Abstract
Breast cancer is the most common cancer in women, with 1.7 million new cases each year. As early diagnosis and prognosis are crucial factors in cancer treatment, we investigated potential DNA methylation biomarkers of the tumour suppressor family Ras-association domain family (RASSF). Promoter hypermethylation of tumour suppressors leads to their inactivation and thereby promotes cancer development and progression. In this study we analysed the tumour suppressors RASSF1A and RASSF10. Our study shows that RASSF10 is expressed in normal breast but inactivated by methylation in breast cancer. We observed a significant inactivating promoter methylation of RASSF10 in primary breast tumours. RASSF10 is inactivated in 63% of primary breast cancer samples but only 4% of normal control breast tissue is methylated (p < 0.005). RASSF1A also shows high promoter methylation levels in breast cancer of 56% vs. 8% of normal tissue (p < 0.005). Interestingly more than 80% of breast cancer samples harboured a hypermethylation of RASSF10 and/or RASSF1A promoter. Matching samples exhibited a strong tumour specific promoter methylation of RASSF10 in comparison to the normal control breast tissue. Demethylation treatment of breast cancer cell lines MCF7 and T47D reversed RASSF10 promoter hypermethylation and re-established RASSF10 expression. In addition, we could show the growth inhibitory potential of RASSF10 in breast cancer cell lines MCF7 and T47D upon exogenous expression of RASSF10 by colony formation. We could further show, that RASSF10 induced apoptotic changes in MCF7 and T47D cells, which was verified by a significant increase in the apoptotic sub G1 fraction by 50% using flow cytometry for MCF7 cells. In summary, our study shows the breast tumour specific inactivation of RASSF10 and RASSF1A due to DNA methylation of their CpG island promoters. Furthermore RASSF10 was characterised by the ability to block growth of breast cancer cell lines by apoptosis induction.
Highlights
Breast cancer is the most common cancer in women
We found that RASSF10 is highly expressed in normal breast tissue and studied breast tumour samples regarding a possible tumour driving RASSF10 inactivation
RASSF10 contains a more than 2 kb large CpG Island covering its promoter, and its methylation status was analysed by COBRA and pyrosequencing
Summary
Breast cancer is the most common cancer in women It is the second most common overall cancer type, accounting for almost 1.7 million new cases among the 14 million total new cancer cases worldwide in 2012 [1]. It ranks fifth as cause of death from cancer overall, due to its favourable prognosis [1]. 70% of breast tumours are positive for the estrogen receptor, which can be targeted by endocrine therapy [4]. Mutations in the BRCA1/2 tumour suppressors are linked to familial predisposition to breast cancer [6]. Aberrant methylation patterns were reported for e.g. the BRCA1 promoter [8]
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