Silencing of the XAF1 gene by promoter hypermethylation in cancer cells and reactivation to TRAIL-sensitization by IFN-beta.
BACKGROUND
XIAP-associated factor 1 (XAF1) is a putative tumor suppressor that exerts its proapoptotic effects through both caspase-dependent and -independent means. Loss of XAF1 expression through promoter methylation has been implicated in the process of tumorigenesis in a variety of cancers. In this report, we investigated the role of basal xaf1 promoter methylation in xaf1 expression and assessed the responsiveness of cancer cell lines to XAF1 induction by IFN-beta.


METHODS
We used the conventional bisulfite DNA modification and sequencing method to determine the methylation status in the CpG sites of xaf1 promoter in glioblastoma (SF539, SF295), neuroblastoma (SK-N-AS) and cervical carcinoma (HeLa) cells. We analysed the status and incidence of basal xaf1 promoter methylation in xaf1 expression in non-treated cells as well as under a short or long exposure to IFN-beta. Stable XAF1 glioblastoma knock-down cell lines were established to characterize the direct implication of XAF1 in IFN-beta-mediated sensitization to TRAIL-induced cell death.


RESULTS
We found a strong variability in xaf1 promoter methylation profile and responsiveness to IFN-beta across the four cancer cell lines studied. At the basal level, aberrant promoter methylation was linked to xaf1 gene silencing. After a short exposure, the IFN-beta-mediated reactivation of xaf1 gene expression was related to the degree of basal promoter methylation. However, in spite of continued promoter hypermethylation, we find that IFN-beta induced a transient xaf1 expression, that in turn, was followed by promoter demethylation upon a prolonged exposure. Importantly, we demonstrated for the first time that IFN-beta-mediated reactivation of endogenous XAF1 plays a critical role in TRAIL-induced cell death since XAF1 knock-down cell lines completely lost their IFN-beta-mediated TRAIL sensitivity.


CONCLUSIONS
Together, these results suggest that promoter demethylation is not the sole factor determining xaf1 gene induction under IFN-beta treatment. Furthermore, our study provides evidence that XAF1 is a crucial interferon-stimulated gene (ISG) mediator of IFN-induced sensitization to TRAIL in cancer.
Micali
O Cristina
OC
Department of Plant-Microbe Interactions, Max-Planck Institute for Plant Breeding Research, Koeln, Germany. micali@mpiz-koeln.mpg.de <micali@mpiz-koeln.mpg.de>
Cheung
Herman H
HH
Plenchette
Stéphanie
S
Hurley
Sandra L
SL
Liston
Peter
P
LaCasse
Eric C
EC
Korneluk
Robert G
RG
D048868:Adaptor Proteins, Signal Transducing; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins; D045744:Cell Line, Tumor; D019175:DNA Methylation; D005786:Gene Expression Regulation; D020868:Gene Silencing; D006801:Humans; D016899:Interferon-beta / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins; D009363:Neoplasm Proteins / Q000235:genetics* / Q000378:metabolism*; D009369:Neoplasms / Q000235:genetics* / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000378:metabolism*; D015533:Transcriptional Activation
D048868:Adaptor Proteins, Signal Transducing; D051017:Apoptosis Regulatory Proteins; D047908:Intracellular Signaling Peptides and Proteins; D009363:Neoplasm Proteins; D053221:TNF-Related Apoptosis-Inducing Ligand; C505653:TNFSF10 protein, human; C417551:XAF1 protein, human; D016899:Interferon-beta