DR5-Cbl-b/c-Cbl-TRAF2 complex inhibits TRAIL-induced apoptosis by promoting TRAF2-mediated polyubiquitination of caspase-8 in gastric cancer cells

Abstract

Ubiquitination of caspase-8 regulates TNF-related apoptosis-inducing ligand (TRAIL) sensitivity in cancer cells, and the preligand assembly complex plays a role in caspase-8 polyubiquitination. However, whether such a complex exists in gastric cancer cells and its role in TRAIL-triggered apoptosis is unclear. In this study, DR5, casitas B-lineage lymphoma-b (Cbl-b)/c-Cbl, and TRAF2 formed a complex in TRAIL-resistant gastric cancer cells, and Cbl-b and c-Cbl were the critical adaptors linking DR5 and TRAF2. Treatment with TRAIL induced caspase-8 translocation into the DR5-Cbl-b/c-Cbl-TRAF2 complex to interact with TRAF2, which then mediated the K48-linked polyubiquitination of caspase-8. The proteasome inhibitor bortezomib markedly enriched the p43/41 products of caspase-8 activated by TRAIL, indicating proteasomal degradation of caspase-8. Moreover, TRAF2 knockdown prevented the polyubiquitination of caspase-8 and thus increased TRAIL sensitivity. In addition, the inhibition of Cbl-b or c-Cbl expression and overexpression of miR-141 targeting Cbl-b and c-Cbl partially reversed TRAIL resistance by inhibiting the interaction between TRAF2 and caspase-8 and the subsequent polyubiquitination of caspase-8. These results indicate that the DR5-Cbl-b/c-Cbl-TRAF2 complex inhibited TRAIL-induced apoptosis by promoting TRAF2-mediated polyubiquitination of caspase-8 in gastric cancer cells.

Keywords: TRAIL; Cbl-b; TRAF2; apoptosis; c-Cbl; caspase-8.

Figure 1

TRAIL did not induce DISC formation in TRAIL‐resistant gastric cancer cells. (A) BGC823 and MGC803 cells were incubated with 100 ng·mL⁻¹ or 5 μg·mL⁻¹ TRAIL for 24 h. MKN45 and HGC27 cells were incubated with 100 ng·mL⁻¹ TRAIL for 24 h. Cell apoptosis was quantified by flow cytometry. *Incubated with TRAIL vs that without TRAIL, P < 0.05. (B) BGC823, MGC803, MKN45, and HGC27 cells were incubated with 100 ng·mL⁻¹ TRAIL for 24 h. Caspase‐8 activities were assayed by absorption spectroscopy and expressed relative to the control, designated as 100. *Incubated with TRAIL vs that without TRAIL, P < 0.05. (C) BGC823, MGC803, and MKN45 cells were incubated with 100 ng·mL⁻¹ TRAIL for 16 h. The interactions between different proteins were detected by western blot and immunoprecipitation. IgG was used as negative control. (D) BGC823, MGC803, and MKN45 cells were treated with 100 ng·mL⁻¹ TRAIL for 30 min, then lysed, and fractionated by the ultracentrifugation. Locations of lipid rafts (lanes 1–2) were determined using caveolin‐1. The indicated proteins were analyzed by western blot.

Figure 2

TRAIL induced K48‐linked polyubiquitination and degradation of caspase‐8 in TRAIL‐resistant gastric cancer cells. (A) BGC823, MGC803, MKN45, and HGC27 cells were incubated with 100 ng·mL⁻¹ TRAIL for 4 h. The K48‐linked polyubiquitination of caspase‐8 was analyzed by western blot and immunoprecipitation. IgG was used as negative control. (B) BGC823 and MGC803 cells were treated with 100 ng·mL⁻¹ TRAIL for 4 h. Then, the cells were stained with anti‐ubiquitin (linkage‐specific K48) rabbit antibody (Alexa Fluor 568) and anti‐caspase‐8 mouse antibody for 1 h, and then incubated overnight at 4 °C. The next day, Alexa Fluor 488 goat anti‐mouse IgG was added and incubated for 1 h at room temperature in the dark. After DAPI nuclear staining, the cells were analyzed by confocal fluorescence microscopy (magnification ×60). (C) BGC823 and MGC803 cells were preincubated with 50 nm bortezomib for 2 h, followed by 100 ng·mL⁻¹ TRAIL for 4 h. The K48‐linked polyubiquitination of caspase‐8 was detected by western blot and immunoprecipitation. (D) The p43/41 products of caspase‐8 were analyzed by western blot.

Figure 3

TRAF2 knockdown prevented K48‐linked polyubiquitination of caspase‐8 in TRAIL‐resistant gastric cancer cells. (A) BGC823 and MGC803 cells were incubated with 100 ng·mL⁻¹ TRAIL for 4 h. The interaction between TRAF2 and caspase‐8 was detected by western blot and immunoprecipitation. IgG was used as negative control. (B) The interaction between TRAF2 and caspase‐8 in BGC823 cells was also analyzed by Duolink in situ PLA. Red, the interaction between TRAF2 and caspase‐8; blue, labeling nucleus using DAPI (magnification ×60). (C) BGC823 and MGC803 cells were transiently transfected with TRAF2 siRNA for 48 h, followed by 100 ng·mL⁻¹ TRAIL for 4 h. The K48‐linked polyubiquitination of caspase‐8 was detected by western blot and immunoprecipitation. (D) TRAF2 WT and TRAF2 Tu31 MT sequences were shown (upper). BGC823 and MGC803 cells were transiently transfected with TRAF2 WT or TRAF2 Tu31 MT for 48 h, followed by 100 ng·mL⁻¹ TRAIL for 4 h. The K48‐linked polyubiquitination of caspase‐8 was detected by western blot and immunoprecipitation.

Figure 4

The depletion of TRAF2 expression increased TRAIL sensitivity in gastric cancer cells. (A) BGC823 and MGC803 cells were transiently transfected with TRAF2 siRNA for 48 h, followed by 100 ng·mL⁻¹ TRAIL for 24 h. Cell apoptosis was quantified by flow cytometry. *Incubated with TRAIL in TRAF2 siRNA vs that in negative control siRNA (NC siRNA), P < 0.05. (B) The expression of protein was analyzed by western blot.

Figure 5

Cbl‐b and c‐Cbl were the critical adaptors linking DR5 and TRAF2 in gastric cancer cells. BGC823 cells were incubated with 100 ng·mL⁻¹ TRAIL for 4 h. (A) The interaction between DR5 and TRAF2, (B) the interaction between DR5 and Cbl‐b or c‐Cbl, (C) the interaction between TRAF2 and Cbl‐b or c‐Cbl, (D) the interaction between caspase‐8 and Cbl‐b or c‐Cbl were detected by Duolink in situ PLA. Red, the interaction between different proteins; blue, labeling nucleus using DAPI (magnification ×60). (E) BGC823 and MGC803 cells were incubated with 100 ng·mL⁻¹ TRAIL for 4 h. The interaction between Cbl‐b and DR5 or TRAF2 and the interaction between c‐Cbl and DR5 or TRAF2 were detected by western blot and immunoprecipitation.

Figure 6

The depletion of Cbl‐b or c‐Cbl expression prevented the interaction between TRAF2 and caspase‐8, and K48‐linked polyubiquitination of caspase‐8 in gastric cancer cells. BGC823 and MGC803 cells were transiently transfected with Cbl‐b or c‐Cbl siRNA for 48 h, followed by 100 ng·mL⁻¹ TRAIL for 4 h. (A) The interaction between TRAF2 and caspase‐8 in BGC823 cells was detected by Duolink in situ PLA. Red, the interaction between different proteins; blue, labeling nucleus using DAPI (magnification ×60). (B) The K48‐linked polyubiquitination of caspase‐8 was detected by western blot and immunoprecipitation. The expression of protein was analyzed by western blot.

Figure 7

The depletion of Cbl‐b or c‐Cbl expression increased TRAIL sensitivity in gastric cancer cells. BGC823 and MGC803 cells were transiently transfected with Cbl‐b or c‐Cbl siRNA for 48 h, followed by 100 ng·mL⁻¹ TRAIL for 24 h. (A) Cell apoptosis was quantified by flow cytometry. *Incubated with TRAIL in Cbl‐b or c‐Cbl siRNA vs that in negative control siRNA (NC siRNA), P < 0.05. (B) The expression of proteins was analyzed by western blot.

Figure 8

Cbl‐b and c‐Cbl were the targets of miR‐141 in gastric cancer cells. (A) The expression of proteins in TRAIL‐resistant and TRAIL‐sensitive gastric cancer cells was analyzed by western blot. (B) The mRNA abundance of Cbl‐b and c‐Cbl was analyzed by RT‐PCR. (C) Affymetrix miRNA chip showed the differential expression of miRNA in BGC823 and MKN45 cells. (D) The differential expression of miR‐141 was analyzed by qRT‐PCR. *miR‐141 expression in HGC27 or MKN45 cells vs that in BGC823 and MGC803 cells, P < 0.05. (E) Potential binding pattern of miR‐141 to the 3′ UTR of Cbl‐b or c‐Cbl, respectively, and the construct information of mutant 3′ UTR of Cbl‐b or c‐Cbl. (F) Luciferase reporters assay through cotransfecting Cbl‐b or c‐Cbl WT‐Luc, or Cbl‐b or c‐Cbl MT‐Luc with a renilla luciferase control plasmid (pRL‐TK), along with negative control mimic (NC mimic) or miR‐141 mimic. The relative luciferase activities normalized to NC mimic group were shown, *P < 0.05.

Figure 9

Overexpression of miR‐141 prevented K48‐linked polyubiquitination of caspase‐8 and increased TRAIL sensitivity in gastric cancer cells. (A) BGC823 and MGC803 cells were transiently transfected with miR‐141 mimic for 48 h, followed by 100 ng·mL⁻¹ TRAIL for 4 h. The K48‐linked polyubiquitination of caspase‐8 was detected by western blot and immunoprecipitation. The expression of protein was analyzed by western blot. (B) The mRNA abundance of Cbl‐b and c‐Cbl was analyzed by RT‐PCR. (C) The interaction between TRAF2 and caspase‐8 was detected by Duolink in situ PLA. Red, the interaction between different proteins; blue, labeling nucleus using DAPI (magnification ×60). (D) BGC823 and MGC803 cells were transiently transfected with miR‐141 mimic for 48 h, followed by 100 ng·mL⁻¹ TRAIL for 24 h. Cell apoptosis was quantified by flow cytometry. *Incubated with TRAIL in miR‐141 mimic vs that in negative control mimic (NC mimic), P < 0.05. (E) The expression of proteins was analyzed by western blot.

Figure 10

Schematic representation of the proposed model. DR5, Cbl‐b/c‐Cbl, and TRAF2 form a complex in TRAIL‐resistant gastric cancer cells, and Cbl‐b and c‐Cbl are the critical adaptors linking DR5 and TRAF2. TRAIL induces FADD and caspase‐8 translocation into the DR5‐Cbl‐b/c‐Cbl‐TRAF2 complex, and the interaction between caspase‐8 and TRAF2, forming the DISC. TRAF2 then mediates the K48‐linked polyubiquitination and degradation of caspase‐8, which blocks the DISC from inducing apoptosis. miR‐141, by targeting Cbl‐b and c‐Cbl, inhibits TRAF2‐mediated K48‐linked polyubiquitination and degradation of caspase‐8, which leads to the induction of apoptosis and hence increases the sensitivity of TRAIL in gastric cancer cells.