Ekaterina B Kuznetsova1, Tatiana V Kekeeva1, Sergei S Larin2, Valeria V Zemlyakova1, Anastasiya V Khomyakova3, Olga V BabenkBabenko1, Marina V Nemtsova1, Dmitry V Zaletayev1, Vladimir V Strelnikov1
1Research Centre for Medical Genetics, Russian Academy of Medical Sciences; Molecular Medicine Institute, I.M. Sechenov Moscow Medical Academy, Moscow, Russia
2Institute of Gene Biology, Russian Academy of Sciences, Moscow, Russia
3Molecular Medicine Institute, I.M. Sechenov Moscow Medical Academy, Moscow, Russia
Loss of BIN1 tumor suppressor expression is abundant in human cancer and its frequency exceeds that of genetic alterations, suggesting the role of epigenetic regulators (DNA methylation). BIN1 re-expression in the DU145 prostate cancer cell line after 5-aza-2′-deoxycytidine treatment was recently reported but no methylation of the BIN1 promoter CpG island was found in DU145.
Methylation-sensitive arbitrarily-primed PCR was used to detect genomic loci abnormally methylated in breast cancer. BIN1 CpG island fragment was identified among the differentially methylated loci as a result of direct sequencing of the methylation-sensitive arbitrarily-primed PCR product and subsequent BLAST alliance. BIN1 CpG island cancer related methylation in breast and prostate cancers was confirmed by bisulphite sequencing and its methylation frequency was evaluated by methylation sensitive PCR. Loss of heterozygosity analysis of the BIN1 region was performed with two introgenic and one closely adjacent extragenic microsatellite markers. BIN1 expression was evaluated by real-time RT-PCR.
We have identified a 3′-part of BIN1 promoter CpG island among the genomic loci abnormally methylated in breast cancer. The fragment proved to be methylated in 18/99 (18%) and 4/46 (9%) breast and prostate tumors, correspondingly, as well as in MCF7 and T47D breast cancer cell lines, but was never methylated in normal tissues and lymphocytes as well as in DU145 and LNCaP prostate cancer cell lines. The 5′-part of the CpG island revealed no methylation in all samples tested. BIN1 expression losses were detected in MCF7 and T47D cells and were characteristic of primary breast tumors (10/13; 77%), while loss of heterozygosity was a rare event in tissue samples (2/22 informative cases; 9%) and was ruled out for MCF7.
BIN1 promoter CpG island is composed of two parts differing drastically in the methylation patterns in cancer. This appears to be a common feature of cancer related genes and demands further functional significance exploration. Although we have found no evidence of the functional role of such a non-core methylation in BIN1 expression regulation, our data do not altogether rule this possibility out.