RESUMO
BACKGROUND AND AIMS: HCC undergoes active metabolic reprogramming. Reactive oxygen species (ROS) are excessively generated in cancer cells and are neutralized by NADPH. Malic enzymes (MEs) are the less studied NADPH producers in cancer. APPROACH AND RESULTS: We found that ME1, but not ME3, was regulated by the typical oxidative stress response pathway mediated by kelch-like ECH associated protein 1/nuclear factor erythroid 2-related factor (NRF2). Surprisingly, ME3 was constitutively induced by superenhancers. Disruption of any ME regulatory pathways decelerated HCC progression and sensitized HCC to sorafenib. Therapeutically, simultaneous blockade of NRF2 and a superenhancer complex completely impeded HCC growth. We show that superenhancers allow cancer cells to counteract the intrinsically high level of ROS through constitutively activating ME3 expression. When HCC cells encounter further episodes of ROS insult, NRF2 allows cancer cells to adapt by transcriptionally activating ME1. CONCLUSIONS: Our study reveals the complementary regulatory mechanisms which control MEs and provide cancer cells multiple layers of defense against oxidative stress. Targeting both regulatory mechanisms represents a potential therapeutic approach for HCC treatment.
Assuntos
Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , Malato Desidrogenase/genética , Álcool Oxidorredutases Dependentes de NAD(+) e NADP(+)/genética , Fator 2 Relacionado a NF-E2/metabolismo , Animais , Carcinoma Hepatocelular/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Hepatócitos , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Neoplasias Hepáticas/genética , Malato Desidrogenase/metabolismo , Metabolômica , Camundongos , Álcool Oxidorredutases Dependentes de NAD(+) e NADP(+)/metabolismo , Estresse Oxidativo/genética , Espécies Reativas de Oxigênio/metabolismo , Ativação Transcricional , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Hepatocellular carcinoma (HCC) is the third most lethal cancer worldwide. Increasing evidence shows that epigenetic alterations play an important role in human carcinogenesis. Deregulation of DNA methylation and histone modifications have recently been characterized in HCC, but the significance of chromatin remodeling in liver carcinogenesis remains to be explored. In this study, by systematically analyzing the expression of chromatin remodeling genes in human HCCs, we found that helicase, lymphoid-specific (HELLS), an SWI2/SNF2 chromatin remodeling enzyme, was remarkably overexpressed in HCC. Overexpression of HELLS correlated with more aggressive clinicopathological features and poorer patient prognosis compared to patients with lower HELLS expression. We further showed that up-regulation of HELLS in HCC was conferred by hyperactivation of transcription factor specificity protein 1 (SP1). To investigate the functions of HELLS in HCC, we generated both gain-of-function and loss-of-function models by the CRISPR activation system, lentiviral short hairpin RNA, and the CRISPR/Cas9 genome editing system. We demonstrated that overexpression of HELLS augmented HCC cell proliferation and migration. In contrast, depletion of HELLS reduced HCC growth and metastasis both in vitro and in vivo. Moreover, inactivation of HELLS led to metabolic reprogramming and reversed the Warburg effect in HCC cells. Mechanistically, by integrating analysis of RNA sequencing and micrococcal nuclease sequencing, we revealed that overexpression of HELLS increased nucleosome occupancy, which obstructed the accessibility of enhancers and hindered formation of the nucleosome-free region (NFR) at the transcription start site. Though this mechanism, up-regulation of HELLS mediated epigenetic silencing of multiple tumor suppressor genes including E-cadherin, FBP1, IGFBP3, XAF1 and CREB3L3 in HCC. Conclusion: Our data reveal that HELLS is a key epigenetic driver of HCC; by altering the nucleosome occupancy at the NFR and enhancer, HELLS epigenetically suppresses multiple tumor suppressor genes to promote HCC progression.
Assuntos
Carcinoma Hepatocelular/enzimologia , DNA Helicases/metabolismo , Neoplasias Hepáticas Experimentais/enzimologia , Nucleossomos/metabolismo , Animais , Antígenos CD/metabolismo , Caderinas/metabolismo , Carcinoma Hepatocelular/etiologia , Linhagem Celular Tumoral , Montagem e Desmontagem da Cromatina , DNA Helicases/genética , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor , Humanos , Neoplasias Hepáticas Experimentais/etiologia , Camundongos Knockout , Camundongos Nus , Metástase Neoplásica , Fator de Transcrição Sp1/metabolismoRESUMO
Hepatocellular carcinoma (HCC) is one of the most prevalent and lethal cancers worldwide which lacks effective treatment. Cancer cells experience high levels of oxidative stress due to increased generation of reactive oxygen species (ROS). Increased antioxidant-producing capacity is therefore found in cancer cells to counteract oxidative stress. The thioredoxin system is a ubiquitous mammalian antioxidant system which scavenges ROS, and we demonstrate that it is vital for HCC growth as it maintains intracellular reduction-oxidation (redox) homeostasis. Transcriptome sequencing in human HCC samples revealed significant overexpression of thioredoxin reductase 1 (TXNRD1), the cytosolic subunit and key enzyme of the thioredoxin system, with significant correlations to poorer clinicopathological features and patient survival. Driven by the transcriptional activation of nuclear factor (erythroid-derived 2)-like 2, the master protector against oxidative stress, TXNRD1 counteracts intracellular ROS produced in human HCC. Inhibition of TXNRD1 through genetic inhibition hindered the proliferation of HCC cells and induced apoptosis in vitro. Administration of the pharmacological TXNRD1 inhibitor auranofin (AUR) effectively suppressed the growth of HCC tumors induced using the hydrodynamic tail vein injection and orthotopic implantation models in vivo. Furthermore, AUR sensitized HCC cells toward the conventional therapeutic sorafenib. Conclusion: Our study highlights the reliance of HCC cells on antioxidants for redox homeostasis and growth advantage; targeting TXNRD1 resulted in dramatic accumulation of ROS, which was found to be an effective approach for the suppression of HCC tumor growth.
Assuntos
Auranofina/uso terapêutico , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Tiorredoxina Redutase 1/metabolismo , Animais , Antineoplásicos/uso terapêutico , Auranofina/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Ensaios de Seleção de Medicamentos Antitumorais , Células Hep G2 , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Neoplasias Hepáticas/tratamento farmacológico , Camundongos , Fator 2 Relacionado a NF-E2/metabolismo , Sorafenibe/uso terapêutico , Tiorredoxina Redutase 1/antagonistas & inibidoresRESUMO
Cancer cells experience an increase in oxidative stress. The pentose phosphate pathway (PPP) is a major biochemical pathway that generates antioxidant NADPH. Here, we show that transketolase (TKT), an enzyme in the PPP, is required for cancer growth because of its ability to affect the production of NAPDH to counteract oxidative stress. We show that TKT expression is tightly regulated by the Nuclear Factor, Erythroid 2-Like 2 (NRF2)/Kelch-Like ECH-Associated Protein 1 (KEAP1)/BTB and CNC Homolog 1 (BACH1) oxidative stress sensor pathway in cancers. Disturbing the redox homeostasis of cancer cells by genetic knockdown or pharmacologic inhibition of TKT sensitizes cancer cells to existing targeted therapy (Sorafenib). Our study strengthens the notion that antioxidants are beneficial to cancer growth and highlights the therapeutic benefits of targeting pathways that generate antioxidants.
Assuntos
Carcinoma Hepatocelular/enzimologia , Carcinoma Hepatocelular/patologia , Estresse Oxidativo , Transcetolase/metabolismo , Animais , Sequência de Bases , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Técnicas de Silenciamento de Genes , Glucose/metabolismo , Glutationa/metabolismo , Glicólise/efeitos dos fármacos , Humanos , Neoplasias Hepáticas/enzimologia , Neoplasias Hepáticas/patologia , Masculino , Metaboloma/efeitos dos fármacos , Camundongos Nus , Dados de Sequência Molecular , Niacinamida/análogos & derivados , Niacinamida/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Via de Pentose Fosfato/efeitos dos fármacos , Peróxidos/farmacologia , Compostos de Fenilureia/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Sorafenibe , Transcetolase/antagonistas & inibidores , Transcetolase/genética , Regulação para Cima/efeitos dos fármacosRESUMO
OBJECTIVE: We investigated the mutational landscape of mammalian target of rapamycin (mTOR) signalling cascade in hepatocellular carcinomas (HCCs) with chronic HBV background, aiming to evaluate and delineate mutation-dependent mechanism of mTOR hyperactivation in hepatocarcinogenesis. DESIGN: We performed next-generation sequencing on human HCC samples and cell line panel. Systematic mutational screening of mTOR pathway-related genes was undertaken and mutant genes were evaluated based on their recurrence. Protein expressions of tuberous sclerosis complex (TSC)1, TSC2 and pRPS6 were assessed by immunohistochemistry in human HCC samples. Rapamycin sensitivity was estimated by colony-formation assay in HCC cell lines and the treatment was further tested using our patient-derived tumour xenograft (PDTX) models. RESULTS: We identified and confirmed multiple mTOR components as recurrently mutated in HBV-associated HCCs. Of significance, we detected frequent (16.2%, n=18/111) mutations of TSC1 and TSC2 genes in the HCC samples. The spectrum of TSC1/2 mutations likely disrupts the endogenous gene functions in suppressing the downstream mTOR activity through different mechanisms and leads to more aggressive tumour behaviour. Mutational disruption of TSC1 and TSC2 was also observed in HCC cell lines and our PDTX models. TSC-mutant cells exhibited reduced colony-forming ability on rapamycin treatment. With the use of biologically relevant TSC2-mutant PDTXs, we demonstrated the therapeutic benefits of the hypersensitivity towards rapamycin treatment. CONCLUSIONS: Taken together, our findings suggest the significance of previously undocumented mutation-dependent mTOR hyperactivation and frequent TSC1/2 mutations in HBV-associated HCCs. They define a molecular subset of HCC having genetic aberrations in mTOR signalling, with potential significance of effective specific drug therapy.
Assuntos
Antibióticos Antineoplásicos/uso terapêutico , Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Sirolimo/uso terapêutico , Serina-Treonina Quinases TOR/metabolismo , Proteínas Supressoras de Tumor/genética , Adulto , Idoso , Animais , Antibióticos Antineoplásicos/farmacologia , Proteína Axina/genética , Carcinoma Hepatocelular/química , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Análise Mutacional de DNA , Proteínas de Ligação a DNA , Feminino , Humanos , Neoplasias Hepáticas/química , Neoplasias Hepáticas/metabolismo , Masculino , Camundongos , Pessoa de Meia-Idade , Taxa de Mutação , Transplante de Neoplasias , Proteínas Nucleares/genética , Transdução de Sinais , Sirolimo/farmacologia , Fatores de Transcrição/genética , Proteína 1 do Complexo Esclerose Tuberosa , Proteína 2 do Complexo Esclerose Tuberosa , Ensaio Tumoral de Célula-Tronco , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor/análise , Adulto Jovem , beta Catenina/genéticaRESUMO
UNLABELLED: A population of stromal cells, myeloid-derived suppressor cells (MDSCs), is present in tumors. Though studies have gradually revealed the protumorigenic functions of MDSCs, the molecular mechanisms guiding MDSC recruitment remain largely elusive. Hypoxia, O2 deprivation, is an important factor in the tumor microenvironment of solid cancers, whose growth often exceeds the growth of functional blood vessels. Here, using hepatocellular carcinoma as the cancer model, we show that hypoxia is an important driver of MDSC recruitment. We observed that MDSCs preferentially infiltrate into hypoxic regions in human hepatocellular carcinoma tissues and that hypoxia-induced MDSC infiltration is dependent on hypoxia-inducible factors. We further found that hypoxia-inducible factors activate the transcription of chemokine (C-C motif) ligand 26 in cancer cells to recruit chemokine (C-X3-C motif) receptor 1-expressing MDSCs to the primary tumor. Knockdown of chemokine (C-C motif) ligand 26 in cancer cells profoundly reduces MDSC recruitment, angiogenesis, and tumor growth. Therapeutically, blockade of chemokine (C-C motif) ligand 26 production in cancer cells by the hypoxia-inducible factor inhibitor digoxin or blockade of chemokine (C-X3-C motif) receptor 1 in MDSCs by chemokine (C-X3-C motif) receptor 1 neutralizing antibody could substantially suppress MDSC recruitment and tumor growth. CONCLUSION: This study unprecedentedly reveals a novel molecular mechanism by which cancer cells direct MDSC homing to primary tumor and suggests that targeting MDSC recruitment represents an attractive therapeutic approach against solid cancers. (Hepatology 2016;64:797-813).
Assuntos
Carcinoma Hepatocelular/metabolismo , Quimiocinas CC/metabolismo , Hipóxia/metabolismo , Neoplasias Hepáticas Experimentais/metabolismo , Células Supressoras Mieloides/fisiologia , Animais , Sequência de Bases , Receptor 1 de Quimiocina CX3C , Linhagem Celular Tumoral , Quimiocina CCL26 , Digoxina , Humanos , Fator 1 Induzível por Hipóxia/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Nus , Dados de Sequência Molecular , Neovascularização Patológica , Receptores de Quimiocinas/antagonistas & inibidores , Microambiente TumoralRESUMO
Transforming growth factor ß (TGF-ß) physiologically induces Epstein-Barr virus (EBV) lytic infection by activating the expression of EBV's latent-lytic switch BZLF1 gene. Liang et al. (J. Biol. Chem. 277:23345-23357, 2002) previously identified a Smad-binding element (SBE) within the BZLF1 promoter, Zp; however, it accounts for only 20 to 30% of TGF-ß-mediated activation of transcription from Zp. Here, we identified additional factors responsible for the rest of this activation. The incubation of EBV-positive MutuI cells with a TGF-ß neutralizing antibody or inhibitors of the TGF-ß type I receptor (TßRI) or Smad3 eliminated the TGF-ß-induced reactivation of EBV. The coexpression of Smad2, Smad3, and Smad4 together with a constitutively active form of TßRI induced 15- to 25-fold transcription from Zp in gastric carcinoma AGS cells. By electrophoretic mobility shift assays, we identified four additional Smad-binding elements, named SBE2 to SBE5. Substitution mutations in individual SBEs reduced Smad-mediated activation of Zp by 20 to 60%; together, these mutations essentially eliminated it. Chromatin immunoprecipitation assays confirmed that Smad4 newly bound the Zp region of the EBV genome following the incubation of MutuI cells with TGF-ß. SBE2 overlaps the ZEB-binding ZV silencing element of Zp. Depending upon posttranslational modifications, Smad4 either competed with ZEB1 for binding or formed a complex with ZEB1 on the Zp ZV element in a cell-free assay system. In transiently transfected cells, exogenously expressed ZEB1 inhibited Smad-mediated transcriptional activation from Zp. We conclude that TGF-ß induces EBV lytic reactivation via the canonical Smad pathway by activating BZLF1 gene expression through multiple SBEs acting in concert.
Assuntos
Herpesvirus Humano 4/fisiologia , Proteínas Smad/fisiologia , Transativadores/genética , Fator de Crescimento Transformador beta/fisiologia , Ativação Viral , Latência Viral , Sequência de Bases , Linhagem Celular Tumoral , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-DirigidaRESUMO
We previously showed that the cellular proteins ZEB1 and ZEB2/SIP1 both play key roles in regulating the latent-lytic switch of Epstein-Barr Virus (EBV) by repressing BZLF1 gene expression. We investigated here the effects of cellular microRNA (miRNA) 200 (miR200) family members on the EBV infection status of cells. We show that miR200b and miR429, but not miR200a, can induce EBV-positive cells into lytic replication by downregulating expression of ZEB1 and ZEB2, leading to production of infectious virus. The levels of miR200 family members in EBV-infected cells strongly negatively correlated with the levels of the ZEBs (e.g., -0.89 [P < 0.001] for miR429 versus ZEB1) and positively correlated with the degree of EBV lytic gene expression (e.g., 0.73 [P < 0.01] for miR429 versus BZLF1). The addition of either miR200b or miR429 to EBV-positive cells led to EBV lytic reactivation in a ZEB-dependent manner; inhibition of these miRNAs led to decreased EBV lytic gene expression. The degree of latent infection by an EBV mutant defective in the primary ZEB-binding site of the EBV BZLF1 promoter was not affected by the addition of these miRNAs. Furthermore, EBV infection of primary blood B cells led to downregulation of these miRNAs and upregulation of ZEB levels. Thus, we conclude that miRNAs 200b and 429 are key regulators via their effects on expression of ZEB1 and ZEB2 of the switch between latent and lytic infection by EBV and, therefore, potential targets for development of new lytic induction therapeutics with which to treat patients with EBV-associated malignancies.
Assuntos
Herpesvirus Humano 4/genética , Herpesvirus Humano 4/fisiologia , MicroRNAs/genética , Sequência de Bases , Linhagem Celular , DNA Viral/genética , Regulação para Baixo , Infecções por Vírus Epstein-Barr/genética , Infecções por Vírus Epstein-Barr/virologia , Genes Virais , Herpesvirus Humano 4/patogenicidade , Proteínas de Homeodomínio/fisiologia , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Modelos Biológicos , Regiões Promotoras Genéticas , Proteínas Repressoras/fisiologia , Transativadores/genética , Fatores de Transcrição/fisiologia , Latência Viral/genética , Latência Viral/fisiologia , Replicação Viral/genética , Replicação Viral/fisiologia , Homeobox 2 de Ligação a E-box com Dedos de Zinco , Homeobox 1 de Ligação a E-box em Dedo de ZincoRESUMO
Sickle cell anemia (SCA) is characterized by decreased red blood cell (RBC) deformability due to polymerization of deoxygenated hemoglobin, leading to abnormal mechanical properties of RBC, increased cellular adhesion, and microcirculatory obstruction. Prior work has demonstrated that NO⢠influences RBC hydration and deformability and is produced at a basal rate that increases under shear stress in normal RBC. Nevertheless, the origin and physiological relevance of nitric oxide (NOâ¢) production and scavenging in RBC remains unclear. We aimed to assess the basal and shear-mediated production of NO⢠in RBC from SCA patients and control (CTRL) subjects. RBCs loaded with a fluorescent NO⢠detector, DAF-FM (4-Amino-5-methylamino- 2',7'-difluorofluorescein diacetate), were imaged in microflow channels over 30-min without shear stress, followed by a 30-min period under 0.5Pa shear stress. We utilized non-specific nitric oxide synthase (NOS) blockade and carbon monoxide (CO) saturation of hemoglobin to assess the contribution of NOS and hemoglobin, respectively, to NO⢠production. Quantification of DAF-FM fluorescence intensity in individual RBC showed an increase in NO⢠in SCA RBC at the start of the basal period; however, both SCA and CTRL RBC increased NO⢠by a similar quantity under shear. A subpopulation of sickle-shaped RBC exhibited lower basal NO⢠production compared to discoid RBC from SCA group, and under shear became more circular in the direction of shear when compared to discoid RBC from SCA and CTRL, which elongated. Both CO and NOS inhibition caused a decrease in basal NO⢠production. Shear-mediated NO⢠production was decreased by CO in all RBC, but was decreased by NOS blockade only in SCA. In conclusion, total NO⢠production is increased and shear-mediated NO⢠production is preserved in SCA RBC in a NOS-dependent manner. Sickle shaped RBC with inclusions have higher NO⢠production and they become more circular rather than elongated with shear.
Assuntos
Anemia Falciforme , Óxido Nítrico , Deformação Eritrocítica , Eritrócitos , Humanos , MicrocirculaçãoRESUMO
Hypoxia is commonly found in cancers. Hypoxia, due to the lack of oxygen (O2) as the electron recipient, causes inefficient electron transfer through the electron transport chain at the mitochondria leading to accumulation of reactive oxygen species (ROS) which could create irreversible cellular damages. Through hypoxia-inducible factor 1 (HIF-1) which elicits various molecular events, cells are able to overcome low O2. Knowledge about the new molecular mechanisms governed by HIF-1 is important for new therapeutic interventions targeting hypoxic tumors. Using hepatocellular carcinoma (HCC) as a model, we revealed that the HIF-1 and the Notch signaling pathways cross-talk to control mitochondrial biogenesis of cancer cells to maintain REDOX balance. From transcriptome sequencing, we found that HEY1, a transcriptional repressor, in the NOTCH pathway was consistently induced by hypoxia in HCC cell lines. We identified a strong hypoxia response element (HRE) in HEY1 by chromatin immunoprecipitation (ChIP) and luciferase reporter assays. Transcriptome and ChIP sequencing further identified PINK1, a gene essential for mitochondrial biogenesis, as a novel transcriptional target of HEY1. HCC cells with HEY1 knockdown re-expressed PINK1. HEY1 and PINK1 expressions inversely correlated in human HCC samples. Overexpression of HEY1 and under-expression of PINK1 were detected in human HCC and associated with poor clinical outcomes. Functionally, we found that overexpression of HEY1 or knockdown of PINK1 consistently reduced mitochondrial cristae, mitochondrial mass, oxidative stress level, and increased HCC growth.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Carcinoma Hepatocelular/metabolismo , Proteínas de Ciclo Celular/metabolismo , Hipóxia Celular/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias Hepáticas/metabolismo , Mitocôndrias/metabolismo , Proteínas Quinases/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Carcinoma Hepatocelular/patologia , Proteínas de Ciclo Celular/genética , Células HeLa , Células Hep G2 , Xenoenxertos , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Nus , Proteínas Quinases/genética , Transfecção , Carga Tumoral/genéticaRESUMO
Hepatocellular carcinoma (HCC), accounting for 90% of primary liver cancer, is a lethal malignancy that is tightly associated with chronic hepatitis B virus (HBV) infection. HBV encodes a viral onco-protein, transactivator protein X (HBx), which interacts with proteins of hepatocytes to promote oncogenesis. Our current study focused on the interaction of HBx with a transcription factor, hypoxia-inducible factor-1α (HIF-1α), which is stabilized by low O2 condition (hypoxia) and is found to be frequently overexpressed in HCC intra-tumorally due to poor blood perfusion. Here, we showed that overexpression of HBx by tetracycline-inducible systems further stabilized HIF-1α under hypoxia in HBV-negative HCC cell lines. Reversely, knockdown of HBx reduced HIF-1α protein stabilization under hypoxia in HBV-positive HCC cell lines. More intriguingly, overexpression of HBx elevated the mRNA and protein expression of a family of HIF-1α target genes, the lysyl oxidase (LOX) family in HCC. The LOX family members function to cross-link collagen in the extracellular matrix (ECM) to promote cancer progression and metastasis. By analyzing the collagens under scanning electron microscope, we found that collagen fibers were significantly smaller in size when incubated with conditioned medium from HBx knockdown HCC cells as compared to control HCC cells in vitro. Transwell invasion assay further revealed that less cells were able to invade through the matrigel which was pre-treated with conditioned medium from HBx knockdown HCC cells as compared to control HCC cells. Orthotopic and subcutaneous HCC models further showed that knockdown of HBx in HCC cells reduced collagen crosslinking and stiffness in vivo and repressed HCC growth and metastasis. Taken together, our in vitro and in vivo studies showed the HBx remodeled the ECM through HIF-1α/LOX pathway to promote HCC metastasis.
RESUMO
Cancer cells preferentially utilize glucose and glutamine, which provide macromolecules and antioxidants that sustain rapid cell division. Metabolic reprogramming in cancer drives an increased glycolytic rate that supports maximal production of these nutrients. The folate cycle, through transfer of a carbon unit between tetrahydrofolate and its derivatives in the cytoplasmic and mitochondrial compartments, produces other metabolites that are essential for cell growth, including nucleotides, methionine, and the antioxidant NADPH. Here, using hepatocellular carcinoma (HCC) as a cancer model, we have observed a reduction in growth rate upon withdrawal of folate. We found that an enzyme in the folate cycle, methylenetetrahydrofolate dehydrogenase 1-like (MTHFD1L), plays an essential role in support of cancer growth. We determined that MTHFD1L is transcriptionally activated by NRF2, a master regulator of redox homeostasis. Our observations further suggest that MTHFD1L contributes to the production and accumulation of NADPH to levels that are sufficient to combat oxidative stress in cancer cells. The elevation of oxidative stress through MTHFD1L knockdown or the use of methotrexate, an antifolate drug, sensitizes cancer cells to sorafenib, a targeted therapy for HCC. Taken together, our study identifies MTHFD1L in the folate cycle as an important metabolic pathway in cancer cells with the potential for therapeutic targeting.
Assuntos
Aminoidrolases/metabolismo , Carcinoma Hepatocelular/enzimologia , Ácido Fólico/metabolismo , Formiato-Tetra-Hidrofolato Ligase/metabolismo , Neoplasias Hepáticas/enzimologia , Metilenotetra-Hidrofolato Desidrogenase (NADP)/metabolismo , Complexos Multienzimáticos/metabolismo , Proteínas de Neoplasias/metabolismo , Aminoidrolases/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Ácido Fólico/genética , Formiato-Tetra-Hidrofolato Ligase/genética , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Metilenotetra-Hidrofolato Desidrogenase (NADP)/genética , Complexos Multienzimáticos/genética , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Proteínas de Neoplasias/genéticaRESUMO
Myeloid-derived suppressor cells (MDSCs) possess immunosuppressive activities, which allow cancers to escape immune surveillance and become non-responsive to immune checkpoints blockade. Here we report hypoxia as a cause of MDSC accumulation. Using hepatocellular carcinoma (HCC) as a cancer model, we show that hypoxia, through stabilization of hypoxia-inducible factor-1 (HIF-1), induces ectoenzyme, ectonucleoside triphosphate diphosphohydrolase 2 (ENTPD2/CD39L1), in cancer cells, causing its overexpression in HCC clinical specimens. Overexpression of ENTPD2 is found as a poor prognostic indicator for HCC. Mechanistically, we demonstrate that ENTPD2 converts extracellular ATP to 5'-AMP, which prevents the differentiation of MDSCs and therefore promotes the maintenance of MDSCs. We further find that ENTPD2 inhibition is able to mitigate cancer growth and enhance the efficiency and efficacy of immune checkpoint inhibitors. Our data suggest that ENTPD2 may be a good prognostic marker and therapeutic target for cancer patients, especially those receiving immune therapy.Myeloid-derived suppressor cells (MDSCs) promote tumor immune escape. Here, the authors show that in hepatocellular carcinoma, hypoxia induces the expression of ENTPD2 on cancer cells leading to elevated extracellular 5'-AMP, which in turn promote the maintenance of MDSCs by preventing their differentiation.
Assuntos
Adenosina Trifosfatases/metabolismo , Carcinoma Hepatocelular/enzimologia , Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias Hepáticas/enzimologia , Células Supressoras Mieloides/enzimologia , Adenosina Trifosfatases/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/fisiopatologia , Diferenciação Celular , Proliferação de Células , Humanos , Hipóxia/enzimologia , Hipóxia/genética , Hipóxia/metabolismo , Hipóxia/fisiopatologia , Fator 1 Induzível por Hipóxia/genética , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/fisiopatologia , Células Supressoras Mieloides/citologia , Células Supressoras Mieloides/metabolismoRESUMO
Arabidopsis thaliana is the most widely-studied plant today. The concerted efforts of over 11 000 researchers and 4000 organizations around the world are generating a rich diversity and quantity of information and materials. This information is made available through a comprehensive on-line resource called the Arabidopsis Information Resource (TAIR) (http://arabidopsis.org), which is accessible via commonly used web browsers and can be searched and downloaded in a number of ways. In the last two years, efforts have been focused on increasing data content and diversity, functionally annotating genes and gene products with controlled vocabularies, and improving data retrieval, analysis and visualization tools. New information include sequence polymorphisms including alleles, germplasms and phenotypes, Gene Ontology annotations, gene families, protein information, metabolic pathways, gene expression data from microarray experiments and seed and DNA stocks. New data visualization and analysis tools include SeqViewer, which interactively displays the genome from the whole chromosome down to 10 kb of nucleotide sequence and AraCyc, a metabolic pathway database and map tool that allows overlaying expression data onto the pathway diagrams. Finally, we have recently incorporated seed and DNA stock information from the Arabidopsis Biological Resource Center (ABRC) and implemented a shopping-cart style on-line ordering system.
Assuntos
Arabidopsis/genética , Bases de Dados Genéticas , Arabidopsis/enzimologia , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Gráficos por Computador , Genoma de Planta , Armazenamento e Recuperação da Informação , Internet , Modelos Biológicos , Análise de Sequência com Séries de Oligonucleotídeos , Polimorfismo GenéticoRESUMO
PURPOSE: Hepatocellular carcinoma (HCC) lacks effective curative therapy. Hypoxia is commonly found in HCC. Hypoxia elicits a series of protumorigenic responses through hypoxia-inducible factor-1 (HIF1). Better understanding of the metabolic adaptations of HCC cells during hypoxia is essential to the design of new therapeutic regimen. EXPERIMENTAL DESIGN: Expressions of genes involved in the electron transport chain (ETC) in HCC cell lines (20% and 1% O2) and human HCC samples were analyzed by transcriptome sequencing. Expression of NDUFA4L2, a less active subunit in complex I of the ETC, in 100 pairs of HCC and nontumorous liver tissues were analyzed by qRT-PCR. Student t test and Kaplan-Meier analyses were used for clinicopathologic correlation and survival studies. Orthotopic HCC implantation model was used to evaluate the efficiency of HIF inhibitor. RESULTS: NDUFA4L2 was drastically overexpressed in human HCC and induced by hypoxia. NDUFA4L2 overexpression was closely associated with tumor microsatellite formation, absence of tumor encapsulation, and poor overall survival in HCC patients. We confirmed that NDUFA4L2 was HIF1-regulated in HCC cells. Inactivation of HIF1/NDUFA4L2 increased mitochondrial activity and oxygen consumption, resulting in ROS accumulation and apoptosis. Knockdown of NDUFA4L2 markedly suppressed HCC growth and metastasis in vivo HIF inhibitor, digoxin, significantly suppressed growth of tumors that expressed high level of NDUFA4L2. CONCLUSIONS: Our study has provided the first clinical relevance of NDUFA4L2 in human cancer and suggested that HCC patients with NDUFA4L2 overexpression may be suitable candidates for HIF inhibitor treatment. Clin Cancer Res; 22(12); 3105-17. ©2016 AACR.
Assuntos
Carcinoma Hepatocelular/patologia , Hipóxia Celular/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias Hepáticas/patologia , NADH Desidrogenase/metabolismo , Estresse Oxidativo/genética , Animais , Apoptose/fisiologia , Carcinoma Hepatocelular/genética , Linhagem Celular Tumoral , Digoxina/farmacologia , Complexo I de Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/metabolismo , Feminino , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Neoplasias Hepáticas/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Repetições de Microssatélites/genética , Pessoa de Meia-Idade , Mitocôndrias/metabolismo , NADH Desidrogenase/genética , Oxirredução , Consumo de Oxigênio/fisiologia , Interferência de RNA , RNA Interferente Pequeno/genética , Espécies Reativas de Oxigênio/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Hepatocellular carcinoma (HCC) is characteristically one of the most rapidly proliferating tumors which outgrows functional blood supply and results in regional oxygen deprivation. Overexpression of PIM1, a serine/threonine kinase, has been identified recently in human cancers. Knowledge on PIM1 in HCC is however, scarce. By immunohistochemical analysis on 56 human primary HCC samples, we observed overexpression of PIM1 in 39% of the cases. In two independent cohorts of paired primary and extra-hepatic metastatic HCC tissues, PIM1 expression was higher (p=0.002) in the extra-hepatic metastatic HCC tissues as compared with the corresponding primary HCCs. PIM1 was markedly up-regulated in multiple HCC cell lines in hypoxic condition (1% O2) versus normoxia (20% O2). Silencing of PIM1 suppressed HCC cell invasion in vitro as compared to non-target control, and decreased HCC cell proliferation in vitro and tumor growth and metastatic potential in vivo. Knockdown of PIM1 significantly reduced glucose uptake by HCC cells and was associated with decreased levels of p-AKT and key molecules in the glycolytic pathway. Taken together, PIM1 is up-regulated by hypoxia in HCC and promotes tumor growth and metastasis through facilitating cancer cell glycolysis. Targeting PIM1 may have potential role in the management of HCC.
Assuntos
Carcinoma Hepatocelular/enzimologia , Glicólise , Neoplasias Hepáticas/enzimologia , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , Transporte Ativo do Núcleo Celular , Idoso , Idoso de 80 Anos ou mais , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/secundário , Hipóxia Celular , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Progressão da Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Masculino , Camundongos Nus , Pessoa de Meia-Idade , Invasividade Neoplásica , Fosforilação , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-pim-1/genética , Interferência de RNA , Transdução de Sinais , Fatores de Tempo , Transfecção , Carga Tumoral , Regulação para CimaRESUMO
Hepatocellular carcinoma (HCC) is an aggressive tumor, with a high mortality rate due to late symptom presentation and frequent tumor recurrences and metastasis. It is also a rapidly growing tumor supported by different metabolic mechanisms; nevertheless, the biological and molecular mechanisms involved in the metabolic reprogramming in HCC are unclear. In this study, we found that pyruvate kinase M2 (PKM2) was frequently over-expressed in human HCCs and its over-expression was associated with aggressive clinicopathological features and poor prognosis of HCC patients. Furthermore, knockdown of PKM2 suppressed aerobic glycolysis and cell proliferation in HCC cell lines in vitro. Importantly, knockdown of PKM2 hampered HCC growth in both subcutaneous injection and orthotopic liver implantation models, and reduced lung metastasis in vivo. Of significance, PKM2 over-expression in human HCCs was associated with a down-regulation of a liver-specific microRNA, miR-122. We further showed that miR-122 interacted with the 3UTR of the PKM2 gene. Re-expression of miR-122 in HCC cell lines reduced PKM2 expression, decreased glucose uptake in vitro, and suppressed HCC tumor growth in vivo. Our clinical data and functional studies have revealed a novel biological mechanism involved in HCC metabolic reprogramming.
Assuntos
Carcinoma Hepatocelular/patologia , Proteínas de Transporte/metabolismo , Neoplasias Hepáticas/patologia , Neoplasias Pulmonares/secundário , Proteínas de Membrana/metabolismo , MicroRNAs/genética , Hormônios Tireóideos/metabolismo , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Regulação Neoplásica da Expressão Gênica , Glicólise , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas Experimentais , Neoplasias Pulmonares/patologia , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Nus , MicroRNAs/metabolismo , Prognóstico , Hormônios Tireóideos/genética , Proteínas de Ligação a Hormônio da TireoideRESUMO
Shuang-Huang-Lian (SHL) is a traditional Chinese formula which comprises of three medicinal herbs: Flos Lonicerae, Radix Scutellariae and Fructus Forsythiae, and is commonly used to treat acute upper respiratory tract infection, acute bronchitis and light pneumonia. A simple, reliable and reproducible rapid resolution liquid chromatography (RRLC) method was developed for the quality control of SHL preparations, which baseline separates the major bioactive compounds within 6min. The method uses a C18-HST column (2.5µm, 100mm×3.0mm) kept at 40°C. The mobile phases consist of 0.1% phosphoric acid aqueous solution and acetonitrile. Flow rate is 1.0ml/min and UV detection is performed at 327nm from 0 to 4min and 229nm from 4 to 7min. This method was further validated according to the ICH guidelines. Eight batches of commercial SHL preparations obtained from different pharmaceutical manufacturers as well as individual herbs were examined and their chromatographic profiles were compared. The stability test revealed that chlorogenic acid is stable only at acidic pH, and hence it is necessary to further evaluate and optimize the preparatory procedures and storage conditions for commercial SHL preparations.
Assuntos
Forsythia/química , Lonicera/química , Plantas Medicinais/química , Scutellaria baicalensis/química , Calibragem , Química Farmacêutica/normas , Ácido Clorogênico/análise , Cromatografia Líquida/métodos , Estabilidade de Medicamentos , Medicamentos de Ervas Chinesas , Guias como Assunto/normas , Humanos , Concentração de Íons de Hidrogênio , Limite de Detecção , Medicina Tradicional Chinesa , Estrutura Molecular , Controle de Qualidade , Padrões de Referência , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Fatores de TempoRESUMO
An RRLC method capable of simultaneous identification and rapid quantification of six biologically active compounds (salidroside, tyrosol, rosarin, rosavin, rosin, rosiridin) in Rhodiola rosea L. and two active compounds (eleutheroside B and eleutheroside E) in Eleutherococcus senticosus Maxim. was developed. The chromatographic analyses were performed on a reversed phase Phenomenex C18 (2)-HST column at 40°C with a neutral mobile phase (purified water and acetonitrile) gradient system at a flow rate of 1.0ml/min and UV detection at 205 and 220nm simultaneously. Baseline separation of eight active compounds was achieved within 8min. This developed method provides good linearity (R>0.9997), precision (RSD<1.99%) and recovery of the bioactive compounds. The RRLC method developed is capable of controlling the quality of R. rosea and E. senticosus raw herbs, commercial extracts, as well as polyherbal formulations containing R. rosea and E. senticosus as ingredients. This RRLC method is accurate and sensitive; in addition, it greatly increases sample analysis throughput with reduced analysis time, which is suitable for routine quality control analysis.
Assuntos
Química Farmacêutica/métodos , Cromatografia Líquida/métodos , Eleutherococcus/metabolismo , Extratos Vegetais/análise , Preparações de Plantas/análise , Rhodiola/metabolismo , Calibragem , Técnicas de Química Analítica , Cromatografia/métodos , Dissacarídeos/análise , Glucosídeos/análise , Lignanas/análise , Fenóis/análise , Álcool Feniletílico/análogos & derivados , Álcool Feniletílico/análise , Fenilpropionatos/análise , Extratos Vegetais/farmacologia , Controle de Qualidade , Reprodutibilidade dos Testes , Resinas Vegetais/análiseRESUMO
A simple, sensitive and reliable reversed phase Rapid Resolution Liquid Chromatography (RRLC) method was developed and validated for six biologically active compounds (salidroside, tyrosol, rosarin, rosavin, rosin and rosiridin) in Rhodiola rosea L. roots and powder extracts. The method uses a Phenomenex C18 (2)-HST column at 40 degrees C with a neutral gradient system mobile phase (H20 and acetonitrile), a flow rate of 1.0 mL/min, and UV detection wavelengths set at 205 and 254 nm, simultaneously. Baseline separation of the six active compounds was achieved within 8 minutes. The average percentages of rosavins (rosarin, rosavin, and rosin) in authentic R. rosea roots and root powder extracts were quantitatively determined and a characteristic R. rosea roots RRLC profile was established. The RRLC method is accurate and sensitive; in addition, it effectively increases the sample analysis throughput compared with conventional HPLC.