In vivo characterization of glutamine metabolism identifies therapeutic targets in clear cell renal cell carcinoma

Targeting metabolic vulnerabilities has been proposed as a therapeutic strategy in renal cell carcinoma (RCC). Here, we analyzed the metabolism of patient-derived xenografts (tumorgrafts) from diverse subtypes of RCC. Tumorgrafts from VHL-mutant clear cell RCC (ccRCC) retained metabolic features of human ccRCC and engaged in oxidative and reductive glutamine metabolism. Genetic silencing of isocitrate dehydrogenase-1 or isocitrate dehydrogenase-2 impaired reductive labeling of tricarboxylic acid (TCA) cycle intermediates in vivo and suppressed growth of tumors generated from tumorgraft-derived cells. Glutaminase inhibition reduced the contribution of glutamine to the TCA cycle and resulted in modest suppression of tumorgraft growth. Infusions with [amide-15N]glutamine revealed persistent amidotransferase activity during glutaminase inhibition, and blocking these activities with the amidotransferase inhibitor JHU-083 also reduced tumor growth in both immunocompromised and immunocompetent mice. We conclude that ccRCC tumorgrafts catabolize glutamine via multiple pathways, perhaps explaining why it has been challenging to achieve therapeutic responses in patients by inhibiting glutaminase

<i>Trans</i>-vaccenic acid reprograms CD8⁺ T cells and anti-tumour immunity

Diet-derived nutrients are inextricably linked to human physiology by providing energy and biosynthetic building blocks and by functioning as regulatory molecules. However, the mechanisms by which circulating nutrients in the human body influence specific physiological processes remain largely unknown. Here we use a blood nutrient compound library-based screening approach to demonstrate that dietary trans-vaccenic acid (TVA) directly promotes effector CD8+ T cell function and anti-tumour immunity in vivo. TVA is the predominant form of trans-fatty acids enriched in human milk, but the human body cannot produce TVA endogenously. Circulating TVA in humans is mainly from ruminant-derived foods including beef, lamb and dairy products such as milk and butter, but only around 19% or 12% of dietary TVA is converted to rumenic acid by humans or mice, respectively. Mechanistically, TVA inactivates the cell-surface receptor GPR43, an immunomodulatory G protein-coupled receptor activated by its short-chain fatty acid ligands. TVA thus antagonizes the short-chain fatty acid agonists of GPR43, leading to activation of the cAMP–PKA–CREB axis for enhanced CD8+ T cell function. These findings reveal that diet-derived TVA represents a mechanism for host-extrinsic reprogramming of CD8+ T cells as opposed to the intrahost gut microbiota-derived short-chain fatty acids. TVA thus has translational potential for the treatment of tumours

p63 and SOX2 Dictate Glucose Reliance and Metabolic Vulnerabilities in Squamous Cell Carcinomas

Squamous cell carcinoma (SCC), a malignancy arising across multiple anatomical sites, is responsible for significant cancer mortality due to insufficient therapeutic options. Here, we identify exceptional glucose reliance among SCCs dictated by hyperactive GLUT1-mediated glucose influx. Mechanistically, squamous lineage transcription factors p63 and SOX2 transactivate the intronic enhancer cluster of SLC2A1. Elevated glucose influx fuels generation of NADPH and GSH, thereby heightening the anti-oxidative capacity in SCC tumors. Systemic glucose restriction by ketogenic diet and inhibiting renal glucose reabsorption with SGLT2 inhibitor precipitate intratumoral oxidative stress and tumor growth inhibition. Furthermore, reduction of blood glucose lowers blood insulin levels, which suppresses PI3K/AKT signaling in SCC cells. Clinically, we demonstrate a robust correlation between blood glucose concentration and worse survival among SCC patients. Collectively, this study identifies the exceptional glucose reliance of SCC and suggests its candidacy as a highly vulnerable cancer type to be targeted by systemic glucose restriction

Amyloid and tau pathology associations with personality traits, neuropsychiatric symptoms, and cognitive lifestyle in the preclinical phases of sporadic and autosomal dominant Alzheimer’s disease

Background Major prevention trials for Alzheimer’s disease (AD) are now focusing on multidomain lifestyle interventions. However, the exact combination of behavioral factors related to AD pathology remains unclear. In 2 cohorts of cognitively unimpaired individuals at risk of AD, we examined which combinations of personality traits, neuropsychiatric symptoms, and cognitive lifestyle (years of education or lifetime cognitive activity) related to the pathological hallmarks of AD, amyloid-β, and tau deposits. Methods A total of 115 older adults with a parental or multiple-sibling family history of sporadic AD (PREVENT-AD [PRe-symptomatic EValuation of Experimental or Novel Treatments for AD] cohort) underwent amyloid and tau positron emission tomography and answered several questionnaires related to behavioral attributes. Separately, we studied 117 mutation carriers from the DIAN (Dominant Inherited Alzheimer Network) study group cohort with amyloid positron emission tomography and behavioral data. Using partial least squares analysis, we identified latent variables relating amyloid or tau pathology with combinations of personality traits, neuropsychiatric symptoms, and cognitive lifestyle. Results In PREVENT-AD, lower neuroticism, neuropsychiatric burden, and higher education were associated with less amyloid deposition (p = .014). Lower neuroticism and neuropsychiatric features, along with higher measures of openness and extraversion, were related to less tau deposition (p = .006). In DIAN, lower neuropsychiatric burden and higher education were also associated with less amyloid (p = .005). The combination of these factors accounted for up to 14% of AD pathology. Conclusions In the preclinical phase of both sporadic and autosomal dominant AD, multiple behavioral features were associated with AD pathology. These results may suggest potential pathways by which multidomain interventions might help delay AD onset or progression

LKB1 and AMPK negatively regulate the Warburg effect in cancer

Many of the common mutations in cancer affect key metabolic signaling pathways. These mutations promote altered cellular metabolism (Hanahan and Weinberg, 2011). This change in metabolism, termed the "Warburg Effect" describes the phenomenon where many cancer cells preferentially use aerobic glycolysis for energy production, despite an adequate oxygen supply. This change in metabolism supplies both the necessary energy and biosynthetic intermediates for proliferation, and confers a selective growth advantage for cancer cells. AMP-activated protein kinase (AMPK) is an evolutionarily conserved central regulator of cellular metabolism and energy homeostasis. Under conditions of energetic stress, AMPK is activated by its upstream kinase LKB1, and initiates a number of biological pathways aimed at preserving cellular energy levels. In addition, AMPK is situated in a signaling cascade of tumour suppressors, its specific role in cancer remains controversial. The work in this thesis characterizes the energy sensing LKB1-AMPK pathway in regulating tumor metabolism. Specifically, the role of AMPK in suppressing MYC-driven tumourigenesis, as well as its negative regulation of the Warburg effect is examined. We further address the previously unidentified role of LKB1 loss in cancer metabolism. In both studies, these metabolic phenotypes were driven by the normoxic stabilization of HIF1α. Mechanistically, we demonstrate that this stabilization is due to chronically elevated levels of mitochondrial ROS in the absence of AMPK. We conclude that LKB1 and AMPK negatively regulate the Warburg effect in cancer.Parmi les mutations qui sont fréquemment présentes dans les cancers, un grand nombre affecte des voies clés de la signalisation métabolique, ce qui provoque généralement une altération du métabolisme cellulaire (Hanahan and Weinberg, 2011). Cette modification du métabolisme, appelée «l’effet Warburg», correspond à un phénomène au cours duquel de nombreuses cellules cancéreuses utilisent de manière préférentielle la glycolyse aérobie afin de produire de l’énergie, et ce malgré un approvisionnement suffisant en oxygène. Ce phénomène permet de fournir l’énergie et les intermédiaires biosynthétiques nécessaires à la prolifération, conférant ainsi un avantage de croissance sélectif aux cellules cancéreuses. La kinase activée par l’AMP (AMPK) est une protéine conservée au cours de l’évolution et qui détient un rôle central dans la régulation du métabolisme cellulaire et l’homéostasie énergétique. LKB1 est la kinase présente en amont d’AMPK et l’active en réponse à des conditions de stress énergétique. Etant donné qu’AMPK se situe aussi dans la cascade de signalisation des suppresseurs de tumeurs, le rôle précis qu’il joue dans le cancer reste controversé. Le travail présenté dans cette thèse caractérise le rôle de la voie de signalisation, LKB1/AMPK dans la régulation du métabolisme cancéreux. Plus précisément, les rôles de la protéine AMPK dans la répression de la tumorigénèse initiée par MYC et de l’effet Warburg sont examinés plus en détails. Nous abordons aussi les conséquences de la perte de LKB1 sur le métabolisme cancéreux, mécanisme qui n’était pas connu au départ. Au cours de ces deux études, les phénotypes métaboliques observés résultent de la stabilisation de HIF-1α dans des conditions de normoxie. Nous démontrons que les mécanismes de cette stabilisation impliquent une augmentation chronique des niveaux de dérivés réactifs de l'oxygène provenant de la mitochondrie en réponse à l’absence d’AMPK. Finalement, nous concluons que les protéines LKB1 et AMPK régulent de manière négative l’effet Warburg au cours du cancer

Resveratrol Prevents Insulin Resistance Caused by Short-Term Elevation of Free Fatty Acids In Vivo

Elevated levels of plasma free fatty acids (FFA), which are commonly found in obesity, induce insulin resistance. FFA activate protein kinases including the proinflammatory IκBα kinase β (IKKβ), leading to serine phosphorylation of insulin receptor substrate 1 (IRS-1) and impaired insulin signaling. In order to test whether resveratrol, a polyphenol found in red wine, prevents FFA-induced insulin resistance, we used a hyperinsulinemic-euglycemic clamp with tracer to assess hepatic and peripheral insulin sensitivity in overnight-fasted Wistar rats infused for 7 hours with either saline, Intralipid plus 20 U/ml heparin (IH, triglyceride emulsion that elevates FFA levels in vivo; 5.5 l/min) with or without resveratrol (3mg kg-1 h-1), or resveratrol alone. Infusion of IH significantly decreased glucose infusion rate (GIR; PThe accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

Metformin Antagonizes Cancer Cell Proliferation by Suppressing Mitochondrial-Dependent Biosynthesis.

Metformin is a biguanide widely prescribed to treat Type II diabetes that has gained interest as an antineoplastic agent. Recent work suggests that metformin directly antagonizes cancer cell growth through its actions on complex I of the mitochondrial electron transport chain (ETC). However, the mechanisms by which metformin arrests cancer cell proliferation remain poorly defined. Here we demonstrate that the metabolic checkpoint kinases AMP-activated protein kinase (AMPK) and LKB1 are not required for the antiproliferative effects of metformin. Rather, metformin inhibits cancer cell proliferation by suppressing mitochondrial-dependent biosynthetic activity. We show that in vitro metformin decreases the flow of glucose- and glutamine-derived metabolic intermediates into the Tricarboxylic Acid (TCA) cycle, leading to reduced citrate production and de novo lipid biosynthesis. Tumor cells lacking functional mitochondria maintain lipid biosynthesis in the presence of metformin via glutamine-dependent reductive carboxylation, and display reduced sensitivity to metformin-induced proliferative arrest. Our data indicate that metformin inhibits cancer cell proliferation by suppressing the production of mitochondrial-dependent metabolic intermediates required for cell growth, and that metabolic adaptations that bypass mitochondrial-dependent biosynthesis may provide a mechanism of tumor cell resistance to biguanide activity

AMPK Maintains Cellular Metabolic Homeostasis through Regulation of Mitochondrial Reactive Oxygen Species

Reactive oxygen species (ROS) are continuously produced as a by-product of mitochondrial metabolism and eliminated via antioxidant systems. Regulation of mitochondrially produced ROS is required for proper cellular function, adaptation to metabolic stress, and bypassing cellular senescence. Here, we report non-canonical regulation of the cellular energy sensor AMP-activated protein kinase (AMPK) by mitochondrial ROS (mROS) that functions to maintain cellular metabolic homeostasis. We demonstrate that mitochondrial ROS are a physiological activator of AMPK and that AMPK activation triggers a PGC-1α-dependent antioxidant response that limits mitochondrial ROS production. Cells lacking AMPK activity display increased mitochondrial ROS levels and undergo premature senescence. Finally, we show that AMPK-PGC-1α-dependent control of mitochondrial ROS regulates HIF-1α stabilization and that mitochondrial ROS promote the Warburg effect in cells lacking AMPK signaling. These data highlight a key function for AMPK in sensing and resolving mitochondrial ROS for stress resistance and maintaining cellular metabolic balance

Loss of the tumor suppressor LKB1 promotes metabolic reprogramming of cancer cells via HIF-1α

One of the major metabolic changes associated with cellular transformation is enhanced nutrient utilization, which supports tumor progression by fueling both energy production and providing biosynthetic intermediates for growth. The liver kinase B1 (LKB1) is a serine/threonine kinase and tumor suppressor that couples bioenergetics to cell-growth control through regulation of mammalian target of rapamycin (mTOR) activity; however, the influence of LKB1 on tumor metabolism is not well defined. Here, we show that loss of LKB1 induces a progrowth metabolic program in proliferating cells. Cells lacking LKB1 display increased glucose and glutamine uptake and utilization, which support both cellular ATP levels and increased macromolecular biosynthesis. This LKB1-dependent reprogramming of cell metabolism is dependent on the hypoxia-inducible factor-1α (HIF-1α), which accumulates under normoxia in LKB1-deficient cells and is antagonized by inhibition of mTOR complex I signaling. Silencing HIF-1α reverses the metabolic advantages conferred by reduced LKB1 signaling and impairs the growth and survival of LKB1-deficient tumor cells under low-nutrient conditions. Together, our data implicate the tumor suppressor LKB1 as a central regulator of tumor metabolism and growth control through the regulation of HIF-1α–dependent metabolic reprogramming