title: DNA Hypermethylation of SHISA3 in Colorectal Cancer: An Independent Predictor of Poor Prognosis.
abstract: BACKGROUND
  Shisa3 is a novel tumor suppressor identified in lung cancer. However, its antitumor activity in other human cancers and the mechanism of gene inactivation remain unknown.
  
  
  METHODS
  SHISA3 expression was measured by reverse transcription-PCR (RT-PCR) and quantitative RT-PCR (RT-qPCR). DNA methylation was determined by bisulfite sequencing and pyrosequencing.
  
  
  RESULTS
  Down-regulation of SHISA3 expression was observed in all of 11 colorectal cancer (CRC) cell lines and was further confirmed in 34 (65.4 %) of 52 colorectal carcinomas by RT-qPCR. Four of six CRC cell lines could restore SHISA3 expression after treatment with 5-aza-2'-deoxycytidine. Tumor-specific methylation of five CpG sites in the first intron of SHISA3 was identified by bisulfite sequencing, and their methylation levels were quantified in 127 pairs of primary CRC tissues by bisulfite pyrosequencing. The methylation levels of SHISA3 in tumors were noticeably higher than that in their matched normal mucosae. In addition, SHISA3 hypermethylation was significantly associated with an increased risk of disease recurrence in patients with stage II and III disease (P = 0.007) and was an independent predictor of poor overall survival [hazard ratio (HR) 2.9, 95 % confidence interval (CI) 1.5-5.8; P = 0.002] and disease-free survival (HR 4.0, 95 % CI 1.6-10.2; P = 0.003) of CRC patients.
  
  
  CONCLUSIONS
  SHISA3 gene is epigenetically inactivated in a substantial fraction of CRC, and its hypermethylation is of prognostic significance in predicting clinical outcome. The quantitative bisulfite pyrosequencing assay established could be a cost-effective tool for providing a potential biomarker of adverse prognosis in CRC.
authors.affiliation: Department of Surgery, Cardinal Tien Hospital, New Taipei City, Taiwan.|School of Medicine, Fu-Jen Catholic University, New Taipei City, Taiwan.
authors.forename: Ming-Hong
authors.initials: MH
authors.lastname: Tsai
authors.affiliation: Department of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan University, Taipei, Taiwan.
authors.forename: Wen-Chi
authors.initials: WC
authors.lastname: Chen
authors.affiliation: Department of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan University, Taipei, Taiwan.|Department of Laboratory Medicine, National Taiwan University Hospital, Taipei, Taiwan.
authors.forename: Sung-Liang
authors.initials: SL
authors.lastname: Yu
authors.affiliation: Department of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan University, Taipei, Taiwan.
authors.forename: Chun-Chieh
authors.initials: CC
authors.lastname: Chen
authors.affiliation: Department of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan University, Taipei, Taiwan.
authors.forename: Tzu-Ming
authors.initials: TM
authors.lastname: Jao
authors.affiliation: Department of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan University, Taipei, Taiwan.
authors.forename: Chi-Yen
authors.initials: CY
authors.lastname: Huang
authors.affiliation: Department of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan University, Taipei, Taiwan.
authors.forename: Sheng-Tai
authors.initials: ST
authors.lastname: Tzeng
authors.affiliation: Department of Surgery, Cardinal Tien Hospital, New Taipei City, Taiwan.
authors.forename: Sou-Jhy
authors.initials: SJ
authors.lastname: Yen
authors.affiliation: Department of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan University, Taipei, Taiwan. ycyangntu@ntu.edu.tw.|Department of Laboratory Medicine, National Taiwan University Hospital, Taipei, Taiwan. ycyangntu@ntu.edu.tw.
authors.forename: Ya-Chien
authors.initials: YC
authors.lastname: Yang
mesh_terms: D000230:Adenocarcinoma / Q000235:genetics* / Q000556:secondary; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D014408:Biomarkers, Tumor / Q000235:genetics*; D016022:Case-Control Studies; D015179:Colorectal Neoplasms / Q000235:genetics* / Q000473:pathology; D018899:CpG Islands; D019175:DNA Methylation; D005260:Female; D005500:Follow-Up Studies; D006801:Humans; D008207:Lymphatic Metastasis; D008297:Male; D008565:Membrane Proteins / Q000235:genetics*; D008875:Middle Aged; D009364:Neoplasm Recurrence, Local / Q000235:genetics* / Q000473:pathology; D009367:Neoplasm Staging; D011379:Prognosis; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013447:Sulfites; D015996:Survival Rate; D014407:Tumor Cells, Cultured
chemical_list: D014408:Biomarkers, Tumor; D008565:Membrane Proteins; D012333:RNA, Messenger; D013447:Sulfites; C042345:hydrogen sulfite