X-linked inhibitor of apoptosis (XIAP)-associated factor 1 (XAF1) is a tumor suppressor that is frequently lost or down-regulated by aberrant promoter hypermethylation in multiple human cancers. To explore XAF1’s candidacy for a suppressor in breast tumorigenesis, we investigated its expression status in tumor cell lines and tissues, effect on ER regulation of cell growth, and the molecular basis for its function.
XAF1 expression was examined by RT-PCR, immunoblot and bisulfite sequencing. Molecular basis for the XAF1 interplay with ERa and BRCA1 was defined using gene transfection, siRNA-mediated depletion, immunoprecipitation, pull-down assays, ubiquitination assay, immunohistochemistry and animal studies.
XAF1 expression was lost or abnormally diminished by promoter hypermethylation in a substantial fraction of cell lines and primary tumors. XAF1 expression shows an inverse correlation with ERα expression. In ERα-expressing cells, restoration of XAF1 expression extremely increased cellular sensitivity to estrogen-induced, ERα-mediated apoptosis. Likewise, in ERα-nonexpressing cells, recovery of ERα restoration led to the recovery of apoptotic response to estrogen in XAF1-dependent fashion. Intriguingly, XAF1 was found to directly bind to and destabilizes ERα and this interaction is crucial for its apoptosis-promoting activity. Mechanistically, XAF1 has the characteristic of binding with E3 ligase, XAF1 interacts with BRCA1 and subsequently stimulates BRCA1 binding to ERα and BRCA1-mediated K48 polyubiquitination of ERα. Using a series of truncated mutants, we determined the domains of XAF1, ERα, and BRCA1 that are required for the assembly formation. Additionally, XAF1 was characterized to be upregulated by estrogen at the transcription level through the p38, JNK, and NF-kB signaling pathway.
XAF1 is promoted by estrogen and its activation directs the apoptotic switch of estrogen function through the assembly of BRCA1-mediated ERα destruction complex. Our study illuminates the mechanistic consequence of epigenetic inactivation of XAF1 in human breast tumorigenesis.
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