LKB1 signaling is activated in CTNNB1 -mutated HCC and positively regulates β-catenin-dependent CTNNB1 -mutated HCC

International audienceCTNNB1‐mutated HCC. They were found to be well‐differentiated, almost never steatotic, and often cholestatic, with a microtrabecular or acinar growth pattern. Here, we investigated whether LKB1, which controls energy metabolism, cell polarity, and cell growth, mediates the specific phenotype of CTNNB1‐mutated HCC. The LKB1 protein was overexpressed in CTNNB1‐mutated HCC and oncogenic activation of β‐catenin in human HCC cells induced the post‐transcriptional accumulation of the LKB1 protein encoded by the LKB1 (STK11) gene. Hierarchical clustering, based on the expression of a murine hepatic liver Lkb1 (Stk11) signature in a human public dataset, identified a HCC cluster, composed of almost all the CTNNB1‐mutated HCC, that expresses a hepatic liver LKB1 program. This was confirmed by RT‐qPCR of an independent cohort of CTNNB1‐mutated HCC and the suppression of the LKB1‐related profile upon β‐catenin silencing of CTNNB1‐mutated human hepatoma cell lines. Previous studies described an epistatic relationship between LKB1 and CTNNB1 in which LKB1 acts upstream of CTNNB1. Thus, we also analyzed the consequences of Lkb1 deletion on the zonation of hepatic metabolism, known to be the hallmark of β‐catenin signaling in the liver. Lkb1 was required for the establishment of metabolic zonation in the mouse liver by positively modulating β‐catenin signaling. We identified positive reciprocal cross talk between the canonical Wnt pathway and LKB1, both in normal liver physiology and during tumorigenesis that likely participates in the amplification of the β‐catenin signaling by LKB1 and the distinctive phenotype of the CTNNB1‐mutated HCC

Study of the response to oxydative stress in β-catenin activated hepatocellular carcinomas

Le carcinome hépatocellulaire (CHC) est la 2nde cause de mortalité par le cancer dans le monde. Notre équipe s’intéresse aux CHC mutés CTNNB1 représentant 30 à 40% des CHC. A l’aide de modèles murins mimant cette carcinogénèse, nous avons montré que ces tumeurs présentent une oxydation lipidique accrue indispensable à la tumorigénèse et au développement des CHC CTNNB1. De plus, ces cellules présentent une respiration mitochondriale accrue. L'activation de la β-caténine induit également une augmentation de l'expression des CYP450 qui, avec une respiration mitochondriale accrue, sont générateurs d'espèces réactives de l'oxygène (ERO). L'accumulation d'ERO mène à un stress oxydant, néfaste pour la cellule, s'il n'est pas détoxifié. Les cellules présentant un taux élevé d'ERO nécessitent donc la mise en place de systèmes antioxydants (SAO) dont elles sont dépendantes pour survivre. L'objectif de mon projet de thèse a été d'étudier le statut redox de ces tumeurs, d'identifier les SAO et de cibler ces mécanismes afin de tester s'ils peuvent être la base d'une approche thérapeutique. Mes résultats montrent que les cellules β-caténine activées pré-néoplasiques génèrent plus d’ERO mais qu’elles sont protégées grâce à la voie de détoxification Nrf2, expliquant ainsi la croissance tumorale dans un contexte délétère. Nous montrons également que l’inhibition de la détoxification Nrf2 par un inhibiteur pharmacologique (Halofuginone) est délétère pour ces cellules pré-néoplasiques. Après avoir montré que les tumeurs β-caténine activées sont également protégées contre le stress oxydant par l’activation du programme Nrf2, nous montrons également que le traitement de ces tumeurs à l’halofuginone engendre une diminution de la croissance tumorale. De façon intéressante, grâce à l’analyse des données du TCGA, nous avons pu montrer qu’une signature Nrf2 est significativement associées au CHC CTNNB1 mutés chez l’homme et que ce programme Nrf2 associé au CHC CTNNB1 mutés aggrave le pronostique de survie des patients en comparaison à des CHC CTNNB1 mutés ne présentant pas de signature Nrf2. Ainsi, l’inhibition de ce programme pourrait alors constituer une stratégie anticancer pour cibler les CHC β-caténine activés. Mes résultats montrent également que le programme Nrf2 participe au phénotype métabolique des hépatocytes β-caténine activés pré-néoplasiques. En effet, l’activation de la β-caténine mène à une oxydation accrue des acides gras au sein de laquelle Nrf2 redirige l’acétyl-CoA vers le cycle de Krebs au détriment de la synthèse des corps cétoniques. D’autre part, de par l’augmentation de l’expression des cytochromes P450 par l’activation de la β-caténine, ces cellules sont sensibles à l'APAP. Des traitements à l’APAP engendrent donc la mort des cellules tumorales β-caténine activées et une diminution de la croissance tumorale. Ainsi cette étude permet de montrer deux potentielles stratégie anti-cancer en ciblant la balance redox.Hepatocellular carcinoma (HCC) is the 2nd leading cause of cancer related death worldwide. For many years, our team has been interested in β-catenin activated HCC which represent 30 to 40% of all HCCs. With murine models mimicking carcinogenesis, we were able to show that these tumors present an increase in fatty acid oxidation. We also showed that this β-oxidation is essential for β-catenin activated tumorigenesis and tumor progression and that they present an increased activity of the mitochondrial respiration chain.β-catenin activated cells also have an increased expression of P450 cytochromes which, with the increase of mitochondrial respiration chain, are known to generate reactive oxygen species (ROS). ROS accumulation can create an oxidative stress which is deleterious for the cell if not taken in charge by an anti-oxidantdefense (AOD). Cancer cells are known to have a high level of ROS that requires the establishment of an AOD in order to survive and proliferate. By changing the AOD, it is possible to modify the redox balance and hence reach a toxic threshold leading to cell death without compromising normal cells.The aims of my PhD were to 1) characterize the redox status of these tumors, 2) to identify the protection mechanisms again oxidative stress and 3) to see if it could be used as a therapeutic approach. My results show that pre-neoplasic β-catenin activated cells generate a bigger amount of ROS but that they are better protected against oxidative stress thanks to the activation of the Nrf2 pathway. This should explain the tumoral growth seen in this deleterious context. We also showed that the inhibition of the Nrf2 program with a pharmacological inhibitor (Halofuginone) is deleterious for these cells. Moreover, after having shown that β-catenin activated tumors are also protected thanks to the Nrf2 pathway, we also demonstrate that Halofuginone treatments lead to a decrease in tumor growth rate. Interestingly, thanks to the analyses of the TCGA data, we were able to show that an Nrf2 signature is associated with CTNNB1-mutated HCCs and that this program worsen the prognosis of CTNNB1 mutated patients. Hence, the inhibition of the Nrf2 program could be an anti-cancer strategy to target β-catenin activated HCCs. My results also showed that the Nrf2 program participate in the metabolic phenotype of pre-neoplasic β-catenin activated hepatocytes. Indeed, the oncogenic activation of the β-catenin lead the an increased β-oxydation in which Nrf2 rewire acetyl coA towards the Krebs cycle at the expense of ketogenesis. Moreover, our studie showed that β-catenin activated cells are sensible to Acetaminophen (APAP) because of the increased expression of cytochromes P450. APAP treatments lead to the death of β-catenin activated tumoral cells and to the decrease of the tumoral growth. In conclusion, this study shows to possible anti-cancer strategies by using the redox balance

La reprogrammation métabolique des carcinomes hépatocellulaires β-caténine mutés

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Cooperation Between the NRF2 Pathway and Oncogenic β‐catenin During HCC Tumorigenesis

International audienceCTNNB1 (catenin beta 1)-mutated hepatocellular carcinomas (HCCs) account for a large proportion of human HCCs. They display high levels of respiratory chain activity. As metabolism and redox balance are closely linked, tumor cells must maintain their redox status during these metabolic alterations. We investigated the redox balance of these HCCs and the feasibility of targeting this balance as an avenue for targeted therapy. We assessed the expression of the nuclear erythroid 2 p45-related factor 2 (NRF2) detoxification pathway in an annotated human HCC data set and reported an enrichment of the NRF2 program in human HCCs with CTNNB1 mutations, largely independent of NFE2L2 (nuclear factor, erythroid 2 like 2) or KEAP1 (Kelch-like ECH-associated protein 1) mutations. We then used mice with hepatocyte-specific oncogenic β-catenin activation to evaluate the redox status associated with β-catenin activation in preneoplastic livers and tumors. We challenged them with various oxidative stressors and observed that the β-catenin pathway activation increased transcription of Nfe2l2, which protects β-catenin-activated hepatocytes from oxidative damage and supports tumor development. Moreover, outside of its effects on reactive oxygen species scavenging, we found out that Nrf2 itself contributes to the metabolic activity of β-catenin-activated cells. We then challenged β-catenin activated tumors pharmacologically to create a redox imbalance and found that pharmacological inactivation of Nrf2 was sufficient to considerably decrease the progression of β-catenin-dependent HCC development. Conclusion: These results demonstrate cooperation between oncogenic β-catenin signaling and the NRF2 pathway in CTNNB1-mediated HCC tumorigenesis, and we provide evidence for the relevance of redox balance targeting as a therapeutic strategy in CTNNB1-mutated HCC

β-catenin-activated hepatocellular carcinomas are addicted to fatty acids

International audienceObjectives: CTNNB1-mutated hepatocellular carcinomas (HCCs) constitute a major part of human HCC and are largely inaccessible to target therapy. Yet, little is known about the metabolic reprogramming induced by β-catenin oncogenic activation in the liver. We aimed to decipher such reprogramming and assess whether it may represent a new avenue for targeted therapy of CTNNB1-mutated HCC.Design: We used mice with hepatocyte-specific oncogenic activation of β-catenin to evaluate metabolic reprogramming using metabolic fluxes on tumourous explants and primary hepatocytes. We assess the role of Pparα in knock-out mice and analysed the consequences of fatty acid oxidation (FAO) using etomoxir. We explored the expression of the FAO pathway in an annotated human HCC dataset.Results: β-catenin-activated HCC were not glycolytic but intensively oxidised fatty acids. We found that Pparα is a β-catenin target involved in FAO metabolic reprograming. Deletion of Pparα was sufficient to block the initiation and progression of β-catenin-dependent HCC development. FAO was also enriched in human CTNNB1-mutated HCC, under the control of the transcription factor PPARα.Conclusions: FAO induced by β-catenin oncogenic activation in the liver is the driving force of the β-catenin-induced HCC. Inhibiting FAO by genetic and pharmacological approaches blocks HCC development, showing that inhibition of FAO is a suitable therapeutic approach for CTNNB1-mutated HCC

AXIN deficiency in human and mouse hepatocytes induces hepatocellular carcinoma in the absence of β-catenin activation

International audienceBackground & aims - The Wnt/β-catenin pathway is the most frequently deregulated pathway in hepatocellular carcinoma (HCC). Inactivating mutations of the gene encoding AXIN1, a known negative regulator of the Wnt/β-catenin signaling pathway, are observed in about 10% of HCCs. Whole-genome studies usually place HCC with AXIN1 mutations and CTNNB1 mutations in the group of tumors with Wnt/β-catenin activated program. However, it has been shown that HCCs with activating CTNNB1 mutations form a group of HCCs, with a different histology, prognosis and genomic signature to those with inactivating biallelic AXIN1 mutations. We aimed to elucidate the relationship between CTNNB1 mutations, AXIN1 mutations and the activation level of the Wnt/β-catenin program. Methods - We evaluated two independent human HCC datasets for the expression of a 23-β-catenin target genes program. We modeled Axin1 loss of function tumorigenesis in two engineered mouse models and performed gene expression profiling. Results - Based on gene expression, we defined three levels of β-catenin program activation: strong, weak or no activation. While more than 80% CTNNB1-mutated tumors were found in the strong or in the weak activation program, most of the AXIN1-mutated tumors (>70%) were found in the subgroup with no activation. We validated this result by demonstrating that mice with a hepatocyte specific AXIN1 deletion developed HCC in the absence of β-catenin induction. We defined a 329-gene signature common in human and mouse AXIN1 mutated HCC that is highly enriched in Notch and YAP oncogenic signatures. Conclusions - AXIN1-mutated HCCs occur independently of the Wnt/β-catenin pathway and involve Notch and YAP pathways. These pathways constitute potentially interesting targets for the treatment of HCC caused by AXIN1 mutations. Lay summary - Liver cancer has a poor prognosis. Defining the molecular pathways involved is important for developing new therapeutic approaches. The Wnt/β-catenin pathway is the most frequently deregulated pathway in hepatocellular carcinoma (HCC). Mutations of AXIN1, a member of this pathway, represent about 10% of HCC mutations. Using both human HCC collections and engineered mouse models of liver cancers with AXIN1 mutation or deletion, we defined a common signature of liver tumors mutated for AXIN1 and demonstrate that these tumors occur independently of the activation of the Wnt/β-catenin pathway

Lkb1 suppresses amino acid-driven gluconeogenesis in the liver. Correction

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Lkb1 suppresses amino acid-driven gluconeogenesis in the liver

International audienceExcessive glucose production by the liver is a key factor in the hyperglycemia observed in type 2 diabetes mellitus (T2DM). Here, we highlight a novel role of liver kinase B1 (Lkb1) in this regulation. We show that mice with a hepatocyte-specific deletion of Lkb1 have higher levels of hepatic amino acid catabolism, driving gluconeogenesis. This effect is observed during both fasting and the postprandial period, identifying Lkb1 as a critical suppressor of postprandial hepatic gluconeogenesis. Hepatic Lkb1 deletion is associated with major changes in whole-body metabolism, leading to a lower lean body mass and, in the longer term, sarcopenia and cachexia, as a consequence of the diversion of amino acids to liver metabolism at the expense of muscle. Using genetic, proteomic and pharmacological approaches, we identify the aminotransferases and specifically Agxt as effectors of the suppressor function of Lkb1 in amino acid-driven gluconeogenesis