Hit-and-run epigenetic editing prevents senescence entry in primary breast cells from healthy donors

Aberrant promoter DNA hypermethylation is a hallmark of cancer; however, whether this is sufficient to drive cellular transformation is not clear. To investigate this question, we use a CRISPR-dCas9 epigenetic editing tool, where an inactive form of Cas9 is fused to DNA methyltransferase effectors. Using this system, here we show simultaneous de novo DNA methylation of genes commonly methylated in cancer, CDKN2A, RASSF1, HIC1 and PTEN in primary breast cells isolated from healthy human breast tissue. We find that promoter methylation is maintained in this system, even in the absence of the fusion construct, and this prevents cells from engaging senescence arrest. Our data show that the key driver of this phenotype is repression of CDKN2A transcript p16 where myoepithelial cells harbour cancer-like gene expression but do not exhibit anchorage-independent growth. This work demonstrates that hit-and-run epigenetic events can prevent senescence entry, which may facilitate tumour initiation

Differential DNA methylation patterns in endo-siRNAs mediated silencing of LINE-1 retrotransposons

Analyzing differences in DNA methylation is a powerful tool for assessing the effect of endo-siRNAs expression in the human genome. Here, we present a simple genome-wide DNA methylation assay that allows for a precise quantitative analysis of difference

Assessment of DNA methylation status in early stages of breast cancer development

BACKGROUND: Molecular pathways determining the malignant potential of premalignant breast lesions remain unknown. In this study, alterations in DNA methylation levels were monitored during benign, premalignant and malignant stages of ductal breast cancer development. METHODS: To study epigenetic events during breast cancer development, four genomic biomarkers (Methylated-IN-Tumour (MINT)17, MINT31, RARβ2 and RASSF1A) shown to represent DNA hypermethylation in tumours were selected. Laser capture microdissection was employed to isolate DNA from breast lesions, including normal breast epithelia (n=52), ductal hyperplasia (n=23), atypical ductal hyperplasia (n=31), ductal carcinoma in situ (DCIS, n=95) and AJCC stage I invasive ductal carcinoma (IDC, n=34). Methylation Index (MI) for each biomarker was calculated based on methylated and unmethylated copy numbers measured by Absolute Quantitative Assessment Of Methylated Alleles (AQAMA). Trends in MI by developmental stage were analysed. RESULTS: Methylation levels increased significantly during the progressive stages of breast cancer development; P-values are 0.0012, 0.0003, 0.012, <0.0001 and <0.0001 for MINT17, MINT31, RARβ2, RASSF1A and combined biomarkers, respectively. In both DCIS and IDC, hypermethylation was associated with unfavourable characteristics. CONCLUSION: DNA hypermethylation of selected biomarkers occurs early in breast cancer development, and may present a predictor of malignant potential