ABSTRACT
Background: Neddylation (NAE) inhibition, affecting posttranslational protein function and turnover, is a promising therapeutic approach to cancer. We report the cytotoxic vulnerability to NAE inhibitors in a subset of glioblastoma (GBM) preclinical models and identify genetic alterations and biological processes underlying differential response. Methods: GBM DNA sequencing and transcriptomic data were queried for genes associated with response to NAE inhibition; candidates were validated by molecular techniques. Multi-omics and functional assays revealed processes implicated in NAE inhibition response. Results: Transcriptomics and shotgun proteomics depict PTEN signaling, DNA replication, and DNA repair pathways as significant differentiators between sensitive and resistant models. Vulnerability to MLN4924, a NAE inhibitor, is associated with elevated S-phase populations, DNA re-replication, and DNA damage. In a panel of GBM models, loss of WT PTEN is associated with resistance to different NAE inhibitors. A NAE inhibition response gene set could segregate the GBM cell lines that are most resistant to MLN4924. Conclusions: Loss of WT PTEN is associated with non-sensitivity to 3 different compounds that inhibit NAE in GBM. A NAE inhibition response gene set largely consisting of DNA replication genes could segregate GBM cell lines most resistant to NAEi and may be the basis for future development of NAE inhibition signatures of vulnerability and clinical trial enrollment within a precision medicine paradigm.
ABSTRACT
DCN1, a critical co-E3 ligase in the neddylation process, mediates the activation of Cullin-RING Ligases (CRLs) by selectively catalyzing cullin neddylation, further regulating the activity of substrate proteins. It has been identified as an important target for human diseases, including cancers, fibrotic diseases, and cardiovascular disorders. This work aims to provide a perspective for the discovery of novel DCN1 inhibitors by the analysis of biological roles, protein structures, structure-activity relationships and design strategy disclosed in recent years. Additionally, we will discuss the current status, challenges and opportunities in hope of offering insights into the development of DCN1 inhibitors for human diseases.
ABSTRACT
BACKGROUND: Gliomas are the most common primary malignant tumours of the central nervous system, and neddylation may be a potential target for the treatment of gliomas. Our study analysed neddylation's potential role in gliomas of different pathological types and its correlation with immunotherapy. METHODS: Genes required for model construction were sourced from existing literature, and their expression data were extracted from the TCGA and CGGA databases. LASSO regression was employed to identify genes associated with the prognosis of glioma patients in TCGA and to establish a clinical prognostic model. Biological changes in glioma cell lines following intervention with hub genes were evaluated using the CCK-8 assay and transwell assay. The genes implicated in the model construction were validated across various cell lines using Western blot. We conducted analyses to examine correlations between model scores and clinical data, tumor microenvironments, and immune checkpoints. Furthermore, we investigated potential differences in molecular functions and mechanisms among different groups. RESULTS: We identified 249 genes from the Reactome database and analysed their expression profiles in the TCGA and CGGA databases. After using LASSO-Cox, four genes (BRCA1, BIRC5, FBXL16 and KLHL25, p < 0.05) with significant correlations were identified. We selected FBXL16 for validation in in vitro experiments. Following FBXL16 overexpression, the proliferation, migration, and invasion abilities of glioma cell lines all showed a decrease. Then, we constructed the NEDD Index for gliomas. The nomogram indicated that this model could serve as an independent prognostic marker. Analysis of the tumour microenvironment and immune checkpoints revealed that the NEDD index was also correlated with immune cell infiltration and the expression levels of various immune checkpoints. CONCLUSION: The NEDD index can serve as a practical tool for predicting the prognosis of glioma patients, and it is correlated with immune cell infiltration and the expression levels of immune checkpoints.
ABSTRACT
Cullin-based RING ligases (CRLs) comprise the largest family of ubiquitin E3 ligases. CRL activity is tightly regulated by cullin neddylation, which has been associated with various diseases. Although inhibitors of CRLs neddylation have been reported, there is a lack of small molecules that can selectively target individual cullins. Here, we identified a natural product, liquidambaric acid (LDA), with relatively selective inhibition properties against cullin (Cul) 2 neddylation, and found that its target, Tumor Necrosis Factor receptor-associated factor 2 (TRAF2) was required for the activity. TRAF2 associates with the Cul2 neddylation complex and regulates the machinery assembly, especially that of E2 (UBC12) and E3 (RBX1) enzymes. In addition, we demonstrated that by intervention of the associations between TRAF2 and the neddylation machinery, LDA disturbed NEDD8 transfer from E1 to E2, therefore blocking Cul2 neddylation. Taken together, we show that TRAF2 plays a positive role in neddylation cascades, and we have identified a small molecule capable of selective modulation of cullin neddylation.
ABSTRACT
Repair of DNA double-strand breaks by the non-homologous end-joining pathway is initiated by the binding of Ku to DNA ends. Multiple Ku proteins load onto linear DNAs in vitro. However, in cells, Ku loading is limited to â¼1-2 molecules per DNA end. The mechanisms enforcing this limit are currently unclear. Here, we show that the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs), but not its protein kinase activity, is required to prevent excessive Ku entry into chromatin. Ku accumulation is further restricted by two mechanisms: a neddylation/FBXL12-dependent process that actively removes loaded Ku molecules throughout the cell cycle and a CtIP/ATM-dependent mechanism that operates in S phase. Finally, we demonstrate that the misregulation of Ku loading leads to impaired transcription in the vicinity of DNA ends. Together, our data shed light on the multiple mechanisms operating to prevent Ku from invading chromatin and interfering with other DNA transactions.
ABSTRACT
The Hippo-YAP signaling pathway plays a central role in many biological processes such as regulating cell fate, organ size, and tissue growth, and its key components are spatiotemporally expressed and posttranslationally modified during these processes. Neddylation is a posttranslational modification that involves the covalent attachment of NEDD8 to target proteins by NEDD8-specific E1-E2-E3 enzymes. Whether neddylation is involved in Hippo-YAP signaling remains poorly understood. Here, we provide evidence supporting the critical role of NEDD8 in facilitating the Hippo-YAP signaling pathway by mediating neddylation of the transcriptional coactivator yes-associated protein 1 (YAP1). Overexpression of NEDD8 induces YAP1 neddylation and enhances YAP1 transactivity, but inhibition of neddylation suppresses YAP1 transactivity and attenuates YAP1 nuclear accumulation. Furthermore, inhibition of YAP1 signaling promotes MLN4924-induced ovarian granulosa cells apoptosis and disruption of nedd8 in zebrafish results in downregulation of yap1-activated genes and upregulation of yap1-repressed genes. Further assays show that the xiap ligase promotes nedd8 conjugates to yap1 and that yap1 neddylation. In addition, we identify lysine 159 as a major neddylation site on YAP1. These findings reveal a novel mechanism for neddylation in the regulation of Hippo-YAP signaling.
ABSTRACT
(1) Background: The neddylation pathway assumes a pivotal role in the initiation and progression of cancer. MLN4924, a potent small-molecule inhibitor of the NEDD8-activating enzyme (NAE), effectively intervenes in the early stages of the neddylation pathway. By instigating diverse cellular responses, such as senescence and apoptosis in cancer cells, MLN4924 also exerts regulatory effects on non-malignant cells within the tumor microenvironment (TME) and tumor virus-infected cells, thereby impeding the onset of tumors. Consequently, MLN4924 has been widely acknowledged as a potent anti-cancer drug. (2) Recent findings: Nevertheless, recent findings have illuminated additional facets of the neddylation pathway, revealing its active involvement in various biological processes detrimental to the survival of cancer cells. This newfound understanding underscores the dual role of MLN4924 in tumor therapy, characterized by both anti-cancer and pro-cancer effects. This dichotomy is herein referred to as the "double-edged effects" of MLN4924. This paper delves into the intricate relationship between the neddylation pathway and cancer, offering a mechanistic exploration and analysis of the causes underlying the double-edged effects of MLN4924-specifically, the accumulation of pro-cancer neddylation substrates. (3) Perspectives: Here, the objective is to furnish theoretical support and novel insights that can guide the development of next-generation anti-cancer drugs targeting the neddylation pathway.
Subject(s)
Antineoplastic Agents , Cyclopentanes , NEDD8 Protein , Neoplasms , Pyrimidines , Cyclopentanes/pharmacology , Cyclopentanes/therapeutic use , Humans , Pyrimidines/pharmacology , Antineoplastic Agents/pharmacology , Neoplasms/drug therapy , Neoplasms/metabolism , Neoplasms/pathology , NEDD8 Protein/metabolism , Animals , Signal Transduction/drug effects , Apoptosis/drug effects , Tumor Microenvironment/drug effects , Ubiquitin-Activating Enzymes/metabolism , Ubiquitin-Activating Enzymes/antagonists & inhibitorsABSTRACT
Plasmodium parasites, the causative agents of malaria, rely on sophisticated cellular mechanisms to survive and proliferate within their hosts. Plasmodium complex life cycle requires posttranslational modifications (PTMs) to control cellular activities. Neddylation is a type of PTM in which NEDD8 is covalently attached to target proteins and plays an important role in cell cycle control and metabolism. Covalent attachment to its substrates requires the Nedd8-activating enzyme, E1; the NEDD8-conjugating enzyme, E2; and the ligase, E3. In Plasmodium, protein neddylation is essential for parasite development during the stage I-II transition from zygote to ookinete differentiation and malaria transmission. Here, we discuss the current understanding of protein neddylation in Plasmodium, which is involved in malaria transmission.
ABSTRACT
Aging is the biggest risk factor for the development of Alzheimer's disease (AD). Here, we performed a whole-genome CRISPR screen to identify regulators of neuronal age and show that the neddylation pathway regulates both cellular age and AD neurodegeneration in a human stem cell model. Specifically, we demonstrate that blocking neddylation increased cellular hallmarks of aging and led to an increase in Tau aggregation and phosphorylation in neurons carrying the APPswe/swe mutation. Aged APPswe/swe but not isogenic control neurons also showed a progressive decrease in viability. Selective neuronal loss upon neddylation inhibition was similarly observed in other isogenic AD and in Parkinson's disease (PD) models, including PSENM146V/M146V cortical and LRRK2G2019S/G2019S midbrain dopamine neurons, respectively. This study indicates that cellular aging can reveal late-onset disease phenotypes, identifies new potential targets to modulate AD progression, and describes a strategy to program age-associated phenotypes into stem cell models of disease.
Subject(s)
Alzheimer Disease , Humans , Alzheimer Disease/pathology , Alzheimer Disease/genetics , Alzheimer Disease/metabolism , Cellular Senescence/genetics , Neurons/metabolism , Neurons/pathology , NEDD8 Protein/metabolism , NEDD8 Protein/genetics , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , tau Proteins/metabolism , tau Proteins/genetics , Parkinson Disease/genetics , Parkinson Disease/pathology , Parkinson Disease/metabolism , Aging/genetics , Aging/pathology , Aging/metabolism , Dopaminergic Neurons/metabolism , Dopaminergic Neurons/pathology , CRISPR-Cas Systems/geneticsABSTRACT
Post-translational modification and fine-tuned protein turnover are of great importance in mammalian early embryo development. Apart from the classic protein degradation promoting ubiquitination, new forms of ubiquitination-like modification are yet to be fully understood. Here, we demonstrate the function and potential mechanisms of one ubiquitination-like modification, neddylation, in mouse preimplantation embryo development. Treated with specific inhibitors, zygotes showed a dramatically decreased cleavage rate and almost all failed to enter the 4-cell stage. Transcriptional profiling showed genes were differentially expressed in pathways involving cell fate determination and cell differentiation, including several down-regulated zygotic genome activation (ZGA) marker genes. A decreased level of phosphorylated RNA polymerase II was detected, indicating impaired gene transcription inside the embryo cell nucleus. Proteomic data showed that differentially expressed proteins were enriched in histone modifications. We confirmed the lowered in methyltransferase (KMT2D) expression and a decrease in histone H3K4me3. At the same time, acetyltransferase (CBP/p300) reduced, while deacetylase (HDAC6) increased, resulting in an attenuation in histone H3K27ac. Additionally, we observed the up-regulation in YAP1 and RPL13 activities, indicating potential abnormalities in the downstream response of Hippo signaling pathway. In summary, we found that inhibition of neddylation induced epigenetic changes in early embryos and led to abnormalities in related downstream signaling pathways. This study sheds light upon new forms of ubiquitination regulating mammalian embryonic development and may contribute to further investigation of female infertility pathology.
Subject(s)
Embryonic Development , Gene Expression Regulation, Developmental , Histones , Zygote , Animals , Mice , Zygote/metabolism , Embryonic Development/genetics , Embryonic Development/drug effects , Histones/metabolism , Female , NEDD8 Protein/metabolism , NEDD8 Protein/genetics , Protein Processing, Post-Translational , Histone Code , Embryo, Mammalian/metabolism , Ubiquitination , Cyclopentanes , PyrimidinesABSTRACT
Cancer affects millions of people and understanding the molecular mechanisms related to disease development and progression is essential to manage the disease. Post-translational modification (PTM) processes such as ubiquitination and neddylation have a significant role in cancer development and progression by regulating protein stability, function, and interaction with other biomolecules. Both ubiquitination and neddylation are analogous processes that involves a series of enzymatic steps leading to the covalent attachment of ubiquitin or NEDD8 to target proteins. Neddylation modifies the CRL family of E3 ligase and regulates target proteins' function and stability. The DCUN1D1 protein is a regulator of protein neddylation and ubiquitination and acts promoting the neddylation of the cullin family components of E3-CRL complexes and is known to be upregulated in several types of cancers. In this review we compare the PTM ubiquitination and neddylation. Our discussion is focused on the neddylation process and the role of DCUN1D1 protein in cancer development. Furthermore, we provide describe DCUN1D1 protein and discuss its role in pathogenesis and signalling pathway in six different types of cancer. Additionally, we explore both the neddylation and DCUN1D1 pathways as potential druggable targets for therapeutic interventions. We focus our analysis on the development of compounds that target specifically neddylation or DCUN1D1. Finally, we provide a critical analysis about the challenges and perspectives in the field of DCUN1D1 and neddylation in cancer research. KEY POINTS: Neddylation is a post-translational modification that regulates target proteins' function and stability. One regulator of the neddylation process is a protein named DCUN1D1 and it is known to have its expression deregulated in several types of cancers. Here, we provide a detailed description of DCUN1D1 structure and its consequence for the development of cancer. We discuss both the neddylation and DCUN1D1 pathways as potential druggable targets for therapeutic interventions and provide a critical analysis about the challenges and perspectives in the field of DCUN1D1 and neddylation in cancer research.
Subject(s)
NEDD8 Protein , Neoplasms , Protein Processing, Post-Translational , Ubiquitination , Humans , Neoplasms/metabolism , Neoplasms/drug therapy , Neoplasms/pathology , NEDD8 Protein/metabolism , NEDD8 Protein/genetics , Signal Transduction/drug effects , Animals , Ubiquitin-Protein Ligases/metabolism , Ubiquitin-Protein Ligases/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Intracellular Signaling Peptides and Proteins/geneticsABSTRACT
BACKGROUND: Paclitaxel (PTX) treatment resistance is an important factor leading to poor prognosis in triple-negative breast cancer (TNBC), therefore there is an urgent need to identify new target for combination therapy. Neddylation is a post-translational process that introduces a ubiquitin-like protein called neural precursor cell expressed developmentally downregulated protein 8 (NEDD8). Previous studies have found that neddylation is activated in multiple tumors, but its relationship with PTX chemotherapy sensitivity has not been reported. METHODS: Differences in UBC12 and NEDD8 expression levels between PTX-sensitive and PTX-insensitive TNBC tissues were validated using public databases and immunohistochemistry. The in vitro and in vivo functional experiments were used to observe the effect of neddylation inhibition combined with PTX therapy on tumor progression. Co-IP, western blot and PCR assays were used to investigate the molecular mechanisms. Molecular docking was used to simulate the protein binding of UBC12 and TRIM25. Molecular dynamics simulation was used to observe the changes in TRIM25 protein conformation. RESULTS: We found that in TNBC that is insensitive to PTX, NEDD8 and NEDD8 conjugating enzyme UBC12 are highly expressed. Treatment with the NEDD8-activating enzyme (NAE) inhibitor mln4924 or knockdown of UBC12 significantly increased the sensitivity of the tumor to PTX, and this increase in sensitivity is related to UBC12-mediated autophagy activation. Mechanistically, UBC12 can transfer NEDD8 to E3 ubiquitin ligase tripartite motif containing 25 (TRIM25) at K117. Molecular dynamics simulations indicate that the neddylation modification of TRIM25 reduces steric hindrance in its RING domain, facilitating the binding of TRIM25 and ubiquitylated substrates. Subsequently, TRIM25 promotes the nuclear translocation of transcription factor EB (TFEB) and transcription of autophagy related genes by increasing K63-polyubiquitination of TFEB, thereby reducing tumor sensitivity to PTX. CONCLUSIONS: Neddylation is activated in PTX-insensitive TNBC. Specifically, autophagy gene transcriptional activation mediated by the UBC12/TRIM25/TFEB axis reduces TNBC sensitivity to PTX. Neddylation suppression combination with PTX treatment shows a synergistic anti-tumor effect.
Subject(s)
Autophagy , NEDD8 Protein , Paclitaxel , Tripartite Motif Proteins , Triple Negative Breast Neoplasms , Ubiquitin-Protein Ligases , Humans , Triple Negative Breast Neoplasms/drug therapy , Triple Negative Breast Neoplasms/metabolism , Triple Negative Breast Neoplasms/pathology , Paclitaxel/pharmacology , Paclitaxel/therapeutic use , Female , Mice , Animals , Autophagy/drug effects , NEDD8 Protein/metabolism , Ubiquitin-Protein Ligases/metabolism , Ubiquitin-Protein Ligases/genetics , Tripartite Motif Proteins/metabolism , Tripartite Motif Proteins/genetics , Cell Line, Tumor , Transcription Factors/metabolism , Transcription Factors/genetics , Cyclopentanes/pharmacology , Drug Resistance, Neoplasm , Xenograft Model Antitumor Assays , Ubiquitin-Conjugating Enzymes/metabolism , Ubiquitin-Conjugating Enzymes/geneticsABSTRACT
BACKGROUND: Neddylation, a post-translational modification process, plays a crucial role in various human neoplasms. However, its connection with kidney renal clear cell carcinoma (KIRC) remains under-researched. METHODS: We validated the Gene Set Cancer Analysis Lite (GSCALite) platform against The Cancer Genome Atlas (TCGA) database, analyzing 33 cancer types and their link with 17 neddylation-related genes. This included examining copy number variations (CNVs), single nucleotide variations (SNVs), mRNA expression, cellular pathway involvement, and methylation. Using Gene Set Variation Analysis (GSVA), we categorized these genes into three clusters and examined their impact on KIRC patient prognosis, drug responses, immune infiltration, and oncogenic pathways. Afterward, our objective is to identify genes that exhibit overexpression in KIRC and are associated with an adverse prognosis. After pinpointing the specific target gene, we used the specific inhibitor MLN4924 to inhibit the neddylation pathway to conduct RNA sequencing and related in vitro experiments to verify and study the specificity and potential mechanisms related to the target. This approach is geared towards enhancing our understanding of the prognostic importance of neddylation modification in KIRC. RESULTS: We identified significant CNV, SNV, and methylation events in neddylation-related genes across various cancers, with notably higher expression levels observed in KIRC. Cluster analysis revealed a potential trade-off in the interactions among neddylation-related genes, where both high and low levels of gene expression are linked to adverse prognoses. This association is particularly pronounced concerning lymph node involvement, T stage classification, and Fustat score. Simultaneously, our research discovered that PSMB10 exhibits overexpression in KIRC when compared to normal tissues, negatively impacting patient prognosis. Through RNA sequencing and in vitro assays, we confirmed that the inhibition of neddylation modification could play a role in the regulation of various signaling pathways, thereby influencing the prognosis of KIRC. Moreover, our results underscore PSMB10 as a viable target for therapeutic intervention in KIRC, opening up novel pathways for the development of targeted treatment strategies. CONCLUSION: This study underscores the regulatory function and potential mechanism of neddylation modification on the phenotype of KIRC, identifying PSMB10 as a key regulatory target with a significant role in influencing the prognosis of KIRC.
ABSTRACT
Cullin RING E3 ligases (CRL) have emerged as key regulators of disease-modifying pathways and therapeutic targets. Cullin3 (Cul3)-containing CRL (CRL3) has been implicated in regulating hepatic insulin and oxidative stress signaling. However, CRL3 function in liver pathophysiology is poorly defined. Here, we report that hepatocyte Cul3 knockout results in rapid resolution of steatosis in obese mice. However, the remarkable resistance of hepatocyte Cul3 knockout mice to developing steatosis does not lead to overall metabolic improvement but causes systemic metabolic disturbances. Liver transcriptomics analysis identifies that CRL3 inactivation causes persistent activation of the nuclear factor erythroid 2-related factor 2 (NRF2) antioxidant defense pathway, which also reprograms the lipid transcriptional network to prevent TG storage. Furthermore, global metabolomics reveals that NRF2 activation induces numerous NAD+-consuming aldehyde dehydrogenases to increase the cellular NADH/NAD+ ratio, a redox imbalance termed NADH reductive stress that inhibits the glycolysis-citrate-lipogenesis axis in Cul3 knockout livers. As a result, this NRF2-induced cellular lipid storage defect promotes hepatic ceramide accumulation, elevates circulating fatty acids, and worsens systemic insulin resistance in a vicious cycle. Hepatic lipid accumulation is restored, and liver injury and hyperglycemia are attenuated when NRF2 activation and NADH reductive stress are abolished in hepatocyte Cul3/Nrf2 double-knockout mice. The resistance to hepatic steatosis, hyperglycemia, and NADH reductive stress are observed in hepatocyte Keap1 knockout mice with NRF2 activation. In summary, our study defines a critical role of CRL3 in hepatic metabolic regulation and demonstrates that the CRL3 downstream NRF2 overactivation causes hepatic metabolic maladaptation to obesity and insulin resistance.
Subject(s)
Fatty Liver , Hyperglycemia , Insulin Resistance , Animals , Mice , Ubiquitin-Protein Ligases/metabolism , Kelch-Like ECH-Associated Protein 1/metabolism , NAD/metabolism , Cullin Proteins/metabolism , NF-E2-Related Factor 2/metabolism , Oxidative Stress , Mice, Knockout , LipidsABSTRACT
Doxorubicin (DOX) is a widely utilized chemotherapeutic agent in clinical oncology for treating various cancers. However, its clinical use is constrained by its significant side effects. Among these, the development of cardiomyopathy, characterized by cardiac remodeling and eventual heart failure, stands as a major concern following DOX chemotherapy. In our current investigation, we have showcased the efficacy of MLN4924 in mitigating doxorubicin-induced cardiotoxicity through direct inhibition of the NEDD8-activating enzyme, NAE. MLN4924 demonstrated the ability to stabilize mitochondrial function post-doxorubicin treatment, diminish cardiomyocyte apoptosis, alleviate oxidative stress-induced damage in the myocardium, enhance cardiac contractile function, mitigate cardiac fibrosis, and impede cardiac remodeling associated with heart failure. At the mechanistic level, MLN4924 intervened in the neddylation process by inhibiting the NEDD8 activating enzyme, NAE, within the murine cardiac tissue subsequent to doxorubicin treatment. This intervention resulted in the suppression of NEDD8 protein expression, reduction in neddylation activity, and consequential manifestation of cardioprotective effects. Collectively, our findings posit MLN4924 as a potential therapeutic avenue for mitigating doxorubicin-induced cardiotoxicity by attenuating heightened neddylation activity through NAE inhibition, thereby offering a viable and promising treatment modality for afflicted patients.
Subject(s)
Cardiotoxicity , Cyclopentanes , Doxorubicin , Myocytes, Cardiac , NEDD8 Protein , Pyrimidines , Animals , Mice , Apoptosis/drug effects , Cardiotoxicity/drug therapy , Cardiotoxicity/pathology , Cardiotoxicity/prevention & control , Cardiotoxicity/etiology , Cardiotoxicity/metabolism , Cyclopentanes/pharmacology , Cyclopentanes/therapeutic use , Doxorubicin/adverse effects , Mice, Inbred C57BL , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/pathology , NEDD8 Protein/metabolism , NEDD8 Protein/antagonists & inhibitors , Oxidative Stress/drug effects , Pyrimidines/pharmacology , Ubiquitin-Activating Enzymes/antagonists & inhibitors , Ubiquitin-Activating Enzymes/metabolism , Ubiquitin-Activating Enzymes/geneticsABSTRACT
UBE2F, a neddylation E2, neddylates CUL5 to activate cullin-RING ligase-5, upon coupling with neddylation E3 RBX2/SAG. Whether and how UBE2F controls pancreatic tumorigenesis is previously unknown. Here, we showed that UBE2F is essential for the growth of human pancreatic cancer cells with KRAS mutation. In the mouse KrasG12D pancreatic ductal adenocarcinoma (PDAC) model, Ube2f deletion suppresses cerulein-induced pancreatitis, and progression of acinar-to-ductal metaplasia (ADM) and pancreatic intraepithelial neoplasia. Mechanistically, Ube2f deletion inactivates the Mapk-c-Myc signals via blocking ubiquitylation of Diras2, a substrate of CRL5Asb11 E3 ligase. Biologically, DIRAS2 suppresses growth and survival of human pancreatic cancer cells harboring mutant KRAS, and Diras2 deletion largely rescues the phenotypes induced by Ube2f deletion. Collectively, Ube2f or Diras2 plays a tumor-promoting or tumor-suppressive role in the mouse KrasG12D PDAC model, respectively. The UBE2F-CRL5ASB11 axis could serve as a valid target for pancreatic cancer, whereas the levels of UBE2F or DIRAS2 may serve as prognostic biomarkers for PDAC patients.
Subject(s)
Carcinoma, Pancreatic Ductal , Pancreatic Neoplasms , Ubiquitin-Conjugating Enzymes , Animals , Humans , Mice , Carcinoma, Pancreatic Ductal/genetics , Carcinoma, Pancreatic Ductal/pathology , Carcinoma, Pancreatic Ductal/metabolism , Cell Line, Tumor , Cell Proliferation , Genes, Tumor Suppressor , Oncogenes/genetics , Pancreatic Neoplasms/pathology , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/metabolism , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/metabolism , Signal Transduction , Ubiquitin-Conjugating Enzymes/metabolism , Ubiquitin-Conjugating Enzymes/genetics , Ubiquitin-Protein Ligases/metabolism , Ubiquitin-Protein Ligases/genetics , UbiquitinationABSTRACT
Adult T-cell leukemia (ATL), caused by HTLV-1, is the most lethal hematological malignancy. NEDD8-activating enzyme (NAE) is a component of the NEDD8 conjunction pathway that regulates cullin-RING ubiquitin ligase (CRL) activity. HTLV-1-infected T cells expressed higher levels of NAE catalytic subunit UBA3 than normal peripheral blood mononuclear cells. NAE1 knockdown inhibited proliferation of HTLV-1-infected T cells. The NAE1 inhibitor MLN4924 suppressed neddylation of cullin and inhibited the CRL-mediated turnover of tumor suppressor proteins. MLN4924 inhibited proliferation of HTLV-1-infected T cells by inducing DNA damage, leading to S phase arrest and caspase-dependent apoptosis. S phase arrest was associated with CDK2 and cyclin A downregulation. MLN4924-induced apoptosis was mediated by the upregulation of pro-apoptotic and downregulation of anti-apoptotic proteins. Furthermore, MLN4924 inhibited NF-κB, AP-1, and Akt signaling pathways and activated JNK. Therefore, neddylation inhibition is an attractive strategy for ATL therapy. Our findings support the use of MLN4924 in ATL clinical trials.
Subject(s)
Apoptosis , Cell Proliferation , Cyclopentanes , Human T-lymphotropic virus 1 , NEDD8 Protein , NF-kappa B , Proto-Oncogene Proteins c-akt , Pyrimidines , Signal Transduction , Transcription Factor AP-1 , Ubiquitin-Activating Enzymes , Humans , Pyrimidines/pharmacology , NF-kappa B/metabolism , Transcription Factor AP-1/metabolism , Cyclopentanes/pharmacology , Signal Transduction/drug effects , Proto-Oncogene Proteins c-akt/metabolism , Apoptosis/drug effects , NEDD8 Protein/metabolism , Cell Proliferation/drug effects , Ubiquitin-Activating Enzymes/antagonists & inhibitors , Ubiquitin-Activating Enzymes/metabolism , Ubiquitin-Activating Enzymes/genetics , T-Lymphocytes/metabolism , T-Lymphocytes/drug effects , Ubiquitins/metabolism , Cullin Proteins/metabolismABSTRACT
Overactivation of neddylation has been found in a number of common human tumor-related diseases. In recent years, targeting the neddylation pathway has become an appealing anti-cancer strategy, and it is critical to find neddylation inhibitors with novel structures and higher efficacy. Here, we present the discovery of novel inhibitors of the NEDD8-activating enzyme (NAE) and their antitumor activity in vitro. All synthesized 1,4-disubstituted piperidine compounds were evaluated for antiproliferative activity against MGC-803, MCF-7, A549, and KYSE-30 cells. Among five representative compounds, III-26 bearing a quinazoline motif was identified as the lead one due to the fact that it significantly hindered the neddylation of Cullin1. Cellular mechanisms elucidated that III-26 inhibited the proliferation, migration, and invasion of UBC12-overexpressed MGC-803 cell lines, as well as induced apoptosis and arrested the cell cycle at G2/M phase. Importantly, III-26 reduced NAE activity, thus selectively preventing neddylation of Cullin3 and Cullin1 over other Cullin members. At a dose of 4 µM, III-26 virtually entirely blocked UBC12-NEDD8 conjugation in MGC-803 cells. Our molecular modeling and kinetic investigation suggested that this compound may function as a non-covalent inhibitor of NAE.
Subject(s)
Neoplasms , Humans , Neoplasms/drug therapy , Cell Line, Tumor , ApoptosisABSTRACT
Post-translational modifications (PTMs) are critical for early development in mice because early cleavage-stage embryos are characterized by transcriptional inactivity. Neddylation is an important ubiquitin-like PTM that regulates multiple biophysical processes. However, the exact roles of neddylation in regulating early embryonic development remain largely unknown. In the present study, we found that inhibition of neddylation by specific inhibitor MLN4924 led to severe arrest of early embryonic development. Transcriptomic analysis showed that neddylation inhibition changed the expression of 3959 genes at the 2-cell stage. Importantly, neddylation inhibition blocked zygotic genome activation and maternal mRNA degradation, thus disrupting the maternal-to-zygotic transition. Moreover, inhibition of neddylation induced mitochondrial dysfunction including aberrant mitochondrial distribution, decreased mitochondrial membrane potential, and reduced ATP content. Further analysis showed that inhibition of neddylation resulted in the accumulation of reactive oxygen species and superoxide anion, thereby resulting in oxidative stress and severe DNA damage at the 2-cell stage. Overall, this study demonstrates that neddylation is vital for early embryonic development in mice. Our findings suggest that proper neddylation regulation is essential for the timely inter-stage transition during early embryonic development.
Subject(s)
Embryonic Development , Protein Processing, Post-Translational , Mice , Animals , MitochondriaABSTRACT
The identification of new therapeutic targets and the development of innovative therapeutic approaches are the most important challenges for osteosarcoma treatment. In fact, despite being relatively rare, recurrence and metastatic potential, particularly to the lungs, make osteosarcoma a deadly form of cancer. In fact, although current treatments, including surgery and chemotherapy, have improved survival rates, the disease's recurrence and metastasis are still unresolved complications. Insights for analyzing the still unclear molecular mechanisms of osteosarcoma development, and for finding new therapeutic targets, may arise from the study of post-translational protein modifications. Indeed, they can influence and alter protein structure, stability and function, and cellular interactions. Among all the post-translational modifications, ubiquitin-like modifications (ubiquitination, deubiquitination, SUMOylation, and NEDDylation), as well as glycosylation, are the most important for regulating protein stability, which is frequently altered in cancers including osteosarcoma. This review summarizes the relevance of ubiquitin-like modifications and glycosylation in osteosarcoma progression, providing an overview of protein stability regulation, as well as highlighting the molecular mediators of these processes in the context of osteosarcoma and their possible targeting for much-needed novel therapy.