ABSTRACT
The majority of oncogenic drivers are intracellular proteins, constraining their immunotherapeutic targeting to mutated peptides (neoantigens) presented by individual human leukocyte antigen (HLA) allotypes1. However, most cancers have a modest mutational burden that is insufficient for generating responses using neoantigen-based therapies2,3. Neuroblastoma is a paediatric cancer that harbours few mutations and is instead driven by epigenetically deregulated transcriptional networks4. Here we show that the neuroblastoma immunopeptidome is enriched with peptides derived from proteins essential for tumorigenesis. We focused on targeting the unmutated peptide QYNPIRTTF discovered on HLA-A*24:02, which is derived from the neuroblastoma-dependency gene and master transcriptional regulator PHOX2B. To target QYNPIRTTF, we developed peptide-centric chimeric antigen receptors (PC-CARs) through a counter panning strategy using predicted potentially cross-reactive peptides. We further proposed that PC-CARs can recognize peptides on additional HLA allotypes when presenting a similar overall molecular surface. Informed by our computational modelling results, we show that PHOX2B PC-CARs also recognize QYNPIRTTF presented by HLA-A*23:01, the most common non-A2 allele in people with African ancestry. Finally, we demonstrate potent and specific killing of neuroblastoma cells expressing these HLAs in vitro and complete tumour regression in mice. These data suggest that PC-CARs have the potential to expand the pool of immunotherapeutic targets to include non-immunogenic intracellular oncoproteins and allow targeting through additional HLA allotypes in a clinical setting.
Subject(s)
Antigens, Neoplasm , Neuroblastoma , Oncogene Proteins , Peptides , Receptors, Chimeric Antigen , Animals , Humans , Mice , Africa/ethnology , Alleles , Amino Acid Sequence , Carcinogenesis , Cross Reactions , HLA-A Antigens/chemistry , HLA-A Antigens/immunology , Neuroblastoma/genetics , Neuroblastoma/immunology , Neuroblastoma/therapy , Oncogene Proteins/antagonists & inhibitors , Oncogene Proteins/immunology , Peptides/antagonists & inhibitors , Peptides/chemistry , Peptides/immunology , Receptors, Chimeric Antigen/immunology , Receptors, Chimeric Antigen/therapeutic useABSTRACT
T-cell acute lymphoblastic leukemia (T-ALL) is a serious hematological tumor derived from early T-cell progenitors, which is extremely resistant to chemotherapy. Classically, doxorubicin (DOX) is an effective first-line drug for the treatment of T-ALL; however, DOX resistance limits its clinical effect. The DEK proto-oncogene (DEK) has been involved in neoplasms but remains unexplored in T-ALL. We silenced DEK on Jurkat cells and detected cell proliferation with cell counting and colony formation assay. Then, we detected DEK's drug sensitivity to DOX with CCK-8, cell cycle, and apoptosis with DOX treatment. Western blot analysis was performed to determine protein expression of apoptosis and cell cycle-related genes, including BCL2L1, caspase-3, and cyclin-dependent kinases (CDK). Finally, the tumorigenic ability of DEK was analyzed using a BALB/C nude mouse model. In this study, DEK was highly expressed in Jurkat cells. Inhibition of DEK can lead to decreased cell proliferation and proportion of S-phase cells in the cell cycle and more cell apoptosis, and the effect is more obvious after DOX treatment. Western blot results showed that DOX treatment leads to cell cycle arrest, reduction of cyclin-dependent kinase 6 (CDK6) protein, accumulation of CDKN1A protein, and DOX-induced apoptosis accompanied by reductions in protein levels of BCL2L1, as well as increases in protein level of caspase-3. Furthermore, DEK-silenced Jurkat cells generated a significantly smaller tumor mass in mice. Our study found that DEK is a novel, potential therapeutic target for overcoming DOX resistance in T-ALL.
Subject(s)
DNA-Binding Proteins , Doxorubicin , Oncogene Proteins , Poly-ADP-Ribose Binding Proteins , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma , Animals , Apoptosis/drug effects , Caspase 3/metabolism , Cell Cycle Checkpoints/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/metabolism , Doxorubicin/pharmacology , Drug Synergism , Humans , Jurkat Cells , Mice , Mice, Inbred BALB C , Oncogene Proteins/antagonists & inhibitors , Oncogene Proteins/metabolism , Poly-ADP-Ribose Binding Proteins/antagonists & inhibitors , Poly-ADP-Ribose Binding Proteins/metabolism , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/pathologyABSTRACT
Molecular targeted therapies are the standard of care for front-line treatment of metastatic non-small-cell lung cancers (NSCLCs) harboring driver gene mutations. However, despite the initial dramatic responses, the emergence of acquired resistance is inevitable. Acquisition of secondary mutations in the target gene (on-target resistance) is one of the major mechanisms of resistance. The mouse pro-B cell line Ba/F3 is dependent on interleukin-3 for survival and proliferation. Upon transduction of a driver gene, Ba/F3 cells become independent of interleukin-3 but dependent on the transduced driver gene. Therefore, the Ba/F3 cell line has been a popular system to generate models with oncogene dependence and vulnerability to specific targeted therapies. These models have been used to estimate oncogenicity of driver mutations or efficacies of molecularly targeted drugs. In addition, Ba/F3 models, together with N-ethyl-N-nitrosourea mutagenesis, have been used to derive acquired resistant cells to investigate on-target resistance mechanisms. Here, we reviewed studies that used Ba/F3 models with EGFR mutations, ALK/ROS1/NTRK/RET fusions, MET exon 14 skipping mutations, or KRAS G12C mutations to investigate secondary/tertiary drug resistant mutations. We determined that 68% of resistance mutations reproducibly detected in clinical cases were also found in Ba/F3 models. In addition, sensitivity data generated with Ba/F3 models correlated well with clinical responses to each drug. Ba/F3 models are useful to comprehensively identify potential mutations that induce resistance to molecularly targeted drugs and to explore drugs to overcome the resistance.
Subject(s)
Drug Resistance, Neoplasm/genetics , Lung Neoplasms/genetics , Models, Biological , Molecular Targeted Therapy , Animals , Cell Line , Drug Resistance, Neoplasm/drug effects , Lung Neoplasms/drug therapy , Mice , Mutation , Oncogene Proteins/antagonists & inhibitors , Oncogene Proteins/genetics , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic useABSTRACT
The majority of oncogenic drivers are intracellular proteins, thus constraining their immunotherapeutic targeting to mutated peptides (neoantigens) presented by individual human leukocyte antigen (HLA) allotypes1. However, most cancers have a modest mutational burden that is insufficient to generate responses using neoantigen-based therapies2,3. Neuroblastoma is a paediatric cancer that harbours few mutations and is instead driven by epigenetically deregulated transcriptional networks4. Here we show that the neuroblastoma immunopeptidome is enriched with peptides derived from proteins that are essential for tumourigenesis and focus on targeting the unmutated peptide QYNPIRTTF, discovered on HLA-A*24:02, which is derived from the neuroblastoma dependency gene and master transcriptional regulator PHOX2B. To target QYNPIRTTF, we developed peptide-centric chimeric antigen receptors (CARs) using a counter-panning strategy with predicted potentially cross-reactive peptides. We further hypothesized that peptide-centric CARs could recognize peptides on additional HLA allotypes when presented in a similar manner. Informed by computational modelling, we showed that PHOX2B peptide-centric CARs also recognize QYNPIRTTF presented by HLA-A*23:01 and the highly divergent HLA-B*14:02. Finally, we demonstrated potent and specific killing of neuroblastoma cells expressing these HLAs in vitro and complete tumour regression in mice. These data suggest that peptide-centric CARs have the potential to vastly expand the pool of immunotherapeutic targets to include non-immunogenic intracellular oncoproteins and widen the population of patients who would benefit from such therapy by breaking conventional HLA restriction.
Subject(s)
Antigens, Neoplasm/immunology , HLA Antigens/immunology , Immunotherapy , Neoplasms/immunology , Neoplasms/therapy , Oncogene Proteins/immunology , Receptors, Chimeric Antigen/immunology , Animals , Antigens, Neoplasm/metabolism , Cell Line , Cell Line, Tumor , Cross Reactions , Cross-Priming , Female , HLA Antigens/metabolism , Homeodomain Proteins/immunology , Homeodomain Proteins/metabolism , Humans , Interferon-gamma/immunology , Mice , Neoplasms/metabolism , Oncogene Proteins/antagonists & inhibitors , Oncogene Proteins/metabolism , T-Lymphocytes/immunology , Transcription Factors/immunology , Transcription Factors/metabolismABSTRACT
Non-genetic mechanisms have recently emerged as important drivers of cancer therapy failure1, where some cancer cells can enter a reversible drug-tolerant persister state in response to treatment2. Although most cancer persisters remain arrested in the presence of the drug, a rare subset can re-enter the cell cycle under constitutive drug treatment. Little is known about the non-genetic mechanisms that enable cancer persisters to maintain proliferative capacity in the presence of drugs. To study this rare, transiently resistant, proliferative persister population, we developed Watermelon, a high-complexity expressed barcode lentiviral library for simultaneous tracing of each cell's clonal origin and proliferative and transcriptional states. Here we show that cycling and non-cycling persisters arise from different cell lineages with distinct transcriptional and metabolic programs. Upregulation of antioxidant gene programs and a metabolic shift to fatty acid oxidation are associated with persister proliferative capacity across multiple cancer types. Impeding oxidative stress or metabolic reprogramming alters the fraction of cycling persisters. In human tumours, programs associated with cycling persisters are induced in minimal residual disease in response to multiple targeted therapies. The Watermelon system enabled the identification of rare persister lineages that are preferentially poised to proliferate under drug pressure, thus exposing new vulnerabilities that can be targeted to delay or even prevent disease recurrence.
Subject(s)
Cell Cycle , Cell Lineage , Neoplasm Recurrence, Local/drug therapy , Neoplasm Recurrence, Local/pathology , Neoplasms/drug therapy , Neoplasms/pathology , Antioxidants/metabolism , Cell Cycle/drug effects , Cell Line, Tumor , Cell Lineage/drug effects , Cell Proliferation/drug effects , Cell Survival/drug effects , Clone Cells/drug effects , Clone Cells/metabolism , Clone Cells/pathology , DNA Barcoding, Taxonomic , Fatty Acids/metabolism , Gene Expression Regulation, Neoplastic , Humans , Lentivirus/genetics , Neoplasm Recurrence, Local/genetics , Neoplasms/genetics , Neoplasms/metabolism , Oncogene Proteins/antagonists & inhibitors , Oxidation-Reduction , Oxidative Stress , Reactive Oxygen Species/metabolism , Transcription, Genetic/drug effectsABSTRACT
Accumulating evidence shows that deregulation of fatty acid (FA) metabolism is associated with the development of cancer. Long-chain acyl-coenzyme A synthases (ACSLs) are responsible for activating long-chain FAs and are frequently deregulated in cancers. Among the five mammalian ACSL family members, ACSL1 is involved in the TNFα-mediated pro-inflammatory phenotype and mainly facilitates cancer progression. ACSL3 is an androgen-responsive gene. High ACSL3 expression has been detected in a variety of cancers, including melanoma, triple-negative breast cancer (TNBC) and high-grade non-small cell lung carcinoma (NSCLC), and correlates with worse prognosis of patients with these diseases. ACSL4 can exert opposing roles acting as a tumor suppressor or as an oncogene depending on the specific cancer type and tissue environment. Moreover, ACSL4 behaves as a crucial regulator in ferroptosis that is defined as a cell death process caused by iron-dependent peroxidation of lipids. ACSL5 is nuclear-coded and expressed in the mitochondria and physiologically participates in the pro-apoptotic sensing of cells. ACSL5 mainly acts as a tumor suppressor in cancers. ACSL6 downregulation has been observed in many forms of cancers, except in colorectal cancer (CRC). Here, we address the differential regulatory mechanisms of the ACSL family members as well as their functions in carcinogenesis. Moreover, we enumerate the clinical therapeutic implications of ACSLs, which might serve as valuable biomarkers and therapeutic targets for precision cancer treatment.
Subject(s)
Antineoplastic Agents/therapeutic use , Coenzyme A Ligases/metabolism , Enzyme Activators/therapeutic use , Enzyme Inhibitors/therapeutic use , Neoplasms/drug therapy , Animals , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Coenzyme A Ligases/antagonists & inhibitors , Coenzyme A Ligases/genetics , Disease Models, Animal , Enzyme Activators/pharmacology , Enzyme Inhibitors/pharmacology , Fatty Acids/metabolism , Gene Expression Regulation, Neoplastic , Humans , Neoplasms/genetics , Neoplasms/metabolism , Neoplasms/pathology , Oncogene Proteins/antagonists & inhibitors , Oncogene Proteins/genetics , Oncogene Proteins/metabolism , Tumor Suppressor Proteins/agonists , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolism , Xenograft Model Antitumor AssaysABSTRACT
BACKGROUND: Ovarian clear cell carcinoma (OCCC) is resistant to platinum chemotherapy and is characterized by poor prognosis. Today, the use of poly (ADP-ribose) polymerase (PARP) inhibitor, which is based on synthetic lethality strategy and characterized by cancer selectivity, is widely used for new types of molecular-targeted treatment of relapsed platinum-sensitive ovarian cancer. However, it is less effective against OCCC. METHODS: We conducted siRNA screening to identify synthetic lethal candidates for the ARID1A mutation; as a result, we identified Cyclin-E1 (CCNE1) as a potential target that affects cell viability. To further clarify the effects of CCNE1, human OCCC cell lines, namely TOV-21G and KOC7c (ARID1A mutant lines), and RMG-I and ES2 (ARID1A wild type lines) were transfected with siRNA targeting CCNE1 or a control vector. RESULTS: Loss of CCNE1 reduced proliferation of the TOV-21G and KOC7c cells but not of the RMG-I and ES2 cells. Furthermore, in vivo interference of CCNE1 effectively inhibited tumor cell proliferation in a xenograft mouse model. CONCLUSION: This study showed for the first time that CCNE1 is a synthetic lethal target gene to ARID1A-mutated OCCC. Targeting this gene may represent a putative, novel, anticancer strategy in OCCC treatment.
Subject(s)
Adenocarcinoma, Clear Cell/genetics , Cyclin E/genetics , DNA-Binding Proteins/genetics , Oncogene Proteins/genetics , Ovarian Neoplasms/genetics , Synthetic Lethal Mutations , Transcription Factors/genetics , Adenocarcinoma, Clear Cell/metabolism , Adenocarcinoma, Clear Cell/pathology , Animals , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Apoptosis/genetics , Cell Cycle/drug effects , Cell Cycle/genetics , Cell Line, Tumor , Cell Proliferation/drug effects , Cisplatin/pharmacology , Cyclin E/antagonists & inhibitors , Cyclin E/metabolism , DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/metabolism , Female , Gene Expression Regulation, Neoplastic , Humans , Mice , Mice, Nude , Molecular Targeted Therapy , Oncogene Proteins/antagonists & inhibitors , Oncogene Proteins/metabolism , Ovarian Neoplasms/metabolism , Ovarian Neoplasms/pathology , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Transcription Factors/antagonists & inhibitors , Transcription Factors/metabolism , Tumor Burden/drug effects , Xenograft Model Antitumor AssaysABSTRACT
A series of novel substituted bisurea 1,4-Diisocyanatobenzene compounds were designed, synthesized and introduced as potent anticancer compounds and screened for their in vitro anti-proliferative activities in human cancer cell lines. The structures of all titled compounds were characterized using Fourier-transform infrared mass spectra, nuclear magnetic resonance spectroscopy, elemental analysis and evaluated their sustainability using biological experiments. A selected group of ten derivatives were apprised for their anti-proliferative activity. The compounds 3d and 3e displayed potent anticancer activity with low IC50 value of 5.40, and 5.89 µM against HeLa cancer cell lines. The observed apoptosis data has demonstrated that compounds 3d and 3e induce the activaties of caspase-9 and caspase-3, the compounds 3d and 3e regulated fungal zone inhibition. Due to promising growth inhibitions, the all synthesized compounds were allowed to campaign includes quantum-polarized-ligand, quantum mechanical and molecular mechanical, docking experiments. The compounds 3d and 3e have exhibited a higher affinity for ERK/MAP kinase and CDK2 proteins. The molecular docking interactions have demonstrated two stage inhibition of cancer cells by binding with ERK/MAP kinase and CDK2 leads to inactivation of cell proliferation,cell cycle progression,cell divisionanddifferentiation, and hypo-phosphorylation of ribosome leading cells to restricts at point boundary of the G1/S phase. The long-range molecular dynamics, 150 ns, simulations were also revealed more consistency by 3d. Our study conclude good binding propensity for active-tunnel of ERK/MAP kinase and CDK2 proteins, by 3d (1,1'-(1,4-phenylene) bis(3-(2-chlorobenzyl)urea)), to suggest that the designed and synthesized 3d is to use as selective novel nuclei in anti-cancer chemotherapeutics.
Subject(s)
Antifungal Agents/pharmacology , Antineoplastic Agents/pharmacology , Benzene Derivatives/pharmacology , Isocyanates/pharmacology , Protein Kinase Inhibitors/pharmacology , Urea/pharmacology , Animals , Antifungal Agents/chemical synthesis , Antifungal Agents/chemistry , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Apoptosis/drug effects , Benzene Derivatives/chemical synthesis , Benzene Derivatives/chemistry , Cell Proliferation/drug effects , Cyclin E/antagonists & inhibitors , Cyclin E/metabolism , Cyclin-Dependent Kinase 2/antagonists & inhibitors , Cyclin-Dependent Kinase 2/deficiency , Cyclin-Dependent Kinase 2/metabolism , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Humans , Isocyanates/chemical synthesis , Isocyanates/chemistry , MAP Kinase Signaling System/drug effects , Mice , Microbial Sensitivity Tests , Molecular Dynamics Simulation , Molecular Structure , Oncogene Proteins/antagonists & inhibitors , Oncogene Proteins/metabolism , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/chemistry , Saccharomyces cerevisiae/drug effects , Signal Transduction/drug effects , Structure-Activity Relationship , Urea/analogs & derivatives , Urea/chemistryABSTRACT
The monopolar spindle 1 ((hMps1/TTK) is a serine/threonine kinase that plays an important role in spindle assembly checkpoint signaling. To explore the possible relationship between TTK inhibition and radiosensitivity, we examined whether TTK inhibition influences cellular susceptibility of radiation. And we further revealed its mechanisms. We found that the expression of TTK was obviously higher in liver cancer tissues compared to the normal liver tissues. Kaplan-Meier Plotter demonstrated that patients with low TTK expression levels had a longer overall survival than patients with high TTK expression levels. TTK inhibitor AZ3146 could simulated liver cancer cells to accumulate in the G2/M phase, which ultimately enhances DNA damage with more γ-H2AX foci and more apoptosis and necrosis induced by radiation, which prompted that TTK inhibition sensitized liver cancer cells to radiation. In addition, TTK inhibition altered cell-cycle progression and exacerbated centrosome abnormalities, resulting in enhanced mitotic catastrophe (MC) induced by radiation in a p21-mediated manner. In this study, we present evidences that the TTK inhibitor promotes the radiosensitivity of liver cancer cells through regulating cell cycle in p21-mediated manner in vitro, indicating that TTK inhibitor may be an attractive radiosensitizer for the patients with liver cancer.
Subject(s)
Cell Cycle Proteins/antagonists & inhibitors , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Liver Neoplasms/metabolism , Liver Neoplasms/radiotherapy , Oncogene Proteins/antagonists & inhibitors , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/antagonists & inhibitors , Radiation Tolerance/drug effects , Apoptosis/drug effects , Apoptosis/radiation effects , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Line, Tumor , Centrosome/drug effects , Centrosome/metabolism , Centrosome/radiation effects , DNA Damage/drug effects , DNA Damage/radiation effects , G2 Phase Cell Cycle Checkpoints/drug effects , G2 Phase Cell Cycle Checkpoints/radiation effects , Histones/metabolism , Humans , Liver Neoplasms/pathology , M Phase Cell Cycle Checkpoints/drug effects , M Phase Cell Cycle Checkpoints/radiation effects , Necrosis/drug therapy , Necrosis/radiotherapy , Prognosis , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Protein-Tyrosine Kinases/genetics , Protein-Tyrosine Kinases/metabolism , Survival AnalysisABSTRACT
The Neuroepithelial transforming gene 1 (Net1) is a RhoA subfamily guanine nucleotide exchange factor that is overexpressed in a number of cancers and contributes to cancer cell motility and proliferation. Net1 also plays a Rho GTPase independent role in mitotic progression, where it promotes centrosomal activation of Aurora A and Pak2, and aids in chromosome alignment during prometaphase. To understand regulatory mechanisms controlling the mitotic function of Net1, we examined whether it was phosphorylated by the mitotic kinase Cdk1. We observed that Cdk1 phosphorylated Net1 on multiple sites in its N-terminal regulatory domain and C-terminus in vitro. By raising phospho-specific antibodies to two of these sites, we also demonstrated that both endogenous and transfected Net1 were phosphorylated by Cdk1 in cells. Substitution of the major Cdk1 phosphorylation sites with aliphatic or acidic residues inhibited the interaction of Net1 with RhoA, and treatment of metaphase cells with a Cdk1 inhibitor increased Net1 activity. Cdk1 inhibition also increased Net1 localization to the plasma membrane and stimulated cortical F-actin accumulation. Moreover, Net1 overexpression caused spindle polarity defects that were reduced in frequency by acidic substitution of the major Cdk1 phosphorylation sites. These data indicate that Cdk1 phosphorylates Net1 during mitosis and suggest that this negatively regulates its ability to signal to RhoA and alter actin cytoskeletal organization.
Subject(s)
CDC2 Protein Kinase/metabolism , Mitosis , Oncogene Proteins/metabolism , rhoA GTP-Binding Protein/metabolism , Actin Cytoskeleton , Actins/metabolism , CDC2 Protein Kinase/antagonists & inhibitors , CDC2 Protein Kinase/genetics , Cell Membrane/metabolism , HeLa Cells , Humans , Mutagenesis, Site-Directed , Oncogene Proteins/antagonists & inhibitors , Oncogene Proteins/genetics , Phosphorylation , Protein Stability , RNA Interference , RNA, Small Interfering/metabolism , Spindle Apparatus/physiology , rhoA GTP-Binding Protein/geneticsABSTRACT
Despite the dramatic advances in cancer research over the decades, effective therapeutic strategies are still urgently needed. Increasing evidence indicates that connective tissue growth factor (CTGF), a multifunctional signaling modulator, promotes cancer initiation, progression, and metastasis by regulating cell proliferation, migration, invasion, drug resistance, and epithelial-mesenchymal transition (EMT). CTGF is also involved in the tumor microenvironment in most of the nodes, including angiogenesis, inflammation, and cancer-associated fibroblast (CAF) activation. In this review, we comprehensively discuss the expression of CTGF and its regulation, oncogenic role, clinical relevance, targeting strategies, and therapeutic agents. Herein, we propose that CTGF is a promising cancer therapeutic target that could potentially improve the clinical outcomes of cancer patients.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , Connective Tissue Growth Factor/antagonists & inhibitors , Neoplasms/drug therapy , Oncogene Proteins/antagonists & inhibitors , Animals , Antibodies, Monoclonal, Humanized/pharmacology , Antibodies, Monoclonal, Humanized/therapeutic use , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Cell Line, Tumor , Cell Movement/drug effects , Cell Movement/genetics , Cell Proliferation/drug effects , Cell Proliferation/genetics , Clinical Trials as Topic , Connective Tissue Growth Factor/genetics , Connective Tissue Growth Factor/metabolism , Curcumin/pharmacology , Curcumin/therapeutic use , Disease Progression , Drug Resistance, Neoplasm/drug effects , Drug Resistance, Neoplasm/genetics , Epithelial-Mesenchymal Transition/drug effects , Epithelial-Mesenchymal Transition/genetics , Extracellular Matrix/metabolism , Gene Expression Regulation, Neoplastic , Gene Knockdown Techniques , Humans , Mice , Neoplasms/genetics , Neoplasms/mortality , Neoplasms/pathology , Oncogene Proteins/genetics , Oncogene Proteins/metabolism , Prognosis , Signal Transduction/drug effects , Survival Rate , Tumor Microenvironment/drug effects , Tumor Microenvironment/genetics , Xenograft Model Antitumor AssaysABSTRACT
This study is to investigate the inhibitory effects and mechanisms of DEK-targeting aptamer (DTA-64) on epithelial mesenchymaltransition (EMT)-mediated airway remodelling in mice and human bronchial epithelial cell line BEAS-2B. In the ovalbumin (OVA)-induced asthmatic mice, DTA-64 significantly reduced the infiltration of eosinophils and neutrophils in lung tissue, attenuated the airway resistance and the proliferation of goblet cells. In addition, DTA-64 reduced collagen deposition, transforming growth factor 1 (TGF-ß1) level in BALF and IgE levels in serum, balanced Th1/Th2/Th17 ratio, and decreased mesenchymal proteins (vimentin and α-SMA), as well as weekend matrix metalloproteinases (MMP-2 and MMP-9) and NF-κB p65 activity. In the in vitro experiments, we used TGF-ß1 to induce EMT in the human epithelial cell line BEAS-2B. DEK overexpression (ovDEK) or silencing (shDEK) up-regulated or down-regulated TGF-ß1 expression, respectively, on the contrary, TGF-ß1 exposure had no effect on DEK expression. Furthermore, ovDEK and TGF-ß1 synergistically promoted EMT, whereas shDEK significantly reduced mesenchymal markers and increased epithelial markers, thus inhibiting EMT. Additionally, shDEK inhibited key proteins in TGF-ß1-mediated signalling pathways, including Smad2/3, Smad4, p38 MAPK, ERK1/2, JNK and PI3K/AKT/mTOR. In conclusion, the effects of DTA-64 against EMT of asthmatic mice and BEAS-2B might partially be achieved through suppressing TGF-ß1/Smad, MAPK and PI3K signalling pathways. DTA-64 may be a new therapeutic option for the management of airway remodelling in asthma patients.
Subject(s)
Aptamers, Nucleotide/pharmacology , Asthma/etiology , Asthma/metabolism , Chromosomal Proteins, Non-Histone/antagonists & inhibitors , Epithelial-Mesenchymal Transition/drug effects , Oncogene Proteins/antagonists & inhibitors , Poly-ADP-Ribose Binding Proteins/antagonists & inhibitors , Signal Transduction/drug effects , Alveolar Epithelial Cells/drug effects , Alveolar Epithelial Cells/metabolism , Animals , Asthma/pathology , Basic Helix-Loop-Helix Transcription Factors/metabolism , Biomarkers , Disease Susceptibility , Epithelial-Mesenchymal Transition/genetics , Female , Gene Silencing , Humans , Immunoglobulin E/immunology , Immunoglobulin E/metabolism , Immunomodulation/drug effects , Lung/immunology , Lung/metabolism , Mice , Mitogen-Activated Protein Kinases/metabolism , NF-kappa B/metabolism , Ovalbumin/immunology , Phosphatidylinositol 3-Kinases/metabolism , Receptors, Aryl Hydrocarbon/metabolism , Smad Proteins/metabolism , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism , Transforming Growth Factor beta1/metabolismABSTRACT
Hepatocellular carcinoma (HCC) is the leading cause of tumor death in China with high mortality since its strong metastatic potential. Currently, treatment against advanced HCC is poorly efficient and thus screening new drugs to prevent the HCC invasion is of great significance to improve the survival rate of patients with HCC. From the results of this study, we concluded that propofol, a widely used anesthetics could prevent the proliferation by MTT assay. The scratch wound and invasion assays showed that migratory property and invasiveness in HCC cells SMMC-7721 was inhibited by propofol. This process was probably mediated by NET1 since NET1 overexpression offset the repressive effect of propofol on the invasiveness and migratory ability of SMMC-7721 cells. Furthermore, propofol treatment also reduced p-ERK1/2 and VEGF level by western blot analysis. Similar observation was found when NET1 was silenced. Thus, the results of this study provided valuable clinical therapy potential of propofol against liver cancer. We also disclosed molecular mechanism underlying the regulation of invasion and migration in HCC cells by NET1.
Subject(s)
Carcinoma, Hepatocellular/drug therapy , Liver Neoplasms/drug therapy , Oncogene Proteins/antagonists & inhibitors , Propofol/pharmacology , Carcinoma, Hepatocellular/pathology , Cell Line, Tumor , Cell Movement/drug effects , Cell Movement/genetics , Down-Regulation/drug effects , Drug Screening Assays, Antitumor , Gene Expression Regulation, Neoplastic/drug effects , Humans , Liver Neoplasms/pathology , MAP Kinase Signaling System/drug effects , Oncogene Proteins/genetics , Oncogene Proteins/metabolism , Propofol/therapeutic use , RNA Interference , Vascular Endothelial Growth Factor A/metabolismABSTRACT
Trefoil factors 1, 2, and 3 (TFFs) are a family of small secretory molecules involved in the protection and repair of the gastrointestinal tract (GI). TFFs maintain and restore epithelial structural integrity via transducing key signaling pathways for epithelial cell migration, proliferation, and invasion. In recent years, TFFs have emerged as key players in the pathogenesis of multiple diseases, especially cancer. Initially recognized as tumor suppressors, emerging evidence demonstrates their key role in tumor progression and metastasis, extending their actions beyond protection. However, to date, a comprehensive understanding of TFFs' mechanism of action in tumor initiation, progression and metastasis remains obscure. The present review discusses the structural, functional and mechanistic implications of all three TFF family members in tumor progression and metastasis. Also, we have garnered information from studies on their structure and expression status in different organs, along with lessons from their specific knockout in mouse models. In addition, we highlight the emerging potential of using TFFs as a biomarker to stratify tumors for better therapeutic intervention.
Subject(s)
Biomarkers, Tumor/metabolism , Neoplasms/pathology , Oncogene Proteins/metabolism , Trefoil Factors/metabolism , Tumor Suppressor Proteins/metabolism , Animals , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Biomarkers, Tumor/agonists , Biomarkers, Tumor/analysis , Biomarkers, Tumor/antagonists & inhibitors , Cell Transformation, Neoplastic/drug effects , Cell Transformation, Neoplastic/pathology , Disease Models, Animal , Disease Progression , Disease-Free Survival , Epigenesis, Genetic/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Humans , Mice , Mucous Membrane/metabolism , Neoplasm Staging , Neoplasms/drug therapy , Neoplasms/genetics , Neoplasms/mortality , Oncogene Proteins/analysis , Oncogene Proteins/antagonists & inhibitors , Prognosis , Protein Domains , Trefoil Factors/agonists , Trefoil Factors/analysis , Trefoil Factors/antagonists & inhibitors , Tumor Suppressor Proteins/agonists , Tumor Suppressor Proteins/analysisABSTRACT
Heterotrimeric G proteins are the core upstream elements that transduce and amplify the cellular signals from G protein-coupled receptors (GPCRs) to intracellular effectors. GPCRs are the largest family of membrane proteins encoded in the human genome and are the targets of about one-third of prescription medicines. However, to date, no single therapeutic agent exerts its effects via perturbing heterotrimeric G protein function, despite a plethora of evidence linking G protein malfunction to human disease. Several recent studies have brought to light that the Gq family-specific inhibitor FR900359 (FR) is unexpectedly efficacious in silencing the signaling of Gq oncoproteins, mutant Gq variants that mostly exist in the active state. These data not only raise the hope that researchers working in drug discovery may be able to potentially strike Gq oncoproteins from the list of undruggable targets, but also raise questions as to how FR achieves its therapeutic effect. Here, we place emphasis on these recent studies and explain why they expand our pharmacological armamentarium for targeting Gq protein oncogenes as well as broaden our mechanistic understanding of Gq protein oncogene function. We also highlight how this novel insight impacts the significance and utility of using G(q) proteins as targets in drug discovery efforts.
Subject(s)
Antineoplastic Agents/pharmacology , Depsipeptides/pharmacology , GTP-Binding Protein alpha Subunits, Gq-G11/antagonists & inhibitors , Oncogene Proteins/antagonists & inhibitors , Animals , Antineoplastic Agents/chemistry , Depsipeptides/chemistry , Drug Discovery/methods , GTP-Binding Protein alpha Subunits, Gq-G11/metabolism , Humans , Oncogene Proteins/metabolism , Signal TransductionABSTRACT
Cholangiocarcinoma (CCA) is a malignant tumor that arises from the epithelial cells of the bile duct and is notorious for its poor prognosis. The clinical outcome remains disappointing, and thus more effective therapeutic options are urgently required. Cordycepin, a traditional Chinese medicine, provides multiple pharmacological strategies in antitumors, but its mechanisms have not been fully elucidated. In this study, we reported that cordycepin inhibited the viability and proliferation capacity of CCA cells in a time- and dose-dependent manner determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and colony formation assay. Flow cytometry and Hoechst dye showed that cordycepin induced cancer cell apoptosis via extracellular signal-regulated kinase (ERK) 1/2 deactivation. Moreover, cordycepin significantly reduced the angiogenetic capabilities of CCA in vitro as examined by tube formation assay. We also discovered that cordycepin inhibited DEK expression by using Western blot assay. DEK serves as an oncogenic protein that is overexpressed in various gastrointestinal tumors. DEK silencing inhibited CCA cell viability and angiogenesis but not apoptosis induction determined by Western blot and flow cytometry. Furthermore, cordycepin significantly inhibited tumor growth and angiogenic capacities in a xenograft model by downregulating the expression of DEK, phosphorylated ERK1/2 CD31 and von Willebrand factor (vWF). Taken together, we demonstrated that cordycepin inhibited CCA cell proliferation and angiogenesis with a DEK interaction via downregulation in ERK signaling. These data indicate that cordycepin may serve as a novel agent for CCA clinical treatment and prognosis improvement. SIGNIFICANCE STATEMENT: Cordycepin provides multiple strategies in antitumors, but its mechanisms are not fully elucidated, especially on cholangiocarcinoma (CCA). We reported that cordycepin inhibited the viability of CCA cells, induced apoptosis via extracellular signal-regulated kinase 1/2 deactivation and DEK inhibition, and reduced the angiogenetic capabilities of CCA both in vivo and in vitro.
Subject(s)
Bile Duct Neoplasms/drug therapy , Cholangiocarcinoma/drug therapy , Chromosomal Proteins, Non-Histone/antagonists & inhibitors , Deoxyadenosines/pharmacology , Extracellular Signal-Regulated MAP Kinases/physiology , MAP Kinase Signaling System/drug effects , Neovascularization, Pathologic/prevention & control , Oncogene Proteins/antagonists & inhibitors , Poly-ADP-Ribose Binding Proteins/antagonists & inhibitors , Animals , Bile Duct Neoplasms/pathology , Cell Line, Tumor , Cell Proliferation/drug effects , Cholangiocarcinoma/pathology , Humans , Male , Mice , Xenograft Model Antitumor AssaysABSTRACT
A critical problem in the fight against bacterial infection is the rising rates of resistance and the lack of new antibiotics. The discovery of new targets or new antibacterial mechanisms is a potential solution but is becoming more difficult. Here we report an antibacterial mechanism that safeguards intestine cells from enteropathogenic Escherichia coli (EPEC) by shutting down an infection-responsive signal of the host intestine cell. A key step in EPEC infection of intestinal cells involves Tir-induced actin reorganization. Nck mediates this event by binding with Tir through its SH2 domain (Nck-SH2) and with WIP through its second SH3 domain (Nck-SH3.2). Here we report the design of a synthetic peptide that reacts precisely with a unique cysteine of the Nck-SH3.2 domain, blocks the binding site of the Nck protein, and prevents EPEC infection of Caco-2 cells. Oral update of this nontoxic peptide before EPEC administration safeguards mice from EPEC infection and diarrhea. This study demonstrates domain-specific blockage of an SH3 domain of a multidomain adaptor protein inside cells and the inhibition of Tir-induced rearrangement of the host actin cytoskeleton as a previously unknown antibacterial mechanism.
Subject(s)
Adaptor Proteins, Signal Transducing/antagonists & inhibitors , Antimicrobial Cationic Peptides/pharmacology , Enteropathogenic Escherichia coli/drug effects , Escherichia coli Infections/prevention & control , Escherichia coli Proteins/antagonists & inhibitors , Host-Pathogen Interactions/drug effects , Oncogene Proteins/antagonists & inhibitors , Receptors, Cell Surface/antagonists & inhibitors , Actins/metabolism , Adaptor Proteins, Signal Transducing/chemistry , Adaptor Proteins, Signal Transducing/metabolism , Animals , Antimicrobial Cationic Peptides/therapeutic use , Caco-2 Cells , Enteropathogenic Escherichia coli/metabolism , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Humans , Intestinal Mucosa/metabolism , Intestinal Mucosa/microbiology , Mice , Mice, Inbred C57BL , Oncogene Proteins/chemistry , Oncogene Proteins/metabolism , Protein Binding , Receptors, Cell Surface/chemistry , Receptors, Cell Surface/metabolism , Signal Transduction , src Homology DomainsABSTRACT
AIMS: To investigate the effects of malignant fibrous histiocytoma amplified sequence 1 (MFHAS1) on cognitive dysfunction, the expression of tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß and amyloid ß peptide (Aß) in the hippocampus, as well as dendritic pathology in the hippocampal CA1 region in sepsis-associated encephalopathy (SAE) rats. MAIN METHODS: The rats were randomly divided into four groups: 1) control group (subjected to sham surgery), 2) control plus Mfhas1 siRNA group (rats received intracerebroventricular injection of Mfhas1 siRNA after sham surgery), 3) CLP plus control siRNA group (rats received intracerebroventricular injection of control siRNA after cecal ligation and puncture (CLP)), 4) CLP plus Mfhas1 siRNA group (rats received intracerebroventricular injection of Mfhas1 siRNA after CLP). The learning and memory capabilities of the rats were examined by means of fear conditioning and Barnes maze test. The concentration of TNF-α and IL-1ß was determined by enzyme-linked immunosorbent assay. The efficiency of siRNA transfection, MFHAS1 and Aß expression were detected by Western blotting. Total branch lengths of pyramidal dendrites of the CA1 basilar trees and spine density were determined by Golgi staining. KEY FINDINGS: We observed that MFHAS1 knock-down by Mfhas1 siRNA intracerebroventricular injection could improve cognitive impairment, reduce the expression of TNF-α, IL-1ß and Aß in the hippocampus induced by CLP, and alleviate the dendritic spinal loss of the pyramidal neurons, as well as increase the dendritic branching of the CA1 basilar trees of septic rats. SIGNIFICANCE: MFHAS1 knock-down can alleviate cognitive impairment, neuroinflammation and dendritic spinal loss in SAE rats.
Subject(s)
Cognitive Dysfunction/prevention & control , Dendrites/drug effects , Disease Models, Animal , Hippocampus/drug effects , Oncogene Proteins/antagonists & inhibitors , RNA, Small Interfering/administration & dosage , Sepsis-Associated Encephalopathy/complications , Animals , Cognitive Dysfunction/etiology , Cognitive Dysfunction/metabolism , Cognitive Dysfunction/pathology , Dendrites/metabolism , Dendrites/pathology , Hippocampus/metabolism , Hippocampus/pathology , Male , Oncogene Proteins/genetics , Oncogene Proteins/metabolism , RNA, Small Interfering/genetics , Rats , Rats, WistarABSTRACT
BACKGROUND: Metformin has been reported to function as the anti-tumor inhibiting the growth of different types of cancers, including bladder cancer. But there are few reports on the roles of Yap1, the key molecule of Hippo pathway, in the metformin induced inhibition of bladder cancer (BLCA). We are wondering if the inhibitory effect of metformin on bladder cancer is fulfilled via Yap1 and exploring the related mechanism. METHODS: MTS and colony formation assays were used to explore the cellular viabilities and proliferation of BLCA cells challenged by metformin at different concentrations, in vitro. Flow Cytometry (FCM) was used to analyze the cell cycle and the cellular apoptosis of the BLCA cells. Western Blot was performed to detect the expressions of AMPKα, Yap1, CCND1, CCNE1/2 and CDK2/4/6 in the metformin-treated BLCA cell lines. RNAi method was used for the related genetic functional analysis. The relationships among Yap1, TEADs and CCNE1/2 were predicted and evaluated using bioinformatics, dual-luciferase reporter and co-immunoprecipitation (Co-IP) assays. For in vivo experiments, a xenograft model was used to investigate the effects of metformin on the proliferation of BLCA cells. And Immunohistochemistry (IHC) assay was performed to assess the expressions of CCNE1/2 and Yap1 proteins in the tumor tissues from the model. RESULTS: Metformin could inhibit the proliferation of the BLCA cells via inducing the G1 cell cycle arrest without apoptosis. And metformin upregulated the phosphorylated AMPKα and decreased the expressions of Yap1 and CCND1, CCNE1/2 and CDK4/6. AMPK inhibition by compound C (CC) restored the cell proliferation and the G1 cell cycle arrest induced by metformin, in vivo. Knockdown of YAP1 inhibited the proliferation of BLCA cells and caused the cell cycle arrest at G1 phase by decreasing the expressions of CCNE1/2 and other G1 phase related molecules, which has been restored by the Yap 5SA mutant. Bioinformatics analysis showed that trans-factor TEAD4 was highly expressed and positively associated with the expressions of CCNE1 and CCNE2 in BLCA and only TEAD4 was precipitated by Yap1 in the BLCA cells. Further studies demonstrated that Yap1 positively regulated both CCNE1 and CCNE2 expressions via forming complex with TEAD4. Furthermore, we observed that metformin inhibited the cell proliferation by decreasing the expressions of Yap1 and both CCNE1 and CCNE2 in xenograft model. CONCLUSIONS: The results of our study reveal a new potential regulatory pathway in which metformin inhibits cell proliferation via AMPKα/Yap1/TEAD4/CCNE1/2 axis in BLCA cells, providing new insights into novel molecular therapeutic targets for BLCA.
Subject(s)
AMP-Activated Protein Kinases/metabolism , Adaptor Proteins, Signal Transducing/antagonists & inhibitors , Cyclin E/antagonists & inhibitors , Cyclins/antagonists & inhibitors , DNA-Binding Proteins/metabolism , Metformin/pharmacology , Muscle Proteins/metabolism , Oncogene Proteins/antagonists & inhibitors , Transcription Factors/antagonists & inhibitors , Transcription Factors/metabolism , Urinary Bladder Neoplasms/drug therapy , Adaptor Proteins, Signal Transducing/biosynthesis , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Animals , Cell Cycle Checkpoints/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Cyclin E/biosynthesis , Cyclin E/genetics , Cyclin E/metabolism , Cyclins/biosynthesis , Cyclins/genetics , Cyclins/metabolism , DNA-Binding Proteins/genetics , Female , G1 Phase/drug effects , Heterografts , Humans , Mice , Mice, Inbred BALB C , Mice, Nude , Muscle Proteins/genetics , Oncogene Proteins/biosynthesis , Oncogene Proteins/genetics , Oncogene Proteins/metabolism , TEA Domain Transcription Factors , Transcription Factors/biosynthesis , Transcription Factors/genetics , Transfection , Urinary Bladder Neoplasms/genetics , Urinary Bladder Neoplasms/metabolism , Urinary Bladder Neoplasms/pathology , YAP-Signaling ProteinsABSTRACT
BACKGROUND: To clarify the effects of cylcin E1 expression on HCC tumor progression, we studied the expression of cyclin E1 and inhibitory efficacy of regorafenib and sorafenib in HCC cells, and investigated a potential therapy that combines regorafenib treatment with cyclin E1 inhibition. METHODS: Western blotting for caspase-3 and Hoechst 33225 staining was used to measure the expression level of apoptosis-related proteins under drug treatment. RESULTS: Our results showed that enhanced expression of cyclin E1 after transfection compromised apoptosis in HCC cells induced by regorafenib or sorafenib. Conversely, down-regulation of cyclin E1 gene expression or inhibition of cyclin E1 by the cyclin-dependent kinase (CDK) inhibitors dinaciclib (DIN) or flavopiridol sensitized HCC cells to regorafenib and sorafenib by inducing apoptosis. The expression of Mcl-1, which is modulated by STAT3, plays a key role in regulating the therapeutic effects of CDK inhibitors. Xenograft experiments conducted to test the efficacy of regorafenib combined with DIN showed dramatic tumor inhibitory effects due to induction of apoptosis. Our results suggested that the level of cyclin E1 expression in HCCs may be used as a pharmacodynamic biomarker to assess the antitumor effects of regorafenib or sorafenib. CONCLUSIONS: Combining regorafenib and CDK inhibitors may enhance the clinical efficiency of the treatment of HCCs.