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Most children with medulloblastoma (MB) achieve remission, but some face very aggressive metastatic tumors. Their dismal outcome highlights the critical need to advance therapeutic approaches that benefit such high-risk patients. Minnelide, a clinically relevant analog of the natural product triptolide, has oncostatic activity in both preclinical and early clinical settings. Despite its efficacy and tolerable toxicity, this compound has not been evaluated in MB. Utilizing a bioinformatic data set that integrates cellular drug response data with gene expression, we predicted that Group 3 (G3) MB, which has a poor 5-year survival, would be sensitive to triptolide/Minnelide. We subsequently showed that both triptolide and Minnelide attenuate the viability of G3 MB cells ex vivo. Transcriptomic analyses identified MYC signaling, a pathologically relevant driver of G3 MB, as a downstream target of this class of drugs. We validated this MYC dependency in G3 MB cells and showed that triptolide exerts its efficacy by reducing both MYC transcription and MYC protein stability. Importantly, Minnelide acted on MYC to reduce tumor growth and leptomeningeal spread, which resulted in improved survival of G3 MB animal models. Moreover, Minnelide improved the efficacy of adjuvant chemotherapy, further highlighting its potential for the treatment of MYC-driven G3 MB.
Assuntos
Diterpenos , Compostos de Epóxi , Meduloblastoma , Fenantrenos , Proteínas Proto-Oncogênicas c-myc , Ensaios Antitumorais Modelo de Xenoenxerto , Fenantrenos/farmacologia , Diterpenos/farmacologia , Compostos de Epóxi/farmacologia , Meduloblastoma/tratamento farmacológico , Meduloblastoma/genética , Meduloblastoma/patologia , Meduloblastoma/metabolismo , Animais , Humanos , Camundongos , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Linhagem Celular Tumoral , Pró-Fármacos/farmacologia , Neoplasias Cerebelares/tratamento farmacológico , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/patologia , Neoplasias Cerebelares/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , OrganofosfatosRESUMO
OBJECTIVE: Progressive hepatic fibrosis can be considered the final stage of chronic liver disease. Hepatic stellate cells (HSC) play a central role in liver fibrogenesis. Thyroid hormones (TH, e.g. thyroxine; T4 and triiodothyronine; T3) significantly affect development, growth, cell differentiation and metabolism through activation of TH receptor α and/or ß (TRα/ß). Here, we evaluated the influence of TH in hepatic fibrogenesis. DESIGN: Human liver tissue was obtained from explanted livers following transplantation. TRα-deficient (TRα-KO) and wild-type (WT) mice were fed a control or a profibrogenic methionine-choline deficient (MCD) diet. Liver tissue was assessed by qRT-PCR for fibrogenic gene expression. In vitro, HSC were treated with TGFß in the presence or absence of T3. HSC with stable TRα knockdown and TRα deficient mouse embryonic fibroblasts (MEF) were used to determine receptor-specific function. Activation of HSC and MEF was assessed using the wound healing assay, Western blotting, and qRT-PCR. RESULTS: TRα and TRß expression is downregulated in the liver during hepatic fibrogenesis in humans and mice. TRα represents the dominant isoform in HSC. In vitro, T3 blunted TGFß-induced expression of fibrogenic genes in HSC and abrogated wound healing by modulating TGFß signalling, which depended on TRα presence. In vivo, TRα-KO enhanced MCD diet-induced liver fibrogenesis. CONCLUSION: These observations indicate that TH action in non-parenchymal cells is highly relevant. The interaction of TRα with TH regulates the phenotype of HSC via the TGFß signalling pathway. Thus, the TH-TR axis may be a valuable target for future therapy of liver fibrosis.
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Fibroblastos , Células Estreladas do Fígado , Animais , Camundongos , Humanos , Células Estreladas do Fígado/metabolismo , Hormônios Tireóideos/metabolismo , Hormônios Tireóideos/farmacologia , Receptores alfa dos Hormônios Tireóideos/genética , Receptores alfa dos Hormônios Tireóideos/metabolismo , Fator de Crescimento Transformador betaRESUMO
Dysregulation of Sonic hedgehog (SHH) signaling drives the growth of distinct cancer subtypes, including medulloblastoma (MB). Such cancers have been treated in the clinic with a number of clinically relevant SHH inhibitors, the majority of which target the upstream SHH regulator, Smoothened (SMO). Despite considerable efficacy, many of these patients develop resistance to these drugs, primarily due to mutations in SMO. Therefore, it is essential to identify druggable, signaling components downstream of SMO to target in SMO inhibitor resistant cancers. We utilized an integrated functional genomics approach to identify epigenetic regulators of SHH signaling and identified a novel complex of Ubiquitin-like with PHD and RING finger domains 1 (UHRF1), DNA methyltransferase 1 (DNMT1), and GLI proteins. We show that this complex is distinct from previously described UHRF1/DNMT1 complexes, suggesting that it works in concert to regulate GLI activity in SHH driven tumors. Importantly, we show that UHRF1/DNMT1/GLI complex stability is targeted by a repurposed FDA-approved therapy, with a subsequent reduction in the growth of SHH-dependent MB ex vivo and in vivo. IMPLICATIONS: This work describes a novel, druggable UHRF1/DNMT1/GLI complex that regulates SHH-dependent tumor growth, and highlights an FDA-approved drug capable of disrupting this complex to attenuate tumor growth.
Assuntos
Neoplasias Cerebelares , Meduloblastoma , Humanos , Proteínas Hedgehog/metabolismo , Receptor Smoothened/genética , Receptor Smoothened/metabolismo , Meduloblastoma/tratamento farmacológico , Meduloblastoma/genética , Meduloblastoma/metabolismo , Transdução de Sinais/genética , Neoplasias Cerebelares/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
SRY (sex determining region Y)-box 2 (SOX2)-labeled cells play key roles in chemoresistance and tumor relapse; thus, it is critical to elucidate the mechanisms propagating them. Single-cell transcriptomic analyses of the most common malignant pediatric brain tumor, medulloblastoma (MB), revealed the existence of astrocytic Sox2+ cells expressing sonic hedgehog (SHH) signaling biomarkers. Treatment with vismodegib, an SHH inhibitor that acts on Smoothened (Smo), led to increases in astrocyte-like Sox2+ cells. Using SOX2-enriched MB cultures, we observed that SOX2+ cells required SHH signaling to propagate, and unlike in the proliferative tumor bulk, the SHH pathway was activated in these cells downstream of Smo in an MYC-dependent manner. Functionally different GLI inhibitors depleted vismodegib-resistant SOX2+ cells from MB tissues, reduced their ability to further engraft in vivo, and increased symptom-free survival. Our results emphasize the promise of therapies targeting GLI to deplete SOX2+ cells and provide stable tumor remission.
Assuntos
Neoplasias Encefálicas , Neoplasias Cerebelares , Meduloblastoma , Neoplasias Cerebelares/genética , Criança , Proteínas Hedgehog/metabolismo , Humanos , Meduloblastoma/genética , Meduloblastoma/patologia , Recidiva Local de Neoplasia , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Transdução de Sinais , Proteína GLI1 em Dedos de Zinco/metabolismoRESUMO
The oncotherapeutic promise of IL-15, a potent immunostimulant, is limited by a short serum t 1/2 The fusion protein N-803 is a chimeric IL-15 superagonist that has a >20-fold longer in vivo t 1/2 versus IL-15. This phase 1 study characterized the pharmacokinetic (PK) profile and safety of N-803 after s.c. administration to healthy human volunteers. Volunteers received two doses of N-803, and after each dose, PK and safety were assessed for 9 d. The primary endpoint was the N-803 PK profile, the secondary endpoint was safety, and immune cell levels and immunogenicity were measures of interest. Serum N-803 concentrations peaked 4 h after administration and declined with a t 1/2 of â¼20 h. N-803 did not cause treatment-emergent serious adverse events (AEs) or grade ≥3 AEs. Injection site reactions, chills, and pyrexia were the most common AEs. Administration of N-803 was well tolerated and accompanied by proliferation of NK cells and CD8+ T cells and sustained increases in the number of NK cells. Our results suggest that N-803 administration can potentiate antitumor immunity.
Assuntos
Linfócitos T CD8-Positivos , Interleucina-15 , Voluntários Saudáveis , Humanos , Proteínas Recombinantes de FusãoRESUMO
Chronic intermittent hypoxia and hedgehog (Hh) pathway dysregulation are associated with nonalcoholic fatty liver disease (NAFLD) progression. In this study, we determined the relationship between obstructive sleep apnea (OSA)/nocturnal hypoxia and Hh signaling in pediatric NAFLD. Adolescents with histologic NAFLD (n = 31) underwent polysomnogram testing, laboratory testing, and Sonic Hh (SHh), Indian hedgehog (IHh), glioblastoma-associated oncogene 2 (Gli2), keratin 7 (K7), α-smooth muscle actin (α-SMA), and hypoxia-inducible factor 1α (HIF-1α) immunohistochemistry. Aspartate aminotransferase (AST) correlated with SHh, r = 0.64; Gli2, r = 0.4; α-SMA, r = 0.55; and K7, r = 0.45 (P < 0.01), as did alanine aminotransferase (ALT) (SHh, r = 0.51; Gli2, r = 0.43; α-SMA, r = 0.51; P < 0.02). SHh correlated with NAFLD activity score (r = 0.39), whereas IHh correlated with inflammation (r = -0.478) and histologic grade (r = -0.43); P < 0.03. Subjects with OSA/hypoxia had higher SHh (4.0 ± 2.9 versus 2.0 ± 1.5), Gli2 (74.2 ± 28.0 versus 55.8 ± 11.8), and α-SMA (6.2 ± 3.3 versus 4.3 ± 1.2); compared to those without (P < 0.03). OSA severity correlated with SHh (r = 0.31; P = 0.09) and Gli2 (r = 0.37; P = 0.04) as did hypoxia severity, which was associated with increasing SHh (r = -0.53), Gli2 (r = -0.52), α-SMA (r = -0.61), and K7 (r = -0.42); P < 0.02. Prolonged O2 desaturations <90% also correlated with SHh (r = 0.55) and Gli2 (r = 0.61); P < 0.05. Conclusion: The Hh pathway is activated in pediatric patients with NAFLD with nocturnal hypoxia and relates to disease severity. Tissue hypoxia may allow for functional activation of HIF-1α, with induction of genes important in epithelial-mesenchymal transition, including SHh, and NAFLD progression.
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BACKGROUND & AIMS: Chronic failure of mechanisms that promote effective regeneration of dead hepatocytes causes replacement of functional hepatic parenchyma with fibrous scar tissue, ultimately resulting in cirrhosis. Therefore, defining and optimizing mechanisms that orchestrate effective regeneration might prevent cirrhosis. We hypothesized that effective regeneration of injured livers requires hepatocytes to evade the growth-inhibitory actions of TGFß, since TGFß signaling inhibits mature hepatocyte growth but drives cirrhosis pathogenesis. METHODS: Wild-type mice underwent 70% partial hepatectomy (PH); TGFß expression and signaling were evaluated in intact tissue and primary hepatocytes before, during, and after the period of maximal hepatocyte proliferation that occurs from 24-72â¯h after PH. To determine the role of Yap1 in regulating TGFß signaling in hepatocytes, studies were repeated after selectively deleting Yap1 from hepatocytes of Yap1flox/flox mice. RESULTS: TGFß expression and hepatocyte nuclear accumulation of pSmad2 and Yap1 increased in parallel with hepatocyte proliferative activity after PH. Proliferative hepatocytes also upregulated Snai1, a pSmad2 target gene that promotes epithelial-to-mesenchymal transition (EMT), suppressed epithelial genes, induced myofibroblast markers, and produced collagen 1α1. Deleting Yap1 from hepatocytes blocked their nuclear accumulation of pSmad2 and EMT-like response, as well as their proliferation. CONCLUSION: Interactions between the TGFß and Hippo-Yap signaling pathways stimulate hepatocytes to undergo an EMT-like response that is necessary for them to grow in a TGFß-enriched microenvironment and regenerate injured livers. LAY SUMMARY: The adult liver has an extraordinary ability to regenerate after injury despite the accumulation of scar-forming factors that normally block the proliferation and reduce the survival of residual liver cells. We discovered that liver cells manage to escape these growth-inhibitory influences by transiently becoming more like fibroblasts themselves. They do this by reactivating programs that are known to drive tissue growth during fetal development and in many cancers. Understanding how the liver can control programs that are involved in scarring and cancer may help in the development of new treatments for cirrhosis and liver cancer.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Transição Epitelial-Mesenquimal/fisiologia , Hepatócitos/fisiologia , Regeneração Hepática/fisiologia , Fosfoproteínas/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Proteínas de Ciclo Celular , Proliferação de Células , Camundongos , Camundongos Knockout , Transdução de Sinais , Proteínas de Sinalização YAPRESUMO
BACKGROUND: Immunotherapy with PD-1 or PD-L1 blockade fails to induce a response in about 80% of patients with unselected non-small cell lung cancer (NSCLC), and many of those who do initially respond then develop resistance to treatment. Agonists that target the shared interleukin-2 (IL-2) and IL-15Rßγ pathway have induced complete and durable responses in some cancers, but no studies have been done to assess the safety or efficacy of these agonists in combination with anti-PD-1 immunotherapy. We aimed to define the safety, tolerability, and activity of this drug combination in patients with NSCLC. METHODS: In this non-randomised, open-label, phase 1b trial, we enrolled patients (aged ≥18 years) with previously treated histologically or cytologically confirmed stage IIIB or IV NSCLC from three academic hospitals in the USA. Key eligibility criteria included measurable disease, eligibility to receive anti-PD-1 immunotherapy, and an Eastern Cooperative Oncology Group performance status of 0 or 1. Patients received the anti-PD-1 monoclonal antibody nivolumab intravenously at 3 mg/kg (then 240 mg when US Food and Drug Administration [FDA]-approved dosing changed) every 14 days (either as new treatment or continued treatment at the time of disease progression) and the IL-15 superagonist ALT-803 subcutaneously once per week on weeks 1-5 of four 6-week cycles for 6 months. ALT-803 was administered at one of four escalating dose concentrations: 6, 10, 15, or 20 µg/kg. The primary endpoint was to define safety and tolerability and to establish a recommended phase 2 dose of ALT-803 in combination with nivolumab. Analyses were per-protocol and included any patients who received at least one dose of study treatment. This trial is registered with ClinicalTrials.gov, number NCT02523469; phase 2 enrolment of patients is ongoing. FINDINGS: Between Jan 18, 2016, and June 28, 2017, 23 patients were enrolled and 21 were treated at four dose levels of ALT-803 in combination with nivolumab. Two patients did not receive treatment because of the development of inter-current illness during enrolment, one patient due to leucopenia and one patient due to pulmonary dysfunction. No dose-limiting toxicities were recorded and the maximum tolerated dose was not reached. The most common adverse events were injection-site reactions (in 19 [90%] of 21 patients) and flu-like symptoms (15 [71%]). The most common grade 3 adverse events, occurring in two patients each, were lymphocytopenia and fatigue. A grade 3 myocardial infarction occurred in one patient. No grade 4 or 5 adverse events were recorded. The recommended phase 2 dose of ALT-803 is 20 µg/kg given once per week subcutaneously in combination with 240 mg intravenous nivolumab every 2 weeks. INTERPRETATION: ALT-803 in combination with nivolumab can be safely administered in an outpatient setting. The promising clinical activity observed with the addition of ALT-803 to the regimen of patients with PD-1 monoclonal antibody relapsed and refractory disease shows evidence of anti-tumour activity for a new class of agents in NSCLC. FUNDING: Altor BioScience (a NantWorks company), National Institutes of Health, and Medical University of South Carolina Hollings Cancer Center.
Assuntos
Antineoplásicos Imunológicos/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Nivolumabe/administração & dosagem , Proteínas/administração & dosagem , Idoso , Antineoplásicos Imunológicos/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Carcinoma Pulmonar de Células não Pequenas/secundário , Feminino , Humanos , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Nivolumabe/efeitos adversos , Proteínas/efeitos adversos , Proteínas Recombinantes de Fusão , Fatores de Tempo , Resultado do Tratamento , Estados UnidosRESUMO
BACKGROUND & AIMS: Cirrhosis results from accumulation of myofibroblasts derived from quiescent hepatic stellate cells (Q-HSCs); it regresses when myofibroblastic HSCs are depleted. Hedgehog signaling promotes transdifferentiation of HSCs by activating Yes-associated protein 1 (YAP1 or YAP) and inducing aerobic glycolysis. However, increased aerobic glycolysis alone cannot meet the high metabolic demands of myofibroblastic HSCs. Determining the metabolic processes of these cells could lead to strategies to prevent progressive liver fibrosis, so we investigated whether glutaminolysis (conversion of glutamine to alpha-ketoglutarate) sustains energy metabolism and permits anabolism when Q-HSCs become myofibroblastic, and whether this is controlled by hedgehog signaling to YAP. METHODS: Primary HSCs were isolated from C57BL/6 or Smoflox/flox mice; we also performed studies with rat and human myofibroblastic HSCs. We measured changes of glutaminolytic genes during culture-induced primary HSC transdifferentiation. Glutaminolysis was disrupted in cells by glutamine deprivation or pathway inhibitors (bis-2-[5-phenylacetamido-1,2,4-thiadiazol-2-yl] ethyl sulfide, CB-839, epigallocatechin gallate, and aminooxyacetic acid), and effects on mitochondrial respiration, cell growth and migration, and fibrogenesis were measured. Hedgehog signaling to YAP was disrupted in cells by adenovirus expression of Cre-recombinase or by small hairpin RNA knockdown of YAP. Hedgehog and YAP activity were inhibited by incubation of cells with cyclopamine or verteporfin, and effects on glutaminolysis were measured. Acute and chronic liver fibrosis were induced in mice by intraperitoneal injection of CCl4 or methionine choline-deficient diet. Some mice were then given injections of bis-2-[5-phenylacetamido-1,2,4-thiadiazol-2-yl] ethyl sulfide to inhibit glutaminolysis, and myofibroblast accumulation was measured. We also performed messenger RNA and immunohistochemical analyses of percutaneous liver biopsies from healthy human and 4 patients with no fibrosis, 6 patients with mild fibrosis, and 3 patients with severe fibrosis. RESULTS: Expression of genes that regulate glutaminolysis increased during transdifferentiation of primary Q-HSCs into myofibroblastic HSCs, and inhibition of glutaminolysis disrupted transdifferentiation. Blocking glutaminolysis in myofibroblastic HSCs suppressed mitochondrial respiration, cell growth and migration, and fibrogenesis; replenishing glutaminolysis metabolites to these cells restored these activities. Knockout of the hedgehog signaling intermediate smoothened or knockdown of YAP inhibited expression of glutaminase, the rate-limiting enzyme in glutaminolysis. Hedgehog and YAP inhibitors blocked glutaminolysis and suppressed myofibroblastic activities in HSCs. In livers of patients and of mice with acute or chronic fibrosis, glutaminolysis was induced in myofibroblastic HSCs. In mice with liver fibrosis, inhibition of glutaminase blocked accumulation of myofibroblasts and fibrosis progression. CONCLUSIONS: Glutaminolysis controls accumulation of myofibroblast HSCs in mice and might be a therapeutic target for cirrhosis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Metabolismo Energético , Glutamina/metabolismo , Proteínas Hedgehog/metabolismo , Células Estreladas do Fígado/metabolismo , Cirrose Hepática/metabolismo , Fígado/metabolismo , Miofibroblastos/metabolismo , Fosfoproteínas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Estudos de Casos e Controles , Proteínas de Ciclo Celular , Proliferação de Células , Transdiferenciação Celular , Células Cultivadas , Reprogramação Celular , Regulação da Expressão Gênica , Glutaminase/metabolismo , Proteínas Hedgehog/genética , Células Estreladas do Fígado/patologia , Humanos , Ácidos Cetoglutáricos/metabolismo , Fígado/patologia , Cirrose Hepática/genética , Cirrose Hepática/patologia , Cirrose Hepática Experimental/genética , Cirrose Hepática Experimental/metabolismo , Cirrose Hepática Experimental/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias Hepáticas/metabolismo , Mitocôndrias Hepáticas/patologia , Miofibroblastos/patologia , Fenótipo , Fosfoproteínas/genética , Interferência de RNA , Ratos , Transdução de Sinais , Receptor Smoothened/genética , Receptor Smoothened/metabolismo , Fatores de Tempo , Fatores de Transcrição , Transfecção , Proteínas de Sinalização YAPRESUMO
BACKGROUND: Obesity is associated with multiple diseases, but it is unclear how obesity promotes progressive tissue damage. Recovery from injury requires repair, an energy-expensive process that is coupled to energy availability at the cellular level. The satiety factor, leptin, is a key component of the sensor that matches cellular energy utilization to available energy supplies. Leptin deficiency signals energy depletion, whereas activating the Hedgehog pathway drives energy-consuming activities. Tissue repair is impaired in mice that are obese due to genetic leptin deficiency. Tissue repair is also blocked and obesity enhanced by inhibiting Hedgehog activity. We evaluated the hypothesis that loss of leptin silences Hedgehog signaling in pericytes, multipotent leptin-target cells that regulate a variety of responses that are often defective in obesity, including tissue repair and adipocyte differentiation. RESULTS: We found that pericytes from liver and white adipose tissue require leptin to maintain expression of the Hedgehog co-receptor, Smoothened, which controls the activities of Hedgehog-regulated Gli transcription factors that orchestrate gene expression programs that dictate pericyte fate. Smoothened suppression prevents liver pericytes from being reprogrammed into myofibroblasts, but stimulates adipose-derived pericytes to become white adipocytes. Progressive Hedgehog pathway decay promotes senescence in leptin-deficient liver pericytes, which, in turn, generate paracrine signals that cause neighboring hepatocytes to become fatty and less proliferative, enhancing vulnerability to liver damage. CONCLUSIONS: Leptin-responsive pericytes evaluate energy availability to inform tissue construction by modulating Hedgehog pathway activity and thus, are at the root of progressive obesity-related tissue pathology. Leptin deficiency inhibits Hedgehog signaling in pericytes to trigger a pericytopathy that promotes both adiposity and obesity-related tissue damage.
Assuntos
Células Estreladas do Fígado/fisiologia , Leptina/genética , Obesidade/fisiopatologia , Receptor Smoothened/genética , Receptor Smoothened/metabolismo , Adipócitos/citologia , Adipócitos/metabolismo , Tecido Adiposo/citologia , Animais , Diferenciação Celular , Movimento Celular , Proliferação de Células , Células Cultivadas , Proteínas Hedgehog/fisiologia , Células Estreladas do Fígado/citologia , Células Estreladas do Fígado/metabolismo , Leptina/deficiência , Leptina/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/fisiologia , Camundongos , Camundongos Obesos , Miofibroblastos/citologia , Miofibroblastos/metabolismo , Obesidade/genética , Comunicação Parácrina/genética , Receptores para Leptina/metabolismo , Receptor Smoothened/agonistasRESUMO
BACKGROUND: After portal vein ligation of 1 side of the liver, the other side regenerates at a slow rate. This slow growth may be accelerated to rapid growth by adding a transection between the 2 sides, i.e., performing portal vein ligation and parenchymal transection. We found that in patients undergoing portal vein ligation and parenchymal transection, portal vein hyperflow in the regenerating liver causes a significant reduction of arterial flow due to the hepatic arterial buffer response. We postulated that the reduction of arterial flow induces hypoxia in the regenerating liver and used a rat model to assess hypoxia and its impact on kinetic growth. METHODS: A rat model of rapid (portal vein ligation and parenchymal transection) and slow regeneration (portal vein ligation) was established. Portal vein flow and pressure data were collected. Liver regeneration was assessed in rats using computed tomography, proliferation with Ki-67, and hypoxia with pimonidazole and HIF-1α staining. RESULTS: The rat model confirmed acceleration of regeneration in portal vein ligation and parenchymal transection as well as the portal vein hyperflow seen in patients. Additionally, tissue hypoxia was observed after portal vein ligation and parenchymal transection, while little hypoxia staining was detected after portal vein ligation. To determine if hypoxia is a consequence or an inciting stimulus of rapid liver regeneration, we used a prolyl-hydroxylase blocker to activate hypoxia signaling pathways in the slow model. This clearly accelerated slow to rapid liver regeneration. Inversely, abrogation of hypoxia led to a blunting of rapid growth to slow growth. The topical application of prolyl-hydroxylase inhibitors on livers in rats induced spontaneous areas of regeneration. CONCLUSION: This study shows that pharmacologically induced hypoxic signaling accelerates liver regeneration similar to portal vein ligation and parenchymal transection. Hypoxia is likely an accelerator of liver regeneration. Also, prolyl-hydroxylase inhibitors may be used to enhance liver regeneration pharmaceutically.
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Regeneração Hepática/fisiologia , Fígado/irrigação sanguínea , Fígado/patologia , Veia Porta , Animais , Hipóxia Celular/fisiologia , Hepatectomia , Ligadura , Masculino , Modelos Animais , Ratos , Ratos WistarRESUMO
UNLABELLED: Adult liver regeneration requires induction and suppression of proliferative activity in multiple types of liver cells. The mechanisms that orchestrate the global changes in gene expression that are required for proliferative activity to change within individual liver cells, and that coordinate proliferative activity among different types of liver cells, are not well understood. Morphogenic signaling pathways that are active during fetal development, including Hedgehog and Hippo/Yes-associated protein 1 (Yap1), regulate liver regeneration in adulthood. Cirrhosis and liver cancer result when these pathways become dysregulated, but relatively little is known about the mechanisms that coordinate and control morphogenic signaling during effective liver regeneration. We evaluated the hypothesis that the Hedgehog pathway controls Yap1 activation during liver regeneration by studying intact mice and cultured liver cells. In cultured hepatic stellate cells (HSCs), disrupting Hedgehog signaling blocked activation of Yap1, and knocking down Yap1 inhibited induction of both Yap1- and Hedgehog-regulated genes that enable HSC to become myofibroblasts (MFs). In mice, disrupting Hedgehog signaling in MFs inhibited liver regeneration after partial hepactectomy (PH). Reduced proliferative activity in the liver epithelial compartment resulted from loss of stroma-derived paracrine signals that activate Yap1 and the Hedgehog pathway in hepatocytes. This prevented hepatocytes from up-regulating Yap1- and Hedgehog-regulated transcription factors that normally promote their proliferation. CONCLUSIONS: Morphogenic signaling in HSCs is necessary to reprogram hepatocytes to regenerate the liver epithelial compartment post-PH. This discovery identifies novel molecules that might be targeted to correct defective repair during cirrhosis and liver cancer. (Hepatology 2016;64:232-244).
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Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Hedgehog/metabolismo , Células Estreladas do Fígado/metabolismo , Regeneração Hepática , Fosfoproteínas/metabolismo , Animais , Proteínas de Ciclo Celular , Desdiferenciação Celular , Proliferação de Células , Hepatectomia , Hepatócitos/fisiologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Comunicação Parácrina , Regulação para Cima , Proteínas de Sinalização YAPRESUMO
INTRODUCTION: Liver fibrosis develops when hepatic stellate cells (HSC) are activated into collagen-producing myofibroblasts. In non-alcoholic steatohepatitis (NASH), the adipokine leptin is upregulated, and promotes liver fibrosis by directly activating HSC via the hedgehog pathway. We reported that hedgehog-regulated osteopontin (OPN) plays a key role in promoting liver fibrosis. Herein, we evaluated if OPN mediates leptin-profibrogenic effects in NASH. METHODS: Leptin-deficient (ob/ob) and wild-type (WT) mice were fed control or methionine-choline deficient (MCD) diet. Liver tissues were assessed by Sirius-red, OPN and αSMA IHC, and qRT-PCR for fibrogenic genes. In vitro, HSC with stable OPN (or control) knockdown were treated with recombinant (r)leptin and OPN-neutralizing or sham-aptamers. HSC response to OPN loss was assessed by wound healing assay. OPN-aptamers were also added to precision-cut liver slices (PCLS), and administered to MCD-fed WT (leptin-intact) mice to determine if OPN neutralization abrogated fibrogenesis. RESULTS: MCD-fed WT mice developed NASH-fibrosis, upregulated OPN, and accumulated αSMA+ cells. Conversely, MCD-fed ob/ob mice developed less fibrosis and accumulated fewer αSMA+ and OPN+ cells. In vitro, leptin-treated HSC upregulated OPN, αSMA, collagen 1α1 and TGFß mRNA by nearly 3-fold, but this effect was blunted by OPN loss. Inhibition of PI3K and transduction of dominant negative-Akt abrogated leptin-mediated OPN induction, while constitutive active-Akt upregulated OPN. Finally, OPN neutralization reduced leptin-mediated fibrogenesis in both PCLS and MCD-fed mice. CONCLUSION: OPN overexpression in NASH enhances leptin-mediated fibrogenesis via PI3K/Akt. OPN neutralization significantly reduces NASH fibrosis, reinforcing the potential utility of targeting OPN in the treatment of patients with advanced NASH.
Assuntos
Leptina/metabolismo , Cirrose Hepática/metabolismo , Fígado/patologia , Hepatopatia Gordurosa não Alcoólica/metabolismo , Osteopontina/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Deleção de Genes , Hepatócitos/metabolismo , Hepatócitos/patologia , Leptina/genética , Fígado/metabolismo , Cirrose Hepática/genética , Cirrose Hepática/patologia , Masculino , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/patologia , Osteopontina/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos Sprague-Dawley , Transdução de Sinais , Regulação para CimaRESUMO
AIMS: Recent studies have suggested that oesophageal submucosal gland (ESMG) ducts harbour progenitor cells that may contribute to oesophageal metaplasia. Our objective was to determine whether histological differences exist between the ESMGs of individuals with and without oesophageal adenocarcinoma (EAC). METHODS AND RESULTS: We performed histological assessment of 343 unique ESMGs from 30 control patients, 24 patients with treatment-naïve high-grade columnar dysplasia (HGD) or EAC, and 23 non-EAC oesophagectomy cases. A gastrointestinal pathologist assessed haematoxylin and eosin-stained ESMG images by using a scoring system that assigns individual ESMG acini to five histological types (mucous, serous, oncocytic, dilated, or ductal metaplastic). In our model, ductal metaplastic acini were more common in patients with HGD/EAC (12.7%) than in controls (3.5%) (P = 0.006). We also identified greater proportions of acini with dilation (21.9%, P < 0.001) and, to a lesser extent, ductal metaplasia (4.3%, P = 0.001) in non-EAC oesophagectomy cases than in controls. Ductal metaplasia tended to occur in areas of mucosal ulceration or tumour. CONCLUSIONS: We found a clear association between ductal metaplastic ESMG acini and HGD/EAC. Non-EAC cases had dilated acini and some ductal dilation. Because ESMGs and ducts harbour putative progenitor cells, these associations could have significance for understanding the pathogenesis of EAC.
Assuntos
Adenocarcinoma/patologia , Neoplasias Esofágicas/patologia , Esôfago/patologia , Inflamação/patologia , Adenocarcinoma/cirurgia , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias Esofágicas/cirurgia , Esofagectomia , Esôfago/cirurgia , Feminino , Humanos , Inflamação/cirurgia , Masculino , Metaplasia/patologia , Metaplasia/cirurgia , Pessoa de Meia-IdadeRESUMO
TNF-like weak inducer of apoptosis (TWEAK) is a growth factor for bipotent liver progenitors that express its receptor, fibroblast growth factor-inducible 14 (Fn14), a TNF receptor superfamily member. Accumulation of Fn14(+) progenitors occurs in severe acute alcoholic steatohepatitis (ASH) and correlates with acute mortality. In patients with severe ASH, inhibition of TNF-α increases acute mortality. The aim of this study was to determine whether deletion of Fn14 improves the outcome of liver injury in alcohol-consuming mice. Wild-type (WT) and Fn14 knockout (KO) mice were fed control high-fat Lieber deCarli diet or high-fat Lieber deCarli diet with 2% alcohol (ETOH) and injected intraperitoneally with CCl4 for 2 wk to induce liver injury. Mice were euthanized 3 or 10 days after CCl4 treatment. Survival was assessed. Liver tissues were analyzed for cell death, inflammation, proliferation, progenitor accumulation, and fibrosis by quantitative RT-PCR, immunoblot, hydroxyproline content, and quantitative immunohistochemistry. During liver injury, Fn14 expression, apoptosis, inflammation, hepatocyte replication, progenitor and myofibroblast accumulation, and fibrosis increased in WT mice fed either diet. Mice fed either diet expressed similar TWEAK/Fn14 levels, but ETOH-fed mice had higher TNF-α expression. The ETOH-fed group developed more apoptosis, inflammation, fibrosis, and regenerative responses. Fn14 deletion did not reduce hepatic TNF-α expression but improved all injury parameters in mice fed the control diet. In ETOH-fed mice, Fn14 deletion inhibited TNF-α induction and increased acute mortality, despite improvement in liver injury. Fn14 mediates wound-healing responses that are necessary to survive acute liver injury during alcohol exposure.
Assuntos
Fígado Gorduroso Alcoólico/metabolismo , Fígado/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Doença Aguda , Animais , Apoptose , Tetracloreto de Carbono , Proliferação de Células , Modelos Animais de Doenças , Etanol , Fígado Gorduroso Alcoólico/etiologia , Fígado Gorduroso Alcoólico/genética , Fígado Gorduroso Alcoólico/patologia , Hidroxiprolina/metabolismo , Mediadores da Inflamação/metabolismo , Fígado/patologia , Cirrose Hepática Alcoólica/etiologia , Cirrose Hepática Alcoólica/metabolismo , Cirrose Hepática Alcoólica/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores do Fator de Necrose Tumoral/deficiência , Receptores do Fator de Necrose Tumoral/genética , Transdução de Sinais , Receptor de TWEAK , Fatores de Tempo , Fator de Necrose Tumoral alfa/metabolismo , CicatrizaçãoRESUMO
BACKGROUND & AIMS: Pro-inflammatory cytokines are important for liver regeneration after partial hepatectomy (PH). Expression of Fibroblast growth factor-inducible 14 (Fn14), the receptor for TNF-like weak inducer of apoptosis (TWEAK), is induced rapidly after PH and remains elevated throughout the period of peak hepatocyte replication. The role of Fn14 in post-PH liver regeneration is uncertain because Fn14 is expressed by liver progenitors and TWEAK-Fn14 interactions stimulate progenitor growth, but replication of mature hepatocytes is thought to drive liver regeneration after PH. METHODS: To clarify the role of TWEAK-Fn14 after PH, we compared post-PH regenerative responses in wild type (WT) mice, Fn14 knockout (KO) mice, TWEAK KO mice, and WT mice treated with anti-TWEAK antibodies. RESULTS: In WT mice, rare Fn14(+) cells localized with other progenitor markers in peri-portal areas before PH. PH rapidly increased proliferation of Fn14(+) cells; hepatocytic cells that expressed Fn14 and other progenitor markers, such as Lgr5, progressively accumulated from 12-8 h post-PH and then declined to baseline by 96 h. When TWEAK/Fn14 signaling was disrupted, progenitor accumulation, induction of pro-regenerative cytokines, hepatocyte and cholangiocyte proliferation, and over-all survival were inhibited, while post-PH liver damage and bilirubin levels were increased. TWEAK stimulated proliferation and increased Lgr5 expression in cultured liver progenitors, but had no effect on either parameter in cultured primary hepatocytes. CONCLUSIONS: TWEAK-FN14 signaling is necessary for the healthy adult liver to regenerate normally after acute partial hepatectomy.
Assuntos
Hepatectomia , Regeneração Hepática , Fígado/metabolismo , Fígado/cirurgia , Receptores do Fator de Necrose Tumoral/metabolismo , Transdução de Sinais , Fatores de Necrose Tumoral/metabolismo , Animais , Anticorpos/farmacologia , Proliferação de Células/efeitos dos fármacos , Citocina TWEAK , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Deleção de Genes , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Fígado/citologia , Regeneração Hepática/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitógenos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Receptor de TWEAK , Inibidores do Fator de Necrose TumoralRESUMO
BACKGROUND: Alcohol consumption promotes hepatocellular carcinoma (HCC). The responsible mechanisms are not well understood. Hepatocarcinogenesis increases with age and is enhanced by factors that impose a demand for liver regeneration. Because alcohol is hepatotoxic, habitual alcohol ingestion evokes a recurrent demand for hepatic regeneration. The alcohol-preferring (P) rat model mimics the level of alcohol consumption by humans who habitually abuse alcohol. Previously, we showed that habitual heavy alcohol ingestion amplified age-related hepatocarcinogenesis in P rats, with over 80% of alcohol-consuming P rats developing HCCs after 18 months of alcohol exposure, compared with only 5% of water-drinking controls. METHODS: Herein, we used quantitative real-time PCR and quantitative immunocytochemistry to compare liver tissues from alcohol-consuming P rats and water-fed P rat controls after 6, 12, or 18 months of drinking. We aimed to identify potential mechanisms that might underlie the differences in liver cancer formation and hypothesized that chronic alcohol ingestion would activate Hedgehog (HH), a regenerative signaling pathway that is overactivated in HCC. RESULTS: Chronic alcohol ingestion amplified age-related degenerative changes in hepatocytes, but did not cause appreciable liver inflammation or fibrosis even after 18 months of heavy drinking. HH signaling was also enhanced by alcohol exposure, as evidenced by increased levels of mRNAs encoding HH ligands, HH-regulated transcription factors, and HH target genes. Immunocytochemistry confirmed increased alcohol-related accumulation of HH ligand-producing cells and HH-responsive target cells. HH-related regenerative responses were also induced in alcohol-exposed rats. Three of these processes (i.e., deregulated progenitor expansion, the reverse Warburg effect, and epithelial-to-mesenchymal transitions) are known to promote cancer growth in other tissues. CONCLUSIONS: Alcohol-related changes in Hedgehog signaling and resultant deregulation of liver cell replacement might promote hepatocarcinogenesis.
Assuntos
Carcinogênese/efeitos dos fármacos , Depressores do Sistema Nervoso Central/efeitos adversos , Etanol/efeitos adversos , Proteínas Hedgehog/metabolismo , Neoplasias Hepáticas Experimentais/induzido quimicamente , Animais , Transição Epitelial-Mesenquimal , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Distribuição Aleatória , RatosRESUMO
UNLABELLED: It is unclear why the histology of pediatric and adult nonalcoholic fatty liver disease (NAFLD) sometimes differs. In adults, severity of portal inflammation and fibrosis correlate with Hedgehog pathway activity. Hedgehog (Hh) signaling regulates organogenesis, but is silent in adult livers until injury reinduces Hh ligand production. During adolescence, liver development is completed and children's livers normally lose cells that produce and/or respond to Hh ligands. We postulated that fatty liver injury interferes with this process by increasing Hh ligand production, and theorized that hepatic responses to Hh ligands might differ among children according to age, gender, and/or puberty status. Using unstained liver biopsy slides from 56 children with NAFLD, we performed immunohistochemistry to assess Hh pathway activation and correlated the results with clinical information obtained at biopsy. Fibrosis stage generally correlated with Hh pathway activity, as demonstrated by the numbers of Hh-ligand-producing cells (P < 0.0001) and Hh-responsive (glioma-associated oncogene 2-positive [Gli2]) cells (P = 0.0013). The numbers of Gli2(+) cells also correlated with portal inflammation grade (P = 0.0012). Two distinct zonal patterns of Hh-ligand production, portal/periportal versus lobular, were observed. Higher portal/periportal Hh-ligand production was associated with male gender. Male gender and prepuberty were also associated with ductular proliferation (P < 0.05), increased numbers of portal Gli2(+) cells (P < 0.017) and portal fibrosis. CONCLUSION: The portal/periportal (progenitor) compartment of prepubescent male livers exhibits high Hh pathway activity. This may explain the unique histologic features of pediatric NAFLD because Hh signaling promotes the fibroductular response.
Assuntos
Fígado Gorduroso/fisiopatologia , Proteínas Hedgehog/fisiologia , Fígado/fisiopatologia , Transdução de Sinais/fisiologia , Actinas/metabolismo , Adolescente , Fatores Etários , Biópsia , Criança , Estudos Transversais , Fígado Gorduroso/metabolismo , Feminino , Humanos , Queratina-7/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Fígado/metabolismo , Fígado/patologia , Masculino , Hepatopatia Gordurosa não Alcoólica , Proteínas Nucleares/metabolismo , Sistema Porta/metabolismo , Sistema Porta/patologia , Fatores Sexuais , Vimentina/metabolismo , Proteína Gli2 com Dedos de ZincoRESUMO
Hepatocellular carcinoma (HCC) typically develops in cirrhosis, a condition characterized by Hedgehog (Hh) pathway activation and accumulation of Hh-responsive myofibroblasts. Although Hh signaling generally regulates stromal-epithelial interactions that support epithelial viability, the role of Hh-dependent myofibroblasts in hepatocarcinogenesis is unknown. Here, we used human HCC samples, a mouse HCC model, and hepatoma cell/myofibroblast cocultures to examine the hypothesis that Hh signaling modulates myofibroblasts' metabolism to generate fuels for neighboring malignant hepatocytes. The results identify a novel paracrine mechanism whereby malignant hepatocytes produce Hh ligands to stimulate glycolysis in neighboring myofibroblasts, resulting in release of myofibroblast-derived lactate that the malignant hepatocytes use as an energy source. This discovery reveals new diagnostic and therapeutic targets that might be exploited to improve the outcomes of cirrhotic patients with HCCs.
Assuntos
Carcinoma Hepatocelular/metabolismo , Proteínas Hedgehog/fisiologia , Neoplasias Hepáticas/metabolismo , Comunicação Parácrina/fisiologia , Animais , Carcinoma Hepatocelular/complicações , Carcinoma Hepatocelular/patologia , Células Cultivadas , Fígado Gorduroso/complicações , Fígado Gorduroso/metabolismo , Fígado Gorduroso/patologia , Glicólise/fisiologia , Proteínas Hedgehog/metabolismo , Células Hep G2 , Humanos , Ácido Láctico/metabolismo , Lipogênese/fisiologia , Neoplasias Hepáticas/complicações , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Knockout , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Hepatopatia Gordurosa não AlcoólicaRESUMO
BACKGROUND & AIMS: The pathogenesis of cirrhosis, a disabling outcome of defective liver repair, involves deregulated accumulation of myofibroblasts derived from quiescent hepatic stellate cells (HSCs), but the mechanisms that control transdifferentiation of HSCs are poorly understood. We investigated whether the Hedgehog (Hh) pathway controls the fate of HSCs by regulating metabolism. METHODS: Microarray, quantitative polymerase chain reaction, and immunoblot analyses were used to identify metabolic genes that were differentially expressed in quiescent vs myofibroblast HSCs. Glycolysis and lactate production were disrupted in HSCs to determine if metabolism influenced transdifferentiation. Hh signaling and hypoxia-inducible factor 1α (HIF1α) activity were altered to identify factors that alter glycolytic activity. Changes in expression of genes that regulate glycolysis were quantified and localized in biopsy samples from patients with cirrhosis and liver samples from mice following administration of CCl(4) or bile duct ligation. Mice were given systemic inhibitors of Hh to determine if they affect glycolytic activity of the hepatic stroma; Hh signaling was also conditionally disrupted in myofibroblasts to determine the effects of glycolytic activity. RESULTS: Transdifferentiation of cultured, quiescent HSCs into myofibroblasts induced glycolysis and caused lactate accumulation. Increased expression of genes that regulate glycolysis required Hh signaling and involved induction of HIF1α. Inhibitors of Hh signaling, HIF1α, glycolysis, or lactate accumulation converted myofibroblasts to quiescent HSCs. In diseased livers of animals and patients, numbers of glycolytic stromal cells were associated with the severity of fibrosis. Conditional disruption of Hh signaling in myofibroblasts reduced numbers of glycolytic myofibroblasts and liver fibrosis in mice; similar effects were observed following administration of pharmacologic inhibitors of Hh. CONCLUSIONS: Hedgehog signaling controls the fate of HSCs by regulating metabolism. These findings might be applied to diagnosis and treatment of patients with cirrhosis.