Altered regulation of DNA ligase IV activity by aberrant promoter DNA methylation and gene amplification in colorectal cancer

Hum Mol Genet. 2014 Apr 15;23(8):2043-54. doi: 10.1093/hmg/ddt599. Epub 2013 Nov 26.

Abstract

Colorectal cancer (CRC) presents as a very heterogeneous disease which cannot sufficiently be characterized with the currently known genetic and epigenetic markers. To identify new markers for CRC we scrutinized the methylation status of 231 DNA repair-related genes by methyl-CpG immunoprecipitation followed by global methylation profiling on a CpG island microarray, as altered expression of these genes could drive genomic and chromosomal instability observed in these tumors. We show for the first time hypermethylation of MMP9, DNMT3A and LIG4 in CRC which was confirmed in two CRC patient groups with different ethnicity. DNA ligase IV (LIG4) showed strong differential promoter methylation (up to 60%) which coincided with downregulation of mRNA in 51% of cases. This functional association of LIG4 methylation and gene expression was supported by LIG4 re-expression in 5-aza-2'-deoxycytidine-treated colon cancer cell lines, and reduced ligase IV amounts and end-joining activity in extracts of tumors with hypermethylation. Methylation of LIG4 was not associated with other genetic and epigenetic markers of CRC in our study. As LIG4 is located on chromosome 13 which is frequently amplified in CRC, two loci were tested for gene amplification in a subset of 47 cases. Comparison of amplification, methylation and expression data revealed that, in 30% of samples, the LIG4 gene was amplified and methylated, but expression was not changed. In conclusion, hypermethylation of the LIG4 promoter is a new mechanism to control ligase IV expression. It may represent a new epigenetic marker for CRC independent of known markers.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biomarkers, Tumor / genetics*
  • Blotting, Western
  • Cell Cycle
  • Cell Proliferation
  • Colon / metabolism
  • Colorectal Neoplasms / genetics*
  • CpG Islands / genetics
  • DNA (Cytosine-5-)-Methyltransferases / genetics
  • DNA Ligase ATP
  • DNA Ligases / genetics*
  • DNA Ligases / metabolism
  • DNA Methylation*
  • Female
  • Gene Amplification*
  • Gene Expression Regulation, Neoplastic*
  • Gene Silencing
  • Humans
  • Male
  • Matrix Metalloproteinase 9 / genetics
  • Middle Aged
  • Neoplasm Staging
  • Prognosis
  • Promoter Regions, Genetic / genetics*
  • RNA, Messenger / genetics
  • Real-Time Polymerase Chain Reaction
  • Reverse Transcriptase Polymerase Chain Reaction
  • Tumor Cells, Cultured

Substances

  • Biomarkers, Tumor
  • LIG4 protein, human
  • RNA, Messenger
  • DNA (Cytosine-5-)-Methyltransferases
  • DNA methyltransferase 3A
  • MMP9 protein, human
  • Matrix Metalloproteinase 9
  • DNA Ligases
  • DNA Ligase ATP