Inhibition of USP2 Enhances TRAIL-Mediated Cancer Cell Death through Downregulation of Survivin

Abstract

Ubiquitin-specific protease 2 (USP2) is a deubiquitinase belonging to the USPs subfamily. USP2 has been known to display various biological effects including tumorigenesis and inflammation. Therefore, we aimed to examine the sensitization effect of USP2 in TRAIL-mediated apoptosis. The pharmacological inhibitor (ML364) and siRNA targeting USP2 enhanced TNF-related apoptosis-inducing ligand (TRAIL)-induced cancer cell death, but not normal cells. Mechanistically, USP2 interacted with survivin, and ML364 degraded survivin protein expression by increasing the ubiquitination of survivin. Overexpression of survivin or USP2 significantly prevented apoptosis through cotreatment with ML364 and TRAIL, whereas a knockdown of USP2 increased sensitivity to TRAIL. Taken together, our data suggested that ML364 ubiquitylates and degrades survivin, thereby increasing the reactivity to TRAIL-mediated apoptosis in cancer cells.

Keywords: ML364; TRAIL; USP2; deubiquitinase; survivin.

Figure 1

Knockdown of USP2 induces survivin downregulation in cancer cells. (A,B) Caki-1 (A), ACHN, DU145, and A549 (B) cells were treated with various concentrations of ML364. (C) Caki-1 cells were transfected with control or USP2 siRNA for 24 h. (D) Caki-1 cells were transfected with Cont siRNA or USP2 siRNA without/with USP2 plasmid for 24 h. The protein expression was detected using Western blotting.

Figure 2

ML364 induces proteasome-mediated survivin degradation. (A,B) Caki-1 cells were treated with 2 μM ML364 for the different times. (C) Caki-1 cells were treated with 2 μM ML364 after pretreatment with 20 μg/mL cycloheximide (CHX) for the different times. (D) Caki-1 cells were treated with 2 μM ML364 after pretreatment with 0.25 μM MG132 for 24 h. (E) Caki-1 cells were transfected with control or USP2 siRNA and then treated with 20 μg/mL CHX for the different times. (E,F) Caki-1 cells were transfected with vector, USP2 WT, or mutant (C276A) plasmid for 24 h and then treated with 20 μg/mL CHX for the different times. The protein expression was detected using Western blotting (A,C–F). The c-FLIP mRNA level was detected using RT-PCR and qPCR (B). The values in the graph represent the mean ± SD of three independent samples. * p < 0.05 compared to the CHX.

Figure 3

USP2 binds and deubiquitinates survivin. (A) Examination of the interaction between proteins using immunoprecipitation (IP). (B) Caki-1 cells were transfected with HA-Ub plasmid and treated with 2 μM ML364 after pretreatment with 0.25 μM MG132 for 24 h. These assays were detected using Western blotting.

Figure 4

ML364 sensitizes TRAIL-mediated apoptosis in cancer cells. (A,B) Cancer (A,B) and normal (B) cells were treated with 2 μM ML364 and/or 50 ng/mL TRAIL for 24 h. The sub-G1 population (A) and protein expression (A,B) were determined using flow cytometry and Western blotting, respectively. Cell morphology was assessed using a microscope. Scale bar: 50 μm (B). Values in the graphs (A,B) represent the mean ± SD of three independent experiments. * p < 0.01 compared to the control.

Figure 5

ML364-induced survivin downregulation contributes to TRAIL sensitization. (A–C) Caki-1 cells were treated with 2 μM ML364 and/or 50 ng/mL TRAIL for 24 h. DNA fragmentation was analyzed using DAPI staining (A) and kit (B). Measurement of DEVDase (caspase-3) activity using substrate (C). (D) Caki-1 cells were treated with combination of 2 μM ML364 and 50 ng/mL TRAIL after pretreatment with 20 μM zVAD for 24 h. (E) Vector and survivin-overexpressed Caki-1 cells were treated with 2 μM ML364 and/or 50 ng/mL TRAIL for 24 h. The sub-G1 population and protein expression were determined using flow cytometry and Western blotting, respectively (D,E). Values in the graphs (B–E) represent the mean ± SD of three independent experiments. * p < 0.01 compared to the control. # p < 0.01 compared to combinations of ML364 and TRAIL. scale bar: 20 μm. White arrows indicate nucleus condensation.

Figure 6

Knockdown of USP2 sensitizes TRAIL-mediated apoptosis. (A) Cancer cells were treated with 50 ng/mL TRAIL after transfection of control or USP2 siRNA for 24 h. (B) Caki-1 cells were treated with combinations of 2 μM ML364 and 50 ng/mL TRAIL after transfection of vector and USP2 plasmids (WT and C276A) for 24 h. The sub-G1 population and protein expression were determined using flow cytometry and Western blotting, respectively (A,B). Values in the graphs (A,B) represent the mean ± SD of three independent experiments. * p < 0.01 compared TRAIL treatment in control siRNA. # p < 0.01 compared to ML364 and TRAIL treatment in vector.