Epigenetic silencing of CRABP2 and MX1 in head and neck tumors.
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.
Calmon
Marilia F
MF
Department of Biology, IBILCE-UNESP, São José do Rio Preto-SP, Brazil.
Rodrigues
Rodrigo V
RV
Kaneto
Carla M
CM
Moura
Ricardo P
RP
Silva
Sabrina D
SD
Mota
Louise Danielle C
LD
Pinheiro
Daniel G
DG
Torres
Cesar
C
de Carvalho
Alex F
AF
Cury
Patrícia M
PM
Nunes
Fabio D
FD
Nishimoto
Ines Nobuko
IN
Soares
Fernando A
FA
da Silva
Adriana M A
AM
Kowalski
Luis P
LP
Brentani
Helena
H
Zanelli
Cleslei F
CF
Silva
Wilson A
WA
Rahal
Paula
P
Tajara
Eloiza H
EH
Carraro
Dirce M
DM
Camargo
Anamaria A
AA
Valentini
Sandro R
SR
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
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