The NEDD8-activating enzyme inhibitor, MLN4924, cooperates with TRAIL to augment apoptosis through facilitating c-FLIP degradation in head and neck cancer cells - PubMed (original) (raw)

The NEDD8-activating enzyme inhibitor, MLN4924, cooperates with TRAIL to augment apoptosis through facilitating c-FLIP degradation in head and neck cancer cells

Liqun Zhao et al. Mol Cancer Ther. 2011 Dec.

Abstract

TNF-related apoptosis-inducing ligand (TRAIL) is a tumor-selective cytokine with potential anticancer activity and is currently under clinical testing. Head and neck squamous cell carcinoma (HNSCC), like other cancer types, exhibits varied sensitivity to TRAIL. MLN4924 is a newly developed investigational small molecule inhibitor of NEDD8-activating enzyme with potent anticancer activity. This study reveals a novel function of MLN4924 in synergizing with TRAIL to induce apoptosis in HNSCC cells. MLN4924 alone effectively inhibited the growth of HNSCC cells and induced apoptosis. When combined with TRAIL, synergistic effects on decreasing the survival and inducing apoptosis of HNSCC cells occurred. MLN4924 decreased c-FLIP levels without modulating death receptor 4 and death receptor 5 expression. Enforced expression of c-FLIP substantially attenuated MLN4924/TRAIL-induced apoptosis. Thus c-FLIP reduction plays an important role in mediating MLN4924/TRAIL-induced apoptosis. Moreover, MLN4924 decreased c-FLIP stability, increased c-FLIP ubiquitination, and facilitated c-FLIP degradation, suggesting that MLN4924 decreases c-FLIP levels through promoting its degradation. MLN4924 activated c-jun-NH(2)-kinase (JNK) signaling, evidenced by increased levels of phospho-c-Jun in MLN4924-treated cells. Chemical inhibition of JNK activation not only prevented MLN4924-induced c-FLIP reduction, but also inhibited MLN4924/TRAIL-induced apoptosis, suggesting that JNK activation mediates c-FLIP downregulation and subsequent enhancement of TRAIL-induced apoptosis by MLN4924. Because knockdown of NEDD8 failed to activate JNK signaling and downregulate c-FLIP, it is likely that MLN4924 reduces c-FLIP levels and enhances TRAIL-induced apoptosis independent of NEDD8 inhibition.

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Figures

Fig. 1

Fig. 1. MLN4924 inhibits the growth (A) and induces apoptosis (B and C) of HNSCC cells

A, The given HNSCC cell lines were seeded on 96-well plates and treated the next day with varied concentrations of MLN4924 as indicated. After 48 h, the cells were subjected to estimation of cell number using the SRB assay. Points, means of four replicate determinations; Bars, ± SDs. B and C, The given cell lines were treated with the indicated concentrations of MLN4924 for 48 h and then harvested for detection of caspase-3 and PARP cleavage (B) and apoptosis (C) with Western blotting and annexin V staining, respectively. Columns, means of duplicate determinations; Bars, ± SDs.

Fig. 2

Fig. 2. MLN4924 and TRAIL combination synergistically decreases cell survival (A), enhances cleavage of caspases (B), and induces apoptosis (C)

A, The given cell lines were plated on 96-well plates and treated the next day with the given doses of TRAIL alone, MLN4924 alone or their combinations. After 24 h, cell numbers were estimated using the SRB assay. Columns, means of four replicated determinations; Bars, ± SDs. B and C, The given cell lines were treated with 40 ng/ml TRAIL alone, 1 µM MLN4924 alone, and their combination. After 24 h, the cells were harvested for preparation of whole-cell protein lysates and subsequent Western blot analysis (B) and for annexin V staining of apoptosis (C). Columns, means of duplicate determinations; Bars, ± SDs. CF, cleaved form.

Fig. 3

Fig. 3. MLN4924 reduces c-FLIP levels without affecting DR4 or DR5 expression

The indicated cell lines were treated with different concentrations of MLN4924 as indicated for 8 h (A) or with 1 µM MLN4924 for the given times (B). The cells were then harvested for preparation of whole-cell protein lysates and subsequent Western blot analysis.

Fig. 4

Fig. 4. Enforced expression of ectopic c-FLIP (A) attenuates the ability of the MLN4924 and TRAIL combination to decrease cell survival (B) and to induce cleavage of caspases (C) and apoptosis (D)

A, Detection of ectopic c-FLIP expression with Western blotting in the given cell lines. B, The indicated transfectants were seeded in 96-well plates and treated the next day with 1 µM MLN4924 (MLN) alone, 25 ng/ml TRAIL alone and their combination. After 24 h, the cells were subjected to estimation of cell number using the SRB assay. Columns, means of four replicate determinations; Bars, ± SDs. C and D, The indicated transfectants were treated with 1 µM MLN4924 alone, 25 ng/ml TRAIL alone and their combinations. After 10 h, the cells were harvested for preparation of whole-cell protein lysates and subsequent Western blot analysis (C) or for detection of apoptosis with Annexin V staining (D). CF, cleaved form; D, DMSO, M, MLN4924; T, TRAIL.

Fig. 5

Fig. 5. MLN4924 decreases c-FLIP levels through ubiquitin/proteasome-mediated protein degradation

A, The proteasome inhibitor MG132 inhibits c-FLIP reduction by MLN4924. SqCC/Y1 cells were pretreated with 20 µM MG132 for 30 minutes prior to the addition of 1 µM MLN4924. After co-treatment for 4 h, the cells were harvested for preparation of whole-cell protein lysates and subsequent Western blot analysis. B, MLN4924 reduces c-FLIP stability. SqCC/Y1 cells were treated with DMSO or 1 µM MLN4924 for 7 h. The cells were then washed with PBS 3 times and refed with fresh medium containing 10 µg/ml CHX. At the indicated times, the cells were harvested for preparation of whole-cell protein lysates and subsequent Western blot analysis. Protein levels were quantitated with NIH Image J software (Bethesda, MA) and were normalized to actin. The results were plotted as the relative c-FLIP levels compared to those at the time 0 of CHX treatment (bottom panel). C, MLN4924 increases c-FLIP ubiquitination. Tr146-FLIPL cells were transfected with HA-ubiquitin plasmid using FuGENE 6 transfection reagent for 24 h. The cells were then pretreated with 20 µM MG132 for 30 minutes and then co-treated with 1 µM MLN4924 for 4 h. Whole-cell protein lysates were then prepared for IP using anti-Flag antibody followed by Western blotting (WB) using anti-HA antibody for detection of ubiquitinated FLIPL (Ub-FLIPL) and anti-Flag antibody for detection of ectopic FLIPL.

Fig. 6

Fig. 6. MLN4924 activates JNK (A and B) that mediates MLN4924-induced c-FLIP downregulation (C) independent of Itch (D) and augmentation of TRAIL-induced apoptosis (E and F)

A and B, The given cell lines were treated with different concentrations of MLN4924 as indicated for 12 h (A) or 1 µM MLN4924 for the indicated times (B). C, The indicated cell lines were pretreated with 20 µM SP600125 for 30 min and then co-treated with 1 µM MLN4924 for another 10 h. D, SqCC/Y1 cells were transfected with control (Ctrl) or Itch siRNA for 48 h and then treated with 1 µM MLN4924 for 10 h. After the aforementioned treatment, the cells were then subjected to preparation of whole-cell protein lysates and subsequent Western blot analysis for detection of the indicated proteins. E and F, SqCC/Y1 cells were pretreated with 20 µM SP600125 for 30 min and then co-treated with 1 µM MLN4924, 25 ng/ml TRAIL and MLN4924 plus TRAIL (F) or with the combination of MLN4924 and TRAIL (E). After 24 h (F) or 16 h (E), the cells were harvested for detection of apoptosis with Annexin V staining (F) or for preparation of whole-cell protein lysates and subsequent Western blot analysis (E). CF, cleaved form.

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