Identification of candidate tumor suppressor genes inactivated by promoter methylation in melanoma.
Tumor suppressor genes (TSGs) are sometimes inactivated by transcriptional silencing through promoter hypermethylation. To identify novel methylated TSGs in melanoma, we carried out global mRNA expression profiling on a panel of 12 melanoma cell lines treated with a combination of 5-Aza-2-deoxycytidine (5AzadC) and an inhibitor of histone deacetylase, Trichostatin A. Reactivation of gene expression after drug treatment was assessed using Illumina whole-genome microarrays. After qRT-PCR confirmation, we followed up 8 genes (AKAP12, ARHGEF16, ARHGAP27, ENC1, PPP1R3C, PPP1R14C, RARRES1, and TP53INP1) by quantitative DNA methylation analysis using mass spectrometry of base-specific cleaved amplification products in panels of melanoma cell lines and fresh tumors. PPP1R3C, ENC1, RARRES1, and TP53INP1, showed reduced mRNA expression in 35-59% of the melanoma cell lines compared to melanocytes and which was correlated with a high proportion of promoter methylation (>40-60%). The same genes also showed extensive promoter methylation in 6-25% of the tumor samples, thus confirming them as novel candidate TSGs in melanoma.
Bonazzi
Vanessa F
VF
Oncogenomics Laboratory, Queensland Institute of Medical Research, Herston, QLD 4006, Australia.
Irwin
Darryl
D
Hayward
Nicholas K
NK
D001374:Azacitidine / Q000031:analogs & derivatives / Q000494:pharmacology; D002352:Carrier Proteins / Q000378:metabolism; D045744:Cell Line, Tumor; D003110:Colonic Neoplasms / Q000235:genetics; D018899:CpG Islands / Q000187:drug effects; D019175:DNA Methylation; D000077209:Decitabine; D004938:Esophageal Neoplasms / Q000235:genetics; D020869:Gene Expression Profiling; D020868:Gene Silencing; D016147:Genes, Tumor Suppressor; D005910:Glioma / Q000235:genetics; D006360:Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins; D008545:Melanoma / Q000235:genetics*; D008565:Membrane Proteins / Q000378:metabolism; D008840:Microfilament Proteins / Q000378:metabolism; D009479:Neuropeptides / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D011401:Promoter Regions, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction
D002352:Carrier Proteins; D006360:Heat-Shock Proteins; D006877:Hydroxamic Acids; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D008840:Microfilament Proteins; D009479:Neuropeptides; D009687:Nuclear Proteins; C491294:PPP1R3C protein, human; C452562:RARRES1 protein, human; C429767:TP53INP1 protein, human; C112302:ectodermal-neural cortex 1 protein; C012589:trichostatin A; D000077209:Decitabine; D010749:Phosphoprotein Phosphatases; D001374:Azacitidine