The NEDD8-Activating Enzyme Inhibitor, MLN4924, Cooperates with TRAIL to Augment Apoptosis through Facilitating c-FLIP Degradation in Head and Neck Cancer Cells

Abstract

Tumor necrosis factor-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 DR4 and DR5 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 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. Since 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.

The Neddylation Inhibitor Pevonedistat (MLN4924) Suppresses and Radiosensitizes Head and Neck Squamous Carcinoma Cells and Tumors

“…Importantly, both the induction of rereplication and lack of apoptotic response in Cal27 or in FaDu cells treated with low pevonedistat concentrations (20-100 nmol/L) reported here is in contrast to the robust apoptosis observed in a previous study in SqCC/Y1 or in Tr146 HNSCC cells (55% and 65% of cells) treated with 5-to 10-fold higher doses of pevonedistat (0.5 and 1 mmol/L, respectively; ref. 67). This significant apoptotic response was attributed to the induction of c-FLIP degradation, which the authors showed to be independent of NEDD8 inhibition (67).…”

“…67). This significant apoptotic response was attributed to the induction of c-FLIP degradation, which the authors showed to be independent of NEDD8 inhibition (67). Furthermore, it is now abundantly clear that hundereds of cellular proteins are regulated through neddylation, and consistently, pevonedistat inhibits several signal transduction pathways, including the NFkB, AKT, and the mTOR signaling pathways, in addition to cullin-signaling (28,29,(63)(64)(65)(66).…”

“…Pevonedistat inhibits all cullin-based E3 ligases and additionally exhibits cullin-independent activity (28,29,(63)(64)(65)(66)(67). However, the results described above suggest that the radiosensitizing activity of pevonedistat is due to its ability to induce rereplication, which can be enhanced by IR.…”

The covalent modifier Nedd8 is critical for the activation of Smurf1 ubiquitin ligase in tumorigenesis

“…In contrast to the tumourpromoting role of Smurf1, Smurf2 has been suggested to function as a tumour suppressor gene since ablation of Smurf2 in mice resulted in increased susceptibility to various types of cancers in aged mice 61 . We noted that the NAE inhibitor MLN4924 has been recently identified as a novel approach to the treatment of acute myeloid leukaemia, head and neck cancer and liver cancer [62][63][64] . In addition, MLN4924 was demonstrated to inhibit tumour growth in lung cancer xenografts 65 .…”

JNK1/2 expression and modulation of STAT3 signaling in oral cancer

“…Previous studies examined the role of JNK in mediating the apoptotic effect of several pharmacological agents or anticancer drugs in HNSCC cell lines, including N-(4-hydroxyphenyl) retinamide (4HPR), pepsin-digested bovine lactoferrin, 5-aminolevulinic acid, MLN4924, 6-(N, N -dimethylamino)-2-(naphthalene-1-yl) -4-quinazolinone, fomitoside-K, mevastatin and AZD8055 (an mTOR inhibitor), and their biological mechanisms of apoptosis (48)(49)(50)(51)(52)(53)(54)(55). Consistent with the present results, an antitumor role of JNK has been proposed upon treatment of HNSCC cells with the JNK inhibitor SP600125, which diminished the aforementioned pharmacological agents-induced apoptosis (47)(48)(49)(50)(51)(52)(53)(54). Similarly, Li and Johnson (56) demonstrated that pharmacological inhibition of JNK with SP600125 markedly inhibited bortezomib-induction of autophagy regulatory proteins and autophagosome formation in HNSCC.…”