Mechanism and consequence of the hypermethylator phenotype in human breast cancer Public Deposited

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  • March 20, 2019
  • Roll, Jacqueline Devon
    • Affiliation: School of Medicine, Department of Pathology and Laboratory Medicine
  • DNA hypermethylation events and other epimutations occur in many neoplasms, producing gene expression changes that contribute to neoplastic transformation, tumorigenesis, and tumor behavior. Some human cancers exhibit a hypermethylator phenotype, characterized by concurrent DNA methylation-dependent silencing of multiple genes. To determine if a hypermethylation defect occurs in breast cancer, the expression profile and promoter methylation status of 66 methylation-sensitive genes were evaluated among 16 breast cancer cell lines. The relationship between gene expression (assessed by RT-PCR and quantitative real-time PCR), promoter methylation (assessed by MSP, bisulfite sequencing, and 5-aza-2'deoxycytidine treatment), and the DNA methyltransferase machinery (total DNMT activity, DNMT1, DNMT3a, and DNMT3b proteins) were examined. Unsupervised cluster analysis of the expression of methylation-sensitive genes revealed two groups of cell lines that possess distinct methylation signatures: (i) hypermethylator cell lines, and (ii) low-frequency methylator cell lines. The hypermethylator cell lines are characterized by high rates of concurrent methylation of nine genes (CDH1, CEACAM6, CST6, ESR1, GNA11, MUC1, MYB, SCNN1A, and TFF3), whereas the low-frequency methylator cell lines typically do not methylate these genes. Hypermethylator cell lines coordinately overexpress total DNMT activity and DNMT3b protein compared to normal breast cells. In contrast, most low-frequency methylator cell lines possess DNMT activity and protein levels that are indistinguishable from normal. Mining of microarray expression data from primary breast cancers identified groups of tumors that express a hypermethylation signature defined by loss of gene expression of seven to nine indicator genes. On average, the hypermethylator breast cancers represent [approximately]23% of tumors, with [approximately]79% of hypermethylator tumors belonging to the basal subtype, and [approximately]58-81% of all basal tumors exhibiting this hypermethylation defect, suggesting that the hypermethylator defect cosegregates with poor prognosis breast cancers. Methylation analysis of 26 primary breast tumors revealed extensive methylation of genes of interest among basal tumors, but low levels of methylation in tumors of other molecular subtypes. RNAi knockdown of DNMT3b in hypermethylator MDA-MB-453 and BT549 cells resulted in reexpression of methylation-silenced indicator genes. These results strongly suggest that overexpressed DNMT3b protein drives aberrant methylation of a concurrent set of epigenetically-regulated genes and typifies a novel hypermethylator phenotype in human breast cancer.
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  • Coleman, William
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