Frequent alteration of XAF1 in human colorectal cancers: implication for tumor cell resistance to apoptotic stresses.
OBJECTIVE
X-linked inhibitor of apoptosis protein-associated factor 1 (XAF1) is a candidate tumor suppressor located at the chromosome 17p13 region, but the molecular basis underlying its inactivation in human tumors and growth-inhibiting function has not been well defined. We explored the candidacy of XAF1 as a suppressor in colorectal tumorigenesis.


METHODS
XAF1 expression was characterized by polymerase chain reaction-based cloning, isoform-specific polymerase chain reaction, ribonuclease protection, and immunoblot assays. Allelic loss of the gene was evaluated by loss of heterozygosity (LOH) assay, and promoter CG dinucleotide (CpG) site methylation was determined using bisulfite sequencing. The effect of XAF1 on tumor growth was examined using flow cytometry, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling, colony formation, and viability assays.


RESULTS
Expression of 5 XAF1 variants including 2 novel transcripts was down-regulated concomitantly in 11 of 20 (55%) cell lines and 26 of 65 (40%) primary tumors. XAF1 reduction was tumor-specific and showed a correlation with advanced stage and high grade of tumor. LOH of the gene was found in 12 of 33 (36%) tumors. Promoter CpG site methylation was observed frequently in both cell lines and tumor tissues including many LOH tumors, suggesting that biallelic inactivation of XAF1 might be common in colorectal cancers. XAF1 expression suppressed tumor cell growth and enhanced cellular response to various apoptotic stimuli, such as 5-fluorouracil, etoposide, H(2)O(2), gamma-irradiation, ultraviolet, and tumor necrosis factor-alpha, whereas knockdown of its expression protected cells from the stresses.


CONCLUSIONS
Genetic and epigenetic alteration of XAF1 is a common event in colorectal tumorigenesis and contributes to the malignant tumor progression by providing survival advantages for tumor cells under various stress conditions.
Chung
Sun-Ku
SK
School of Life Sciences and Biotechnology, Korea University, Seoul, Korea.
Lee
Min-Goo
MG
Ryu
Byung-Kyu
BK
Lee
Jin-Hee
JH
Han
Jikhyon
J
Byun
Do-Sun
DS
Chae
Kwon-Seok
KS
Lee
Kil Yeon
KY
Jang
Jae-Young
JY
Kim
Hyo-Jong
HJ
Chi
Sung-Gil
SG
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins; D045744:Cell Line, Tumor; D002470:Cell Survival; D015179:Colorectal Neoplasms / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology*; D018899:CpG Islands / Q000235:genetics; D019175:DNA Methylation; D015536:Down-Regulation; D015870:Gene Expression; D020868:Gene Silencing; D014644:Genetic Variation; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D019656:Loss of Heterozygosity; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*; D009367:Neoplasm Staging; D011401:Promoter Regions, Genetic; D013268:Stimulation, Chemical; D014158:Transcription, Genetic
D048868:Adaptor Proteins, Signal Transducing; D051017:Apoptosis Regulatory Proteins; D047908:Intracellular Signaling Peptides and Proteins; D009363:Neoplasm Proteins; C417551:XAF1 protein, human