RESUMO
Cervical cancer (CC) remains a significant global health challenge. A deeper understanding of the molecular mechanisms driving CC progression is crucial for developing improved therapeutic strategies. CSN5 is vital in cell functions and cancer, but its role in CC is unclear. This study aims to explore CSN5's role and its target gene in CC progression, assessing their potential as therapeutic targets. We employed an integrated approach combining bioinformatics analysis, proteomic profiling, and in vitro and in vivo functional assays. Immunohistochemistry was used to analyze CSN5 and ENO3 expression in CC and normal tissues. CSN5 upregulation was associated with advanced clinical stage and poor differentiation. Furthermore, CSN5 overexpression enhanced cellular proliferation and glycolytic metabolism but, paradoxically, suppressed migration, invasion, and the epithelial-mesenchymal transition (EMT). These pro-tumorigenic effects were confirmed in vivo, and the glycolytic inhibitor 2-DG was found to reverse the phenotypes induced by CSN5. Protein sequencing highlighted ENO3's role in CSN5-mediated tumorigenesis, regulating EMT and glycolysis by stabilizing its ubiquitination degradation through CSN5. Crucially, silencing ENO3 attenuated the oncogenic effects of CSN5 both in vitro and in vivo. Our findings unveil a novel mechanistic paradigm in which CSN5 promotes CC progression by co-opting ENO3 to enhance glycolytic flux while concurrently suppressing cell motility. This study not only deepens the understanding of CC pathogenesis but also identifies the CSN5-ENO3 axis as a promising target for novel therapeutic interventions.
Assuntos
Complexo do Signalossomo COP9 , Glicólise , Peptídeos e Proteínas de Sinalização Intracelular , Fosfopiruvato Hidratase , Neoplasias do Colo do Útero , Humanos , Feminino , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/patologia , Neoplasias do Colo do Útero/metabolismo , Glicólise/genética , Complexo do Signalossomo COP9/genética , Complexo do Signalossomo COP9/metabolismo , Transição Epitelial-Mesenquimal/genética , Animais , Progressão da Doença , Proliferação de Células/genética , Movimento Celular/genética , Linhagem Celular Tumoral , Camundongos , Regulação Neoplásica da Expressão Gênica , Fosfopiruvato Hidratase/metabolismo , Fosfopiruvato Hidratase/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos Nus , Camundongos Endogâmicos BALB C , Peptídeo HidrolasesRESUMO
Immune checkpoint inhibitors have been approved for various solid tumor treatments but have shown poor efficacy on colon cancer. Curcumin has been proven as an anti-tumor agent that inhibits cell cycle and tumor cell proliferation. Moreover, curcumin has also been reported to have the ability to inhibit PD-L1 expression, which might benefit the therapeutic efficacy of immune checkpoint inhibitors. Therefore, we proposed using antibody-drug conjugate (ADC) could effectively inhibit tumor proliferation and reverse the immunosuppression in colon cancer. We prepared an anti-PD-L1 conjugated curcumin with a ROS-responsive linker of phenylboronic acid carbamate, which provides chemo-drug active targeting ability and tumor environment-responsive release. Both in vitro and in vivo data confirm the improved cytotoxicity of anti-PD-L1-PBA-Cur and inhibited cell invasion. More importantly, the PD-L1 expression on the tumor surface was significantly reduced after being treated with ADC. The in vivo inhibition of tumor progression and PD-L1 expression was confirmed in both subcutaneous and in-suit mouse models. This study provides an effective colon treatment strategy with the advantages of high tumor targeting efficiency and immunopotentiation potential.
Assuntos
Antineoplásicos , Antígeno B7-H1 , Neoplasias do Colo , Curcumina , Imunoconjugados , Curcumina/farmacologia , Curcumina/química , Animais , Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/metabolismo , Antígeno B7-H1/imunologia , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/patologia , Neoplasias do Colo/metabolismo , Humanos , Camundongos , Linhagem Celular Tumoral , Antineoplásicos/farmacologia , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Imunoconjugados/farmacologia , Imunoconjugados/química , Ensaios Antitumorais Modelo de Xenoenxerto , Camundongos Endogâmicos BALB CRESUMO
Promoting tumor cell senescence arrests the cell cycle of tumor cells and activates the immune system to eliminate these senescent cells, thereby suppressing tumor growth. Nevertheless, PD-L1 positive senescent tumor cells resist immune clearance and possess the ability to secret various cytokines and inflammatory factors that stimulate the growth of tumor cells. Consequently, drugs capable of both triggering senescence in tumor cells and concurrently diminishing the expression of PD-L1 to counteract immune evasion are urgently needed. Here, a berberine derivative B68 is developed, which specifically induces tumor cell senescence by targeting BMI1. B68 also involves the degradation of PD-L1 by targeting CSN5, thereby disrupting the immunosuppressive PD-1/PD-L1 interaction and enabling rapid clearance of senescent tumor cells. This approach simultaneously inhibits tumor progression and activates T cell immunity, as evidenced by the robust antitumor response following B68-induced immunization of senescent cancer cells. Moreover, the synergistic effect of B68 with anti-CTLA4 therapy further enhances antitumor immunity, and its ability to induce senescence in cancer cells triggers a strong protective response by dendritic and CD8+ T cells. These findings provide a scientific basis for developing a new tumor treatment strategy based on senescence induction and lay the foundation for further preclinical research.
RESUMO
Multiple Endocrine Neoplasia 1 gene (MEN1), which is known to be a tumor suppressor gene in lung tissues, encodes a 610 amino acid protein menin. Previous research has proven that MEN1 deficiency promotes the malignant progression of lung cancer. However, the biological role of this gene in the immune microenvironment of lung cancer remains unclear. In this study, we found that programmed cell death-ligand 1 (PD-L1) is upregulated in lung-specific KrasG12D mutation-induced lung adenocarcinoma in mice, after Men1 deficiency. Simultaneously, CD8+ and CD3+ T cells are depleted, and their cytotoxic effects are suppressed. In vitro, PD-L1 is inhibited by the overexpression of menin. Mechanistically, we found that MEN1 inactivation promotes the deubiquitinating activity of COP9 signalosome subunit 5 (CSN5) and subsequently increases the level of PD-L1.
Assuntos
Antígeno B7-H1 , Neoplasias Pulmonares , Proteínas Proto-Oncogênicas , Evasão Tumoral , Animais , Antígeno B7-H1/metabolismo , Antígeno B7-H1/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Camundongos , Humanos , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Evasão Tumoral/genética , Complexo do Signalossomo COP9/genética , Complexo do Signalossomo COP9/metabolismo , Microambiente Tumoral/imunologia , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/imunologia , Adenocarcinoma de Pulmão/patologia , Adenocarcinoma de Pulmão/metabolismo , Linhagem Celular Tumoral , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Ubiquitinação , MutaçãoRESUMO
The constitutive photomorphogenesis 9 (COP9) signalosome (CSN) is a deNEDDylase controlling ubiquitination activity of cullin-RING-E3 ligases (CRLs) and thus the levels of key cellular proteins. While the CSN and its catalytic subunit CSN5 have been extensively studied in cancer, its role in inflammatory and neurological diseases is less understood. Following verification that CSN5 is expressed in mouse and human brain, here we studied the role of the CSN in neuroinflammation and ischemic neuronal damage employing models of relevant brain-resident cell types, an ex vivo organotypic brain slice culture model, and the CRL NEDDylation state-modifying drugs MLN4924 and CSN5i-3, which mimic and inhibit, respectively, CSN5 deNEDDylase activity. Untargeted mass spectrometry-based proteomics revealed that MLN4924 and CSN5i-3 substantially alter the microglial proteome, including inflammation-related proteins. Applying these drugs and mimicking microglial and endothelial inflammation as well as ischemic neuronal stress by TNF and oxygen-glucose-deprivation/reoxygenation (OGD/RO) treatment, respectively, we could link CSN5/CSN-mediated cullin deNEDDylation to reduction of microglial inflammation, attenuated cerebral endothelial inflammation, improved barrier integrity, as well as protection from ischemic stress-induced neuronal cell death. Specifically, MLN4924 reduced phagocytic activity, motility, and inflammatory cytokine expression of microglial cells, and this was linked to inhibition of inflammation-induced NF-κB and Akt signaling. Inversely, Csn5 knockdown and CSN5i-3 increased NF-κB signaling. Moreover, MLN4924 abrogated TNF-induced NF-κB signaling in cerebral microvascular endothelial cells (hCMECs) and rescued hCMEC monolayers from OGD/RO-triggered barrier leakage, while CSN5i-3 exacerbated permeability. In an ex vivo organotypic brain slice model of ischemia/reperfusion stress, MLN4924 protected from neuronal death, while CSN5i-3 impaired neuronal survival. Neuronal damage was attributable to microglial activation and inflammatory cytokines, as indicated by microglial shape tracking and TNF-blocking experiments. Our results indicate a protective role of the CSN in neuroinflammation via brain-resident cell types involved in ischemic brain disease and implicate CSN activity-mimicking deNEDDylating drugs as potential therapeutics.
Assuntos
NF-kappa B , Doenças Neuroinflamatórias , Humanos , Animais , Camundongos , Complexo do Signalossomo COP9 , Proteínas Culina , Células Endoteliais , Encéfalo , Inflamação/tratamento farmacológico , CitocinasRESUMO
Cop9 signalosome (CSN) regulates the function of cullin-RING E3 ubiquitin ligases (CRLs) by deconjugating the ubiquitin-like protein NEDD8 from the cullin subunit. To understand the physiological impact of CSN function on the CRL network and cell proliferation, we combined quantitative mass spectrometry and genome-wide CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) screens to identify factors that modulate cell viability upon inhibition of CSN by the small molecule CSN5i-3. CRL components and regulators strongly modulated the antiproliferative effects of CSN5i-3, and in addition we found two pathways involved in genome integrity, SCFFBXO5-APC/C-GMNN and CUL4DTL-SETD8, that contribute substantially to the toxicity of CSN inhibition. Our data highlight the importance of CSN-mediated NEDD8 deconjugation and adaptive exchange of CRL substrate receptors in sustaining CRL function and suggest approaches for leveraging CSN inhibition for the treatment of cancer.
Assuntos
Replicação do DNA , Ubiquitina-Proteína Ligases , Azepinas/metabolismo , Complexo do Signalossomo COP9/antagonistas & inibidores , Complexo do Signalossomo COP9/genética , Complexo do Signalossomo COP9/metabolismo , Sobrevivência Celular , Proteínas Culina/genética , Proteínas Culina/metabolismo , Imidazóis/metabolismo , Proteína NEDD8/metabolismo , Pirazóis/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
BACKGROUND: Despite the understanding of the COP9 signalosome subunit 5 (CSN5) in tumor genesis, there is no conclusive evidence on its value to predict the survival and prognosis of digestive system tumor patients. Hence this study aimed to evaluate the impact of CSN5 levels on the survival and clinicopathological parameters of digestive system neoplasm patients. METHODS: First, a comprehensive search was conducted in four databases. We utilized the Hazard Ratio (HR) with a 95% confidence interval (CI) to evaluate the prognostic value of CSN5 for the overall survival (OS) and recurrence-free survival (RFS) of patients. Then, we estimated the connection between CSN5 and the clinicopathological parameters based on the Odds Ratio (OR) with the corresponding 95% CI. RESULTS: This meta-analysis included 22 studies and 2193 patients diagnosed with digestive system tumors. High expression of CSN5 was correlated to poorer OS (HR = 2.28, 95% CI: 1.71-3.03; p < 0.00001). Additionally, high CSN5 levels were correlated with worse invasion depth (OR = 0.49, 95% CI: 0.25-0.96, p = 0.04), positive lymphatic metastasis (OR = 0.28, 95% CI: 0.16-0.47, p = 0.00001), positive distant metastasis (OR = 0.32, 95% CI: 0.13-0.76, p = 0.01) and poorer differentiation degree (OR = 0.34, 95% CI: 0.19-0.60, p = 0.0003). However, we did not detect a correlation between CSN5 expression and age, gender, tumor stage, tumor size or vascular invasion. Furthermore, no significant publication bias was detected. CONCLUSION: This meta-analysis demonstrated that the overexpression of CSN5 level might foresee poorer OS in digestive system cancer patients. Additionally, CSN5 levels might be related to the prognosis of digestive system tumors.
Assuntos
Biomarcadores Tumorais , Neoplasias do Sistema Digestório , Biomarcadores Tumorais/metabolismo , Neoplasias do Sistema Digestório/diagnóstico , Humanos , Metástase Linfática , Prognóstico , Modelos de Riscos ProporcionaisRESUMO
The immune checkpoint blockade (ICB) targeting on PD-1/PD-L1 has shown remarkable promise in treating cancers. However, the low response rate and frequently observed severe side effects limit its broad benefits. It is partially due to less understanding of the biological regulation of PD-L1. Here, we systematically and comprehensively summarized the regulation of PD-L1 from nuclear chromatin reorganization to extracellular presentation. In PD-L1 and PD-L2 highly expressed cancer cells, a new TAD (topologically associating domain) (chr9: 5,400,000-5,600,000) around CD274 and CD273 was discovered, which includes a reported super-enhancer to drive synchronous transcription of PD-L1 and PD-L2. The re-shaped TAD allows transcription factors such as STAT3 and IRF1 recruit to PD-L1 locus in order to guide the expression of PD-L1. After transcription, the PD-L1 is tightly regulated by miRNAs and RNA-binding proteins via the long 3'UTR. At translational level, PD-L1 protein and its membrane presentation are tightly regulated by post-translational modification such as glycosylation and ubiquitination. In addition, PD-L1 can be secreted via exosome to systematically inhibit immune response. Therefore, fully dissecting the regulation of PD-L1/PD-L2 and thoroughly detecting PD-L1/PD-L2 as well as their regulatory networks will bring more insights in ICB and ICB-based combinational therapy.
RESUMO
TNBC is characterized by high incidence of visceral metastasis and lacks effective clinical targets. This study aims to delineate the molecular mechanisms of SENP1 in TNBC invasion and metastasis. By using IHC to test the SENP1 expression in TNBC tissues, we analyzed the relationship between SENP1 expression and TNBC prognosis. We showed that SENP1 expression was higher in TNBC tumor tissues and related to TNBC prognosis, supporting SENP1 as an independent risk factor. High expression of SENP1 was significantly associated with histologic grade and tumor lymph node invasion. Intriguingly, the expression levels of SENP1 in TNBC tumors were significantly correlated with that of CSN5, GATA1 and ZEB1. Importantly, SENP1 promoted TNBC cell migration and invasion by regulating ZEB1 deubiquitination and expression through CSN5. Further studies showed that deSUMOylation at lysine residue K137 of GATA1 enhanced the binding of GATA1 to the CSN5 promoter and transactivated CSN5 expression. In addition, we showed that ZEB1 is deubiquitinated at lysine residue K1108. Our in vivo studies also indicated that reduction in SENP1 expression upregulated GATA1 SUMOylation, and thus resulted in decreased expression of CSN5 and ZEB1 in the tumor microenvironment, which decelerated TNBC progression and metastasis. SENP1 promoted CSN5-mediated ZEB1 protein degradation via deSUMOylation of GATA1, and thus influenced TNBC progression. These findings suggest that SENP1 could be utilized as a potential target for blockade of TNBC development and thus provide a totally new approach for TNBC treatment.
Assuntos
Neoplasias de Mama Triplo Negativas , Complexo do Signalossomo COP9 , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Fator de Transcrição GATA1/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Lisina/metabolismo , Peptídeo Hidrolases , Neoplasias de Mama Triplo Negativas/metabolismo , Microambiente TumoralRESUMO
Gastric cancer (GC) originates from the stomach and is a prevalent human malignancy. Dysfunction of death associated protein kinase 1 (DAPK1) has been identified as a major regulator involved in the development and progression of GC. However, there's limited data regarding the regulatory mechanism of GC. Herein, we investigated role of DAPK1 in natural killer (NK) cell killing ability and immune evasion of GC cells and mediated pathway. Samples from GC-related gene expression profile and clinical samples from 67 patients with GC were collected to determine the expression of DAPK1, IκB kinase ß (IKKß), programmed death receptor-ligand 1 (PD-L1), and photomorphogenesis 9 (COP9) signalosome 5 (CSN5). The binding affinity among DAPK1, IKKß, CSN5, and PD-L1 was characterized to verify the underlying mechanism. GC lines were transfected with overexpressed plasmid or siRNA to determine the effect of DAPK1/IKKß/CSN5/PD-L1 axis on NK cell killing ability and immune evasion of GC cells. GC cells and tissues presented low expression of DAPK1 and high expression of IKKß, CSN5 and PD-L1. IKKß, negatively regulated by DAPK1, was capable of activating CSN5 and upregulating PD-L1 expression. Overexpression of DAPK1 promoted NK cell killing ability and reduced immune evasion, coupled with reduction of NK cell apoptosis and increases in levels of TNF-α, IFN-γ, CD107a, and Granzyme B cytokines. The tumor-suppressing properties of DAPK1 through downregulation of IKKß/CSN5/PD-L1 axis in GC were further confirmed in vivo. In summary, overexpression of DAPK1 promoted the NK cell killing ability and restrained immune evasion of GC cells, providing a potential therapeutic strategy for GC treatment by modulating immune evasion.
Assuntos
Antígeno B7-H1/metabolismo , Complexo do Signalossomo COP9/metabolismo , Proteínas Quinases Associadas com Morte Celular/metabolismo , Quinase I-kappa B/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Células Matadoras Naturais/imunologia , Peptídeo Hidrolases/metabolismo , Neoplasias Gástricas/imunologia , Neoplasias Gástricas/metabolismo , Animais , Antígeno B7-H1/genética , Complexo do Signalossomo COP9/genética , Linhagem Celular Tumoral , Proteínas Quinases Associadas com Morte Celular/genética , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Xenoenxertos , Humanos , Quinase I-kappa B/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Células Matadoras Naturais/metabolismo , Camundongos , Camundongos Nus , Modelos Biológicos , Peptídeo Hidrolases/genética , Fosforilação , Prognóstico , Neoplasias Gástricas/genética , Evasão Tumoral/genética , Evasão Tumoral/imunologia , Ubiquitinação , Regulação para CimaRESUMO
BACKGROUND: CSN5, a member of Cop9 signalosome, is essential for protein neddylation. It has been supposed to serve as an oncogene in some cancers. However, the role of CSN5 has not been investigated in cervical cancer yet. METHODS: Data from TCGA cohorts and GEO dataset was analyzed to examine the expression profile of CSN5 and clinical relevance in cervical cancers. The role of CSN5 on cervical cancer cell proliferation was investigated in cervical cancer cell lines, Siha and Hela, through CSN5 knockdown via CRISPR-CAS9. Western blot was used to detect the effect of CSN5 knockdown and overexpression. The biological behaviors were analyzed by CCK8, clone formation assay, 3-D spheroid generation assay and cell cycle assay. Besides, the role CSN5 knockdown in vivo was evaluated by xenograft tumor model. MLN4924 was given in Siha and Hela with CSN5 overexpression. RESULTS: We found that downregulation of CSN5 in Siha and Hela cells inhibited cell proliferation in vitro and in vivo, and the inhibitory effects were largely rescued by CSN5 overexpression. Moreover, deletion of CSN5 caused cell cycle arrest rather than inducing apoptosis. Importantly, CSN5 overexpression confers resistance to the anti-cancer effects of MLN4924 (pevonedistat) in cervical cancer cells. CONCLUSIONS: Our findings demonstrated that CSN5 functions as an oncogene in cervical cancers and may serve as a potential indicator for predicting the effects of MLN4924 treatment in the future.
RESUMO
Protein Disulfide Isomerase Family A Member 6 (PDIA6) is an endoplasmic reticulum protein that is capable of catalyzing protein folding and disulfide bond formation. Abnormally elevated expression of PDIA6 has been reported to predict poor outcomes in various cancers. Herein, gain-of- and loss-of-function experiments were performed to investigate how PDIA6 participated in the carcinogenesis of pancreatic cancer (PC). By analyzing the protein expression of PDIA6 in 28 paired PC and para carcinoma specimens, we first found that PDIA6 expression was higher in PC samples. Both the overall survival and disease-free survival rates of PC patients with higher PDIA6 expression were poorer than those with lower PDIA6 (n = 178). Furthermore, knockdown of PDIA6 impaired the malignancies of PC cells - suppressed cell proliferation, invasion, migration, cisplatin resistance, and xenografted tumor growth. PDIA6-silenced PC cells were more sensitive to cytotoxic natural killer (NK) cells. Overexpression of PDIA6 had opposite effects on PC cells. Interestingly, COP9 signalosome subunit 5 (CSN5), a regulator of E3 ubiquitin ligases known to promote deubiquitination of its downstream targets, was demonstrated to interact with PDIA6, and its expression was increased in PC cells overexpressing PDIA6. Additionally, PDIA6 overexpression promoted deubiquitination of ß-catenin and PD-L1 and subsequently upregulated their expression in PC cells. These alterations were partly reversed by CSN5 shRNA. Collectively, the above results demonstrate that PDIA6 contributes to PC progression, which may be associated with CSN5-regulated deubiquitination of ß-catenin and PD-L1. Our findings suggest PDIA6 as a potential target for the treatment of PC.
Assuntos
Antígeno B7-H1/metabolismo , Complexo do Signalossomo COP9/biossíntese , Peptídeos e Proteínas de Sinalização Intracelular/biossíntese , Neoplasias Pancreáticas/metabolismo , Peptídeo Hidrolases/biossíntese , Isomerases de Dissulfetos de Proteínas/biossíntese , Evasão Tumoral/fisiologia , beta Catenina/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Antígeno B7-H1/genética , Complexo do Signalossomo COP9/genética , Enzimas Desubiquitinantes/genética , Enzimas Desubiquitinantes/metabolismo , Progressão da Doença , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Gradação de Tumores/métodos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/imunologia , Peptídeo Hidrolases/genética , Isomerases de Dissulfetos de Proteínas/genética , beta Catenina/genética , CamundongosRESUMO
Targeting immune checkpoints such as programmed cell death protein 1 (PD-1) and programmed death ligand-1 (PD-L1) have been approved for treating melanoma, gastric cancer (GC) and bladder cancer with clinical benefit. Nevertheless, many patients failed to respond to anti-PD-1/PD-L1 treatment, so it is necessary to seek an alternative strategy for traditional PD-1/PD-L1 targeting immunotherapy. Here with the data from The Cancer Genome Atlas (TCGA) and our in-house tissue library, PD-L1 expression was found to be positively correlated with the expression of ubiquitin-specific processing protease 7 (USP7) in GC. Furthermore, USP7 directly interacted with PD-L1 in order to stabilize it, while abrogation of USP7 attenuated PD-L1/PD-1 interaction and sensitized cancer cells to T cell killing in vitro and in vivo. Besides, USP7 inhibitor suppressed GC cells proliferation by stabilizing P53 in vitro and in vivo. Collectively, our findings indicate that in addition to inhibiting cancer cells proliferation, USP7 inhibitor can also downregulate PD-L1 expression to enhance anti-tumor immune response simultaneously. Hence, these data posit USP7 inhibitor as an anti-proliferation agent as well as a novel therapeutic agent in PD-L1/PD-1 blockade strategy that can promote the immune response of the tumor.
RESUMO
We previously reported that the evolutionary conserved transcriptional cofactor Jab1/Cops5 is critical for mouse chondrocyte differentiation by selectively repressing BMP signaling. In this study, we first uncovered that the endogenous Jab1 interacts with endogenous Smad1/5/8. Furthermore, although Jab1 did not directly interact with Acvr1 (Alk2), a key Type I BMP receptor, the interaction between endogenous Smad1/5/8 and Acvr1 was increased in Jab1-null chondrocytes. Thus, Jab1 might negatively regulate BMP signaling during chondrocyte differentiation in part by sequestering Smad1/5/8 away from Acvr1. Next, to identity Jab1 downstream targets in chondrocytes, we performed RNA-sequencing analysis of Jab1-null chondrocytes and discovered a total of 1993 differentially expressed genes. Gene set enrichment analysis revealed that key targets inhibited by Jab1 includes p53, BMP/transforming growth factor beta, and apoptosis pathways. We confirmed that endogenous Jab1 interacts with endogenous p53. There was significantly elevated p53 reporter activity, an enhanced expression of phospho-p53, and an increased expression of a key p53 downstream target, Puma, in Jab1-null chondrocytes. Moreover, treatments with a p53-specific inhibitor and/or a BMP Type I receptor-specific inhibitor reversed the elevated p53 and BMP signaling activities in Jab1-null chondrocytes and partially restored columnar growth plate structure in E17.5 Jab1-null mouse tibia explant cultures. Finally, we demonstrated that the chondrocyte-specific Jab1 overexpression in mice resulted in smaller-sized embryos with disorganized growth plates. In conclusion, our data showed that the delicate Jab1-mediated crosstalk between BMP and p53 pathways is crucial to maintain proper chondrocyte survival and differentiation. Moreover, the appropriate Jab1 expression level is essential for proper skeletal development.
Assuntos
Complexo do Signalossomo COP9/metabolismo , Diferenciação Celular/fisiologia , Condrócitos/metabolismo , Peptídeo Hidrolases/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Condrogênese/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Fator de Crescimento Transformador beta/metabolismoRESUMO
COP9 signalosome subunit 5 (CSN5) plays a key role in carcinogenesis of multiple cancers and contributes to the stabilization of target proteins through deubiquitylation. However, the underlying role of CSN5 in thyroid carcinoma has not been reported. In this research, our data showed that CSN5 was overexpressed in thyroid carcinoma tissues compared with paracancerous tissues. Furthermore, a series of gain/loss functional assays were performed to demonstrate the role of CSN5 in facilitating thyroid carcinoma cell proliferation and metastasis. Additionally, we found there was a positive correlation between CSN5 and angiopoietin-like protein 2 (ANGPTL2) protein levels in thyroid carcinoma tissues and that CSN5 promoted thyroid carcinoma cell proliferation and metastasis through ANGPTL2. We also identified the underlying mechanism that CSN5 elevated ANGPTL2 protein level by directly binding it, decreasing its ubiquitination and degradation. Overall, our results highlight the significance of CSN5 in promoting thyroid carcinoma carcinogenesis and implicate CSN5 as a promising candidate for thyroid carcinoma treatment.
Assuntos
Proteínas Semelhantes a Angiopoietina/fisiologia , Complexo do Signalossomo COP9/fisiologia , Carcinogênese/genética , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Peptídeo Hidrolases/fisiologia , Neoplasias da Glândula Tireoide/genética , Proteína 2 Semelhante a Angiopoietina , Proteínas Semelhantes a Angiopoietina/metabolismo , Animais , Células Cultivadas , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Processamento de Proteína Pós-Traducional/genética , Proteólise , Transdução de Sinais/genética , Neoplasias da Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/patologia , Ubiquitinação/genéticaRESUMO
As an extremely virulent pathogen, white spot syndrome virus (WSSV) greatly threatens shrimp aquaculture worldwide. The interaction between virus and host is important for viral infection. In the present study, a yeast two-hybrid (Y2H) library was constructed to clarify the functions of wsv006, and the interaction between wsv006 and shrimp Litopenaeus vannamei (L. vannamei) was analyzed. Furthermore, we explored the role of the wsv006-interacting molecule L. vannamei COP9 constitutive photomorphogenic-like protein subunit 5 (LvCSN5) in WSSV infection. Y2H assay showed that wsv006 interacted with LvCSN5, and co-immunoprecipitation (Co-IP) assay confirmed such interaction. Multiple alignments of amino acid sequences with other species revealed that the LvCSN5 had high identity with Penaeusmonodon CSN5 (PmCSN5). LvCSN5 was mainly expressed in intestine, eye and hepatopancreas. In addition, the relative expression of LvCSN5 was significantly up-regulated both in intestine and hepatopancreas following the WSSV challenge. Besides, the relative expressions of IE1 and VP28, as well as the viral copy numbers were significantly increased in the LvCSN5-silenced shrimp. Our findings suggested that LvCSN5 was involved in WSSV infection by interacting with wsv006.
Assuntos
Proteínas de Artrópodes , Complexo do Signalossomo COP9 , Infecções por Vírus de DNA , Hepatopâncreas , Intestinos , Penaeidae , Proteínas Virais , Vírus da Síndrome da Mancha Branca 1 , Animais , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/metabolismo , Complexo do Signalossomo COP9/genética , Complexo do Signalossomo COP9/metabolismo , Infecções por Vírus de DNA/imunologia , Infecções por Vírus de DNA/metabolismo , Hepatopâncreas/metabolismo , Interações Hospedeiro-Patógeno , Proteínas Imediatamente Precoces/metabolismo , Imunidade Inata , Intestinos/metabolismo , Penaeidae/imunologia , Ligação Proteica , RNA Interferente Pequeno/genética , Técnicas do Sistema de Duplo-Híbrido , Regulação para Cima , Proteínas do Envelope Viral/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Replicação Viral , Vírus da Síndrome da Mancha Branca 1/fisiologiaRESUMO
Programmed cell death-1 (PD-1)/programmed cell death ligand-1 (PD-L1) blocking therapy has become a major pillar of cancer immunotherapy. Compared with antibodies targeting, small-molecule checkpoint inhibitors which have favorable pharmacokinetics are urgently needed. Here we identified berberine (BBR), a proven anti-inflammation drug, as a negative regulator of PD-L1 from a set of traditional Chinese medicine (TCM) chemical monomers. BBR enhanced the sensitivity of tumour cells to co-cultured T-cells by decreasing the level of PD-L1 in cancer cells. In addition, BBR exerted its antitumor effect in Lewis tumor xenograft mice through enhancing tumor-infiltrating T-cell immunity and attenuating the activation of immunosuppressive myeloid-derived suppressor cells (MDSCs) and regulatory T-cells (Tregs). BBR triggered PD-L1 degradation through ubiquitin (Ub)/proteasome-dependent pathway. Remarkably, BBR selectively bound to the glutamic acid 76 of constitutive photomorphogenic-9 signalosome 5 (CSN5) and inhibited PD-1/PD-L1 axis through its deubiquitination activity, resulting in ubiquitination and degradation of PD-L1. Our data reveals a previously unrecognized antitumor mechanism of BBR, suggesting BBR is small-molecule immune checkpoint inhibitor for cancer treatment.
RESUMO
Allogeneic mesenchymal stem cells (MSCs) from young and healthy donors are reported to hold the potential to treat several immunological and degenerative disorders. However, recent data from animal studies and clinical trials demonstrate that immunogenicity and poor survival of transplanted MSCs impaired the efficacy of cells for regenerative applications. It is reported that initially immunoprivileged under in vitro conditions, MSCs are targeted by the host immune system after transplantation in the ischemic tissues in vivo. We performed in vitro (in MSCs) and in vivo (in the rat model of myocardial infarction [MI]) studies to elucidate the mechanisms responsible for the change in the immunophenotype of MSCs from immunoprivileged to immunogenic under ischemic conditions. We have recently reported that a soluble factor prostaglandin E2 (PGE2) preserves the immunoprivilege of allogeneic MSCs. In the current study, we found that PGE2 levels, which were elevated during normoxia, decreased in MSCs following exposure to hypoxia. Further, we found that proteasome-mediated degradation of cyclooxygenase-2 (COX2, rate-limiting enzyme in PGE2 biosynthesis) in hypoxic MSCs is responsible for PGE2 decrease and loss of immunoprivilege of MSCs. While investigating the mechanisms of COX2 degradation in hypoxic MSCs, we found that in normoxic MSCs, COP9 signalosome subunit 5 (CSN5) binds to COX2 and prevents its degradation by the proteasome. However, exposure to hypoxia leads to a decrease in CSN5 levels and its binding to COX2, rendering COX2 protein susceptible to proteasome-mediated degradation. This subsequently causes PGE2 downregulation and loss of immunoprivilege of MSCs. Maintaining COX2 levels in MSCs preserves immunoprivilege in vitro and improves the survival of transplanted MSCs in a rat model of MI. These data provide novel mechanistic evidence that PGE2 is downregulated in hypoxic MSCs which is responsible for the post-transplantation rejection of allogeneic MSCs. Therefore, our data suggest that the new strategies that target CSN5-COX2 signaling may improve survival and utility of transplanted allogeneic MSCs in the ischemic heart.
Assuntos
Ciclo-Oxigenase 2/química , Hipóxia/fisiopatologia , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/citologia , Infarto do Miocárdio/imunologia , Animais , Masculino , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Infarto do Miocárdio/terapia , Ratos , Ratos Sprague-Dawley , Transplante HomólogoRESUMO
BACKGROUND: Many cancers evade immune surveillance by overexpressing PD-L1. PD-L1 interacted with its receptor PD-1, resulting in reduction of T cell proliferation and activation and thereafter cancer cell death mediated by T-lymphocyte. Understanding the mechanisms that regulate PD-L1 was of vital importance for immune checkpoint blockade therapy (ICBT). METHODS: Human non-small cell lung cancer cells and 293FT cells were used to investigate the function of USP22 upon PD-L1 and CSN5 by WB, Immunoprecipitation, Immunofluorescence and Flow cytometry analysis. B16-F10 cells were used to explore the role of USP22 on tumorigenesis and T cell cytotoxicity. The relationship between USP22 and PD-L1 expression was investigated by Immunohistochemistry analysis in human non-small cell lung cancer samples. RESULTS: Our data showed that USP22 interacted with PD-L1 and promoted its stability. USP22 deubiquitinated PD-L1 and inhibited its proteasome degradation. Moreover, USP22 also interacted with CSN5 and stabilized CSN5 through deubiquitination. Either USP22 or CSN5 could facilitate the interaction of PD-L1 with the other one. Furthermore, USP22 removed K6, K11, K27, K29, K33 and K63-linked ubiquitin chain of both CSN5 and PD-L1. In addition, USP22 depletion inhibited tumorigenesis and promoted T cell cytotoxicity. Besides, USP22 expression positively correlated with PD-L1 expression in human non-small cell lung cancer samples. CONCLUSIONS: Here, we suggested that USP22 is a new regulator for PD-L1. On the one hand, USP22 could directly regulate PD-L1 stability through deubiquitination. On the other hand, USP22 regulated PD-L1 protein level through USP22-CSN5-PD-L1 axis. In addition, USP22 depletion inhibited tumorigenesis and promoted T cell cytotoxicity. Besides, USP22 expression positively correlated with PD-L1 expression in human non-small cell lung cancer samples. Together, we identified a new regulator of PD-L1 and characterized the important role of USP22 in PD-L1 mediated immune evasion. Targeting USP22 might be a new solution to ICBT. Video abstract.
Assuntos
Antígeno B7-H1/metabolismo , Proteólise , Ubiquitina Tiolesterase/metabolismo , Animais , Complexo do Signalossomo COP9/metabolismo , Carcinogênese/metabolismo , Carcinogênese/patologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Citotoxicidade Imunológica , Células HEK293 , Humanos , Terapia de Imunossupressão , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias Pulmonares/metabolismo , Camundongos , Peptídeo Hidrolases/metabolismo , Ligação Proteica , Estabilidade Proteica , Linfócitos T/imunologia , UbiquitinaçãoRESUMO
Recent studies have showed that α5 nicotinic acetylcholine receptor (α5-nAChR) is closely associated with nicotine-related lung cancer. Our previous studies also demonstrated that α5-nAChR mediates nicotine-induced lung carcinogenesis. However, the mechanism by which α5-nAChR functions in lung carcinogenesis remains to be elucidated. Jab1/Csn5 is a key regulatory factor in smoking-induced lung cancer. In this study, we explored the underlying mechanisms linking the α5-nAChR-Jab1/Csn5 axis with lung cancer epithelial-mesenchymal transition (EMT) and metastasis, which may provide potential therapeutic targets for future lung cancer treatments. Our results demonstrated that the expression of α5-nAChR was correlated with the expression of Jab1/Csn5 in lung cancer tissues and lung cancer cells. α5-nAChR expression is associated with Jab1/Csn5 expression in lung tumour xenografts in mice. In vitro, the expression of α5-nAChR mediated Stat3 and Jab1/Csn5 expression, significantly regulating the expression of the EMT markers, N-cadherin and Vimentin. In addition, the down-regulation of α5-nAChR or/and Stat3 reduced Jab1/Csn5 expression, while the silencing of α5-nAChR or Jab1/Csn5 inhibited the migration and invasion of NSCLC cells. Mechanistically, α5-nAChR contributes to EMT and metastasis by regulating Stat3-Jab1/Csn5 signalling in NSCLC, suggesting that α5-nAChR may be a potential target in NSCLC diagnosis and immunotherapy.