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Background: Tumor cells are highly dependent on glucose even in the presence of oxygen. This concept called the Warburg effect is a hallmark of cancer and strategies are considered to therapeutically exploit the phenomenon such as ketogenic diets. The success of such strategies is dependent on a profound understanding of tumor cell metabolism. With new techniques it is now possible to thoroughly analyze the metabolic responses to the withdrawal of substrates and their substitution by others. In the present study we used gas chromatography coupled to mass spectrometry (GC-MS) to analyze how glioblastoma brain tumor cells respond metabolically when glucose is withdrawn and substituted by pyruvate. Methods: Glioblastoma brain tumor cells were cultivated in medium with high (25 mM), medium (11 mM) or low (5.5 mM) glucose concentration or with pyruvate (5 mM). After 24 h GC-MS metabolite profiling was performed. Results: The abundances of most metabolites were dependent on the supply of glucose in tendency but not in a linear manner indicating saturation at high glucose. Noteworthy, a high level of sorbitol production and release was observed at high concentrations of glucose and high release of alanine, aspartate and citrate were observed when glucose was substituted by pyruvate. Intermediates of the TCA cycle were present under all nutritional conditions and evidence was found that cells may perform gluconeogenesis from pyruvate. Conclusions: Our experiments reveal a high plasticity of glioblastoma cells to changes in nutritional supply which has to be taken into account in clinical trials in which specific diets are considered for therapy.

Lactate Dehydrogenase-A (LDH-A) is up-regulated in a broad array of cancers and is associated with poor prognosis. Involved in the hypoxic response, LDH-A is a HIF-1 target and is responsible for the enzymatic reduction of pyruvate to lactate. This is important for several reasons, chiefly (1) the regeneration of NAD+ which feeds back into earlier glycolytic stages and (2) the depletion of intracellular pyruvate concentrations. High intracellular pyruvate is known to inhibit HDACs and is associated with increased apoptosis. LDH-A is also known to be controlled by oncogenes such as c-Myc suggesting an oncogenic role. Studies have shown that the knock-out of LDH-A reduces proliferation and tumourgenicity, and stimulates the mitochondria. This thesis therefore had three aims: firstly, to validate LDH-A inhibition and elucidate its full nature in terms of the implications for tumour survival; secondly, to ascertain the role of LDH-B in order to determine whether selectivity towards LDH-A would be a necessary feature of any small molecule; lastly, to recapitulate siRNA mediated LDH-A inhibition with small molecule inhibitors that had the potential for clinical application. The thesis examined both clinical data and a broad panel of cultured cancer cell types in order to select appropriate model in which to validate siRNA mediated inhibition of LDH-A and LDH-B. After it was demonstrated that LDH-A inhibition reduced the growth of cultured cells, a range of techniques were used to quantify this reduced growth in terms of cell death and changes in metabolism. Further to this, literature studies had proposed a role for LDH-B in maintaining lactate fuelled tumour growth; however, this thesis shows that in the cell lines studied, lactate-fuelled tumour growth was an LDH-A dependent phenomenon. Finally, a high throughput assay system was designed and validated and a library of small molecules was selected, synthesized, and screened in order to identify selective inhibitors of LDH-A.

Dans la plupart des pays industrialisés, le cancer du sein est la principale cause de décès chez les femmes. Le microenvironnement tumoral (TME) joue un rôle important dans la progression du cancer du sein. Le TME est un tissu complexe composé d’une matrice extracellulaire remaniée, de fibroblastes, de cellules inflammatoires et endothéliales. Récemment un nouveau composant cellulaire du TMA a été identifié. Il est formé par des adipocytes modifiés situés en regard de cellules cancéreuses appelées "adipocytes associés au cancer" (CAA). Ces constituants ajoutent à la complexité du TME. La protéase matricielle Matrix Metalloproteinase-11 (MMP-11) est une protéine du TME, elle est sécrétée par les «fibroblastes associés au cancer» (CAF) au centre de la tumeur et par les CAA à la périphérie de la tumeur (le front d’invasion). Soutenant l'idée que la MMP11 contribue à la progression tumorale, des études antérieures ont montré qu’une expression élevée était associée à une survie sans récidive plus courte des patientes atteintes d'un cancer du sein. Cependant, le mécanisme d'action spécifique de cette protéase est resté mal compris. Des études plus récentes ont montré que la MMP-11 est un régulateur négatif du développement du tissu adipeux et qu’elle module le métabolisme énergétique. Ces observations suggéraient que l'expression de MMP-11 dans le TME pourrait participer directement à la progression de la tumeur en modulant le métabolisme du tissu adipeux au profit des cellules cancéreuses. Cependant, la façon dont la MMP-11 agit, notamment à l'interface entre les cellules cancéreuses du sein et les CAAs, reste largement inconnue. Pour l’étudier, nous avons développé des modèles précliniques de cancer de la glande mammaire chez la souris par génie génétique. Tout d'abord, des souris déficientes (perte de fonction-LOF) ou surexprimant MMP-11 (Gain de Fonction-GOF) ont été croisées avec un modèle génétique de tumeurs mammaires (MMTV-PyMT). Des résultats cohérents ont été obtenus en utilisant les deux modèles. La MMP11 favorise la progression tumorale précoce, en augmentant la prolifération et en réduisant l'apoptose des cellules cancéreuses. De plus, l’expression de la MMP-11 a été associée à un changement métabolique dans la tumeur et à une altération significative de l’Unfolded Protein Response mitochondriale (UPRmt) et à une activation du stress du réticulum endoplasmique (UPRER). Ces données confortent l'idée selon laquelle la MMP-11 contribue à une réponse métabolique adaptative favorisant la croissance du cancer. Deuxièmement, pour aborder directement la fonction de la MMP-11 produite par le tissu adipeux sur la progression du cancer, nous avons généré une lignée de souris transgénique (appelée aP2-MMP11-IRES-GFP) dans laquelle l'expression de MMP-11 est contrôlée par un promoteur spécifique du tissu adipeux. L’implantation directe de cellules cancéreuses syngéniques dans le coussinet mammaire de ces souris a montré que l'expression de la MMP11 favorisait la croissance tumorale. Finalement, nos données soutiennent le concept selon lequel l'expression de MMP-11 par les adipocytes associés au cancer (CAA) contribuerait à une réponse métabolique adaptative favorisant la croissance du cancer. Ils renforcent aussi l’intérêt que représente la MMP-11 comme cible pour le traitement du cancer. / Breast cancer is the most common leading cause of death in women. The tumor microenvironment (TME) plays an important role in breast cancer progression. The TME is a complex tissue composed of extracellular matrix proteins, fibroblasts, inflammatory and endothelial cells. Recently modified adipocytes called “Cancer-Associated Adipocytes” (CAAs) were identified as emerging components of the TME adding into the complexity of this tumor component. Matrix Metalloproteinase-11 (MMP-11) is a protein from the TME, it is secreted by "Cancer-Associated Fibroblasts" (CAFs) in the center of the tumor and by CAAs in the tumor periphery also qualified as the “invasive front”. Previous studies showed that elevated MMP11 expression is associated with a poorer outcome in breast cancer patients supporting the idea that MMP11 contributes to tumor progression but the mechanism of action remained unclear. Recent studies showed that MMP-11 is a negative regulator of adipose tissue development and controls energy metabolism. These observations suggested that MMP-11 expression in the TME may directly participate in breast tumor progression by modulating the adipose tissue metabolism at the benefit of cancer cells. However, how MMP-11 acts in the TME notably at the interface of breast cancer cells and CAAs remains largely unknown. To study the role of MMP-11 on breast cancer progression, we developed a series of preclinical mouse mammary gland tumour models by genetic engineering. First, mice either deficient- (Loss of Function-LOF) or overexpressing- MMP-11 were crossed with a genetic model of spontaneous mammary tumors (MMTV-PyMT). Consistent results were obtained using GOF and LOF, showing that MMP11 favored early tumor progression, by increasing proliferation and reducing apoptosis of cancer cells. Of interest, MMP-11 was associated with a metabolic switch in the tumor and the activation of the mitochondrial unfolded protein response (UPRmt) and endoplasmic reticulum stress (UPRER). These data support the idea that MMP-11 contributes to an adaptive metabolic response favoring cancer growth. Second, to directly address the function of MMP-11 produced by the adipose tissue on cancer progression, we generated a transgenic mouse line (named aP2-MMP11-IRES-GFP) in which MMP-11 expression is controlled by an adipose tissue-specific promoter. Direct grafting of syngeneic cancer cells in the mammary fad-pad of these mice showed that MMP11 expression favored tumor growth. Altogether our data support the idea that MMP-11 expression by cancer associated adipocytes contributes to an adaptive metabolic response, named metabolic flexibility, favoring cancer growth. They further substantiate the potential of MMP-11 as a target for cancer therapy.

Hexavalent chromium, Cr(VI), is an established human carcinogen that is a worldwide environmental health concern. It is well understood that reactive oxygen species, genomic instability, and DNA damage repair deficiency are important contributors to Cr(VI)-induced carcinogenesis. After decades of research some cancer hallmarks remain understudied for the mechanism of Cr(VI) carcinogenesis. Dysregulated cellular energetics have been established as a hallmark of cancer. Energy pathways that become dysregulated in cancer include mitochondrial respiration, lipogenesis, pentose phosphate pathway, one carbon metabolism, and increased anaerobic glycolysis in the presence of oxygen or ‘Warburg effect’. To investigate metabolism changes in Cr(VI) carcinogenesis, we exposed human lung epithelial cells (BEAS-2B cells) to Cr(VI) for six months and isolated a colony from soft agar. To confirm the results in the BEAS-2B cells, we used two other sets of Cr(VI)-transformed cells, human lung epithelial cells (BEP2D cells) and human lung fibroblasts (WTHBF-6 cells). We found increased lipogenesis related protein expressions including: ATP citrate lyase (ACLY), acetyl-CoA carboxylase 1 (ACC1), and fatty acid synthase (FASN) in Cr(VI)-transformed cells as compared to passage-matched control cells. We also observed increased palmitic acid levels, confirming that Cr(VI)-transformed cells were making more lipids. Cr(VI)-transformed BEAS-2B cells had decreased colony formation in soft agar and decreased cell growth when treated with a FASN inhibitor (C75). ACLY, ACC1, and FASN protein expressions were also increased in chromate-induced lung tumors in human tissue samples. We also observed that Cr(VI)-transformed human lung cells (BEAS-2B, BEP2D, and WTHBF-6 cells) had no major changes in their mitochondrial respiration as measured by the Seahorse Analyzer when compared to their passage-matched control cells. Conversely, xenograft tumor-derived cells had mitochondrial respiratory dysfunction. Interestingly, we also found that Cr(VI)-transformed human lung cells (BEAS-2B, BEP2D, and WTHBF-6 cells) had no major changes in their glycolytic function as measured by the Seahorse Analyzer when compared to their passage-matched control cells. Similarly, these cells did not have changes in glycolytic enzymes or extracellular L-lactate levels. Moreover, xenograft tumor-derived cells showed no changes in glycolytic endpoints or L-lactate levels. This indicates these cells did not undergo the ‘Warburg effect’. These data demonstrate that increased lipogenesis is important to Cr(VI)-induced lung carcinogenesis and are consistent with the cancer literature which reports that increased lipogenesis proteins occur during carcinogenesis. Additionally, our results indicate mitochondrial respiratory dysfunction is likely a result of the tumor microenvironment and a later step during Cr(VI) carcinogenesis. Lastly, we observed the ‘Warburg effect’ is not required for Cr(VI)-induced carcinogenesis in vitro. However, it remains to be shown if the ‘Warburg effect’ is still a consequence or contributing factor for tumorigenesis. Future studies are needed to investigate other metabolic pathways in Cr(VI)-induced carcinogenesis. In conclusion, some metabolism pathways are important to Cr(VI)-induced carcinogenesis, while others appear not to be.

Le catabolisme exacerbé du glucose et de la glutamine est actuellement reconnu comme une caractéristique des cellules cancéreuses, qui leur procure un avantage prolifératif via la production et l’accumulation de plusieurs métabolites au niveau du microenvironnement. Parmi ces métabolites, l’acide lactique représente une molécule de signalisation clé, favorisant la migration et les métastases. Mon projet de thèse s’inscrit dans le contexte d’une étude du métabolisme glycolytique associé aux cellules tumorales à division rapide. Durant ce projet, nous nous sommes intéressés à la caractérisation génétique et fonctionnelle des transporteurs MCT (MonoCarboxylate Transporters) 1 et 4, qui sont des symporteurs H+/lactate dont l’expression membranaire et la fonctionnalité requièrent la liaison avec une protéine chaperonne : CD147/BASIGINE (BSG). Afin de mieux explorer la physiologie des complexes MCT/BSG, et valider le ciblage de l’export d’acide lactique comme une nouvelle approche anti-cancer, nous avons développé une stratégie visant à invalider le gène BSG et/ou MCT4, en utilisant la technologie des Zinc Finger Nucleases (ZFN), dans des lignées cellulaires cancéreuses humaines de côlon, poumon et glioblastome. D’abord, nous avons démontré, que l’effet pro-tumoral majeur de BSG est lié à son action directe sur la stabilisation des MCTs au niveau des tumeurs glycolytiques et non pas à la production des metalloprotéases. Ensuite, nous avons démontré pour la première fois que l’inhibition concomitante de MCT1 et MCT4 est nécessaire pour induire une baisse significative de la tumorigénécité in vivo. / Enhanced glucose and glutamine catabolism has become a recognized feature of cancer cells, leading to accumulation of metabolites in the tumour microenvironment, which offers growth advantages to tumours. Among these metabolites is emerging as a key signalling molecule that plays a pivotal role in cancer cell migration and metastasis. In this thesis, we focused on the genetic and functional characterization of monocarboxylate transporters (MCT) 1 and 4, which are H+/lactate symporters that require an interaction with an ancillary protein, CD147/BASIGIN (BSG), for their plasma membrane expression and function. To further explore the physiology of MCT/BSG complexes and validate the blockade of lactic acid export as an anti-cancer strategy, we designed experiments using Zinc Finger Nuclease mediated BSG and/or MCT4 gene knockouts in human colon adenocarcinoma, lung carcinoma and glioblastoma cell lines. First of all, we demonstrated that the major protumoural action of BSG is to control the energetics of glycolytic tumours via MCT1/4 activity and not to produce matrix metalloproteases. Second, we showed for the first time that combined inhibition of both MCT1 and MCT4 transporters is required to achieve a significant reduction in the tumour growth in vivo. Moreover, our findings reported that disruption of the BSG gene dramatically reduced the plasma membrane expression and lactate transport activity of both MCT1 and MCT4, leading to increased accumulation of intracellular pools of lactic and pyruvic acids, decreased intracellular pH and reduced rate of glycolysis.

Dissolution dynamic nuclear polarisation is an emerging clinical technique which enables the metabolism of hyperpolarised 13C-labelled molecules to be dynamically and non- invasively imaged in tissue. The first molecule to gain clinical approval is [1-13C]pyruvate, the conversion of which to [1-13C]lactate has been shown to detect early treatment re- sponse in cancers and correlate with tumour grade. As the technique has recently been translated into humans, accurate and reliable quantitative methods are required in order to detect, analyse and compare regions of altered metabolism in patients. Furthermore, there is a requirement to understand the biological processes which govern lactate pro- duction in tumours in order to draw reliable conclusions from this data. This work begins with a comprehensive analysis of the quantitative methods which have previously been applied to hyperpolarised 13C data and compares these to some novel approaches. The most appropriate kinetic model to apply to hyperpolarised data is determined and some simple, robust quantitative metrics are identified which are suitable for clinical use. A means of automatically segmenting 5D hyperpolarised imaging data using a fuzzy Markov random field approach is presented in order to reliably identify regions of abnormal metabolic activity. The utility of the algorithm is demonstrated on both in silico and animal data. To gain insight into the processes driving lactate metabolism, a mathematical model is developed which is capable of simulating tumour growth and treatment response under a range of metabolic and tissue conditions, focusing on the interaction between tumour and stroma. Finally, hyperpolarised 13C-pyruvate imaging data from the first human subjects to be imaged in Cambridge is analysed. The ability to detect and quantify lactate production in patients is demonstrated through application of the methods derived in earlier chapters. The mathematical approaches presented in this work have the potential to inform both the analysis and interpretation of clinical hyperpolarised 13C imaging data and to aid in the clinical translation of this technique.

L’être humain est exposé quotidiennement et simultanément à des dizaines de polluants environnementaux, pourtant il n’existe encore que peu ou pas d’études sur les effets de ces mélanges. Des études épidémiologiques et transcriptomiques montrent que les polluants peuvent perturber le métabolisme glucidique et lipidique. Le lien entre métabolisme et cancer a été démontré depuis plusieurs décennies, en effet, une dérégulation du métabolisme oxydatif, appelée effet Warburg, et commune à presque toutes les cellules tumorales se caractérise par une déviation du métabolisme oxydatif mitochondrial vers une glycolyse anaérobie entrainant une augmentation de la production de lactate. Pour ce travail les effets d’un mélange de deux polluants organiques persistants ayant des voies de signalisation différentes sont étudiés. Le mélange associe la TCDD, une substance cancérigène, à un pesticide l’α-endosulfan, afin d’évaluer les effets combinés de ces deux polluants sur le métabolisme énergétique de cellules cancéreuses coliques humaines Caco2. Le traitement des cellules pendant 48 heures par la TCDD (25 nM) et l’α-endosulfan (10µM) conduit à une diminution de l’oxydation du glucose, corrélée à une augmentation de la production de lactate, alors que chaque polluant seul exerce un effet peu significatif. Le mélange diminue l’activité globale de la mitochondrie caractérisée par une diminution de la respiration cellulaire, et de la production d’ATP sans toutefois modifier l’intégrité mitochondriale. L’étude des mécanismes impliqués dans ces effets indique l’implication de l’AMPK et du complexe PDH, deux enzymes clés régulant de façon très importante la glycolyse cellulaire ; en effet l’inhibition de l’AMPK abolit les effets des polluants. Des modifications du calcium intracellulaire qui régule entre autre l’activité de ces deux enzymes sont observées et l’inhibition du calcium abolit également les effets des polluants. Ces travaux montrent que le mélange induit une aggravation de la dérégulation du phénotype métabolique des cellules Caco2 à l’état prolifératif. Cela pourrait signifier une synergie de l’activité de ces polluants qui pourrait accentuer ce phénotype dans un contexte tumorale via un mécanisme impliquant le calcium. / During tumorigenesis most of cancer cells exhibit an altered metabolism that is characterized by an elevated uptake of glucose and an increased glycolytic rate; this phenomenon is known as the Warburg effect. Compelling recent evidences suggest that alteration of cellular metabolism is critical during cancer development and constitutes a major feature of aggressive tumour. Considering the recent observations on the impact of persistent organic pollutants (POPs) on cell metabolism, we hypothesize that POPs could exert their carcinogenic effects by promoting metabolic alterations that could converge to a metabolic shift supporting a tumoral phenotype. Proliferating colon cancer cells (Caco2) were treated with TCDD (25 nM) or/and α-endosulfan (10 µM), two environmental pollutants mainly produced by human activities and designated by the International Agency for Research on Cancer as probably or well-established carcinogenic to humans. A significant decrease of glucose and glutamine oxidation (60%) was observed after a treatment for 48 hours with the two pollutants while each pollutant alone had no significant effect. These observations are correlated with an increased lactate production by two fold. These effects are maintained in the presence of antioxidative NAC (10 mM), suggesting that they are independent of the oxidative status of the cell. We also observed a decreased incorporation of glucose in total lipids (50%). The ATP production and the cell respiration level were significantly decreased by the mixture by about 50% and 80%, respectively. In the same conditions, the glycogen production and the NADPH/NADPH,H+ ratio were unchanged. Taken together, these results suggest that POPs could worsen the metabolic phenotype of cancer cells. The molecular mechanisms underlying the POPs-induced metabolic reprograming are under investigation and should provide a better understanding of the signalling pathways involved in POPs action on the regulation of the energetic metabolism balance and their consequence on cancer.

Energy production is a very important function for the cells to maintain homeostasis, survive and proliferate. Cellular energy can be produced either through oxidative phosphorylation (OXPHOS) in the presence of oxygen or glycolysis in its absence. Cancer cells, even in the presence of oxygen prefer to produce energy through glycolysis and this confers them a survival advantage. Energy metabolism has recently attracted the interest of several laboratories as targeting the pathways for energy production in cancer cells could be an efficient anticancer treatment. For that purpose the role of various transcription factors in determining the pathway of energy production has been investigated extensively and there is evidence to suggest that oncogenic transcription factors promote glycolysis whereas tumour suppressors demote it. In line with this notion, the master regulator of cellular response to hypoxia, the Hypoxia Inducible Factor 1 (HIF-1) has been shown to induce the expression of a variety of genes encoding enzymes involved in glucose metabolism as well as OXPHOS favouring energy production through glucose metabolism in hypoxic cells. The tumour suppressor p53 on the other hand inhibits glycolysis and stimulates OXPHOS. One of the pathways through which p53 exerts these effects, is by inducing the inhibitor of glycolysis TIGAR and the cytochrome c oxidase assembly factor SCO2 gene expression under DNA damage conditions. However, the regulation of the expression of these genes in hypoxic conditions has been only partially elucidated. We hypothesised that under hypoxic conditions, TIGAR and SCO2 gene expression might be differentially regulated in cells bearing mutated p53 and in these cells the involvement of HIF-1 could be crucial. Indeed under hypoxia mimicking conditions, the TIGAR and SCO2 protein and mRNA levels were found to be modulated differentially in p53 wild type and mutant cell lines. The bioinformatics analysis revealed the presence of hypoxia responsive elements (HREs) within the regulatory region of the promoters of TIGAR and SCO2 genes. Firefly reporter assays and chromatin immunoprecipitation (ChIP) assays have indicated that HIF-1 plays a crucial role in the regulation of TIGAR gene expression. The direct involvement of HIF-1 in the regulation of SCO2 gene expression requires further investigation. We and others have recently reported that PCAF is a common cofactor for p53 and HIF-1α, regulating the protein stability and transcription target selectivity of both transcription factors thereby orchestrating the balance between life and death in cancer cells. We hypothesised that PCAF plays a similar role in the regulation of cellular energy metabolism by differentially targeting HIF-1α and p53 to the promoter of TIGAR and SCO2 genes. In this study we present evidence to support the notion that PCAF plays an import role in the regulation of TIGAR and SCO2 gene expression under hypoxic mimicking conditions. This conclusion was supported by assessing the functional consequences of PCAFwt and PCAFΔHAT overexpression on the intracellular lactate production, cellular oxygen consumption, NAD+/NADH ratio and ROS generation in cells under hypoxia mimicking conditions.