Death Receptor-Induced Apoptosis Signalling Regulation by Ezrin Is Cell Type Dependent and Occurs in a DISC-Independent Manner in Colon Cancer Cells

International audienceEzrin belongs to the ERM (ezrin-radixin-moesin) protein family and has been demonstrated to regulate early steps of Fas receptor signalling in lymphoid cells, but its contribution to TRAIL-induced cell death regulation in adherent cancer cells remains unknown. In this study we report that regulation of FasL and TRAIL-induced cell death by ezrin is cell type depen-dant. Ezrin is a positive regulator of apoptosis in T-lymphoma cell line Jurkat, but a negative regulator in colon cancer cells. Using ezrin phosphorylation or actin-binding mutants, we provide evidence that negative regulation of death receptor-induced apoptosis by ezrin occurs in a cytoskeleton-and DISC-independent manner, in colon cancer cells. Remarkably, inhibition of apoptosis induced by these ligands was found to be tightly associated with regulation of ezrin phosphorylation on serine 66, the tumor suppressor gene WWOX and activation of PKA. Deficiency in WWOX expression in the liver cancer SK-HEP1 or the pancreatic Mia PaCa-2 cell lines as well as WWOX silencing or modulation of PKA activation by pharmacological regulators, in the colon cancer cell line SW480, abrogated regulation of TRAIL signalling by ezrin. Altogether our results show that death receptor pro-apoptotic signalling regulation by ezrin can occur downstream of the DISC in colon cancer cells

Ezrin-mediated TRAIL inhibition involves WWOX.

<p>(A) WWOX expression levels in indicated cell lines were analyzed by immunoblot. HSC70 was used here as a loading control. (B) VSV-ezrin WT was expressed ectopically in WWOX-deficient cells SK-HEP-1 and MIA PaCa-2. Expression levels were analysed by immunoblot and apoptosis in the corresponding cell lines after FasL (100 ng/ml) or TRAIL (500 ng/ml) stimulation was analysed by Hoechst staining. (C) S66A and S66D ezrin mutants were expressed in MIA PaCa-2. Expression levels were analysed by immunoblot as above and cell sensitivity to TRAIL-induced cell death was quantified by methylene blue. (D) SW480 cells expressing either ezrin wt or ezrin S66A or S66D were transfected with scramble (Scr) or WWOX (WOXX) si-RNAs for 4 hours and sensitivity to apoptosis induced by TRAIL was quantified by Hoechst staining. Data represent the mean ± SD of at least three different experiments. (**P<0.01; *P<0.05 respective to Scr siRNA tranfected cells; ns stands for not statistically relevant).</p

Ezrin inhibits TRAIL-induced apoptosis downstream of the DISC.

<p>(A) HCT116 or (B) SW480 cells, expressing or not VSV-ezrin were treated for 6 hours with Fas ligand (100 ng/ml) or His-TRAIL (500 ng/ml) or 16 hours with 1μM staurosporin (STS). Apoptosis was quantified by Hoechst staining. Data represent the mean ± SD of at least three different experiments. (*P<0.05; **P<0.01 respective to control cells). Ezrin ectopic expression levels were analyzed by immunoblotting using an anti-VSV antibody in control or ezrin WT-expressing HCT116 and SW480 cells. HSC70 was used as a loading control. (C) HCT116 or (D) SW480 cells, were transfected ezrin or scramble (Scr) siRNAs. 72 h after transfection cells were stimulated for 6 hours with Fas ligand (100 ng/ml) or His-TRAIL (500 ng/ml) and apoptosis was quantified after staining with APO2.7 antibody by flow cytometry. Data represent the mean ± SD of at least three different experiments. (*P<0.05; **P<0.01 respective to control cells). Ezrin expression levels were analyzed by immunoblotting. Actin was used as a loading control. (E) Analysis of TRAIL DISC formation. HCT116 and SW480 cells were stimulated or not with 5 μg/ml Flag-TRAIL cross-linked with 10 μg/ml anti-Flag (M2) antibody. Cells were lysed, and the DISC was immunoprecipitated and analyzed by western blot. One of three independent experiments is shown. (F) HCT116 cells were stimulated or not with 5 μg/ml His-TRAIL for 20 and 60 minutes. After cell lysis, GAPDH antibody was added to the cell lysates and immunoprecipitates were analyzed by western blot.</p

Ezrin inhibits TRAIL-induced cell death at the mitochondria level.

<p>(A) Immunoblot analysis of caspase activation, in SW480 cells expressing either ezrin S66A, S66D, R579A or WT, 16 or 6 hours after His-TRAIL (20 or 200 ng/ml) stimulation, respectively. (B) Analysis of TRAIL DISC formation in SW480 cells expressing either S66A, S66D, or WT ezrin. Cells were stimulated with 5 μg/ml Flag-TRAIL cross-linked with 10 μg/ml anti-Flag (M2) antibody. After cell lysis, the DISC was immunoprecipitated and analyzed by western blot. (C) Analysis of Bax activation. Ezrin S66A, S66D, WT and Mock-infected SW480 cells were left untreated or stimulated with His-TRAIL (20 or 200 ng/ml) for 16 h, then permeabilized and stained with an antibody recognizing active Bax before analysis by flow cytometry. The effect of two different concentrations of TRAIL (20 or 200 ng/ml, gray and black lines, respectively) were compared to unstimulated cells (gray filled curve). The percentage of cells containing active Bax after TRAIL stimulation is shown (upper and lower value, respectively).</p

Ezrin phosphorylation on serine 66 differentially affects its ability to inhibit TRAIL-induced apoptosis.

<p>(A) Immunoblot analysis of phospho-ezrin (Thr567) expression levels in SW480 cells after stimulation with His-TRAIL, Fas ligand or orthovanadate (NaV). Percentage of relative phospho-ezrin (Thr567) intensities were determined as follows: intensity of specific band in stimulated cells divided by the normalized intensity of unstimulated cells, normalized to HSC70. (B) SW480 cells were stimulated with 500 ng/ml His-TRAIL or 100 ng/ml Fas ligand for 15 minutes or left untreated. After cell lysis in NP40-containing buffer, ezrin was immunoprecipitated with an anti-ezrin antibody (clone 3C12). The level of ezrin phosphorylation was determined by western blot using anti-phospho-ezrin targeting tyrosines 353 and 145, anti-phospho-ERM recognizing phosphorylated-ezrin on threonine 567,-moesin on threonine 558 and-radixin on threonine 564 and an anti-pan phosphoserine. (C) Schematic representation of ezrin domains and phosphorylation sites within the protein. (D) SW480 cells were infected with an empty pMSCV retroviral vector (Mock) or with a pMSCV vector encoding ezrin WT, ezrin S66A, ezrin S66D, ezrin Y145F, ezrin Y145D, ezrin Y353F, ezrin Y353D, ezrin T567A, ezrin T567D and ezrin R579A. Expression levels of ezrin constructs were determined by immunoblot from NP40 cell extracts using an anti-VSV antibody. Actin was used here as a loading control. Data shown is representative of three independent experiments. (E) Selected cell extracts obtained after lysis in SDS were analyzed by immunoblot as above. (F) Effect of ezrin WT and ezrin phosphomutants ectopic expression on TRAIL-induced cell death in SW480 cells. Cells were stimulated with TRAIL 500 ng/ml for 6 hours. Apoptosis was measured by APO2.7 staining by flow cytometry. (G) Cell viability in the SW480 cells expressing ezrin S66A, ezrin S66D or ezrin R579A as compared to Mock infected cells was evaluated by methylene blue assay 24h after treatment using increasing concentrations of His-TRAIL. (H) Percent inhibitory TRAIL concentration curves, in ng/ml, from SW480 cells expressing ectopically the indicated ezrin mutants were obtained by methylen blue staining 16h after increasing His-TRAIL concentrations. Corresponding IC25, IC50 and IC90, inducing 25, 50 and 90% cell death, were obtained using CompuSyn. Data represent mean ± SD of at least 3 independent experiments. *P<0.05; **P<0.01; ***P<0.001 respective to Mock control cells.</p