title: Hypomethylation of long interspersed nuclear element-1 (LINE-1) leads to activation of proto-oncogenes in human colorectal cancer metastasis.
abstract: OBJECTIVE
  Hypomethylation of LINE-1 elements has emerged as a distinguishing feature in human cancers. Limited evidence indicates that some LINE-1 elements encode an additional internal antisense promoter, and increased hypomethylation of this region may lead to inadvertent activation of evolutionarily methylation-silenced downstream genes. However, the significance of this fundamental epigenetic mechanism in colorectal cancer (CRC) has not been investigated previously.
  
  
  METHODS
  We analysed tissue specimens from 77 CRC patients with matched sets of normal colonic mucosa, primary CRC tissues (PC), and liver metastasis tissues (LM). LINE-1 methylation levels were determined by quantitative bisulfite pyrosequencing. MET, RAB3IP and CHRM3 protein expression was determined by western blotting and IHC. MET proto-oncogene transcription and 5-hydroxymethylcytosine (5-hmc) were evaluated by quantitative real-time-PCR.
  
  
  RESULTS
  Global LINE-1 methylation levels in LM were significantly lower compared with the matched PC (PC=66.2% vs LM=63.8%; p<0.001). More importantly, we observed that specific LINE-1 sequences residing within the intronic regions of multiple proto-oncogenes, MET (p<0.001), RAB3IP (p=0.05) and CHRM3 (p=0.01), were significantly hypomethylated in LM tissues compared with corresponding matched PC. Furthermore, reduced methylation of specific LINE-1 elements within the MET gene inversely correlated with induction of MET expression in CRC metastases (R=-0.44; p<0.0001). Finally, increased 5-hmc content was associated with LINE-1 hypomethylation.
  
  
  CONCLUSIONS
  Our results provide novel evidence that hypomethylation of specific LINE-1 elements permits inadvertent activation of methylation-silenced MET, RAB3IP and CHRM3 proto-oncogenes in CRC metastasis. Moreover, since 5-hmc content inversely correlated with LINE-1 hypomethylation in neoplastic tissues, our results provide important mechanistic insights into the fundamental processes underlying global DNA hypomethylation in human CRC.
authors.affiliation: Gastrointestinal Cancer Research Laboratory, Baylor Research Institute and Sammons Cancer Center, Baylor University Medical Center, , Dallas, Texas, USA.
authors.forename: Keun
authors.initials: K
authors.lastname: Hur
authors.forename: Paloma
authors.initials: P
authors.lastname: Cejas
authors.forename: Jaime
authors.initials: J
authors.lastname: Feliu
authors.forename: Juan
authors.initials: J
authors.lastname: Moreno-Rubio
authors.forename: Emilio
authors.initials: E
authors.lastname: Burgos
authors.forename: C Richard
authors.initials: CR
authors.lastname: Boland
authors.forename: Ajay
authors.initials: A
authors.lastname: Goel
mesh_terms: D015153:Blotting, Western; D016022:Case-Control Studies; D045744:Cell Line, Tumor; D003106:Colon / Q000378:metabolism; D015179:Colorectal Neoplasms / Q000235:genetics* / Q000503:physiopathology; D019175:DNA Methylation / Q000502:physiology*; D015972:Gene Expression Regulation, Neoplastic / Q000502:physiology; D006801:Humans; D020084:Long Interspersed Nucleotide Elements / Q000235:genetics* / Q000502:physiology; D009362:Neoplasm Metastasis / Q000235:genetics / Q000503:physiopathology; D000090063:Proto-Oncogene Mas; D019859:Proto-Oncogene Proteins c-met / Q000378:metabolism; D011519:Proto-Oncogenes / Q000502:physiology*; D060888:Real-Time Polymerase Chain Reaction; D020692:rab3 GTP-Binding Proteins / Q000378:metabolism
chemical_list: C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; C491743:MET protein, human; D019859:Proto-Oncogene Proteins c-met; D020692:rab3 GTP-Binding Proteins
keywords: Colorectal Neoplasia; Methylation; Oncogenes