients (including 49 paired tumor/normal neighbor samples) with pyrosequencing methylation assay in the promoter region. To confirm the role of epigenetic mechanisam in the inactivation of RASSF1A through detection of the changes of methylation level and reexpression of RASSF1A mRNA by demethylation treatment with 5-Aza-dC and/or trichostatin A. There was a high frenquency of hypermethylation in RASSF1A promoter region in breast tumor tissues and the methylation level increased in ER-positive, big size (T>2cm) and low histological grade of the tumor. Some degree of RASSF1A methylation was observed in normal adjacent breast tissues in paired samples and it increased with the patient’s age and primary tumor size. For the change of methylation level in each CpG site, it had a direct correlation with the cancer recurrent and had an inverse correlation with the lymph node metastasis. The change in each CpG site in paired samples was horizontal and no specificity of the CpG site. As an in vitro model, the lost of RASSF1A expression of the hypermethylated cell line MDA-MB-330 can be recovered by methylation inhibitor 5-Aza-dC after 72 hours treatment. In conclusion, we have used pyrosequencing technology to demonstrate that there is an aberrant hypermethylation of RASSF1A promoter in breast cancer, suggesting that epigenetic inactivation of RASSF1A may play a critical role in breast carcinogenesis. The methylation level of RASSF1A has a correlation with tumor size, histological grade, estrogen receptor status and the disease recurrence. Our data also indicate that RASSF1A may undergo methylation in normal breast tissues in an age-dependent pattern. RASSF1A methylation starts in normal breast tissues as a function of age and remarkably increased in tumor. A large paired breast cancer population and some normal breast tissues from non-cancer people is need for future study.