title: Epigenetic silencing of Aristaless-like homeobox-4, a potential tumor suppressor gene associated with lung cancer.
abstract: Using genome-wide methylation screening, we found Aristaless-like homeobox-4 (ALX4) preferentially methylated in lung cancer. ALX4 is a putative transcription factor that belongs to the family of paired-class homeoproteins involved in epithelial development. However, the role of ALX4 in tumorigenesis remains largely unclear. Here, we analyzed its epigenetic regulation, biological functions and related molecular mechanisms in lung cancer. CpG island methylation and expression of ALX4 were evaluated by methylation-specific polymerase chain reaction (PCR), bisulfite genomic sequencing, reverse-transcription PCR and Western blotting. ALX4 functions were determined by cell viability, colony formation, flow cytometry and in vivo tumorigenicity assays. ALX4 hypermethylation was detected in 55% (54/98) of primary lung cancers compared to none (0/20) of the normal lung tissue samples tested (p < 0.01). ALX4 was readily expressed in normal lung tissues with an unmethylated status, but downregulated or silenced in 90% (9/10) of lung cancer cell lines with a hypermethylation status. Demethylation experiments further confirmed that loss of ALX4 expression was regulated by CpG island hypermethylation. Re-expression of ALX4 in lung cancer cell lines suppressed cell viability, colony formation and migration, whereas it induced apoptosis and G1/S arrest and restrained the tumorigenicity in nude mice. These effects were associated with upregulation of proapoptotic proteins caspase-7, -8 and -9, and downregulation of Bcl-2. On the other hand, knockdown of ALX4 expression by siRNA increased cell viability and proliferation, whereas it inhibited apoptosis and cell cycle arrest. In conclusion, our results suggest that ALX4 is a novel putative tumor suppressor with epigenetic silencing in lung carcinogenesis.
authors.affiliation: Institute of Toxicology, College of Preventive Medicine, Third Military Medical University, Chongqing, China; Key Laboratory of Medical Protection for Electromagnetic Radiation, Ministry of Education of China, Chongqing, China.
authors.forename: Wen-Bin
authors.initials: WB
authors.lastname: Liu
authors.forename: Fei
authors.initials: F
authors.lastname: Han
authors.forename: Xing-Hua
authors.initials: XH
authors.lastname: Du
authors.forename: Xiao
authors.initials: X
authors.lastname: Jiang
authors.forename: Yong-Hong
authors.initials: YH
authors.lastname: Li
authors.forename: Yong
authors.initials: Y
authors.lastname: Liu
authors.forename: Hong-Qiang
authors.initials: HQ
authors.lastname: Chen
authors.forename: Lin
authors.initials: L
authors.lastname: Ao
authors.forename: Zhi-Hong
authors.initials: ZH
authors.lastname: Cui
authors.forename: Jia
authors.initials: J
authors.lastname: Cao
authors.forename: Jin-Yi
authors.initials: JY
authors.lastname: Liu
mesh_terms: D000818:Animals; D017209:Apoptosis; D014408:Biomarkers, Tumor / Q000235:genetics / Q000378:metabolism; D015153:Blotting, Western; D020169:Caspases / Q000378:metabolism; D002448:Cell Adhesion; D002453:Cell Cycle; D002465:Cell Movement; D049109:Cell Proliferation; D002478:Cells, Cultured; D018899:CpG Islands; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D044127:Epigenesis, Genetic; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D020868:Gene Silencing; D006801:Humans; D008168:Lung / Q000378:metabolism / Q000473:pathology; D008175:Lung Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D009367:Neoplasm Staging; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014157:Transcription Factors / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D014410:Tumor Stem Cell Assay
chemical_list: C416676:ALX4 protein, human; D014408:Biomarkers, Tumor; D004268:DNA-Binding Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D014157:Transcription Factors; D020169:Caspases
keywords: Aristaless-like homeobox-4; DNA methylation; lung cancer; tumor suppressor