Tobacco smoke induces polycomb-mediated repression of Dickkopf-1 in lung cancer cells.
Limited information is available about epigenetic mechanisms by which cigarette smoke enhances the initiation and progression of lung cancer. To examine this issue, A549 and Calu-6 lung cancer cells were cultured in normal media with or without tobacco smoke condensate (TSC) under clinically relevant exposure conditions. Ten-day TSC exposure dramatically increased the tumorigenicity of lung cancer cells in nude mice. Microarray and quantitative reverse transcription-PCR (RT-PCR) experiments revealed that this phenomenon coincided with diminished expression of Dickkopf-1 (Dkk-1). Western blot, chromatin immunoprecipitation, methylation-specific PCR, and pyrosequencing experiments showed that repression of Dkk-1 coincided with decreased H4K16Ac, increased H3K27me3, and recruitment of SirT1, EZH2, SUZ12, and Bmi1 without DNA hypermethylation within the Dkk-1 promoter despite prolonged TSC exposures. Removal of TSC from culture media resulted in loss of promoter-associated polycomb repressor complexes and reexpression of Dkk-1. siRNA-mediated knockdown of EZH2 and SirT1 partially abrogated TSC-mediated inhibition of Dkk-1 expression. Western blot and quantitative RT-PCR array experiments showed that TSC exposure as well as knockdown of Dkk-1 activated Wnt signaling and significantly up-regulated Wnt5a in lung cancer cells. Knockdown of Dkk-1 recapitulated the dramatic protumorigenic effects of TSC exposure in Calu-6 cells. Despite the transient nature of Dkk-1 repression following TSC exposure in vitro, Dkk-1 remained silenced in tumor xenografts derived from TSC-treated Calu-6 cells. Collectively, these data provide evidence that cigarette smoke directly engages polycomb machinery to activate a signaling network implicated in maintenance of cancer stem cells.
Thoracic Oncology Section, Surgery Branch, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland 20892-1201, USA.
Mustafa
M
Hussain
Mahadev
M
Rao
Ashley E
AE
Humphries
Julie A
JA
Hong
Fang
F
Liu
Maocheng
M
Yang
Diana
D
Caragacianu
David S
DS
Schrump
D000107:Acetylation; D000818:Animals; D045744:Cell Line, Tumor; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000235:genetics; D015536:Down-Regulation; D000071221:Enhancer of Zeste Homolog 2 Protein; D020869:Gene Expression Profiling; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D008175:Lung Neoplasms / Q000209:etiology* / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D051379:Mice; D008819:Mice, Nude; D063151:Polycomb Repressive Complex 2; D063146:Polycomb-Group Proteins; D011401:Promoter Regions, Genetic; D034741:RNA, Small Interfering / Q000235:genetics; D012097:Repressor Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction; D056564:Sirtuin 1; D037761:Sirtuins / Q000235:genetics; D012906:Smoke; D014157:Transcription Factors / Q000235:genetics; D014183:Transplantation, Heterologous; D051153:Wnt Proteins / Q000378:metabolism
C501126:DKK1 protein, human; D004268:DNA-Binding Proteins; D036341:Intercellular Signaling Peptides and Proteins; D063146:Polycomb-Group Proteins; D034741:RNA, Small Interfering; D012097:Repressor Proteins; D012906:Smoke; D014157:Transcription Factors; D051153:Wnt Proteins; C498409:EZH2 protein, human; D000071221:Enhancer of Zeste Homolog 2 Protein; D063151:Polycomb Repressive Complex 2; C447939:SIRT1 protein, human; D056564:Sirtuin 1; D037761:Sirtuins