ABSTRACT
Hepatocellular carcinoma (HCC)-the most common form of liver cancer-is an aggressive malignancy with few effective treatment options1. Lenvatinib is a small-molecule inhibitor of multiple receptor tyrosine kinases that is used for the treatment of patients with advanced HCC, but this drug has only limited clinical benefit2. Here, using a kinome-centred CRISPR-Cas9 genetic screen, we show that inhibition of epidermal growth factor receptor (EGFR) is synthetic lethal with lenvatinib in liver cancer. The combination of the EGFR inhibitor gefitinib and lenvatinib displays potent anti-proliferative effects in vitro in liver cancer cell lines that express EGFR and in vivo in xenografted liver cancer cell lines, immunocompetent mouse models and patient-derived HCC tumours in mice. Mechanistically, inhibition of fibroblast growth factor receptor (FGFR) by lenvatinib treatment leads to feedback activation of the EGFR-PAK2-ERK5 signalling axis, which is blocked by EGFR inhibition. Treatment of 12 patients with advanced HCC who were unresponsive to lenvatinib treatment with the combination of lenvatinib plus gefitinib (trial identifier NCT04642547) resulted in meaningful clinical responses. The combination therapy identified here may represent a promising strategy for the approximately 50% of patients with advanced HCC who have high levels of EGFR.
Subject(s)
Antineoplastic Agents/pharmacology , Carcinoma, Hepatocellular/metabolism , Liver Neoplasms/metabolism , Phenylurea Compounds/pharmacology , Quinolines/pharmacology , Animals , Carcinoma, Hepatocellular/drug therapy , Cell Line, Tumor , Drug Resistance, Neoplasm , ErbB Receptors/antagonists & inhibitors , ErbB Receptors/metabolism , Female , Gefitinib/pharmacology , Humans , Liver Neoplasms/drug therapy , Male , Mice , Mice, Inbred BALB C , Mice, Nude , Receptors, Fibroblast Growth Factor , Signal Transduction , Xenograft Model Antitumor AssaysABSTRACT
BACKGROUND & AIMS: Despite the increasing number of treatment options available for liver cancer, only a small proportion of patients achieve long-term clinical benefits. Here, we aim to develop new therapeutic approaches for liver cancer. METHODS: A compound screen was conducted to identify inhibitors that could synergistically induce senescence when combined with cyclin-dependent kinase (CDK) 4/6 inhibitor. The combination effects of CDK4/6 inhibitor and exportin 1 (XPO1) inhibitor on cellular senescence were investigated in a panel of human liver cancer cell lines and multiple liver cancer models. A senolytic drug screen was performed to identify drugs that selectively killed senescent liver cancer cells. RESULTS: The combination of CDK4/6 inhibitor and XPO1 inhibitor synergistically induces senescence of liver cancer cells in vitro and in vivo. The XPO1 inhibitor acts by causing accumulation of RB1 in the nucleus, leading to decreased E2F signaling and promoting senescence induction by the CDK4/6 inhibitor. Through a senolytic drug screen, cereblon (CRBN)-based proteolysis targeting chimera (PROTAC) ARV-825 was identified as an agent that can selectively kill senescent liver cancer cells. Up-regulation of CRBN was a vulnerability of senescent liver cancer cells, making them sensitive to CRBN-based PROTAC drugs. Mechanistically, we find that ubiquitin specific peptidase 2 (USP2) directly interacts with CRBN, leading to the deubiquitination and stabilization of CRBN in senescent liver cancer cells. CONCLUSIONS: Our study demonstrates a striking synergy in senescence induction of liver cancer cells through the combination of CDK4/6 inhibitor and XPO1 inhibitor. These findings also shed light on the molecular processes underlying the vulnerability of senescent liver cancer cells to CRBN-based PROTAC therapy.
Subject(s)
Adaptor Proteins, Signal Transducing , Cellular Senescence , Cyclin-Dependent Kinase 4 , Cyclin-Dependent Kinase 6 , Exportin 1 Protein , Karyopherins , Liver Neoplasms , Protein Kinase Inhibitors , Receptors, Cytoplasmic and Nuclear , Ubiquitin-Protein Ligases , Humans , Cellular Senescence/drug effects , Cyclin-Dependent Kinase 6/antagonists & inhibitors , Cyclin-Dependent Kinase 6/metabolism , Cyclin-Dependent Kinase 4/antagonists & inhibitors , Cyclin-Dependent Kinase 4/metabolism , Karyopherins/antagonists & inhibitors , Karyopherins/metabolism , Receptors, Cytoplasmic and Nuclear/antagonists & inhibitors , Receptors, Cytoplasmic and Nuclear/metabolism , Ubiquitin-Protein Ligases/metabolism , Liver Neoplasms/drug therapy , Liver Neoplasms/pathology , Liver Neoplasms/metabolism , Cell Line, Tumor , Protein Kinase Inhibitors/pharmacology , Adaptor Proteins, Signal Transducing/metabolism , Adaptor Proteins, Signal Transducing/antagonists & inhibitors , Animals , Retinoblastoma Binding Proteins/metabolism , Retinoblastoma Binding Proteins/genetics , Drug Synergism , Senotherapeutics/pharmacology , Xenograft Model Antitumor Assays , Signal Transduction/drug effects , Proteolysis/drug effects , Hydrazines/pharmacology , Hydrazines/therapeutic use , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Hep G2 Cells , Mice , Piperazines , Pyridines , TriazolesABSTRACT
Liver cancer remains difficult to treat, owing to a paucity of drugs that target critical dependencies1,2; broad-spectrum kinase inhibitors such as sorafenib provide only a modest benefit to patients with hepatocellular carcinoma3. The induction of senescence may represent a strategy for the treatment of cancer, especially when combined with a second drug that selectively eliminates senescent cancer cells (senolysis)4,5. Here, using a kinome-focused genetic screen, we show that pharmacological inhibition of the DNA-replication kinase CDC7 induces senescence selectively in liver cancer cells with mutations in TP53. A follow-up chemical screen identified the antidepressant sertraline as an agent that kills hepatocellular carcinoma cells that have been rendered senescent by inhibition of CDC7. Sertraline suppressed mTOR signalling, and selective drugs that target this pathway were highly effective in causing the apoptotic cell death of hepatocellular carcinoma cells treated with a CDC7 inhibitor. The feedback reactivation of mTOR signalling after its inhibition6 is blocked in cells that have been treated with a CDC7 inhibitor, which leads to the sustained inhibition of mTOR and cell death. Using multiple in vivo mouse models of liver cancer, we show that treatment with combined inhibition of of CDC7 and mTOR results in a marked reduction of tumour growth. Our data indicate that exploiting an induced vulnerability could be an effective treatment for liver cancer.
Subject(s)
Apoptosis/drug effects , Cellular Senescence/drug effects , Liver Neoplasms/drug therapy , Liver Neoplasms/pathology , Molecular Targeted Therapy , Sertraline/pharmacology , Animals , Cell Cycle Proteins/antagonists & inhibitors , Cell Line, Tumor , Disease Models, Animal , Female , Liver Neoplasms/genetics , Liver Neoplasms/metabolism , Male , Mice , Mice, Inbred BALB C , Mutation , Protein Serine-Threonine Kinases/antagonists & inhibitors , Sertraline/therapeutic use , Signal Transduction/drug effects , TOR Serine-Threonine Kinases/antagonists & inhibitors , Tumor Suppressor Protein p53/geneticsABSTRACT
Polypyrimidine tract-binding protein 1 (PTBP1) plays an essential role in splicing and post-transcriptional regulation. Moreover, PTBP1 has been implicated as a causal factor in tumorigenesis. However, the involvement of PTBP1 in cellular senescence, a key biological process in aging and cancer suppression, remains to be clarified. Here, it is shown that PTBP1 is associated with the facilitation of tumor growth and the prognosis in lung adenocarcinoma (LUAD). PTBP1 exhibited significantly increased expression in various cancer types including LUAD and showed consistently decreased expression in multiple cellular senescence models. Suppression of PTBP1 induced cellular senescence in LUAD cells. In terms of molecular mechanisms, the silencing of PTBP1 enhanced the skipping of exon 3 in F-box protein 5 (FBXO5), resulting in the generation of a less stable RNA splice variant, FBXO5-S, which subsequently reduces the overall FBXO5 expression. Additionally, downregulation of FBXO5 was found to induce senescence in LUAD. Collectively, these findings illustrate that PTBP1 possesses an oncogenic function in LUAD through inhibiting senescence, and that targeting aberrant splicing mediated by PTBP1 has therapeutic potential in cancer treatment.
ABSTRACT
Low-level light therapy (LLLT), also known as photo biomodulation (PBM), is a type of optical therapy that uses red or near-infrared lasers or light-emitting diodes (LEDs) for medical treatment. The laser wavelengths involved in PBM typically range between 600-700 nm and 780-1100 nm, with power densities ranging between 5 mW/cm2 and 5 W/cm2. PBM is a series of biochemical cascades exhibited by biological tissues after absorbing a certain amount of energy from light. PBM has been widely used in clinical practice in the past 20 years, and numerous clinical trials have demonstrated its biological efficacy. However, the underlying mechanisms have not yet been fully explored. In this paper, we have summarized the research into PBM over the past two decades, to identify the important mechanisms of the biological effects of PBM from the perspective of molecular mechanisms, cellular levels, and tissue changes. We hope our study provide a theoretical basis for future investigations into the underlying mechanisms.
Subject(s)
Lasers , Low-Level Light Therapy , LightABSTRACT
Almost all currently approved systemic therapies for hepatocellular carcinoma (HCC) failed to achieve satisfactory therapeutic effect. Exploring tailored treatment strategies for different individuals provides an approach with the potential to maximize clinical benefit. Previously, multiple studies have reported that hepatoma cell lines belonging to different molecular subtypes respond differently to the same treatment. However, these studies only focused on a small number of typical chemotherapy or targeted drugs across limited cell lines due to time and cost constraints. To compensate for the deficiency of previous experimental researches as well as link molecular classification with therapeutic response, we conducted a comprehensive in silico screening, comprising nearly 2000 compounds, to identify compounds with subclass-specific efficacy. Here, we first identified two transcriptome-based HCC subclasses (AS1 and AS2) and then made comparison of drug response between two subclasses. As a result, we not only found that some agents previously considered to have low efficacy in HCC treatment might have promising therapeutic effects for certain subclass, but also identified novel therapeutic compounds that were not routinely used as anti-tumor drugs in clinic. Discovery of agents with subclass-specific efficacy has potential in changing the status quo of population-based therapies in HCC and providing new insights into precision oncology.
Subject(s)
Antineoplastic Agents/therapeutic use , Carcinoma, Hepatocellular , Gene Expression Regulation, Neoplastic , Liver Neoplasms , Transcriptome , Antineoplastic Agents/classification , Carcinoma, Hepatocellular/drug therapy , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/metabolism , Drug Screening Assays, Antitumor , Humans , Liver Neoplasms/drug therapy , Liver Neoplasms/genetics , Liver Neoplasms/metabolism , Precision MedicineABSTRACT
BACKGROUND: In recent years, the application of functional genetic immuno-oncology screens has showcased the striking ability to identify potential regulators engaged in tumor-immune interactions. Although these screens have yielded substantial data, few studies have attempted to systematically aggregate and analyze them. METHODS: In this study, a comprehensive data collection of tumor immunity-associated functional screens was performed. Large-scale genomic data sets were exploited to conduct integrative analyses. RESULTS: We identified 105 regulator genes that could mediate resistance or sensitivity to immune cell-induced tumor elimination. Further analysis identified MON2 as a novel immune-oncology target with considerable therapeutic potential. In addition, based on the 105 genes, a signature named CTIS (CRISPR screening-based tumor-intrinsic immune score) for predicting response to immune checkpoint blockade (ICB) and several immunomodulatory agents with the potential to augment the efficacy of ICB were also determined. CONCLUSION: Overall, our findings provide insights into immune oncology and open up novel opportunities for improving the efficacy of current immunotherapy agents.
Subject(s)
CRISPR-Cas Systems , Clustered Regularly Interspaced Short Palindromic Repeats , Genetic Testing/methods , Genomics/methods , Medical Oncology , Antineoplastic Agents, Immunological/administration & dosage , Antineoplastic Agents, Immunological/adverse effects , Antineoplastic Agents, Immunological/therapeutic use , Clinical Decision-Making , Computational Biology/methods , Disease Management , Gene Expression Profiling , Gene Regulatory Networks , Humans , Immunotherapy/methods , Immunotherapy/standards , Medical Oncology/methods , Medical Oncology/standards , Prognosis , Transcriptome , Treatment OutcomeABSTRACT
BACKGROUND AND AIMS: Peroxisome proliferator-activated receptor-gamma (PPARγ) coactivator-1α (PGC1α) is a key regulator of mitochondrial biogenesis and respiration. PGC1α is involved in the carcinogenesis, progression, and metabolic state of cancer. However, its role in the progression of hepatocellular carcinoma (HCC) remains unclear. APPROACH AND RESULTS: In this study, we observed that PGC1α was down-regulated in human HCC. A clinical study showed that low levels of PGC1α expression were correlated with poor survival, vascular invasion, and larger tumor size. PGC1α inhibited the migration and invasion of HCC cells with both in vitro experiments and in vivo mouse models. Mechanistically, PGC1α suppressed the Warburg effect through down-regulation of pyruvate dehydrogenase kinase isozyme 1 (PDK1) mediated by the WNT/ß-catenin pathway, and inhibition of the WNT/ß-catenin pathway was induced by activation of PPARγ. CONCLUSIONS: Low levels of PGC1α expression indicate a poor prognosis for HCC patients. PGC1α suppresses HCC metastasis by inhibiting aerobic glycolysis through regulating the WNT/ß-catenin/PDK1 axis, which depends on PPARγ. PGC1α is a potential factor for predicting prognosis and a therapeutic target for HCC patients.
Subject(s)
Biomarkers, Tumor/metabolism , Carcinoma, Hepatocellular/secondary , Liver Neoplasms/pathology , Lung Neoplasms/secondary , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha/metabolism , Biomarkers, Tumor/blood , Carcinogenesis/genetics , Carcinogenesis/pathology , Carcinoma, Hepatocellular/blood , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/mortality , Cell Line, Tumor , Cell Movement/genetics , Cell Proliferation/genetics , Disease Progression , Female , Gene Expression Regulation, Neoplastic , Humans , Liver/pathology , Liver/surgery , Liver Neoplasms/blood , Liver Neoplasms/genetics , Liver Neoplasms/mortality , Male , Middle Aged , Neoplasm Invasiveness/genetics , Neoplasm Invasiveness/pathology , PPAR gamma/metabolism , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha/blood , Prognosis , Pyruvate Dehydrogenase Acetyl-Transferring Kinase/metabolism , Warburg Effect, Oncologic , Wnt Signaling Pathway/genetics , Xenograft Model Antitumor AssaysABSTRACT
OBJECTIVES: Hepatocellular carcinoma (HCC) is one of the most frequent malignancies and a major leading cause of cancer-related deaths worldwide. Several therapeutic options like sorafenib and regorafenib provide only modest survival benefit to patients with HCC. This study aims to identify novel druggable candidate genes for patients with HCC. DESIGN: A non-biased CRISPR (clustered regularly interspaced short palindromic repeats) loss-of-function genetic screen targeting all known human kinases was performed to identify vulnerabilities of HCC cells. Whole-transcriptome sequencing (RNA-Seq) and bioinformatics analyses were performed to explore the mechanisms of the action of a cyclin-dependent kinase 12 (CDK12) inhibitor in HCC cells. Multiple in vitro and in vivo assays were used to study the synergistic effects of the combination of CDK12 inhibition and sorafenib. RESULTS: We identify CDK12 as critically required for most HCC cell lines. Suppression of CDK12 using short hairpin RNAs (shRNAs) or its inhibition by the covalent small molecule inhibitor THZ531 leads to robust proliferation inhibition. THZ531 preferentially suppresses the expression of DNA repair-related genes and induces strong DNA damage response in HCC cell lines. The combination of THZ531 and sorafenib shows striking synergy by inducing apoptosis or senescence in HCC cells. The synergy between THZ531 and sorafenib may derive from the notion that THZ531 impairs the adaptive responses of HCC cells induced by sorafenib treatment. CONCLUSION: Our data highlight the potential of CDK12 as a drug target for patients with HCC. The striking synergy of THZ531 and sorafenib suggests a potential combination therapy for this difficult to treat cancer.
Subject(s)
Anilides/pharmacology , Antineoplastic Agents/pharmacology , Carcinoma, Hepatocellular/pathology , Cyclin-Dependent Kinases/antagonists & inhibitors , Liver Neoplasms/pathology , Pyrimidines/pharmacology , Sorafenib/pharmacology , Carcinoma, Hepatocellular/drug therapy , Cell Culture Techniques , Cell Line, Tumor , Humans , Liver Neoplasms/drug therapyABSTRACT
Metastasis is the main reason for high recurrence and poor survival of hepatocellular carcinoma (HCC). The molecular mechanism underlying HCC metastasis remains unclear. In this study, we found that argininosuccinate synthase 1 (ASS1) expression was significantly decreased and down-regulation of ASS1 was closely correlated with poor prognosis in HCC patients. DNA methylation led to the down-regulation of ASS1 in HCC. Stable silencing of ASS1 promoted migration and invasion of HCC cells, whereas overexpression of ASS1-inhibited metastasis of HCC cells in vivo and in vitro. We also revealed that ASS1-knockdown increased the phosphorylation level of S727STAT3, which contributed to HCC metastasis by up-regulation of inhibitor of differentiation 1 (ID1). These findings indicate that ASS1 inhibits HCC metastasis and may serve as a target for HCC diagnosis and treatment.
Subject(s)
Argininosuccinate Synthase/physiology , Carcinoma, Hepatocellular/pathology , Liver Neoplasms/pathology , STAT3 Transcription Factor/antagonists & inhibitors , Animals , Argininosuccinate Synthase/antagonists & inhibitors , Argininosuccinate Synthase/genetics , Carcinoma, Hepatocellular/enzymology , Cell Line, Tumor , Cell Movement , DNA Methylation , Humans , Inhibitor of Differentiation Protein 1/genetics , Liver Neoplasms/enzymology , Mice , Neoplasm Invasiveness , Neoplasm Metastasis/prevention & control , STAT3 Transcription Factor/physiology , Signal Transduction/physiologyABSTRACT
BACKGROUND & AIMS: Treatment of liver cancer remains challenging because of a paucity of drugs that target critical dependencies. Sorafenib is a multikinase inhibitor that is approved as the standard therapy for patients with advanced hepatocellular carcinoma, but it only provides limited survival benefit. In this study we aimed to identify potential combination therapies to improve the clinical response to sorafenib. METHODS: To investigate the cause of the limited therapeutic effect of sorafenib, we performed a CRISPR-Cas9 based synthetic lethality screen to search for kinases whose knockout synergizes with sorafenib. Synergistic effects of sorafenib and selumetinib on cell apoptosis and phospho-ERK (p-ERK) were analyzed by caspase-3/7 apoptosis assay and western blot, respectively. p-ERK was measured by immunochemical analysis using a tissue microarray containing 78 liver cancer specimens. The in vivo effects of the combination were also measured in two xenograft models. RESULT: We found that suppression of ERK2 (MAPK1) sensitizes several liver cancer cell lines to sorafenib. Drugs inhibiting the MEK (MEK1/2 [MAP2K1/2]) or ERK (ERK1/2 [MAPK1/3]) kinases reverse unresponsiveness to sorafenib in vitro and in vivo in a subset of liver cancer cell lines characterized by high levels of active p-ERK, through synergistic inhibition of ERK kinase activity. CONCLUSION: Our data provide a combination strategy for treating liver cancer and suggest that tumors with high basal p-ERK levels, which are seen in approximately 30% of liver cancers, are most likely to benefit from such combinatorial treatment. LAY SUMMARY: Sorafenib is approved as the standard therapy for patients with advanced hepatocellular carcinoma, but only provides limited survival benefit. Herein, we found that inhibition of the kinase ERK2 increases the response to sorafenib in liver cancer. Our data indicate that a combination of sorafenib and a MEK inhibitor is most likely to be effective in tumors with high basal phospho-ERK levels.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Extracellular Signal-Regulated MAP Kinases/metabolism , Liver Neoplasms/drug therapy , Mitogen-Activated Protein Kinase Kinases/antagonists & inhibitors , Sorafenib/administration & dosage , Biomarkers , Drug Synergism , Extracellular Signal-Regulated MAP Kinases/antagonists & inhibitors , Humans , PhosphorylationABSTRACT
BACKGROUND & AIMS: Individuals with Down syndrome have a low risk for many solid tumors, prompting the search for tumor suppressor genes on human chromosome 21 (HSA21). We aimed to identify and explore potential mechanisms of tumor suppressors on HSA21 in hepatocellular carcinoma (HCC). METHODS: We compared expression of HSA21 genes in 14 pairs of primary HCC and adjacent noncancer liver tissues using the Affymetrix HG-U133 Plus 2.0 array (Affymetrix, Santa Clara, CA). HCC tissues and adjacent normal liver tissues were collected from 108 patients at a hospital in China for real-time polymerase chain reaction and immunohistochemical analyses; expression levels of regulator of calcineurin 1 (RCAN1) isoform 4 (RCAN1.4) were associated with clinical features. We overexpressed RCAN1.4 from lentiviral vectors in MHCC97H and HCCLM3 cells and knocked expression down using small interfering RNAs in SMMC7721 and Huh7 cells. Cells were analyzed in proliferation, migration, and invasion assays. HCC cells that overexpressed RCAN1.4 or with RCAN1.4 knockdown were injected into livers or tail veins of nude mice; tumor growth and numbers of lung metastases were quantified. We performed bisulfite pyrosequencing and methylation-specific polymerase chain reaction analyses to analyze CpG island methylation. We measured phosphatase activity of calcineurin in HCC cells. RESULTS: RCAN1.4 mRNA and protein levels were significantly decreased in primary HCC compared with adjacent noncancer liver tissues. Reduced levels of RCAN1.4 mRNA were significantly associated with advanced tumor stages, poor differentiation, larger tumor size, and vascular invasion. Kaplan-Meier survival analysis showed that patients with HCCs with lower levels of RCAN1.4 mRNA had shorter time of overall survival and time to recurrence than patients whose tumors had high levels of RCAN1.4 mRNA. In HCC cell lines, expression of RCAN1.4 significantly reduced proliferation, migration, and invasive activity. HCC cells that overexpressed RCAN1.4 formed smaller xenograft tumors, with fewer metastases and blood vessels, than control HCC cells. In HCC cells, RCAN1.4 inhibited expression of insulin-like growth factor 1 and vascular endothelial growth factor A by reducing calcineurin activity and blocking nuclear translocation of nuclear factor of activated T cells (NFAT1). HCC cells incubated with the calcineurin inhibitor cyclosporin A had decreased nuclear level of NFAT1. HCC cells had hypermethylation of a CpG island in the 5' regulatory region of RCAN1.4, which reduced its expression. CONCLUSIONS: RCAN1.4 is down-regulated in HCC tissues, compared with non-tumor liver tissues. RCAN1.4 prevents cell proliferation, migration, and invasion in vitro; overexpressed RCAN1.4 in HCC cells prevents growth, angiogenesis, and metastases of xenograft tumors by inhibiting calcineurin activity and nuclear translocation of NFAT1.
Subject(s)
Calcineurin/metabolism , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/pathology , Intracellular Signaling Peptides and Proteins/genetics , Liver Neoplasms/genetics , Liver Neoplasms/pathology , Muscle Proteins/genetics , NFATC Transcription Factors/metabolism , RNA, Messenger/analysis , Adult , Aged , Animals , Carcinoma, Hepatocellular/chemistry , Carcinoma, Hepatocellular/secondary , Cell Line, Tumor , Cell Movement , Cell Proliferation , Chromosomes, Human, Pair 21 , CpG Islands/genetics , DNA Methylation , DNA-Binding Proteins , Disease-Free Survival , Down-Regulation , Female , Gene Expression , Humans , Insulin-Like Growth Factor I/genetics , Insulin-Like Growth Factor I/metabolism , Intracellular Signaling Peptides and Proteins/analysis , Liver/chemistry , Liver Neoplasms/chemistry , Male , Mice , Middle Aged , Muscle Proteins/analysis , NFATC Transcription Factors/genetics , Neoplasm Grading , Neoplasm Invasiveness , Neoplasm Staging , Neoplasm Transplantation , Protein Isoforms/genetics , Protein Transport/drug effects , Regulatory Sequences, Nucleic Acid , Signal Transduction , Survival Rate , Tumor Burden , Vascular Endothelial Growth Factor A/metabolismABSTRACT
BACKGROUND & AIMS: Desmoplasia and poor vascularity cause severe metabolic stress in pancreatic ductal adenocarcinomas (PDACs). Serotonin (5-HT) is a neuromodulator with neurotransmitter and neuroendocrine functions that contributes to tumorigenesis. We investigated the role of 5-HT signaling in the growth of pancreatic tumors. METHODS: We measured the levels of proteins that regulate 5-HT synthesis, packaging, and degradation in pancreata from KrasG12D/+/Trp53R172H/+/Pdx1-Cre (KPC) mice, which develop pancreatic tumors, as well as in PDAC cell lines and a tissue microarray containing 81 human PDAC samples. We also analyzed expression levels of proteins involved in 5-HT synthesis and degradation by immunohistochemical analysis of a tissue microarray containing 311 PDAC specimens, and associated expression levels with patient survival times. 5-HT level in 14 matched PDAC tumor and non-tumor tissues were analyzed by ELISA. PDAC cell lines were incubated with 5-HT and cell survival and apoptosis were measured. We analyzed expression of the 5-HT receptor HTR2B in PDAC cells and effects of receptor agonists and antagonists, as well as HTR2B knockdown with small hairpin RNAs. We determined the effects of 5-HT stimulation on gene expression profiles of BxPC-3 cells. Regulation of glycolysis by 5-HT signaling via HTR2B was assessed by immunofluorescence and immunoprecipitation analyses, as well as by determination of the extracellular acid ratio, glucose consumption, and lactate production. Primary PDACs, with or without exposure to SB204741 (a selective antagonist of HTR2B), were grown as xenograft tumors in mice, and SB204741 was administered to tumor-bearing KPC mice; tumor growth and metabolism were measured by imaging analyses. RESULTS: In immunohistochemical analysis of a tissue microarray of PDAC specimens, increased levels of TPH1 and decreased level of MAOA, which regulate 5-HT synthesis and degradation, correlated with stage and size of PDACs and shorter patient survival time. We found levels of 5-HT to be increased in human PDAC tissues compared with non-tumor pancreatic tissues, and PDAC cell lines compared with non-transformed pancreatic cells. Incubation of PDAC cell lines with 5-HT increased proliferation and prevented apoptosis. Agonists of HTR2B, but not other 5-HT receptors, promoted proliferation and prevented apoptosis of PDAC cells. Knockdown of HTR2B in PDAC cells, or incubation of cells with HTR2B inhibitors, reduced their growth as xenograft tumors in mice. We observed a correlation between 5-HT and glycolytic flux in PDAC cells; levels of metabolic enzymes involved in glycolysis, the phosphate pentose pathway, and hexosamine biosynthesis pathway increased significantly in PDAC cells following 5-HT stimulation. 5-HT stimulation led to formation of the HTR2B-LYN-p85 complex, which increased PI3K-Akt-mTOR signaling and the Warburg effect by increasing protein levels of MYC and HIF1A. Administration of SB204741 to KPC mice slowed growth and metabolism of established pancreatic tumors and prolonged survival of the mice. CONCLUSIONS: Human PDACs have increased levels of 5-HT, and PDAC cells increase expression of its receptor, HTR2B. These increases allow for tumor glycolysis under metabolic stress and promote growth of pancreatic tumors and PDAC xenograft tumors in mice.
Subject(s)
Carcinoma, Pancreatic Ductal/metabolism , Pancreatic Neoplasms/metabolism , Receptor, Serotonin, 5-HT2B/metabolism , Serotonin/metabolism , Aged , Animals , Apoptosis/drug effects , Carcinoma, Pancreatic Ductal/chemistry , Carcinoma, Pancreatic Ductal/drug therapy , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Female , Gene Silencing , Glucose/metabolism , Glycolysis/drug effects , Humans , Indoles/therapeutic use , Lactic Acid/biosynthesis , Male , Mice , Middle Aged , Monoamine Oxidase/analysis , Neoplasm Transplantation , Pancreas/chemistry , Pancreatic Neoplasms/chemistry , Pancreatic Neoplasms/drug therapy , Phosphatidylinositol 3-Kinase/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Proto-Oncogene Proteins c-myc/metabolism , Receptor, Serotonin, 5-HT2B/genetics , Serotonin/analysis , Serotonin/pharmacology , Serotonin 5-HT2 Receptor Agonists/pharmacology , Serotonin 5-HT2 Receptor Antagonists/pharmacology , Serotonin 5-HT2 Receptor Antagonists/therapeutic use , Signal Transduction , Stress, Physiological , TOR Serine-Threonine Kinases/metabolism , Tissue Array Analysis , Transcriptome , Tryptophan Hydroxylase/analysis , Urea/analogs & derivatives , Urea/therapeutic use , src-Family Kinases/metabolismABSTRACT
Chronic hepatitis B virus infection is a major risk factor for hepatocellular carcinoma (HCC). Hepatitis B virus X protein (HBx) is a hepatitis B virus protein that has multiple cellular functions, but its role in HCC pathogenesis has been controversial. Farnesoid X receptor (FXR) is a nuclear receptor with activities in anti-inflammation and inhibition of hepatocarcinogenesis. However, whether or how FXR can impact hepatitis B virus/HBx-induced hepatocarcinogenesis remains unclear. In this study, we showed that HBx can interact with FXR and function as a coactivator of FXR. Expression of HBx in vivo enhanced FXR-responsive gene regulation. HBx also increased the transcriptional activity of FXR in a luciferase reporter gene assay. The HBx-FXR interaction was confirmed by coimmunoprecipitation and glutathione S-transferase pull-down assays, and the FXR activation function 1 domain was mapped to bind to the third α helix in the C terminus of HBx. We also found that the C-terminally truncated variants of HBx, which were found in clinical HCC, were not effective at transactivating FXR. Interestingly, recruitment of the full-length HBx, but not the C-terminally truncated HBx, enhanced the binding of FXR to its response element. In vivo, FXR ablation markedly sensitized mice to HBx-induced hepatocarcinogenesis. CONCLUSIONS: We propose that transactivation of FXR by full-length HBx may represent a protective mechanism to inhibit HCC and that this inhibition may be compromised upon the appearance of C-terminally truncated HBx or when the expression and/or activity of FXR is decreased. (Hepatology 2017;65:893-906).
Subject(s)
Carcinoma, Hepatocellular/genetics , Hepatitis C/complications , RNA-Binding Proteins/metabolism , Trans-Activators/genetics , Transcriptional Activation/genetics , Animals , Carcinogenesis/genetics , Carcinoma, Hepatocellular/pathology , Disease Models, Animal , Gene Expression Regulation, Neoplastic , Gene Silencing , Hepatitis B virus/genetics , Hepatitis C/pathology , Humans , Immunohistochemistry , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Promoter Regions, Genetic , Random Allocation , Viral Regulatory and Accessory ProteinsABSTRACT
Reprogrammed metabolism has been identified as an emerging hallmark in cancer cells. It has been demonstrated that fructose-1, 6-bisphosphatase 1 (FBP1) as a rate-limiting enzyme in gluconeogenesis plays critical roles in tumor initiation and progression in several cancer types. However, function of FBP1 in hepatocellular carcinoma (HCC) is still not clear. In this study, we observed that the expression of FBP1 was obviously downregulated in the cell lines and tissues of HCC. Downregulation of FBP1 in HCC tissues was correlated with a lower overall survival rate and had a relatively higher tendency of tumor recurrence (n = 224). Silencing FBP1 could significantly promote colony formation, proliferation and metastasis of HCC cells, while ectopic overexpression of FBP1 resulted in impaired abilities of colony formation, proliferation and metastasis in vitro and in vivo. Mechanistically, silencing FBP1 facilitated glycolysis in HCC cell lines, which may be responsible for aggressiveness of HCC cells. We further found that targeting the Warburg effect using the specific inhibitor FX11 could suppress the aggressiveness of HCC cells which was mediated by loss of FBP1. These findings indicate that FBP1 appears to be a tumor suppressor in HCC. Strategies to restore the levels and activities of FBP1 might be developed to treat patients with HCC.
Subject(s)
Carcinoma, Hepatocellular/genetics , Cell Transformation, Neoplastic/genetics , DNA Helicases/genetics , DNA-Binding Proteins/genetics , Liver Neoplasms/genetics , Animals , Carcinoma, Hepatocellular/drug therapy , Carcinoma, Hepatocellular/pathology , Cell Proliferation/genetics , DNA Helicases/antagonists & inhibitors , DNA-Binding Proteins/antagonists & inhibitors , Disease Progression , Gene Expression Regulation, Neoplastic , Gene Silencing , Gluconeogenesis/drug effects , Gluconeogenesis/genetics , Hep G2 Cells , Humans , Liver Neoplasms/drug therapy , Liver Neoplasms/pathology , Mice , Naphthalenes/administration & dosage , Neoplasm Metastasis , RNA-Binding Proteins , Xenograft Model Antitumor AssaysABSTRACT
BACKGROUND: Several of the thousands of human long noncoding RNAs (lncRNAs) have been functionally characterized, yet their potential involvement in hepatocellular carcinoma (HCC) remains poorly understood. METHODS: LncRNA-HOXD-AS1 was identified by microarray and validated by real-time PCR. The clinicopathological significance of HOXD-AS1 was analyzed by Kaplan-Meier method. Chromatin immunoprecipitation was conducted to examine the mechanism of HOXD-AS1 upregulation. The role of HOXD-AS1 in HCC cells was assessed both in vitro and in vivo. ceRNA function of HOXD-AS1 was evaluated by RNA immunoprecipitation and biotin-coupled miRNA pull down assays. RESULTS: In this study, we found that HOXD-AS1 was significantly upregulated in HCC tissues. Clinical investigation demonstrated high expression level of HOXD-AS1 was associated with poor prognosis and high tumor node metastasis stage of HCC patients, and was an independent risk factor for survival. Moreover, our results revealed that STAT3 could specifically interact with the promoter of HOXD-AS1 and activate HOXD-AS1 transcription. Knockdown of HOXD-AS1 significantly inhibited migration and invasion of HCC cells in vitro and distant lung metastasis in vivo. Additionally, HOXD-AS1 was enriched in the cytoplasm, and shared miRNA response elements with SOX4. Overexpression of HOXD-AS1 competitively bound to miR-130a-3p that prevented SOX4 from miRNA-mediated degradation, thus activated the expression of EZH2 and MMP2 and facilitated HCC metastasis. CONCLUSIONS: In summary, HOXD-AS1 is a prognostic marker for HCC patients and it may play a pro-metastatic role in hepatocarcinogenesis.
Subject(s)
Liver Neoplasms/metabolism , RNA, Long Noncoding/metabolism , RNA/metabolism , SOXC Transcription Factors/metabolism , STAT3 Transcription Factor/metabolism , Cell Line, Tumor , Female , Gene Expression Profiling , Homeodomain Proteins/genetics , Humans , Liver Neoplasms/genetics , Liver Neoplasms/pathology , Male , Middle Aged , Neoplasm Metastasis/genetics , RNA/genetics , RNA, Long Noncoding/analysis , RNA, Long Noncoding/genetics , SOXC Transcription Factors/genetics , STAT3 Transcription Factor/genetics , Up-Regulation/geneticsABSTRACT
Emerging evidence has indicated that deregulation of long non-coding RNAs (lncRNAs) can contribute to the progression and metastasis of human cancer, including hepatocellular carcinoma (HCC). However, the roles of most lncRNAs in HCC remain largely unknown. Here we found a long noncoding RNA termed SchLAH (seven chromosome locus associated with HCC; also called BC035072) was generally downregulated in HCC. Low expression of SchLAH was significantly correlated with shorter overall survival of HCC patients. In vitro and in vivo assays indicated that overexpression of SchLAH inhibited the migration and lung metastasis of HCC cells. Knockdown of SchLAH by siRNA pool promoted the migration of HCC cells. RNA pull-down and RNA immunoprecipitation assays demonstrated SchLAH physically interacted with fused in sarcoma (FUS). PCR array analysis showed that RhoA and Rac1 were the downstream effector molecules of SchLAH during HCC metastasis. Knockdown of FUS rescued the mRNA levels of RhoA and Rac1 that were repressed by SchLAH. These results suggest that SchLAH may suppress the metastasis of HCC cells by interacting with FUS, which indicates potential of SchLAH for the prognosis and treatment of HCC.
Subject(s)
Carcinoma, Hepatocellular/genetics , Gene Expression Regulation, Neoplastic , Liver Neoplasms/genetics , RNA, Long Noncoding/genetics , RNA-Binding Protein FUS/genetics , Animals , Blotting, Western , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Cell Line, Tumor , Cell Movement/genetics , Hep G2 Cells , Humans , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Mice, Inbred BALB C , Mice, Nude , Neoplasm Metastasis , Prognosis , Protein Binding , RNA Interference , RNA, Long Noncoding/metabolism , RNA-Binding Protein FUS/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Transplantation, Heterologous , rac1 GTP-Binding Protein/genetics , rac1 GTP-Binding Protein/metabolism , rhoA GTP-Binding Protein/genetics , rhoA GTP-Binding Protein/metabolismABSTRACT
The capacity of liver regeneration is critical for patients with liver diseases. However, cellular and molecular mechanisms of liver regeneration are still incompletely defined. Here, we assessed roles of LASS2 in liver regeneration following partial hepatectomy (PHx) in mice. Our results showed that protein level of LASS2 remarkably increased during liver regeneration after PHx in wildtype (WT) mice. Comparing to WT mice, liver regeneration index after PHx was significantly decreased from day 1 to day 5 in liver-specific LASS2 knockout (LASS2-LKO) mice. Interestingly, liver mass of LASS2-LKO mice could sufficiently recover at day 14 after PHx. Immunohistochemistry (IHC) and western blot analyses revealed that proliferation markers, such as PCNA and Ki67, were potently reduced during liver regeneration in LASS2-LKO mice. In addition, several cell cycle related molecules, such as cyclin A, CDK2 and p-Rb, were decreased in LASS2-LKO mice after PHx. Co-immunoprecipitation assay further revealed a decreased formation of CDK4/cyclin D1 complex after PHx in LASS2-LKO mice. However, phosphorylation of Akt was significantly activated from day 2 after PHx in LASS2-LKO mice when compared with that in WT mice, which may explain the recovery of liver mass at the late stage of liver regeneration in LASS2-LKO mice. Taken together, we conclude that LASS2 plays an important role in efficient liver regeneration in response to PHx.
Subject(s)
Hepatectomy/methods , Liver Regeneration/physiology , Sphingosine N-Acyltransferase/genetics , Animals , Cell Cycle/physiology , Cell Proliferation , Cell Size , Hepatocytes/cytology , Hepatocytes/metabolism , Mice, Knockout , Mice, Transgenic , Proto-Oncogene Proteins c-akt/metabolism , Sphingosine N-Acyltransferase/metabolismABSTRACT
Cancer immunotherapy has shown great promise as a new standard cancer therapeutic modality. However, the response rates are limited for current approach that depends on enhancing spontaneous antitumor immune responses. Therefore, increasing tumor immunogenicity by expressing appropriate cytokines should further improve the current immunotherapy. IL-33 is a member of the IL-1 family of cytokines and is released by necrotic epithelial cells or activated innate immune cells and is thus considered a "danger" signal. The role of IL-33 in promoting type 2 immune responses and tissue inflammation has been well established. However, whether IL-33 drives antitumor immune responses is controversial. Our previous work established that IL-33 promoted the function of CD8(+) T cells. In this study, we showed that the expression of IL-33 in two types of cancer cells potently inhibited tumor growth and metastasis. Mechanistically, IL-33 increased numbers and IFN-γ production by CD8(+) T and NK cells in tumor tissues, thereby inducing a tumor microenvironment favoring tumor eradication. Importantly, IL-33 greatly increased tumor Ag-specific CD8(+) T cells. Furthermore, both NK and CD8(+) T cells were required for the antitumor effect of IL-33. Moreover, depletion of regulatory T cells worked synergistically with IL-33 expression for tumor elimination. Our studies established "alarmin" IL-33 as a promising new cytokine for tumor immunotherapy through promoting cancer-eradicating type 1 immune responses.
Subject(s)
Breast Neoplasms/immunology , CD8-Positive T-Lymphocytes/immunology , Interleukin-33/immunology , Killer Cells, Natural/immunology , Melanoma, Experimental/immunology , Animals , Breast Neoplasms/pathology , Cell Line, Tumor , Cell Proliferation , Female , Histocompatibility Antigens Class II/biosynthesis , Immunotherapy , Inflammation/immunology , Interferon-gamma/biosynthesis , Interleukin-12/biosynthesis , Interleukin-33/biosynthesis , Melanoma, Experimental/pathology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Neoplasm Metastasis , T-Lymphocytes, Regulatory/immunology , Tumor Microenvironment/immunologyABSTRACT
Heat shock proteins (HSPs) are highly conserved proteins, which are expressed at low levels under normal conditions, but significantly induced in response to cellular stresses. As molecular chaperones, HSPs play crucial roles in protein homeostasis, apoptosis, invasion and cellular signaling transduction. The induction of HSPs is an important part of heat shock response, which could help cancer cells to adapt to stress conditions. Because of the constant stress condition in tumor microenvironment, HSPs overexpression is widely reported in many human cancers. In light of the significance of HSPs for cancer cells to survive and obtain invasive phenotype under stress condition, HSPs are often associated with poor prognosis and treatment resistance in many types of human cancers. It has been described that upregulation of HSPs may serve as diagnostic and prognostic markers in hepatocellular carcinoma (HCC). Targeting HSPs with specific inhibitor alone or in combination with chemotherapy regimens holds promise for the improvement of outcomes for HCC patients. In this review, we summarize the expression profiles, functions and molecular mechanisms of HSPs (HSP27, HSP70 and HSP90) as well as a HSP-like protein (clusterin) in HCC. In addition, we address progression and challenges in targeting these HSPs as novel therapeutic strategies in HCC.