Targeting mitochondria and post-traslational modifications: new promising approaches for the treatment of liver disease

108 p. El contenido de los capítulos 5, 6 y 7 están sujetos a confidencialidadEn esta tesis doctoral hemos corroborado que tanto una correcta función mitocondrial como la homeostasis de proteínas son fundamentales para el correcto funcionamiento del hígado y que alteraciones en ambos procesos están asociados con la lesión hepática. Hemos descubierto nuevos mecanismos subyacentes de la enfermedad hepática, a nivel mitocondrial y de modificaciones post-traduccionales que podrían ayudar al desarrollo de nuevos fármacos y terapias para su tratamiento. Concretamente, hemos descrito que MCJ es un regulador negativo del metabolismo mitocondrial en hígado y que su expresión está altamente implicada en el daño hepático por fármacos y en hígado graso no alcohólico. De manera importante, hemos demostrado que la deficiencia en MCJ podría ser una terapia prometedora para estas enfermedades, ya que su silenciamiento en modelos experimentales animales ha resultado muy efectivo, manteniendo las mitocondrias activas y evitando el daño hepático y la progresión de la enfermedad. Por otro lado hemos descrito que la proteína mitocondrial PHB1 tiene un papel en colestasis, ya que sus niveles se encuentran disminuidos específicamente en pacientes con enfermedades colestásicas y que su deficiencia predispone al daño por ligadura del conducto biliar. Hemos visto que en colestasis, a parte de su función mitocondrial, bajos niveles de PHB1 regulan la actividad del proteosoma e inducen cambios transcripcionales a través de la sobreexpresión y localización nuclear de HDAC4. En este respecto, hemos observado que la disminución de la expresión de HDAC4 mejora el daño hepático asociado a la deficiencia de PHB1. Finalmente, hemos presentado evidencias sobre el importante papel de la nedilización en el desarrollo del CHC y hemos identificado LKB1 y Akt, conocidas oncoquinasas, como nuevas dianas de Nedd8. Tanto el grado de nedilización como los niveles de LKB1 y Akt se asocian a un peor pronóstico. Además, hemos demostrado que inhibir la nedilización bloquea el crecimiento tumoral y mejora el estado del hígado pudiendo ser una buena terapia para pacientes con carcinoma hepatocelular

Targeting mitochondria and post-traslational modifications: new promising approaches for the treatment of liver disease

108 p. El contenido de los capítulos 5, 6 y 7 están sujetos a confidencialidadEn esta tesis doctoral hemos corroborado que tanto una correcta función mitocondrial como la homeostasis de proteínas son fundamentales para el correcto funcionamiento del hígado y que alteraciones en ambos procesos están asociados con la lesión hepática. Hemos descubierto nuevos mecanismos subyacentes de la enfermedad hepática, a nivel mitocondrial y de modificaciones post-traduccionales que podrían ayudar al desarrollo de nuevos fármacos y terapias para su tratamiento. Concretamente, hemos descrito que MCJ es un regulador negativo del metabolismo mitocondrial en hígado y que su expresión está altamente implicada en el daño hepático por fármacos y en hígado graso no alcohólico. De manera importante, hemos demostrado que la deficiencia en MCJ podría ser una terapia prometedora para estas enfermedades, ya que su silenciamiento en modelos experimentales animales ha resultado muy efectivo, manteniendo las mitocondrias activas y evitando el daño hepático y la progresión de la enfermedad. Por otro lado hemos descrito que la proteína mitocondrial PHB1 tiene un papel en colestasis, ya que sus niveles se encuentran disminuidos específicamente en pacientes con enfermedades colestásicas y que su deficiencia predispone al daño por ligadura del conducto biliar. Hemos visto que en colestasis, a parte de su función mitocondrial, bajos niveles de PHB1 regulan la actividad del proteosoma e inducen cambios transcripcionales a través de la sobreexpresión y localización nuclear de HDAC4. En este respecto, hemos observado que la disminución de la expresión de HDAC4 mejora el daño hepático asociado a la deficiencia de PHB1. Finalmente, hemos presentado evidencias sobre el importante papel de la nedilización en el desarrollo del CHC y hemos identificado LKB1 y Akt, conocidas oncoquinasas, como nuevas dianas de Nedd8. Tanto el grado de nedilización como los niveles de LKB1 y Akt se asocian a un peor pronóstico. Además, hemos demostrado que inhibir la nedilización bloquea el crecimiento tumoral y mejora el estado del hígado pudiendo ser una buena terapia para pacientes con carcinoma hepatocelular

The levels of the RNA binding protein Hu antigen R determine the druggability of the neddylation pathway in liver cancer

Hepatocellular carcinoma (HCC), the most common liver cancer, is an important leading cause of death worldwide. Neddylation is a post-translational modification involved in several processes such as cell growth, viability and development. Importantly, the neddylation pathway is upregulated in liver cancer and specifically enriched in patients with poor prognosis. Hu antigen R (HuR), is a RNA-binding protein that stabilizes target mRNAs involved in hepatocyte proliferation, differentiation and malignant transformation. And notably, HuR levels are highly representative in liver and colon cancer. A ground-breaking knowledge about HCC has been to identify that neddylation plays a role in HCC by regulating the liver oncogenic driver HuR. In addition, the neddylation inhibitor MLN4924 has shown antitumoral effects in vitro and in vivo in liver cancer, partly through HuR destabilization. Importantly, high levels of HuR made hepatoma cells more resistant to neddylation inhibition while low levels of HuR sensitized cells to the treatment, suggesting that the levels of HuR determine the druggability of the neddylation pathway in HCC. Overall, our findings highlight the impact that neddylation plays in liver cancer and open a completely new area of research, paving the way for novel therapeutical approaches

Regulatory network and interplay of hepatokines, stellakines, myokines and adipokines in nonalcoholic fatty liver diseases and nonalcoholic steatohepatitis

Fatty liver disease is a spectrum of liver pathologies ranging from simple hepatic steatosis to non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), and culminating with the development of cirrhosis or hepatocellular carcinoma (HCC). The pathogenesis of NAFLD is complex and diverse, and there is a lack of effective treatment measures. In this review, we address hepatokines identified in the pathogenesis of NAFLD and NASH, including the signaling of FXR/RXR, PPARα/RXRα, adipogenesis, hepatic stellate cell activation/liver fibrosis, AMPK/NF-κB, and type 2 diabetes. We also highlight the interaction between hepatokines, and cytokines or peptides secreted from muscle (myokines), adipose tissue (adipokines), and hepatic stellate cells (stellakines) in response to certain nutritional and physical activity. Cytokines exert autocrine, paracrine, or endocrine effects on the pathogenesis of NAFLD and NASH. Characterizing signaling pathways and crosstalk amongst muscle, adipose tissue, hepatic stellate cells and other liver cells will enhance our understanding of interorgan communication and potentially serve to accelerate the development of treatments for NAFLD and NASH

Boosting mitochondria activity by silencing MCJ overcomes cholestasis-induced liver injury

Background & Aims: Mitochondria are the major organelles for the formation of reactive oxygen species (ROS) in the cell, and mitochondrial dysfunction has been described as a key factor in the pathogenesis of cholestatic liver disease. The methylation-controlled J-protein (MCJ) is a mitochondrial protein that interacts with and represses the function of complex I of the electron transport chain. The relevance of MCJ in the pathology of cholestasis has not yet been explored. Methods: We studied the relationship between MCJ and cholestasis-induced liver injury in liver biopsies from patients with chronic cholestatic liver diseases, and in livers and primary hepatocytes obtained from WT and MCJ-KO mice. Bile duct ligation (BDL) was used as an animal model of cholestasis, and primary hepatocytes were treated with toxic doses of bile acids. We evaluated the effect of MCJ silencing for the treatment of cholestasis-induced liver injury. Results: Elevated levels of MCJ were detected in the liver tissue of patients with chronic cholestatic liver disease when compared with normal liver tissue. Likewise, in mouse models, the hepatic levels of MCJ were increased. After BDL, MCJ-KO animals showed significantly decreased inflammation and apoptosis. In an in vitro model of bile-acid induced toxicity, we observed that the loss of MCJ protected mouse primary hepatocytes from bile acid-induced mitochondrial ROS over-production and ATP depletion, enabling higher cell viability. Finally, the in vivo inhibition of the MCJ expression, following BDL, showed reduced liver injury and a mitigation of the main cholestatic characteristics. Conclusions: We demonstrated that MCJ is involved in the progression of cholestatic liver injury, and our results identified MCJ as a potential therapeutic target to mitigate the liver injury caused by cholestasis. Lay summary: In this study, we examine the effect of mitochondrial respiratory chain inhibition by MCJ on bile acid-induced liver toxicity. The loss of MCJ protects hepatocytes against apoptosis, mitochondrial ROS overproduction, and ATP depletion as a result of bile acid toxicity. Our results identify MCJ as a potential therapeutic target to mitigate liver injury in cholestatic liver diseases.Acknowledgements: We thank MINECO for the Severo Ochoa Excellence Accreditation of CIC bioGUNE [SEV-2016-0644]

Stabilization of LKB1 and Akt by neddylation regulates energy metabolism in liver cancer

The current view of cancer progression highlights that cancer cells must undergo through a post-translational regulation and metabolic reprogramming to progress in an unfriendly environment. In here, the importance of neddylation modification in liver cancer was investigated. We found that hepatic neddylation was specifically enriched in liver cancer patients with bad prognosis. In addition, the treatment with the neddylation inhibitor MLN4924 in Phb1-KO mice, an animal model of hepatocellular carcinoma showing elevated neddylation, reverted the malignant phenotype. Tumor cell death in vivo translating into liver tumor regression was associated with augmented phosphatidylcholine synthesis by the PEMT pathway, known as a liver-specific tumor suppressor, and restored mitochondrial function and TCA cycle flux. Otherwise, in protumoral hepatocytes, neddylation inhibition resulted in metabolic reprogramming rendering a decrease in oxidative phosphorylation and concomitant tumor cell apoptosis. Moreover, Akt and LKB1, hallmarks of proliferative metabolism, were altered in liver cancer being new targets of neddylation. Importantly, we show that neddylation-induced metabolic reprogramming and apoptosis were dependent on LKB1 and Akt stabilization. Overall, our results implicate neddylation/signaling/metabolism, partly mediated by LKB1 and Akt, in the development of liver cancer, paving the way for novel therapeutic approaches targeting neddylation in hepatocellular carcinoma

Stabilization of LKB1 and Akt by neddylation regulates energy metabolism in liver cancer

The current view of cancer progression highlights that cancer cells must undergo through a post-translational regulation and metabolic reprogramming to progress in an unfriendly environment. In here, the importance of neddylation modification in liver cancer was investigated. We found that hepatic neddylation was specifically enriched in liver cancer patients with bad prognosis. In addition, the treatment with the neddylation inhibitor MLN4924 in Phb1-KO mice, an animal model of hepatocellular carcinoma showing elevated neddylation, reverted the malignant phenotype. Tumor cell death in vivo translating into liver tumor regression was associated with augmented phosphatidylcholine synthesis by the PEMT pathway, known as a liver-specific tumor suppressor, and restored mitochondrial function and TCA cycle flux. Otherwise, in protumoral hepatocytes, neddylation inhibition resulted in metabolic reprogramming rendering a decrease in oxidative phosphorylation and concomitant tumor cell apoptosis. Moreover, Akt and LKB1, hallmarks of proliferative metabolism, were altered in liver cancer being new targets of neddylation. Importantly, we show that neddylation-induced metabolic reprogramming and apoptosis were dependent on LKB1 and Akt stabilization. Overall, our results implicate neddylation/signaling/metabolism, partly mediated by LKB1 and Akt, in the development of liver cancer, paving the way for novel therapeutic approaches targeting neddylation in hepatocellular carcinoma

miR-873-5p targets mitochondrialGNMT-Complex II interface contributing tonon-alcoholic fatty liver disease

Objective:Non-alcoholic fatty liver disease (NAFLD) is a complex pathology in which several dysfunctions, including alterations in metabolicpathways, mitochondrial functionality and unbalanced lipid import/export, lead to lipid accumulation and progression to inflammation andfibrosis.The enzyme glycine N-methyltransferase (GNMT), the most important enzyme implicated in S-adenosylmethionine catabolism in the liver, isdownregulated during NAFLD progression. We have studied the mechanism involved in GNMT downregulation by its repressor microRNA miR-873-5p and the metabolic pathways affected in NAFLD as well as the benefit of recovery GNMT expression.Methods:miR-873-5p and GNMT expression were evaluated in liver biopsies of NAFLD/NASH patients. Differentin vitroandin vivoNAFLD murinemodels were used to assess miR-873-5p/GNMT involvement in fatty liver progression through targeting of the miR-873-5p as NAFLD therapy.Results:We describe a new function of GNMT as an essential regulator of Complex II activity in the electron transport chain in the mitochondria.In NAFLD, GNMT expression is controlled by miR-873-5p in the hepatocytes, leading to disruptions in mitochondrial functionality in a preclinicalmurine non-alcoholic steatohepatitis (NASH) model. Upregulation of miR-873-5p is shown in the liver of NAFLD/NASH patients, correlating withhepatic GNMT depletion. Importantly, NASH therapies based on anti-miR-873-5p resolve lipid accumulation, inflammation andfibrosis byenhancing fatty acidb-oxidation in the mitochondria. Therefore, miR-873-5p inhibitor emerges as a potential tool for NASH treatment.Conclusion:GNMT participates in the regulation of metabolic pathways and mitochondrial functionality through the regulation of Complex II activityin the electron transport chain. In NAFLD, GNMT is repressed by miR-873-5p and its targeting arises as a valuable therapeutic option for treatment

miR-873-5p targets mitochondrial GNMT-Complex II interface contributing to non-alcoholic fatty liver disease

Objective: Non-alcoholic fatly liver disease (NAFLD) is a complex pathology in which several dysfunctions, including alterations in metabolic pathways, mitochondrial functionality and unbalanced lipid import/export, lead to lipid accumulation and progression to inflammation and fibrosis. The enzyme glycine N-methyltransferase (GNMT), the most important enzyme implicated in S-adenosylmethionine catabolism in the liver, is downregulated during NAFLD progression. We have studied the mechanism involved in GNMT downregulation by its repressor microRNA miR-873-5p and the metabolic pathways affected in NAFLD as well as the benefit of recovery GNMT expression. Methods: miR-873-5p and GNMT expression were evaluated in liver biopsies of NAFLD/NASH patients. Different in vitro and in vivo NAFLD murine models were used to assess miR-873-5p/GNMT involvement in fatty liver progression through targeting of the miR-873-5p as NAFLD therapy. Results: We describe a new function of GNMT as an essential regulator of Complex II activity in the electron transport chain in the mitochondria. In NAFLD, GNMT expression is controlled by miR-873-5p in the hepatocytes, leading to disruptions in mitochondria! functionality in a preclinical murine non-alcoholic steatohepatitis (NASH) model. Upregulation of miR-873-5p is shown in the liver of NAFLD/NASH patients, correlating with hepatic GNMT depletion. Importantly, NASH therapies based on anti-miR-873-5p resolve lipid accumulation, inflammation and fibrosis by enhancing fatty acid beta-oxidation in the mitochondria. Therefore, miR-873-5p inhibitor emerges as a potential tool for NASH treatment. Conclusion: GNMT participates in the regulation of metabolic pathways and mitochondria! functionality through the regulation of Complex II activity in the electron transport chain. In NAFLD, GNMT is repressed by miR-873-5p and its targeting arises as a valuable therapeutic option for treatment. (C) 2019 The Authors. Published by Elsevier GmbH.This work was supported by grants from NIH (US Department of Health and Human services)-R01AT001576 (to S.C.L., J.M.M., and M.L.M.-C.), Ministerio de Economia, Industria y Competitividad: SAF2017-87301-R (to M.L.M.-C.), SAF2015-64352-R (to P.A.), Gobierno Vasco-Departamento de Salud 2013111114 (to M.L.M.-C.), Gobierno Vasco-Departamento de Educacion IT-336-10 (to PA), BIOEF (Basque Foundation for Innovation and Health Research: EiTB Maratoia BIO15/CA/016/BD (M.L.M.-C.), ELKARTEK 2016, Departamento de Industria del Gobierno Vasco (to M.L.M.-C), Asociacion Espanola contra el Cancer (to T.C.D., P.F.-T. and M.L.M.-C.), Mitotherapeutix (to M.L.M.-C.), Daniel Alagille award from EASL (to T.C.D), Fundacion Cientifica de la Asociacion Espanola Contra el Cancer (AECC Scientific Foundation) Rare Tumor Calls 2017 (to M.L.M.-C.), La Caixa Foundation Program (to M.L.M.-C.), Ayudas Fundacion BBVA a Equipos de Investigacion Cientifica 2019 (to M.L.M.-C.). Ciberehd_ISCIII_MINECO is funded by the Instituto de Salud Carlos III. We thank this work produced with the support of a 2017 Leonardo Grant for Researchers and Cultural Creators, BBVA Foundation (to M.V.R.). This work was supported by Fonds National de la Recherche Luxembourg and the Deutsche Forschungsgemeinschaft (C12/BM/3975937, FL/997/7-1, Inter "HepmiRSTAT", to I.B. and F.L.). We thank MINECO for the Severo Ochoa Excellence Accreditation (SEV2016-0644)