title: Epigenetic silencing of CRABP2 and MX1 in head and neck tumors.
abstract: Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous disease affecting the epithelium of the oral cavity, pharynx and larynx. Conditions of most patients are diagnosed at late stages of the disease, and no sensitive and specific predictors of aggressive behavior have been identified yet. Therefore, early detection and prognostic biomarkers are highly desirable for a more rational management of the disease. Hypermethylation of CpG islands is one of the most important epigenetic mechanisms that leads to gene silencing in tumors and has been extensively used for the identification of biomarkers. In this study, we combined rapid subtractive hybridization and microarray analysis in a hierarchical manner to select genes that are putatively reactivated by the demethylating agent 5-aza-2'-deoxycytidine (5Aza-dC) in HNSCC cell lines (FaDu, UM-SCC-14A, UM-SCC-17A, UM-SCC-38A). This combined analysis identified 78 genes, 35 of which were reactivated in at least 2 cell lines and harbored a CpG island at their 5' region. Reactivation of 3 of these 35 genes (CRABP2, MX1, and SLC15A3) was confirmed by quantitative real-time polymerase chain reaction (PCR; fold change, >or=3). Bisulfite sequencing of their CpG islands revealed that they are indeed differentially methylated in the HNSCC cell lines. Using methylation-specific PCR, we detected a higher frequency of CRABP2 (58.1% for region 1) and MX1 (46.3%) hypermethylation in primary HNSCC when compared with lymphocytes from healthy individuals. Finally, absence of the CRABP2 protein was associated with decreased disease-free survival rates, supporting a potential use of CRABP2 expression as a prognostic biomarker for HNSCC patients.
authors.affiliation: Department of Biology, IBILCE-UNESP, São José do Rio Preto-SP, Brazil.
authors.forename: Marilia F
authors.initials: MF
authors.lastname: Calmon
authors.forename: Rodrigo V
authors.initials: RV
authors.lastname: Rodrigues
authors.forename: Carla M
authors.initials: CM
authors.lastname: Kaneto
authors.forename: Ricardo P
authors.initials: RP
authors.lastname: Moura
authors.forename: Sabrina D
authors.initials: SD
authors.lastname: Silva
authors.forename: Louise Danielle C
authors.initials: LD
authors.lastname: Mota
authors.forename: Daniel G
authors.initials: DG
authors.lastname: Pinheiro
authors.forename: Cesar
authors.initials: C
authors.lastname: Torres
authors.forename: Alex F
authors.initials: AF
authors.lastname: de Carvalho
authors.forename: Patrícia M
authors.initials: PM
authors.lastname: Cury
authors.forename: Fabio D
authors.initials: FD
authors.lastname: Nunes
authors.forename: Ines Nobuko
authors.initials: IN
authors.lastname: Nishimoto
authors.forename: Fernando A
authors.initials: FA
authors.lastname: Soares
authors.forename: Adriana M A
authors.initials: AM
authors.lastname: da Silva
authors.forename: Luis P
authors.initials: LP
authors.lastname: Kowalski
authors.forename: Helena
authors.initials: H
authors.lastname: Brentani
authors.forename: Cleslei F
authors.initials: CF
authors.lastname: Zanelli
authors.forename: Wilson A
authors.initials: WA
authors.lastname: Silva
authors.forename: Paula
authors.initials: P
authors.lastname: Rahal
authors.forename: Eloiza H
authors.initials: EH
authors.lastname: Tajara
authors.forename: Dirce M
authors.initials: DM
authors.lastname: Carraro
authors.forename: Anamaria A
authors.initials: AA
authors.lastname: Camargo
authors.forename: Sandro R
authors.initials: SR
authors.lastname: Valentini
mesh_terms: D001374:Azacitidine / Q000031:analogs & derivatives / Q000494:pharmacology; D014408:Biomarkers, Tumor / Q000235:genetics / Q000378:metabolism; D002294:Carcinoma, Squamous Cell / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D018899:CpG Islands / Q000235:genetics; D019175:DNA Methylation; D000077209:Decitabine; D044127:Epigenesis, Genetic; D019204:GTP-Binding Proteins / Q000235:genetics*; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006258:Head and Neck Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D006801:Humans; D007150:Immunohistochemistry; D053208:Kaplan-Meier Estimate; D064547:Myxovirus Resistance Proteins; D020411:Oligonucleotide Array Sequence Analysis; D011379:Prognosis; D018168:Receptors, Retinoic Acid / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D046888:Tissue Array Analysis
chemical_list: D014408:Biomarkers, Tumor; D064547:Myxovirus Resistance Proteins; D018168:Receptors, Retinoic Acid; C082928:retinoic acid binding protein II, cellular; D000077209:Decitabine; D019204:GTP-Binding Proteins; D001374:Azacitidine