Functional modulation of IGF-binding protein-3 expression in melanoma.
IGF-binding protein-3 (IGFBP3) is a member of the IGFBP family, which regulates mitogenic and antiapoptotic effects of IGFs. In this report we evaluated the role of IGFBP3 in melanoma. Quantitative real-time PCR (qRT-PCR), western blot, and ELISA analyses indicated a significant downregulation of IGFBP3 expression in melanoma cell lines as compared with a normal melanocyte cell line. Melanoma cell lines treated with the demethylating agent 5-AZA-2'-deoxycytidine reexpressed IGFBP3 at the mRNA and protein levels. Chromatin immunoprecipitation assays revealed enrichment of acetylated histones H3 and H4, and H3 di- and tri-methylated lysine 4 on the unmethylated IGFBP3 promoter. The IGFBP3 promoter region was highly methylated in human melanoma samples as compared with normal nevi. Overexpression of IGFBP3 in melanoma cells in vitro suppressed tumor cell survival, induced apoptosis, reduced colony formation and invasion, and induced expression of the proapoptotic genes p21, PUMA, and BAX. IGFBP3 overexpression also resulted in cleavage of caspase 3 and reduced expression of phosphorylated AKT. Stable overexpression of IGFBP3 suppressed tumor cell growth in vivo. Our study results indicate that silencing of IGFBP3 in melanoma is due to the methylation of its promoter, and that overexpression of IGFBP3 induces apoptosis and suppresses cell survival and growth.
Dar
Altaf A
AA
Auerback Melanoma Research Laboratory, Comprehensive Cancer Center, University of California, San Francisco, San Francisco, California, USA.
Majid
Shahana
S
Nosrati
Mehdi
M
de Semir
David
D
Federman
Scot
S
Kashani-Sabet
Mohammed
M
D017209:Apoptosis / Q000502:physiology; D001374:Azacitidine / Q000031:analogs & derivatives / Q000494:pharmacology; D001483:Base Sequence; D053148:Caspase 3 / Q000378:metabolism; D002455:Cell Division / Q000502:physiology; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000502:physiology; D002843:Chromatin / Q000187:drug effects / Q000502:physiology; D019175:DNA Methylation / Q000187:drug effects / Q000502:physiology; D000077209:Decitabine; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects / Q000502:physiology; D020868:Gene Silencing; D006801:Humans; D018972:Insulin-Like Growth Factor Binding Protein 3; D018969:Insulin-Like Growth Factor Binding Proteins / Q000235:genetics* / Q000378:metabolism; D008545:Melanoma / Q000235:genetics* / Q000473:pathology* / Q000503:physiopathology; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000502:physiology; D012333:RNA, Messenger / Q000378:metabolism; D012878:Skin Neoplasms / Q000235:genetics* / Q000473:pathology* / Q000503:physiopathology
D002843:Chromatin; D004791:Enzyme Inhibitors; C515497:IGFBP3 protein, human; D018972:Insulin-Like Growth Factor Binding Protein 3; D018969:Insulin-Like Growth Factor Binding Proteins; D012333:RNA, Messenger; D000077209:Decitabine; C505407:CASP3 protein, human; D053148:Caspase 3; D001374:Azacitidine