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<p>Normal cellular energy metabolism is fundamentally altered in cancer cells to facilitate rapid production of new cellular components, thereby enabling uncontrolled cell growth. Specifically, cancer cells rely on glycolysis and alternative pathways such as lipid and glutamine metabolism for energy, while diverting substrates away from oxidative metabolism regardless of the prevalence of oxygen in the microenvironment. This hallmark of cancer cells is referred to as the Warburg effect, the precise regulation of which is poorly understood despite several decades of research. In comparing the global gene expression profiles of ovarian cancer cells to those that overexpress Ets-1, we have revealed that this transcription factor is involved, at least in part, to this cancer-associated metabolic switch. To support the validity of these findings, we have shown that Ets-1 functionally regulates glycolytic dependence in ovarian and breast cancer cells, while concomitantly displaying a decreased capacity for oxidative phosphorylation. Reactive oxygen species are a normal byproduct of metabolism, and are produced excessively in cancer cells leading to oxidative stress. Interestingly, our genomic pathway analyses uncovered enrichments in antioxidant pathways associated with increased Ets-1 expression. Accordingly, we have also observed that Ets-1 regulates increased intracellular glutathione levels, and induces the activity of key antioxidant enzymes under oxidative stress. Sulfasalazine, an agent that restricts cystine uptake, was shown to be effective for decreasing these high glutathione levels during oxidative stress. These results are clinically relevant because high glutathione levels are associated with iii therapeutic resistance in cancer cells. Collectively, the evidence presented has identified a novel role for the transcription factor Ets-1 in the regulation of cancer energy metabolism, as well as the response to oxidative stress. We have also described a mechanism for Ets- 1-mediated therapeutic resistance, suggesting that this transcription factor may be a promising novel target to enhance conventional cancer therapies.</p> / Doctor of Philosophy (Medical Science)

This dissertation is a compilation of published works and a manuscript that seek to understand the possible role of polyamines in the regulation of capsule in Streptococcus pneumoniae (Spn, pneumococcus). Spn remains a major health risk worldwide while the capsule is widely recognized as the principal virulence factor. Polyamines on the other hand are small hydrocarbon molecules known to regulate a number of cellular processes in bacteria. This work investigates the impact of deletion of polyamine biosynthesis gene, SP_0916 (cadA, lysine decarboxylase at the time of first and second publication), on protein expression and the capsule biosynthesis of virulent pneumococcal serotype 4 (TIGR4). We identify loss of capsular polysaccharide (CPS) in the deletion strain and based on proteomics results, we hypothesized that a shift in metabolism that favors the pentose phosphate pathway (PPP) over glycolytic pathway, that could reduce the availability of precursors for CPS had occurred. Comparison of transcriptomic and untargeted metabolomics profile of ∆SP_0916 with TIGR4 shows impaired glycolysis and Leloir pathways that provide CPS precursors, in the mutant strain. Furthermore, gene expression changes indicate possible reduction of common polyamines (cadaverine, putrescine, spermidine and spermine). Targeted metabolomics analysis confirmed reduced levels of polyamines in SP_0916. However, the result suggests that SP_0916 encodes an arginine decarboxylase, contrary to its existing annotation as a lysine decarboxylase in many bioinformatics databases. Biochemical characterization of the purified protein encoded by SP_0916 confirms that it is indeed catalyzes arginine decarboxylation, and exogenous supplementation of agmatine, the product of the reaction, successfully restores capsule biosynthesis. This study fixes an error in annotation of the TIGR4 genome and further establishes the essentiality of agmatine, a product of arginine decarboxylation as the key polyamine molecule modulating pneumococcal capsule. We later compared the impact of deletion of polyamine synthesis by gene deletion (ΔSP_0916) with chemical inhibition of synthesis using α- difluoromethylornithine (DFMO), in multiple pneumococcal serotypes. Results of this dissertation confirmed that pneumococcal pathways impacted by the disruption of polyamine biosynthesis either by gene deletion or chemical intervention are conserved and could regulate capsule synthesis.

Glioblastomas continue to carry poor prognoses for patients despite advances in surgical, chemotherapeutic, and radiation regimens. One feature of glioblastoma associated with poor prognosis is the degree of hypoxia and elevated expression levels of hypoxia-inducible factor-1 á (HIF-1á). HIF-1á expression allows metabolic adaptation to low oxygen availability, partly through upregulation of vascular endothelial growth factor (VEGF) and increased tumor angiogenesis as well as induction of anaerobic glycolysis. In this study, we demonstrate an induced level of astrocyte-elevated gene-1 (AEG-1) by hypoxia in glioblastoma cells. AEG-1 has the capacity to promote anchorage-independent growth and cooperates with Ha-ras in malignant transformation. In addition, AEG-1 was recently demonstrated to serve as an oncogene and can induce angiogenesis and autophagy in glioblastoma. Results from in vitro studies show that hypoxic induction of AEG-1 is dependent on HIF-1á stabilization during hypoxia and that phosphatidylinositol 3-kinase (PI3K) inhibition abrogates AEG-1 induction during hypoxia through loss of HIF-1á stability. Furthermore, we show that AEG-1 is induced by glucose deprivation and that prevention of intracellular reactive oxygen species (ROS) production prevents this induction. Additionally, AEG-1 knockdown results in increased ROS production and increased glucose deprivation-induced cytotoxicity, whereas AEG-1 overexpression prevents ROS production and decreases glucose deprivation-induced cytotoxicity, indicating that AEG-1 induction is necessary for cells to survive this type of cell stress. From studies examining the expression of enzymes involved in glucose metabolism, we demonstrate that AEG-1 alters the tumor metabolic profile in a partially 5'-adenosine monophosphate (AMP)-activated protein kinase (AMPK)-dependent manner. Moreover, glycolytic inhibition modulates the metabolic effects induced by AEG-1, and AEG-1 knockdown reduces the growth and alters the metabolic phenotype of glioblastoma subcutaneous xenografts. These observations link AEG-1 overexpression observed in glioblastoma with hypoxia and glucose metabolic signaling, and targeting these physiological pathways may lead to therapeutic advances in the treatment of glioblastoma in the future. Recent studies have reported the detection of the human neurotropic virus, JC Virus (JCV), in a significant population of brain tumors, including medulloblastomas. Accordingly, expression of the JCV early protein, T-antigen, which has transforming activity in cell culture and in transgenic mice, results in the development of a broad range of tumors of neural crest and glial origin. Evidently, the association of T-antigen with a range of tumor-suppressor proteins, including p53 and pRb, and signaling molecules, such as â-catenin and IRS-1, play a role in the oncogenic function of JCV T-antigen. We demonstrate that T-antigen expression is suppressed by glucose deprivation in medulloblastoma cells that endogenously express T-antigen. Mechanistic studies indicate that glucose deprivation-mediated suppression of T-antigen is partly influenced by AMPK, a critical sensor of the AMP/ATP ratio in cells. We have found that AMPK activation inhibits T-antigen expression, whereas AMPK inhibition prevents glucose deprivation-mediated T-antigen suppression. In addition, glucose deprivation-induced cell cycle arrest in the G1 phase is blocked with AMPK inhibition, which also prevents T-antigen downregulation. Furthermore, T-antigen-expressing medulloblastoma cells, as compared to those which do not express T-antigen, exhibit less G1 arrest and an increased percentage of cells in the G2 phase of the cell cycle during glucose deprivation. On a functional level, T-antigen downregulation is partially dependent on ROS production during glucose deprivation. Additionally, studies indicate that T-antigen prevents ROS induction, loss of ATP production, and cytotoxicity induced by glucose deprivation. We have also found that T-antigen is downregulated by the glycolytic inhibitor, 2-deoxy-D-glucose (2-DG), and the pentose phosphate inhibitors, 6-aminonicotinamde (6-AN) and oxythiamine (OT). Enzyme expression studies also indicate that T-antigen upregulates the expression of the pentose phosphate enzyme, transaldolase-1 (TALDO1), demonstrating a potential link between T-antigen and glucose metabolic regulation. These studies highlight the potential involvement of JCV T-antigen in the proliferation and metabolic phenotype of medulloblastoma and may enhance our understanding of the role of viral proteins in tumor glycolytic metabolism, thus implicating these proteins as potential targets for the treatment of virus-associated tumors. / Biomedical Neuroscience

Acute myeloid leukemia (AML) develops following oncogenic alterations to hematopoietic stem (HSC) and progenitor cells (HSPCs) in the bone marrow, resulting in dysregulated proliferation of immature myeloid progenitors that interferes with normal hematopoiesis. Understanding the mechanisms of HSPC protection against damage and excessive division, and how these pathways are altered during leukemic progression, is vital for establishing effective therapies. Here, we show that autophagy, a lysosomal degradation pathway, is increased in HSPCs using a novel imaging flow cytometry autophagy assay. Loss of hematopoietic autophagy following deletion of key gene Atg5 resulted in increased HSC proliferation, leading to HSC exhaustion and bone marrow failure. Although erythrocyte and lymphocyte populations were negatively impacted by autophagy loss, myeloid cells showing immature characteristics were expanded. Deletion of Atg5 in an AML model resulted in increased proliferation under metabolic stress, dependent on the glycolytic pathway, and aberrant upstream mTOR signaling. Moreover, modulation of Atg5 altered leukemic response to culture with stromal cells. Finally, primary AML cells displayed multiple markers of decreased autophagy. These data suggest a role for autophagy in preserving HSC function, partially through suppression of HSPC proliferation, and indicate that decreased autophagy may benefit AML cells. We postulate that modulation of autophagy could help maintain stem cell function, for example during transplantation, and aid AML therapy in a setting-specific manner.

L'exposition à la lumière des solutions de nutrition parentérale (NP) génère des peroxydes tels que l'H2O2 et l'ascorbylperoxyde (AscOOH). Cette absence de photo-protection provoque une augmentation des triglycérides (TG) plasmatique chez les enfants prématurés et chez un modèle animal, ayant un stress oxydatif et une stéatose hépatique indépendante de l’exposition au H2O2. Nous pensons que l'AscOOH est l'agent actif conduisant à l'élévation des TG. Le but est d'investiguer le rôle de l'AscOOH sur les métabolismes du glucose et des lipides à l'aide d'un modèle animal néonatal de NP. / The light exposure of parenteral nutritive solutions generates peroxides such as H2O2 and ascorbylperoxide. This absence of photo-protection is associated with higher plasma triacylglycerol concentration (TG) in premature infants and, in animals, with oxidative stress and a H2O2 independent hepatic steatosis. We hypothesized that ascorbylperoxide is the active agent leading to high TG. The aim was to investigate the role of ascorbylperoxide on glucose and lipid metabolism in an animal model of neonatal parenteral nutrition.

High aerobic glycolytic capacity is correlated with poor prognosis and increased tumour aggressiveness. 6Phosphofructo-1-kinase catalyses the first irreversible step of glycolysis, and is activated by fructose-2,6-bisphosphate, a product of the kinase activity of four bifunctional isoenzymes, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase (PFK-2/FBPase-2:PFKFB1-4). These are potential anti-tumour targets, but their individual and collective role requires further investigation. This thesis had three aims; to validate the PFK-2/FBPase-2 isoenzymes as anti-cancer targets, to investigate the requirement for isoenzyme-specific targeting, and to initiate assay development, enabling future identification of novel inhibitors. A panel of cancer cell lines was examined and PFKFB3 and PFKFB4 were confirmed to be the most strongly induced isoenzymes in hypoxia, regulated by HIF-1&alpha;. Basal and hypoxic relative PFKFB3/PFKFB4 expression varied markedly, and three cell lines with varying expression ratios (MCF-7, U87, PC3) were selected for further study. siRNA knockdown of each isoenzyme individually, markedly reduced 2D and 3D cell growth. The effect of PFKFB3 knockdown was consistently more pronounced, particularly in hypoxia. Double PFKFB3/PFKFB4 knockdown was significantly less effective than PFKFB3 knockdown alone. Direct antagonism of PFKFB3 and PFKFB4 on F-2,6-BP concentration was observed, with PFKFB3 exhibiting high kinase activity, as anticipated, and PFKFB4 exhibiting high bisphosphatase activity. The degree of antagonism was dependent on the relative PFKFB3/PFKFB4 expression ratio. Extensive efforts were made to examine the wider metabolic effect of PFKFB3/PFKFB4 on flux towards glycolysis or the pentose phosphate pathway (PPP), including using metabolite, lipid droplet, ¹³C NMR and mass spectrometry assays. No significant change in metabolic flux was detected, the evidence presented therefore suggesting the impact of the antagonistic effects of the isoenzymes on [F-2,6-BP] extends beyond regulation of metabolic flux alone. This study concluded that the most effective therapeutic strategy will be one that involves a PFKFB3-specific inhibitor, preferably hypoxia-targeted. Accordingly, steps were taken to validate and optimise a robust medium-throughput assay system.

Sepsis é uma disfunção de órgãos causada por uma resposta desregulada do hospedeiro em decorrência de uma infecção e que eventualmente leva a morte. A identificação de moléculas que minimizem este processo pode fornecer alvos terapêuticos para prevenir a falência de órgãos durante a sepse. O supressor de sinalização de citocinas 1 (SOCS1) é conhecido por regular negativamente a sinalização de receptores de citocinas e de receptores do tipo Toll (TLRs). No entanto, os alvos celulares e mecanismos moleculares envolvidos nas ações de SOCS1 durante a sepse são desconhecidos. Para determinar o papel de SOCS1 durante a sepse polimicrobiana, camundongos C57BL/6 foram tratados com um peptídeo inibidor do domínio KIR (kinase inhibitor region) do SOCS1 (iKIR) e submetidos à CLP (ligação e perfusão do ceco). O tratamento com iKIR aumentou a mortalidade, a carga bacteriana e a produção de citocinas inflamatórias induzida pela CLP. Além disso, observou-se que animais deficientes de SOCS1 nas células mielóides (SOCS1?myel) também tiveram aumento na carga bacteriana e na produção de citocinas proinflamatórias, quando comparados com camundongos SOCS1fl. O aumento na susceptibilidade a sepse foi acompanhado pelo aumento da via glicolítica nas células peritoneias e no pulmão desses animais. Assim, foi observado aumento da produção de ácido láctico e da expressão de enzimas glicolíticas como hexoquinase-1 (Hk1), lactato desidrogenase A (Ldha) e o transportador de glicose 1 (Glut-1) em camundongos sépticos tratados com iKIR ou SOCS1?myel. A expressão desses genes da via glicolítica foi dependente da via de ativação STAT3/HIF-1?. O tratamento com 2-deoxiglicose (inibidor da via glicolítica) diminuiu a susceptibilidade à sepse em camundongos tratados com iKIR. Estes resultados indicam um papel até agora desconhecido de SOCS1, como um regulador de reprogramação metabólica que reduz a resposta inflamatória exacerbada e o dano de órgãos durante a sepse. / Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection. Identification of pleiotropic molecular brakes might provide therapeutic targets to prevent organ failure during sepsis. Suppressor of cytokine signaling 1 (SOCS1) is known to negatively regulate signaling by cytokine and Tolllike receptors (TLRs). However, the cellular targets and molecular mechanisms involved in SOCS1 actions during sepsis are unknown. To address this in a cecal ligation puncture (CLP) model of sepsis, we treated C57BL/6 mice with an antagonist peptide (iKIR) that blocks the kinase inhibitory region (KIR) domain of SOCS1 and prevents its actions. iKIR treatment increased mortality, bacterial burden and inflammatory cytokine production induced after CLP. We also found that myeloid cell-specific SOCS1 deletion (SOCS1?myel) rendered mice more susceptible to sepsis, shown by higher bacterial loads and inflammatory cytokines than SOCS1fl littermate control mice. O aumento na susceptibilidade a sepse foi acompanhado pelo aumento da via glicolítica nas células peritoneias e pulmão desses animais. These effects were accompanied by increase of glycolysis function in peritoneal cells and lung of SOCS1?myel. Thus, it was observed increased expression of the glycolytic enzymes, hexoquinase-1 (Hk1), lactate dehydrogenase A (Ldha), and glucose transporter 1 (Glut-1) in iKIR-treated or SOCS1?myel septic mice. These events were dependent on the activation of STAT3/HIF-1? pathway. Blocking glycolysis with 2-deoxyglucose ameliorated the increased susceptibility to sepsis in iKIR-treated CLP mice. Together, we unveiled a heretofore unknown role of SOCS1 as a regulator of metabolic reprograming that reduces overwhelming inflammatory response and organ damage during sepsis.

The metabolic state of an immune cell directly influences its ability to function and differentiate, ultimately affecting immunity, inflammation and tolerance. Different immune cell subsets have differing metabolic requirements. Macrophages, as the frontline, tissue-resident cells of the innate immune system, undergo profound metabolic reprogramming in response to environmental stimuli. To date, there has been little consideration how macrophage metabolism might be affected by humoral immunity. IgG antibodies are the soluble effector molecules of the adaptive humoral immune system. Fcγ receptors (FcγRs) mediate the cellular functions of IgG antibodies and are expressed on most immune cells including macrophages. FcγR cross-linking induced by IgG immune complexes (ICs) is important for defence against some infections but can also play a pathogenic role in autoimmunity. Here, I studied the metabolic reprogramming induced in macrophages by IgG IC ligation of FcγRs. I first investigated how FcγRs cross-linking might impact glucose metabolism. We show that macrophages undergo a switch to glycolysis in response to IgG IC stimulation. FcγR-associated glycolysis was dependent on the mammalian target of rapamycin (mTOR) and hypoxia-inducible factor (HIF)1α. Moreover, this glycolytic switch was required to generate a number of pro-inflammatory mediators and cytokines. Inhibition of glycolysis, or genetic depletion of HIF1α in macrophages resulted in the attenuation of IL1β and other inflammatory mediators produced in response to IgG IC in vitro. To determine the relevance of these observations to responses to IgG IC in vivo and, in particular, to IC-associated tissue inflammation in autoimmune diseases such as system lupus erythematosus (SLE), I developed three models to interrogate tissue macrophages. Following administration of IC to peritoneal macrophages, I observed IL1β-associated neutrophil recruitment that was abrogated by inhibiting glycolysis, or in the presence of HIF-1a deficiency. Similarly, following administration of intravenous IC, or nephrotoxic serum, kidney macrophage activation was abrogated by glycolysis inhibition or by myeloid HIF-1a deficiency. Together my data reveal the cellular molecular mechanisms required for FcγR-mediated metabolic reprogramming in macrophages and define a novel therapeutic strategy in autoantibody-induced inflammation. In the final part of the thesis I identified additional metabolic pathways that were altered by FcγR ligation, including cholesterol biosynthesis and fatty acid biosynthesis. This has important implications for protective immune responses and autoimmune susceptibility, since a number of intermediates in these pathways can directly regulate and contribute to immune responses. In summary, I have demonstrated the metabolic alterations triggered by FcγR ligation, reveal the cellular molecular mechanisms required for FcγR-mediated cellular respiration reprogramming in macrophages and define a potential therapeutic target in autoimmunity.

De plus en plus de preuves montrent que le transport axonal rapide (FAT) joue un rôle crucial au cours des maladies neurodégénératives (NDs). La maladie de Huntington est une maladie neurodégénérative causée par une expansion anormale de polyglutamines dans la partie Nterminale de la protéine huntingtine (HTT) : une grande protéine d’échafaudage impliquée dans la régulation du transport. La présence de HTT mutante comme l’absence de la HTT induisent des défauts de transport chez les mammifères. Chez la Drosophile, la HTT mutante reproduit le phénotype observée chez les mammifères, cependant la fonction conservée de la HTT chez la Drosophile melanogaster (DmHTT) n’est pas encore clairement établie. Ici nous mettons en évidence que DmHTT s’associe aux vésicules, aux microtubules et intéragit avec la proteine dynéine. Dans les neurones corticaux de rat, DmHTT remplace partiellement la HTT de mammifère dans le transport axonal rapide, et les drosophiles invalidées pour la HTT montrent des défauts de transport axonal in vivo. Ces résultats suggèrent que la fonction de la HTT est conservée dans le modèle Drosophile.Le FAT est un processus qui requiert un apport constant d’énergie. Les mitochondries sont les principales sources de production d’ATP de la cellule. Cependant nous avons démontré que le FAT ne dépend non pas de cette source d’énergie là, contrairement à ce que l’on pensait, mais de l’ATP glycolytique produit par les vésicules. La dérégulation de GAPDH ou de PK, les deux enzymes glycolytiques productrices d’ATP, ralentit le transport vésiculaire. Néanmoins, l’invalidation de GAPDH n’affecte pas le transport mitochondrial. En outre, toutes les enzymes glycolytiques sont associées à des vésicules dynamiques et sont capables de produire leur propre ATP. Enfin nous montrons que l’ATP produit est suffisant pour assurer leur propre transport, prouvant l’autonomie énergétique des vésicules pour le transport. / Growing evidence support the idea that impairments in Fast Axonal Transport (FAT) play a crucial role in Neurodegenerative Diseases (NDs). Huntington’s Disease is neurodegenerative disorder caused by an abnormal polyglutamine expansion in the N-Terminal part of huntingtin (HTT), a large scaffold protein implicated in transport regulation. Both the presence of the mutated HTT as the loss of HTT leads to transport defects in mammals. In the fruit fly overexpression of the mutant HTT recapitulates the phenotype observed in mammals. However, it is still unclear whether HTT’s function is conserved in D. melanogaster. Here, we show that D. melanogaster HTT (DmHTT) associates with vesicles, microtubules, and interacts with dynein. In rat cortical neurons, DmHTT partially replaces mammalian HTT in fast axonal transport, and DmHTT KO flies show axonal transport defects in vivo. These results suggest that HTT function in transport is conserved in D. melanogaster.FAT is a process that requires a constant supply of energy. Mitochondria are the main producers of ATP in the cell. However, we have demonstrated that FAT does not depend on this source of energy, as previously thought, but it depends on glycolytic ATP produced on vesicles. Perturbing GAPDH or PK, the two ATP generating glycolytic enzymes, slows down vesicular transport. However, knocking down GAPDH does not affect mitochondrial transport. Furthermore, all of the glycolytic enzymes are associated with dynamic vesicles, and are capable of producing their own ATP. Finally, we show that this ATP production is sufficient to sustain their own transport, demonstrating the energetical autonomy of vesicles for transport.