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

BACKGROUND: DNA 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. METHODS: Methylome 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. RESULTS: The 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). CONCLUSIONS: ALKBH3 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.

Notch1 Pathway Activation Results from the Epigenetic Abrogation of Notch-Related MicroRNAs in Mycosis Fungoides.

Notch is a family of transmembrane receptors that participate in the regulation of cell differentiation, proliferation, and stemness. Notch pathway activation has also been found associated with different human cancers including primary cutaneous T-cell lymphomas (CTCL). The elucidation of the mechanisms driving Notch activation in these particular diseases has remained elusive. Here we studied the possibility that DNA methylation at Notch pathway gene promoters and/or deregulation of Notch-associated microRNAs contribute to activate Notch in mycosis fungoides (MF). By genome-wide DNA methylation analysis, we failed to detect any consistent methylation at the Notch1, the Notch-ligand Jagged1, or the Notch-target Hes1 gene promoters, but found a significant methylation of the Notch-related microRNAs, in particular miR-200c and miR-124. Downregulation of miR-200c is associated with overexpression of Jagged1, concomitant to Notch1 activation. CTCL cell lines were infected with lentiviral vector encoding for miR-200c and ectopic expression of miR-200c in CTCL lines resulted in Jagged1 protein downregulation associated with a reduction in the levels of active Notch1. Our study deciphers an epigenetic mechanism regulating the Notch pathway in (MF) that might contribute to the future design of more specific therapeutic strategies.

BRCA1 as a tumor suppressor linked to the regulation of epigenetic states: keeping oncomiRs under control.

BRCA1 is a tumor suppressor gene known to be implicated in the development of a subset of breast and ovarian cancers. The tumor suppressor properties of BRCA1 are generally thought to be linked to the gene's critical roles in the network of DNA damage response. In a recent report, BRCA1-mediated epigenetic repression at the promoter region of miRNA-155 was identified as a novel mechanism by which BRCA1 carries out its tumor suppressor functions.

Tetraploidy in BRCA2 breast tumours.

Tetraploidy and aneuploidy can be caused by cell division errors and are frequently observed in many human carcinomas. We have recently reported delayed cytokinesis in primary human fibroblasts from BRCA2 mutation carriers, implying a function for the BRCA2 tumour suppressor in completion of cell division. Here, we address ploidy aberrations in breast tumours derived from BRCA2 germline mutation carriers. Ploidy aberrations were evaluated from flow cytometry histograms on selected breast tumour samples (n=236), previously screened for local BRCA mutations. The ploidy between BRCA2-mutated (n=71) and matched sporadic (n=165) cancers was compared. Differences in ploidy distribution were examined with respect to molecular tumour subtypes, previously defined by immunohistochemistry on tissue microarray sections. Tetraploidy was significantly 3 times more common in BRCA2 breast cancers than sporadic. However, no differences were found in the overall ploidy distribution between BRCA2-mutation carriers and non-carriers. In BRCA2 cancers, tetraploidy was associated with luminal characteristics. The increased frequency of tetraploidy in BRCA2 associated cancers may be linked to cell division errors, particularly cytokinesis. Additionally, tetraploidy emerges predominantly in BRCA2 breast cancers displaying luminal rather than triple-negative phenotypes.

EZH2-mediated epigenetic repression of DNA repair in promoting breast tumor initiating cells.

Members of the Polycomb-group (PcG) family of proteins, including EZH2 (enhancer of zeste homolog 2), are involved in establishing epigenetic silencing of developmental genes in adult and embryonic stem cells, and their deregulation has been implicated in cancer. In a recent report, EZH2-mediated epigenetic repression of DNA damage repair in breast tumor initiating cells (BTICs) was identified as a mechanism that could promote expansion of BTICs, and may contribute to cancer progression.

CpG island hypermethylation of BRCA1 and loss of pRb as co-occurring events in basal/triple-negative breast cancer.

Triple-negative breast cancer (TNBC) occurs in approximately 15% of all breast cancer patients, and the incidence of TNBC is greatly increased in BRCA1 mutation carriers. This study aimed to assess the impact of BRCA1 promoter methylation with respect to breast cancer subtypes in sporadic disease. Tissue microarrays (TMAs) were constructed representing tumors from 303 patients previously screened for BRCA1 germline mutations, of which a subset of 111 sporadic tumors had previously been analyzed with respect to BRCA1 methylation. Additionally, a set of eight tumors from BRCA1 mutation carriers were included on the TMAs. Expression analysis was performed on TMAs by immunohistochemistry (IHC) for BRCA1, pRb, p16, p53, PTEN, ER, PR, HER2, CK5/6, EGFR, MUC1 and Ki-67. Data on BRCA1 aberrations and IHC expression was examined with respect to breast cancer-specific survival. The results demonstrate that CpG island hypermethylation of BRCA1 significantly associates with the basal/triple-negative subtype. Low expression of pRb, and high/intense p16, were associated with BRCA1 promoter hypermethylation, and the same effects were seen in BRCA1 mutated tumors. The expression patterns of BRCA1, pRb, p16 and PTEN were highly correlated, and define a subgroup of TNBCs characterized by BRCA1 aberrations, high Ki-67 (≥ 40%) and favorable disease outcome. In conclusion, our findings demonstrate that epigenetic inactivation of the BRCA1 gene associates with RB/p16 dysfunction in promoting TNBCs. The clinical implications relate to the potential use of targeted treatment based on PARP inhibitors in sporadic TNBCs, wherein CpG island hypermethylation of BRCA1 represents a potential marker of therapeutic response.

Genomic profiling of breast tumours in relation to BRCA abnormalities and phenotypes.

INTRODUCTION: Germline mutations in the BRCA1 and BRCA2 genes account for a considerable fraction of familial predisposition to breast cancer. Somatic mutations in BRCA1 and BRCA2 have not been found and the involvement of these genes in sporadic tumour development therefore remains unclear. METHODS: The study group consisted of 67 primary breast tumours with and without BRCA1 or BRCA2 abnormalities. Genomic alterations were profiled by high-resolution (~7 kbp) comparative genome hybridisation (CGH) microarrays. Tumour phenotypes were analysed by immunohistochemistry on tissue microarrays using selected biomarkers (ER, PR, HER-2, EGFR, CK5/6, CK8, CK18). RESULTS: Classification of genomic profiles through cluster analysis revealed four subgroups, three of which displayed high genomic instability indices (GII). Two of these GII-high subgroups were enriched with either BRCA1- or BRCA2-related tumours whereas the third was not BRCA-related. The BRCA1-related subgroup mostly displayed non-luminal phenotypes, of which basal-like were most prominent, whereas the other two genomic instability subgroups BRCA2- and GII-high-III (non-BRCA), were almost entirely of luminal phenotype. Analysis of genome architecture patterns revealed similarities between the BRCA1- and BRCA2 subgroups, with long deletions being prominent. This contrasts with the third instability subgroup, not BRCA-related, where small gains were more prominent. CONCLUSIONS: The results suggest that BRCA1- and BRCA2-related tumours develop largely through distinct genetic pathways in terms of the regions altered while also displaying distinct phenotypes. Importantly, we show that the development of a subset of sporadic tumours is similar to that of either familial BRCA1- or BRCA2 tumours. Despite their differences, we observed clear similarities between the BRCA1- and BRCA2-related subgroups reflected in the type of genomic alterations acquired with deletions of long DNA segments being prominent. This suggests similarities in the mechanisms promoting genomic instability for BRCA1- and BRCA2-associated tumours, possibly relating to deficiency in DNA repair through homologous recombination. Indeed, this feature characterized both familial and sporadic tumours displaying BRCA1- or BRCA2-like spectrums of genomic alterations. The importance of these findings lies in the potential benefit from targeted therapy, through the use of agents leading to DNA double-strand breaks such as PARP inhibitors (olaparib) and cisplatin, for a much larger group of patients than the few BRCA1 and BRCA2 germline mutation carriers.