Rôle du métabolisme du glucose dans le phénotype tumoral hépatocytaire

Le carcinome hépatocellulaire (CHC) est une forme de cancer hétérogène tant d’un point de vue morphologique que d’un point de vue fonctionnel. Depuis maintenant plusieurs décennies, l’implication du métabolisme du glucose au sein des cellules tumorales a été abondamment étudiée et plusieurs constats importants à ce sujet ont été notés comme la présence d’une consommation exacerbée en glucose, d’une production accrue en lactate, ou encore d’un « switch » métabolique plus connu sous le nom d'effet Warburg. La notion d'adaptabilité des cellules cancéreuses face à la stringence du microenvironnement devient équivoque dès lors que leur aptitude à pouvoir modifier aisément leur métabolisme s'opère sous l'effet de ces variations. D'autre part, l'intense activité métabolique retrouvée dans le foie rend difficilement détectable la présence de foyers tumoraux primitifs par tomographie par émission de positron (TEP). Cette technique d'imagerie médicale, reposant principalement sur la capacité substantielle des cellules néoplasiques à consommer d'importantes quantités de glucose, ne permet pas ou très laborieusement la discrimination d'une cellule d'hépatocarcinome d’une cellule saine. Les recherches touchant de près au métabolisme de ces cellules comportent alors un grand intérêt non seulement pour étudier leur dépendance au glucose mais également apprécier leur adaptabilité face à un microenvironnement hostile. L'objectif principal de ce projet a donc été de caractériser fonctionnellement les cellules provenant de CHC en comparant leur métabolisme à celui de cellules non tumorales. Dans cette optique, nous avons premièrement étudié les hépatocytes en culture primaire de façon à pouvoir les utiliser en tant que cellules contrôles. Cependant, nous avons constaté que des altérations métaboliques majeures s'opéraient au sein de ces cellules non seulement au fil des différentes étapes de l'isolation mais également après 48 heures in vitro lorsque comparé aux hépatocytes du foie in situ. En effet, une diminution significative des métabolites impliqués dans la réponse face au stress oxydant, à l'activité du cycle de Krebs (TCA) et à la production d'énergie a alors été notée, accompagnée également d'une capacité respiratoire fortement réduite. Parallèlement, l'étude d'une lignée cellulaire isolée au laboratoire, la Dt81Hepa1-6, a clairement montré une plus grande tumorigénicité in vivo par rapport à la lignée mère Hepa1-6 dont elles sont issues. Nous avons ainsi pu, par comparaison avec les Hepa1-6, démontrer que le potentiel tumorigénique des Dt81Hepa1-6 reposait en partie sur leur forte dépendance au glucose environnant mais également sur leur capacité à pouvoir utiliser leur réserve en acides gras en l'absence de glucose. En ciblant cette reprogrammation métabolique par ajout d'oxamate de sodium, un inhibiteur du lactate déshydrogénase (LDH), nous avons non seulement pu mettre en évidence une diminution significative de leur tumorigénicité mais aussi observer une mortalité accrue lorsque couplé au cisplatinium (CP). De plus, nous avons mis en évidence une association entre la surexpression de gènes glycolytiques et le pronostic clinique de patients atteints de CHC : ceci nous a amené à envisager la possibilité de pouvoir combiner des agents ciblant le métabolisme de ces cellules à des agents de chimiothérapie conventionnels dans le traitement du CHC. L'analyse métabolomique nous a également permis de constater qu'outre cette aptitude à pouvoir efficacement s'adapter à leur microenvironnement, les cellules issues d'hépatocarcinome mettaient en place une réelle stratégie métabolique en sollicitant préférentiellement certaines voies métaboliques par rapport à d'autres. Nous avons ainsi pu établir une signature métabolique visant à démontrer que, de l'in vitro vers l'in vivo en passant par l'humain, les voies de la glycolyse et de la réponse liée à l'hypoxie étaient essentielles au maintien de la tumorigénicité des cellules d'hépatocarcinome.Hepatocellular carcinoma (HCC) is a morphologically and functionally heterogeneous cancer. For several decades, the involvement of glucose metabolism in tumor cells has been widely studied and several key features have been observed including increased glucose consumption, increased lactate production, or a "metabolic switch" more commonly known as the Warburg effect. The adaptability of cancer cells in a nutrient-restricted microenvironment becomes evident since they can easily modify their metabolic phenotype under different conditions. On the other hand, the intense metabolic activity displayed by the liver complicates the detection of primary tumor foci by clinical imaging especially by positron emission tomography (PET). This medical imaging technique, based on the substantial ability of neoplastic cells to consume large amounts of glucose, does not efficiently allow to differentiate between healthy and HCC tumor cells. Therefore, studies closely related to the metabolism of HCC cells are of great interest not only to understand their glucose dependence but also their capacity to adapt to a harsh microenvironment. The main objective of this project was to functionally characterize HCC cells by comparing their metabolism to that of non-tumor cells. In this context, we first studied primary cultures of normal hepatocytes as control cells: we demonstrated that major metabolic alterations occurred immediately after hepatocytes are removed from the liver and that these changes could persist or increase during culture. Indeed, a drastic decrease in metabolites related to antioxidative stress, Krebs cycle activity (TCA) and energy production was noted, accompanied by a significant reduced respiratory capacity when compared to liver cells in situ. The study of HCC Dt81Hepa1-6 cells, a derivative of Hepa1-6 cells, demonstrated enhanced tumorigenicity in vivo when compared to their parental cell line. Furthermore, we showed that the tumorigenic potential of these Dt81Hepa1-6 cells was partly based on their capacity to uptake surrounding glucose but also on their ability to use their stored fatty acids under glucose-restricted conditions. Targeting HCC Dt81Hepa1-6 cell metabolic reprogramming by sodium oxamate, a known inhibitor of lactate dehydrogenase (LDH), not only confirmed their greater metabolic plasticity with decreased tumorigenicity but also increased mortality when combined with cisplatinium (CP). Moreover, the association of glycolytic gene overexpression with increased tumorigenicity and mortality in patients with HCC led us to consider the possibility of targeting the metabolic processes used by highly tumorigenic HCC cells to potentiate the effectiveness of current chemotherapeutic drugs. Metabolomic analysis also allowed us to note that besides the intrinsic ability of Dt81Hepa1-6 cells to efficiently adapt to their microenvironment, HCC cells rapidly display a metabolic strategy by preferentially activating some specific metabolic pathways that favors their tumorigenicity both in vitro and in vivo. Therefore, we have been able to identify a metabolic signature associated with increased tumorigenicity in HCC that heralds glycolytic and hypoxia pathways as being critical

Fam65b Phosphorylation Relieves Tonic RhoA Inhibition During T Cell Migration

We previously identified Fam65b as an atypical inhibitor of the small G protein RhoA. Using a conditional model of a Fam65b-deficient mouse, we first show that Fam65b restricts spontaneous RhoA activation in resting T lymphocytes and regulates intranodal T cell migration in vivo. We next aimed at understanding, at the molecular level, how the brake that Fam65b exerts on RhoA can be relieved upon signaling to allow RhoA activation. Here, we show that chemokine stimulation phosphorylates Fam65b in T lymphocytes. This post-translational modification decreases the affinity of Fam65b for RhoA and favors Fam65b shuttling from the plasma membrane to the cytosol. Functionally, we show that the degree of Fam65b phosphorylation controls some cytoskeletal alterations downstream active RhoA such as actin polymerization, as well as T cell migration in vitro. Altogether, our results show that Fam65b expression and phosphorylation can finely tune the amount of active RhoA in order to favor optimal T lymphocyte motility

Tumor Microenvironment: A Metabolic Player that Shapes the Immune Response

Immune cells survey and patrol throughout the body and sometimes take residence in niche environments with distinct cellular subtypes and nutrients that may fluctuate from those in which they matured. Rooted in immune cell physiology are metabolic pathways and metabolites that not only deliver substrates and energy for growth and survival, but also instruct effector functions and cell differentiation. Unlike cancer cells, immune cells are not subject to a “Darwinian evolutionary pressure” that would allow them to adapt to developing tumors but are often irrevocably affected to local nutrient deprivation. Thus, immune cells must metabolically adapt to these changing conditions in order to perform their necessary functions. On the other hand, there is now a growing appreciation that metabolic changes occurring in cancer cells can impact on immune cell functionality and contribute to tumor immune evasion, and as such, there is a considerable and growing interest in developing techniques that target metabolism for immunotherapy. In this review, we discuss the metabolic plasticity displayed by innate and adaptive immune cells and highlight how tumor-derived lactate and tumor acidity restrict immunity. To our knowledge, this review outlines the most recent insights on how tumor microenvironment metabolically instructs immune responsiveness

Warburg and Beyond: The Power of Mitochondrial Metabolism to Collaborate or Replace Fermentative Glycolysis in Cancer

A defining hallmark of tumor phenotypes is uncontrolled cell proliferation, while fermentative glycolysis has long been considered as one of the major metabolic pathways that allows energy production and provides intermediates for the anabolic growth of cancer cells. Although such a vision has been crucial for the development of clinical imaging modalities, it has become now evident that in contrast to prior beliefs, mitochondria play a key role in tumorigenesis. Recent findings demonstrated that a full genetic disruption of the Warburg effect of aggressive cancers does not suppress but instead reduces tumor growth. Tumor growth then relies exclusively on functional mitochondria. Besides having fundamental bioenergetic functions, mitochondrial metabolism indeed provides appropriate building blocks for tumor anabolism, controls redox balance, and coordinates cell death. Hence, mitochondria represent promising targets for the development of novel anti-cancer agents. Here, after revisiting the long-standing Warburg effect from a historic and dynamic perspective, we review the role of mitochondria in cancer with particular attention to the cancer cell-intrinsic/extrinsic mechanisms through which mitochondria influence all steps of tumorigenesis, and briefly discuss the therapeutic potential of targeting mitochondrial metabolism for cancer therapy

From in vivo to in vitro: Major metabolic alterations take place in hepatocytes during and following isolation.

The liver plays a key role in maintaining physiological homeostasis and hepatocytes are largely responsible for this. The use of isolated primary hepatocytes has become an essential tool for the study of nutrient physiology, xenobiotic metabolism and several liver pathologies. Since hepatocytes are removed from their normal environment, the isolation procedure and in vitro culture of primary hepatocytes is partially known to induce undesired metabolic changes. We aimed to perform a thorough metabolic profiling of primary cells before, during and after isolation using state-of-the-art techniques. Extensive metabolite measurements using HPLC were performed in situ in the liver, during hepatocyte isolation using the two-step collagenase perfusion method and during in vitro cell culture for up to 48 hours. Assessment of mitochondrial respiratory capacity and ATP-linked respiration of isolated primary hepatocytes was performed using extracellular flux analysis. Primary hepatocytes displayed a drastic decrease in antioxidative-related metabolites (NADPH, NADP, GSH and GSSG) during the isolation procedure when compared to the in situ liver (P<0.001). Parallel assessment of citric acid cycle activity showed a significant decrease of up to 95% in Acetyl-CoA, Isocitrate/Citrate ratio, Succinate, Fumarate and Malate in comparison to the in situ liver (P<0.001). While the levels of several cellular energetic metabolites such as Adenosine, AMP, ADP and ATP were found to be progressively reduced during the isolation procedure and cell culture (P<0.001), higher ATP/ADP ratio and energy charge level were observed when primary cells were cultured in vitro compared to the in situ liver (P<0.05). In addition, a significant decrease in the respiratory capacity occurred after 24 hours in culture. Interestingly, this was not associated with a significant modification of ATP-linked respiration. In conclusion, major metabolic alterations occur immediately after hepatocytes are removed from the liver. These changes persist or increase during in vitro culture. These observations need to be taken into account when using primary hepatocytes for the study of metabolism or liver physiopathology

Highly tumorigenic hepatocellular carcinoma cell line with cancer stem cell-like properties.

There are limited numbers of models to study hepatocellular carcinoma (HCC) in vivo in immunocompetent hosts. In an effort to develop a cell line with improved tumorigenicity, we derived a new cell line from Hepa1-6 cells through an in vivo passage in C57BL/6 mice. The resulting Dt81Hepa1-6 cell line showed enhanced tumorigenicity compared to Hepa1-6 with more frequent (28±12 vs. 0±0 lesions at 21 days) and more rapid tumor development (21 (100%) vs. 70 days (10%)) in C57BL/6 mice. The minimal Dt81Hepa1-6 cell number required to obtain visible tumors was 100,000 cells. The Dt81Hepa1-6 cell line showed high hepatotropism with subcutaneous injection leading to liver tumors without development of tumors in lungs or spleen. In vitro, Dt81Hepa1-6 cells showed increased anchorage-independent growth (34.7±6.8 vs. 12.3±3.3 colonies; P<0.05) and increased EpCAM (8.7±1.1 folds; P<0.01) and β-catenin (5.4±1.0 folds; P<0.01) expression. A significant proportion of Dt81Hepa1-6 cells expressed EpCAM compared to Hepa1-6 (34.8±1.1% vs 0.9±0.13%; P<0.001). Enriched EpCAM+ Dt81Hepa1-6 cells led to higher tumor load than EpCAM- Dt81Hepa1-6 cells (1093±74 vs 473±100 tumors; P<0.01). The in vivo selected Dt81Hepa1-6 cell line shows high liver specificity and increased tumorigenicity compared to Hepa1-6 cells. These properties are associated with increased expression of EpCAM and β-catenin confirming that EpCAM+ HCC cells comprise a subset with characteristics of tumor-initiating cells with stem/progenitor cell features. The Dt81Hepa1-6 cell line with its cancer stem cell-like properties will be a useful tool for the study of hepatocellular carcinoma in vivo

Metabolic Rewiring toward Oxidative Phosphorylation Disrupts Intrinsic Resistance to Ferroptosis of the Colon Adenocarcinoma Cells

Glutathione peroxidase 4 (GPX4) has been reported as one of the major targets for ferroptosis induction, due to its pivotal role in lipid hydroperoxide removal. However, recent studies pointed toward alternative antioxidant systems in this context, such as the Coenzyme Q-FSP1 pathway. To investigate how effective these alternative pathways are in different cellular contexts, we used human colon adenocarcinoma (CRC) cells, highly resistant to GPX4 inhibition. Data obtained in the study showed that simultaneous pharmacological inhibition of GPX4 and FSP1 strongly compromised the survival of the CRC cells, which was prevented by the ferroptosis inhibitor, ferrostatin-1. Nonetheless, this could not be phenocopied by genetic deletion of FSP1, suggesting the development of resistance to ferroptosis in FSP1-KO CRC cells. Considering that CRC cells are highly glycolytic, we used CRC Warburg-incompetent cells, to investigate the role metabolism plays in this phenomenon. Indeed, the sensitivity to inhibition of both anti-ferroptotic axes (GPx4 and FSP1) was fully revealed in these cells, showing typical features of ferroptosis. Collectively, data indicate that two independent anti-ferroptotic pathways (GPX4-GSH and CoQ10-FSP1) operate within the overall physiological context of cancer cells and in some instances, their inhibition should be coupled with other metabolic modulators, such as inhibitors of glycolysis/Warburg effect

Decrease in antioxidative stress-related metabolites during the isolation procedure and cell culture.

<p>Evaluation of total intracellular: A) NADPH, B) NADP, C) GSH and D) GSSG levels during the hepatocyte isolation procedure from <i>in situ</i> to the washing step in L-15 media and during cell culture over a period of up to 48 hours. Values are ±SEM of 3 independent experiments. Asterisks indicate significance when compared to the <i>in situ</i> liver <i>(*P</i><0.05, <i>**P</i><0.01, <i>***P</i><0.001).</p