Down-regulation of XIAP associated factor 1 (XAF1) through Hsf-1 binding in the promoter region

Abstract

Objective: The heat-shock transcription factors (HSFs) were initially implicated in upregulating or suppressing various gene expression to prevent cell from stress-induced apoptosis. XIAP associated factor 1 (XAF1) was a pro-apoptotic gene with the effect of antagonizing the cytoprotection role of XIAP. The expression of XAF1 was absent or only at low level in most of cancer cell with the mechanism not clear. In the present study, we reported that stress stimuli down-regulated XAF1 expression in GI cancer cells through HSF1. Material and Methods: The expression HSF1 and XAF1 was detected by RT-PCR and Western Blot. The transcription starting site of XAF1 gene was determined by 5’ Rapid Amplification of cDNA END (RACE). The transcriptional regulation of XAF1 gene by stress stimuli was detected by dual luciferase assay after cloning various length of 5’flanking region of XAF1 gene into luciferase reporter vector, pGL3. Putative HSF binding sequences was identified by Electrophoresis Mobility Shift Assay (EMSA). Results: HSF1 was expressed at higher level in cancer than in normal tissue using protein extracts from matched colon cancer and neighbor normal tissues. Heat, hypo-osmolarity and H2O2 stresses induced nuclear accumulation of HSF1 in gastrointestinal (GI) cancer cell lines as well as down-regulated XAF1 expression. The transcription-starting site of XAF1 located at -26nt upstream of the ATG translation starting codon. Dual luciferase assay revealed the existence of a transcription silencer between -592 and -1414nt region. This region contained two putative HSF-1 binding sequences that were rich of nGAAn/nTTCn elements. Electrophoresis mobility shift assay (EMSA) defined the existence of a genuine HSF-1 binding element within the -862/-821 area. The inactivation of this element by either site-directed mutation or pretreatment with a HSF-1 inhibitor, pifithrin-a, restored the promoter activity of the silencer structure. Pretreatment of cancer cell with antioxidants suppressed the HSF-1 binding activity, increased the transcriptional activity and up-regulated the expression of XAF1 in GI cancer cell lines. Conclusion: Our findings suggested that endogenous stress pressure in cancer cells suppressed XAF1 expression through HSF-1 mediated transcription repression, thus, implicated the synergized effect of two anti-apoptotic protein family, HSP and IAPs, in the procession of cytoprotection under stress circumstance.