CpG promoter methylation of the ALKBH3 alkylation repair gene in breast cancer

Abstract

BackgroundDNA repair of alkylation damage is defective in various cancers. This occurs through somatically acquired inactivation of the MGMT gene in various cancer types, including breast cancers. In addition to MGMT, the two E. coli AlkB homologs ALKBH2 and ALKBH3 have also been linked to direct reversal of alkylation damage. However, it is currently unknown whether ALKBH2 or ALKBH3 are found inactivated in cancer.MethodsMethylome datasets (GSE52865, GSE20713, GSE69914), available through Omnibus, were used to determine whether ALKBH2 or ALKBH3 are found inactivated by CpG promoter methylation. TCGA dataset enabled us to then assess the impact of CpG promoter methylation on mRNA expression for both ALKBH2 and ALKBH3. DNA methylation analysis for the ALKBH3 promoter region was carried out by pyrosequencing (PyroMark Q24) in 265 primary breast tumours and 30 proximal normal breast tissue samples along with 8 breast-derived cell lines. ALKBH3 mRNA and protein expression were analysed in cell lines using RT-PCR and Western blotting, respectively. DNA alkylation damage assay was carried out in cell lines based on immunofluorescence and confocal imaging. Data on clinical parameters and survival outcomes in patients were obtained and assessed in relation to ALKBH3 promoter methylation.ResultsThe ALKBH3 gene, but not ALKBH2, undergoes CpG promoter methylation and transcriptional silencing in breast cancer. We developed a quantitative alkylation DNA damage assay based on immunofluorescence and confocal imaging revealing higher levels of alkylation damage in association with epigenetic inactivation of the ALKBH3 gene (P = 0.029). In our cohort of 265 primary breast cancer, we found 72 cases showing aberrantly high CpG promoter methylation over the ALKBH3 promoter (27%; 72 out of 265). We further show that increasingly higher degree of ALKBH3 promoter methylation is associated with reduced breast-cancer specific survival times in patients. In this analysis, ALKBH3 promoter methylation at >20% CpG methylation was found to be statistically significantly associated with reduced survival (HR = 2.3; P = 0.012). By thresholding at the clinically relevant CpG methylation level (>20%), we find the incidence of ALKBH3 promoter methylation to be 5% (13 out of 265).ConclusionsALKBH3 is a novel addition to the catalogue of DNA repair genes found inactivated in breast cancer. Our results underscore a link between defective alkylation repair and breast cancer which, additionally, is found in association with poor disease outcome.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-017-3453-8) contains supplementary material, which is available to authorized users.

Genome-wide association study of Stayability and Heifer Pregnancy in Red Angus cattle

“…This specific SNP was located within an intron in Alkylated DNA Repair Protein AlkB Homolog 3 (ALKBH3). This gene has been specifically associated with cell survival in various forms of cancer in humans (Tasaki et al, 2011;Stefansson et al, 2017). This SNP was also located in a region described by Peters et al (2012) that contained several SNP windows associated with various forms of ADG calculated from birth to weaning, weaning to yearling, and birth to yearling in Brangus heifers.…”

ALKBH4 Functions as a Suppressor of Colorectal Cancer Metastasis via Competitively Binding to WDR5

“…ALKBH4 belongs to the AlkB family of non-heme Fe (II)/a-ketoglutarate-dependent dioxygenases, whose function have been implicated in the repair of methylation damage in DNA and RNA (Lee et al, 2005). Available evidences also indicate that several members of AlkB family, such as ALKBH3 and ALKBH5, are closely linked to the inhibition of tumorigenesis and progression in various human cancers (Stefansson et al, 2017;Wang et al, 2018;Zhu et al, 2019). ALKBH4 has been previously illustrated in mediating demethylation of a monomethylated site in actin and depletion of ALKBH4 contributes to defects in cytokinesis and cell motility (Li et al, 2013), however, the underlying molecular mechanisms of ALKBH4 in tumor remain unknown.…”

DNA alkylation lesion repair: outcomes and implications in cancer chemotherapy

“…Nevertheless, considering that ALKBH2 or ALKBH3 is found to be overexpressed in many types of cancer, including prostate adenocarcinoma, non-small cell lung carcinoma, and head and neck cancer, chemotherapy developed from ALKBH-dependent alkylation repair may be promising (Konishi et al, 2005;Gilljam et al, 2009;Tasaki et al, 2011;Pilžys et al, 2019). Additionally, Stefansson et al (2017) suggested that promoter methylation status of the ALKBH3 gene in several breast cancers correlates with poor prognosis. In addition, a positive correlation exists between the promoter methylation and cellular N 3 meC levels in several breast cancer cell lines, suggesting a potential direction for alkylation chemotherapy (Stefansson et al, 2017).…”