XIAP inhibits mature Smac-induced apoptosis by degrading it through ubiquitination in NSCLC

Abstract

X-linked inhibitor of apoptosis protein (XIAP) and second mitochondrial-derived activator of caspase (Smac) are two important prognostic biomarkers for cancer. They are negatively correlated in many types of cancer. However, their relationship is still unknown in lung cancer. In the present study, we found that there was a negative correlation between Smac and XIAP at the level of protein but not mRNA in NSCLC patients. However, XIAP overexpression had no effect on degrading endogenous Smac in lung cancer cell lines. Therefore, we constructed plasmids with full length of Smac (fSmac) and mature Smac (mSmac) which located in cytoplasm instead of original mitochondrial location, and was confirmed by immunofluorescence. Subsequently, we found that mSmac rather than fSmac was degraded by XIAP and inhibited cell viability. CHX chase assay and ubiquitin assay were performed to illustrate XIAP degraded mSmac through ubiquitin pathway. Overexpression of XIAP partially reverted apoptotic induction and cell viability inhibition by mSmac, which was due to inhibiting caspase-3 activation. In nude mouse xenograft experiments, mSmac inhibited Ki-67 expression and slowed down lung cancer growth, while XIAP partially reversed the effect of mSmac by degrading it. In conclusion, XIAP inhibits mature Smac-induced apoptosis by degrading it through ubiquitination in NSCLC.

Figure 1

XIAP and Smac in NSCLC tumor tissues and matched lung tissues. (A) RT-PCR and (B) western blot analysis of Smac and XIAP in cancer (T) and matched para-tumor tissues (N) are shown. The bands were quantified by densitometry and normalized to GAPDH. Values are depicted as mean ± SD, ^*P<0.05.

Figure 2

XIAP did not degrade endogenous Smac in lung cancer cell lines. (A) Myc-XIAP and empty vector (EV) were double digested by restriction enzymes BamHI and NotI. (B) The expression of XIAP and Smac were evaluated by western blotting in the A549 and H460 cells following transfection with Myc-XIAP or EV for 48 h. β-actin was used as loading control.

Figure 3

The location and function of fSmac and mSmac. (A) The structure of fSmac and Ub-mature Smac fusion protein (Ub-mSmac). (B) Flag-fSmac and Flag-Ub-mSmac were double digested by restriction enzymes BamHI and NotI. (C) The Localization of fSmac and Ub-mSmac were evaluated by immunofluorescence staining in the U2OS cells following transfection with Flag-fSmac or Flag-Ub-mSmac for 48 h. (D) Cell viability was measured by MTT assay following transfected with fSmac or Ub-mSmac in A549 and H460 cells for 48 h. (E) Cells were transfected with Myc-XIAP and Flag-fSmac or Flag-Ub-mSmac for 48 h and exogenous Smac and XIAP were evaluated by western blotting with anti-Flag or anti-Myc antibody. β-actin was used as loading control.

Figure 4

XIAP bound to mSmac and promoted the ubiquitin-mediated proteolysis of mSmac. (A) Flag-Ub-mSmac and Myc-XIAP plasmids were transfected into H460 cells. Ub-mSmac or XIAP was immunoprecipitated with anti-Flag or anti-Myc antibody. Mature Smac and XIAP coimmunoprecipitated with each other. (B) The half-life of mSmac was analyzed following treatment with cycloheximide (CHX). The degradation of mSmac was faster in XIAP overexpressing H460 cells, and treatment with MG132 inhibited XIAP induced mSmac degradation. (C) The intensity of mSmac was normalized to β-actin and depicted in a line chart. (D) Flag-Ub-mSmac was precipitated from Flag-Ub-mSmac overexpressing H460 cells by immunoprecipitation using an anti-Flag antibody. Smac ubiquitination was detected by western blotting.

Figure 5

Mature Smac-induced H460 cell apoptosis is partially reverted by XIAP. (A) H460 cells successfully expressed Flag-Ub-mSmac and/or Myc-XIAP by transfection. (B) Cell apoptosis was measured by flow cytometry and (C) the apoptotic rates are provided as mean ± SD. XIAP overexpression decreased the percentage of apoptotic cells in Ub-mSmac transfected H460 cells. (D) XIAP was found to enhance the viability of mSmac overexpressing H460 cells (MTT assay). (E) The activity of caspase-3 in mSmac overexpressing H460 cells was decreased by Myc-XIAP transfection. Values are depicted as the mean ± SD. ^*P<0.05.

Figure 6

XIAP partially abolishes the mSmac inhibition of tumor growth. (A) Control H460 cells, Ub-mSmac overexpressed H460 cells and Ub-mSmac/Myc-XIAP co-expressing H460 cells, respectively, were implanted into nude mice via subcutaneous injection. Tumor nodules were measured using a caliper at different times after implantation. (B) Tumor nodules were subjected to immunohistochemical staining for XIAP, Smac and Ki-67. (C) The percentage of XIAP, Smac and Ki-67 positive cells in tumors were depicted by quantitative analysis. Values are depicted as the mean ± SD. ^*P<0.05.