title: Promoter hypermethylation and silencing of tissue factor pathway inhibitor-2 in oral squamous cell carcinoma.
abstract: BACKGROUND
  The treatment of oral squamous cell carcinoma (OSCC) following early detection is associated with good outcomes. Therefore, the survival and prognosis of OSCC patients could be hugely improved by identifying reliable biomarkers for the early diagnosis of the disease. Our previous methylation microarray analysis results have suggested that the gene encoding tissue factor pathway inhibitor-2 (TFPI-2) is a potential clinical predictor as well as a key regulator involved in OSCC malignancy.
  
  
  METHODS
  Methylation of the TFPI-2 promoter in oral tissue specimens was evaluated by bisulfite sequencing assay, quantitative methylation-specific PCR, and pyrosequencing assay. The differences in methylation levels among the groups were compared using the Mann-Whitney U test. The area under the receiver operating characteristic curve (AUROC) was used to evaluate the discrimination ability for detecting OSCC. Cellular TFPI-2 expression was analyzed by quantitative reverse-transcription PCR before and after treatment with 5'-aza-2'-deoxycytidine and trichostatin A, to confirm whether TFPI-2 was epigenetically silenced in OSCC cells. We investigated whether TFPI-2 plays a role as a tumor suppressor by establishing TFPI-2-overexpressing OSCC cells and subjecting them to in vitro cellular proliferation, migration, and invasion assays, as well as an in vivo metastasis assay.
  
  
  RESULTS
  TFPI-2 was hypermethylated in OSCC tissues versus normal oral tissues (P < 0.0001), with AUROC = 0.91, when using a pyrosequencing assay to quantify the methylation level. TFPI-2 silencing in OSCC was regulated by both DNA methylation and chromatin histone modification. Restoration of TFPI-2 counteracted the invasiveness of OSCC by inhibiting the enzymatic activity of matrix metalloproteinase-2, and consequently interfered with OSCC metastasis in vivo.
  
  
  CONCLUSIONS
  Our data suggest strongly that TFPI-2 is a down-regulated tumor suppressor gene in OSCC, probably involving epigenetic silencing mechanisms. The loss of TFPI-2 expression is a key event for oral tumorigenesis, especially in the process of tumor metastasis.
authors.forename: Yi-Hui
authors.initials: YH
authors.lastname: Lai
authors.forename: Ru-Yin
authors.initials: RY
authors.lastname: He
authors.forename: Jian-Liang
authors.initials: JL
authors.lastname: Chou
authors.forename: Michael W-Y
authors.initials: MW
authors.lastname: Chan
authors.affiliation: Department of Life Science and Institutes of Molecular Biology and Biomedical Science, National Chung Cheng University, Min-Hsiung, Chia-Yi, Taiwan. yufenli@mail.cmu.edu.tw.
authors.forename: Yu-Fen
authors.initials: YF
authors.lastname: Li
authors.forename: Chien-Kuo
authors.initials: CK
authors.lastname: Tai
mesh_terms: D000818:Animals; D014408:Biomarkers, Tumor / Q000235:genetics / Q000378:metabolism; D002294:Carcinoma, Squamous Cell / Q000175:diagnosis / Q000235:genetics* / Q000473:pathology; D019175:DNA Methylation; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D020868:Gene Silencing; D006023:Glycoproteins / Q000235:genetics* / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D009062:Mouth Neoplasms / Q000175:diagnosis / Q000235:genetics* / Q000473:pathology; D011379:Prognosis; D011401:Promoter Regions, Genetic
chemical_list: D014408:Biomarkers, Tumor; D006023:Glycoproteins; C021240:tissue-factor-pathway inhibitor 2