pevonedistat and Liver-Neoplasms

Topics: Apoptosis; beta-Transducin Repeat-Containing Proteins; Cell Line, Tumor; Cycloheximide; Cyclopentanes; Humans; Inflammation; Liver Neoplasms; NF-KappaB Inhibitor alpha; Pyrimidines; RNA, Messenger; Ubiquitin-Activating Enzymes; Ubiquitins; Xenograft Model Antitumor Assays

Hepatitis B virus (HBV) infection is a major public health problem. The high levels of HBV DNA and HBsAg are positively associated with the development of secondary liver diseases, including hepatocellular carcinoma (HCC). Current treatment with nucleos(t)ide analogues mainly reduces viral DNA, but has minimal, if any, inhibitory effect on the viral antigen. Although IFN reduces both HBV DNA and HBsAg, the serious associated side effects limit its use in clinic. Thus, there is an urgent demanding for novel anti-HBV therapy. In our study, viral parameters were determined in the supernatant of HepG2.2.15 cells, HBV-expressing Huh7 and HepG2 cells which transfected with HBV plasmids and in the serum of HBV mouse models with hydrodynamic injection of pAAV-HBV1.2 plasmid. RT-qPCR and Southern blot were performed to detect 35kb mRNA and cccDNA. RT-qPCR, Luciferase assay and Western blot were used to determine anti-HBV effects of MLN4924 and the underlying mechanisms. We found that treatment with MLN4924, the first-in-class neddylation inhibitor currently in several phase II clinical trials for anti-cancer application, effectively suppressed production of HBV DNA, HBsAg, 3.5kb HBV RNA as well as cccDNA. Mechanistically, MLN4924 blocks cullin neddylation and activates ERK to suppress the expression of several transcription factors required for HBV replication, including HNF1α, C/EBPα and HNF4α, leading to an effective blockage in the production of cccDNA and HBV antigen. Our study revealed that neddylation inhibitor MLN4924 has impressive anti-HBV activity by inhibiting HBV replication, thus providing sound rationale for future MLN4924 clinical trial as a novel anti-HBV therapy.

Topics: Animals; Blotting, Southern; Carcinoma, Hepatocellular; Cyclopentanes; Hep G2 Cells; Hepatitis B virus; Hepatocyte Nuclear Factor 4; Humans; Liver Neoplasms; Mice; Pyrimidines; RNA, Messenger; Transcription Factors

Activation of sterol regulatory element-binding protein 1 (SREBP-1), a master lipogenic transcription factor, is associated with cancer metabolism and metabolic disorders. Neddylation, the process of adding NEDD8 to its substrate, contributes to diverse biological processes. Here, we identified SREBP-1 as a substrate for neddylation by UBC12 and explored its impact on tumor aggressiveness. In cell-based assays, SREBP-1 neddylation prolonged SREBP-1 stability with a decrease in ubiquitination. Consequently, NEDD8 overexpression facilitated proliferation, migration, and invasion of SK-Hep1 liver tumor cells. MLN4924 (an inhibitor of the NEDD8-activating enzyme-E1) treatment or UBC12 knockdown prevented SREBP-1 neddylation and tumor cell phenotype change. This effect was corroborated in an in vivo xenograft model. In human specimens, SREBP-1, UBC12, and NEDD8 were all upregulated in hepatocellular carcinoma (HCC) compared to nontumorous regions. Moreover, SREBP-1 levels positively correlated with UBC12. In GEO database analyses, SREBP-1 levels were greater in metastatic HCC samples accompanying UBC12 upregulation. In HCC analysis, tumoral SREBP-1 and UBC12 levels discriminated overall patient survival rates. Additionally, MLN4924 treatment destabilized SREBP-1 in MDA-MB-231 breast cancer cells and in the tumor cell xenograft. SREBP-1 and UBC12 were also highly expressed in human breast cancer tissues. Moreover, most breast cancers with lymph node metastasis displayed predominant SREBP-1 and UBC12 expressions, which compromised overall patient survival rates. In summary, SREBP-1 is neddylated by UBC12, which may contribute to HCC and breast cancer aggressiveness through SREBP-1 stabilization, and these events can be intervented by MLN4924 therapy. Our findings may also provide potential reliable prognostic markers for tumor metastasis.

Topics: Animals; Breast; Breast Neoplasms; Carcinoma, Hepatocellular; Cell Line, Tumor; Cyclopentanes; Female; Humans; Liver; Liver Neoplasms; Lymphatic Metastasis; Mice; NEDD8 Protein; Prognosis; Protein Stability; Pyrimidines; Sterol Regulatory Element Binding Protein 1; Survival Rate; Ubiquitin-Activating Enzymes; Ubiquitin-Conjugating Enzymes; Ubiquitination; Up-Regulation; Xenograft Model Antitumor Assays

Sorafenib remains the standard care for patients with hepatocellular carcinoma (HCC) even though it has low antitumor efficacy. Protein neddylation is abnormally activated in many types of human cancer. However, whether dysregulation of neddylation is involved in HCC progression and whether targeting neddylation sensitizes HCC cells to sorafenib need to be ascertained. In the present study, it was demonstrated that high expression of neddylation components, neural precursor cell expressed, developmentally downregulated 8 (NEDD8) and NEDD8‑activating enzyme 1 (NAE1), were associated with poor survival of patients with HCC. Inhibition of neddylation by MLN4924, a small‑molecule inhibitor of NAE1, significantly inhibited HCC growth, reduced clonogenic survival, increased apoptosis, and decreased migration capacity. Sorafenib alone exhibited minimal anticancer efficacy. However, a combination of sorafenib with MLN4924 at a low concentration significantly enhanced the inhibition of cell proliferation and migration as well as the induction of apoptosis induced by sorafenib. In vivo HCC xenograft mouse models also showed that MLN4924 increased the antitumor efficacy of sorafenib. Mechanistically, MLN4924 enhanced the antitumor activity of sorafenib in HCC cells via upregulation of cullin‑RING E3 ubiquitin ligase (CRL)/Skp1‑Cullin1‑F box (SCF) E3 ubiquitin ligase substrates p21, p27, Deptor and IκBɑ. Taken together, these findings suggest that combination therapy of MLN4924 with sorafenib appears to present an additive effect with a maximal in the treatment of HCC.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Carcinoma, Hepatocellular; Cell Movement; Cell Proliferation; Cyclopentanes; Female; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Male; Mice; Middle Aged; NEDD8 Protein; Neoplasm Proteins; NF-KappaB Inhibitor alpha; Pyrimidines; Sorafenib; Ubiquitin-Activating Enzymes; Ubiquitins; Xenograft Model Antitumor Assays

The current view of cancer progression highlights that cancer cells must undergo through a post-translational regulation and metabolic reprogramming to progress in an unfriendly environment. In here, the importance of neddylation modification in liver cancer was investigated. We found that hepatic neddylation was specifically enriched in liver cancer patients with bad prognosis. In addition, the treatment with the neddylation inhibitor MLN4924 in Phb1-KO mice, an animal model of hepatocellular carcinoma showing elevated neddylation, reverted the malignant phenotype. Tumor cell death in vivo translating into liver tumor regression was associated with augmented phosphatidylcholine synthesis by the PEMT pathway, known as a liver-specific tumor suppressor, and restored mitochondrial function and TCA cycle flux. Otherwise, in protumoral hepatocytes, neddylation inhibition resulted in metabolic reprogramming rendering a decrease in oxidative phosphorylation and concomitant tumor cell apoptosis. Moreover, Akt and LKB1, hallmarks of proliferative metabolism, were altered in liver cancer being new targets of neddylation. Importantly, we show that neddylation-induced metabolic reprogramming and apoptosis were dependent on LKB1 and Akt stabilization. Overall, our results implicate neddylation/signaling/metabolism, partly mediated by LKB1 and Akt, in the development of liver cancer, paving the way for novel therapeutic approaches targeting neddylation in hepatocellular carcinoma.

Topics: AMP-Activated Protein Kinase Kinases; Animals; Blotting, Western; Carcinoma, Hepatocellular; Cyclopentanes; Energy Metabolism; Hep G2 Cells; Hepatocytes; Humans; Liver Neoplasms; Male; Mice, Inbred C57BL; Mice, Knockout; Mice, Nude; NEDD8 Protein; Prohibitins; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; Pyrimidines; Repressor Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Signal Transduction; Transplantation, Heterologous; Ubiquitins

Liver cancer is the second-most frequent cause of cancer death in the world and is highly treatment resistant. We reported previously that inhibition of neddylation pathway with specific NAE inhibitor MLN4924, suppressed the malignant phenotypes of liver cancer. However, during the process, MLN4924 induces pro-survival autophagy as a mechanism of drug resistance. Here, we report that blockage of autophagy with clinically-available autophagy inhibitors (e.g. chloroquine) significantly enhanced the efficacy of MLN4924 on liver cancer cells by triggering apoptosis. Mechanistically, chloroquine enhanced MLN4924-induced up-regulation of pro-apoptotic proteins (e.g. NOXA) and down-regulation of anti-apoptotic proteins. Importantly, the down-regulation of NOXA expression via siRNA silencing substantially attenuated apoptosis of liver cancer cells. Further mechanistic studies revealed that blockage of autophagy augmented MLN4924-induced DNA damage and reactive oxygen species (ROS) generation. The elimination of DNA damage or blockage of ROS production significantly reduced the expression of NOXA, and thereby attenuated apoptosis and reduced growth inhibition of liver cancer cells. Moreover, blockage of autophagy enhanced the efficacy of MLN4924 in an orthotopic model of human liver cancer, with induction of NOXA and apoptosis in tumor tissues. These findings provide important preclinical evidence for clinical investigation of synergistic inhibition of neddylation and autophagy in liver cancer.

Topics: Antineoplastic Agents; Apoptosis Regulatory Proteins; Autophagy; Carcinoma, Hepatocellular; Cell Line, Tumor; Chloroquine; Cyclopentanes; DNA Damage; Drug Synergism; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Macrolides; Molecular Targeted Therapy; NEDD8 Protein; Neoplasm Proteins; Protein Processing, Post-Translational; Proto-Oncogene Proteins c-bcl-2; Pyrimidines; Random Allocation; RNA, Small Interfering; Transfection; Ubiquitins; Xenograft Model Antitumor Assays

Posttranslational neddylation of cullins in the Cullin-Ring E3 ligase (CRL) complexes is needed for proteolytic degradation of CRL substrates, whose accumulation induces cell-cycle arrest, apoptosis, and senescence. The Nedd8-activating enzyme (NAE) is critical for neddylation of CRL complexes and their growth-promoting function. Recently, the anticancer small molecule MLN4924 currently in phase I trials was determined to be an inhibitor of NAE that blocks cullin neddylation and inactivates CRL, triggering an accumulation of CRL substrates that trigger cell-cycle arrest, apoptosis, and senescence in cancer cells. Here, we report that MLN4924 also triggers autophagy in response to CRL inactivation and that this effect is important for the ability of MLN4924 to suppress the outgrowth of liver cancer cells in vitro and in vivo. MLN4924-induced autophagy was attributed partially to inhibition of mTOR activity, due to accumulation of the mTOR inhibitory protein Deptor, as well as to induction of reactive oxygen species stress. Inhibiting autophagy enhanced MLN4924-induced apoptosis, suggesting that autophagy is a survival signal triggered in response to CRL inactivation. In a xenograft model of human liver cancer, MLN4924 was well-tolerated and displayed a significant antitumor effect characterized by CRL inactivation and induction of autophagy and apoptosis in liver cancer cells. Together, our findings support the clinical investigation of MLN4924 for liver cancer treatment and provide a preclinical proof-of-concept for combination therapy with an autophagy inhibitor to enhance therapeutic efficacy.

Topics: Animals; Apoptosis; Autophagy; Base Sequence; Cyclopentanes; Humans; Liver Neoplasms; Mice; Mice, Nude; NEDD8 Protein; Pyrimidines; RNA, Small Interfering; Ubiquitins