54739

XAF1

BIRC4BP|HSXIAPAF1|XIAPAF1;XIAP associated factor 1;XAF1;XIAP associated factor 1

BIRC4 binding protein|BIRC4-binding protein|XIAP-associated factor 1

17p13.1

protein-coding

X-linked inhibitor of apoptosis (XIAP) is the most potent member of the IAP family that exerts antiapoptotic effects by interfering with the activities of caspases. Recently, XIAP-associated factor 1 (XAF1) and two mitochondrial proteins, Smac/DIABLO and HtrA2, have been identified to negatively regulate the caspase-inhibiting activity of XIAP. To explore the candidacy of XAF1, Smac/DIABLO, and HtrA2 as a tumor suppressor in gastric tumorigenesis, we investigated the expression and mutation status of the genes in 123 gastric tissues and 15 cancer cell lines. Whereas Smac/DIABLO and HtrA2 transcripts were normally expressed in all cancer specimens we examined, XAF1 transcript was not expressed or present at extremely low levels in 40% (6 of 15) of cancer cell lines and in 23% (20 of 87) of primary carcinomas. Abnormal reduction of XAF1 expression showed a strong correlation with stage and grade of tumors, and a tumor-specific down-regulation of XAF1 was observed in 45% (9 of 20) of matched sets. Unlike XAF1, XIAP expression exhibited no detectable alteration in cancers. Whereas loss of heterozygosity within the XAF1 region or somatic mutations of the gene was not detected, expression of XAF1 transcript was reactivated in all nonexpressor cell lines after 5-aza-2-deoxycytidine treatment. The 5 upstream region of the XAF1 gene encompasses no gastric cell-rich region that rigorously satisfies the formal criteria for CpG islands. However, bisulfite DNA sequencing analysis for 34 CpG sites in the promoter region revealed a strong association between hypermethylation and gene silencing. Moreover, transcriptional silencing of XAF1 was tightly associated with hypermethylation of seven CpGs located in the 5 proximal region (nucleotides -23 to -234). Additionally, loss or abnormal reduction of XAF1 expression was found to inversely correlate with p53 mutations, suggesting that epigenetic inactivation of XAF1 and mutational alteration of p53 might be mutually exclusive events in gastric tumorigenesis. Collectively, our study suggests that epigenetic silencing of XAF1 by aberrant promoter methylation may contribute to the malignant progression of human gastric tumors.

PURPOSE: The aim of this study was to investigate whether mRNA expression of the apoptosis-associated genes, XAF1 and XIAP, in bladder cancer patients correlates with response to neoadjuvant treatment. METHODS: gene expression was analyzed by a real-time quantitative PCR method in paired samples from 14 bladder cancer patients treated with a combination of neoadjuvant gemcitabine and cisplatin. The prognostic significance of XAF1 and XIAP mRNA expression as well as the correlation with several clinical and pathological findings were evaluated. RESULTS: The clinical response in the XAF1-high subset (n = 5) was remarkably higher compared with the XAF1-low subset (n = 9) (100% vs. 44.4%; P = 0.038). These results translated into a notably improvement of progression-free survival (PFS) in the XAF1-high subset (log-rank P = 0.012). In addition, patients in the XAF1-high subset had a 3.9-fold decreased chance of dying from the disease (hazard ratio for death (HR), 0.257; (CI 95%), 0.043-1.536, P = 0.036). When we evaluated the expression of XIAP, although an inverse correlation was found between expression and pathological response, there were no statistically significant associations with the clinical response, the length of PFS, and OS. CONCLUSIONS: This is one of the few studies to address the role of XAF1 in a clinical setting. The data presented here identify XAF1 as a novel predictive and prognostic factor in bladder cancer patients. Furthermore, our observations are in line with previous studies, which point towards XAF1 as a tumor-suppressor gene. Nonetheless, additional studies, both mechanistic and translational, are warranted and may help not only in corroborating the role of XAF1 as a prognostic marker, but also as a potential target for anticancer therapy.

OBJECTIVE: To investigate the effect of 5-azacytidine on XAF1 expression in myeloma cell lines RPMI8226 and XG-7 and the in vitro anti-myeloma activity of 5-azacytidine. METHODS: XAF1 mRNA and protein expression was detected by semi-quantitative reverse transcriptase PCR and Western blot, respectively. Methylation specific PCR (MSP) was used to detect methylation status of XAF1 promoter CpG islands. RPMI8226 and XG-7 cells were treated with 0-5 micromol/L of 5-azacytidine and Cell Counting Kit-8 colorimetric assay was used to evaluate the growth inhibitory effect. Cell apoptosis was determined with Annexin V-PE/7-AAD staining by flow cytometry. RESULTS: Untreated RPMI8226 cells expressed XAF1 mRNA isoforms 1 and 2, and untreated XG-7 cells had no XAF1 expression. Hypermethylation of XAF1 promoter CpG islands was detected in both the cell lines. After treated with 2.5 micromol/L 5-azacytidine for 72 h, both the cell lines expressed full-length XAF1 transcript and protein. 5-azacytidine treatment led to XAF1 promoter CpG islands hypomethylation and showed anti-myeloma activity in a time- and concentration-dependent manner with IC50 of 2.4 micromol/L and 2.6 micromol/L at 48 h for RPMI8226 and XG-7 cell lines, respectively. CONCLUSIONS: Lack of XAF1 expression and abnormal expression of XAF1 in myeloma cell lines are associated with XAF1 gene promoter CpG islands hypermethylation. 5-azacytidine treatment can induce XAF1 mRNA and protein expression and exerts anti-myeloma activity via apoptosis at clinically achievable concentrations.