Co-methylation in the concerted regulation of a group of apoptotic genes in breast cancer

Abstract

Background. According to the latest global statistics, breast cancer (BC) has surpassed lung cancer in the incidence among women. Epigenetic changes at the earliest stages of normal cell transformation into tumor cells allow us to predict the possibility of breast cancer in advance, which reflects the advantages of epigenetics in preventing precancerous diseases and early diagnosis. In addition, epigenetic modifications are reversible, while the genetic regulation is irreversible, so the development of drugs aimed at reversible processes is more attractive for the treatment of BC.

The aim of the study was to determine the role of aberrant methylation of the apoptosis-related genes, APAF1, BAK1, BAX, BCL2, BIM, and DAPK1, in the BC progression and the role of co-methylation and co-expression in the co-operative regulation and functions.

Methods. Breast tumor samples were collected and clinically characterized at the Blokhin National Medical Research Center of Oncology. High-molecular DNA was isolated from the tissue using a standard method. The level of methylation of the APAF1, BAK1, BAX, BCL2, BIM, and DAPK1 genes was assessed by bisulfite DNA conversion and quantitative real-time MS-PCR with the ready-made reaction mixture qPCRmix-HS SYBR (Eurogen). Statistical analysis of the expression was performed in the R software environment using the nonparametric Mann-Whitney U test. Correlation analysis was performed with the Spearman rank correlation test, and the significance level was calculated. Differences were considered statistically significant at p<0.05. Expression and methylation parameters of the studied genes were also analyzed in BC using the MethMarkerDB (https://methmarkerdb.hzau.edu.cn/) and GEPIA (http://gepia.cancer-pku.cn/) databases, and data on processes and signaling pathways were analyzed using the ncPath database (http://ncpath.pianlab.cn/#/Home).

Results. Aberrantly methylated APAF1, BAK1, BAX, BIM, DAPK1 genes were identified in BC. A significant (p<0.05) increase in the methylation level was shown at a later stage (III) compared with earlier stages (I-II) of BC; with larger tumor sizes for the APAF1, BAK1, DAPK1 genes; and in the presence of metastases in the lymph nodes for the APAF1 and BIM genes. For the first time, co-methylated apoptosis-related genes were identified in BC: BCL2 – BIM, APAF1 – DAPK1, APAF1 – BIM, and APAF1 – BCL2 (rs{0.30 – 0.46}; p<0.05).

Conclusion. Studying molecular mechanisms of BC development and progression, as well as a new insight into the methylation patterns of apoptosis-related genes will bring us closer to the discovery of new markers and promising targets for targeted therapy of BC.