XIAP-associated factor 1 (XAF1) negatively regulates the function of the X-linked inhibitor of apoptosis protein (XIAP), a member of the IAP family that exerts antiapoptotic effects. The extracellular signal-regulated kinase (ERK) pathway is thought to increase cell proliferation and to protect cells from apoptosis. The aim of the study was to investigate the correlation between the ERK1/2 signaling pathway and XAF1 in colon cancer.
Four human colon cancer cell lines, HCT1116 and Lovo (wildtype p53), DLD1 and SW1116 (mutant p53), were used. Lovo stable transfectants with XAF1 sense and antisense were established. The effects of dominant-negative MEK1 (DN-MEK1) and MEK-specific inhibitor U0126 on the ERK signaling pathway and expression of XAF1 and XIAP proteins were determined. The transcription activity of core XAF1 promoter was assessed by dual luciferase reporter assay. Cell proliferation was measured by MTT assay. Apoptosis was determined by Hoechst 33258 staining.
U0126 increased the expression of XAF1 in a time- and dose-dependent manner. A similar result was obtained in cells transfected with DN-MEK1 treatment. Conversely, the expression of XIAP was down-regulated. Activity of the putative promoter of the XAF1 gene was significantly increased by U0126 treatment and DN-MEK1 transient transfection. rhEGF-stimulated phosphorylation of ERK appeared to have little or no effect on XAF1 expression. Overexpression of XAF1 was more sensitive to U0126-induced apoptosis, whereas down-regulation of XAF1 by antisense reversed U0126-induced inhibition of cell proliferation.
XAF1 expression was up-regulated by inhibition of the ERK1/2 pathway through transcriptional regulation, which required de novo protein synthesis. The results suggest that XAF1 mediates apoptosis induced by the ERK1/2 pathway in colon cancer. Cancer 2007. © 2007 American Cancer Society.