Cancer cells undergo a multifaceted rewiring of cellular metabolism to support their biosynthetic needs. Although the major determinants of this metabolic transformation have been elucidated, their broad biological implications and clinical relevance are unclear. In this study, I systematically analysed the expression of metabolic genes across 20 different cancer types and investigated their impact on clinical outcome. I found that cancers undergo a tissue-specific metabolic rewiring, which converges towards a common metabolic landscape. Of note, downregulation of mitochondrial genes is associated with the worst clinical outcome across all cancer types and correlates with the expression of epithelial-to-mesenchymal transition (EMT) gene signature, a feature of invasive and metastatic cancers. Consistently, suppression of mitochondrial genes is identified as key metabolic signature of metastatic melanoma and renal cancer, and metastatic cell lines. This comprehensive analysis reveals unexpected facets of cancer metabolism, with important implications for cancer patients stratification, prognosis, and therapy. I then investigated how mitochondrial dysfunction could affect cell behaviour. I capitalised on a recently developed in vitro cell model with increasing levels of m.8993T > G mutation heteroplasmy. I found that impaired utilisation of reduced nicotinamide adenine dinucleotide (NADH) by the mitochondrial respiratory chain leads to cytosolic reductive carboxylation of glutamine as a new mechanism for cytosol-confined NADH recycling supported by malate dehydrogenase 1 (MDH1). This metabolic coupling is facilitated by the formation of a multienzymatic complex between MDH1 and GAPDH. Importantly, such metabolic coupling between glutamine metabolism and cytosolic NADH recycling is able to support increased glycolytic flux, an important hallmark of cells with dysfunctional mitochondria, as well as cancer cells. Finally, increased glycolysis in cells with mitochondrial dysfunction is associated with enhanced cell migration, in an MDH1-dependent fashion. These results describe a novel link between glycolysis and mitochondrial dysfunction, and uncover potential targets for cells that rely on aerobic glycolysis for proliferation and migration, such as cancer cells.

INTRODUCTION: In breast cancer patients with the most common metabolic disorder, Type 2 Diabetes (T2D), it has been established that the mortality rate is increased when juxtaposed to breast cancer patients without T2D. The mortality rate further increases when the T2D diagnosis occurs on or post the breast cancer diagnosis. Amongst the breast cancer types, Triple Negative Breast Cancer (TNBC) is a negative diagnosis, resulting from the lack of expression of receptors, and is by far the most heterogenous type of breast cancer and exhibits the greatest difficulties for treatment and worst outcomes. Although solid tumors have been studied by pathologists, surgeons, molecular biologists and other experts for many decades, important questions remain about the role of metabolism and tumor microenvironment in the emergence of cancer metastasis, which is the focus of this thesis. METHODS: The 4T1 cells were cultured in RPMI media at 11 mM glucose concentration and at RPMI at 4 mM glucose, to mimic the diabetic and non-diabetic condition respectively. The cells in both glucose concentrations are cultured parallelly in 10 cm dishes until they reach 60-70% confluence, Nile reed was used to stain the lipid in the cells and was observed under fluorescent microscope. The cells were then treated once with exosomes collected from mature, differentiated, insulin sensitive (IS) and insulin resistant (IR) 3T3-L1 cells, in 6cm dishes and grown for 3 days. They were then plated in a Seahorse plate and analyzed using Seahorse Cell Mito-stress test assay to measure the real-time change in their Oxygen Consumption Rate (OCR) and Extracellular Acidification Rate (ECR), to study the effect the exosome treatment and their response to acute injections of Oligomycin, FCCP and Antimycin A during the assay. Cells were also parallelly extracted with SDS-PAGE sample buffer, separated by electrophoresis and transferred to PVDF membrane for western blotting after SDS PAGE electrophoresis. Phosphorylated and unphosphorylated acetyl-CoA carboxylase (ACC), AMP-activated protein kinase (AMPK) and Ki67 (nuclear protein- biomarker of proliferation) were detected with specific rabbit antibodies (Cell Signaling). Protein bands were detected on a GE LAS-4000 gel imager using enhanced chemiluminescence. Bands were analyzed using ImageJ software (Schneider, Rasband and Eliceiri 2012) and the intensity of the bands was measured and analyzed with Adobe photoshop. RESULTS: There was a noticeable loss of lipid accumulation when the 4T1 cells were shifted from 11 mM glucose to 4 mM glucose. The cell count in both 11 mM glucose and 4 mM glucose was significantly higher in the IR treatment group when compared to the other groups. In 11 mM glucose cells, the treatment with IR exosomes drove up the baseline OCR and overall response to the injections when compared to the controls. In 4 mM glucose cells, the IR exosome treatment seems to have inhibited the baseline OCR and the overall response to the injections. Western blot analysis, it revealed that the IR exosome treatment increased the phosphorylation of AMPK in 11 mM glucose cells whereas it drastically reduced it in 4 mM glucose cells. The levels of phosphorylation of ACC and AKT remained mostly unchanged amongst all treatment groups of 11 mM and 4 mM glucose cells whereas the IR treatment ramped up the expression of Ki67 only in 11 mM glucose cells and not in 4 mM glucose cells. CONCLUSION: The results obtained reveal that there may be a correlation with the lipid stores that are found in the 11 mM glucose cells that may have played a role in the effectiveness of the exosome treatment, in terms of the intensity and direction of the effect. Similarly, the 4 mM glucose cells respond in a different way possibly due to a difference in the metabolism of the exosomes themselves. 4 mM glucose cells are depleted of the lipid stores that the 11 mM glucose cells have and the exosomes that are used for the treatment originate from the adipocytes, indicating that the cross-talk that is implied between the adipose tissues surrounding the breast cancer cells does more than just deliver possible nutrients but is greatly involved in the metabolic reprogramming that is long standing and possibly irreversible making the breast cancer cells of the triple negative type in T2D patients proliferate rapidly, indicating more metastasis and rendering it even more dangerous.

Metabolic imaging with hyperpolarized 13C-labeled cell substrates is a promising technique for imaging tissue metabolism in vivo. However, the transient nature of the hyperpolarization - and its depletion following excitation - limits the imaging time and the number of excitation pulses that can be used. A single-shot 3D imaging sequence has been developed and it is shown in this thesis to generate 13C MR images in tumour-bearing mice injected with hyperpolarized [1-13C]pyruvate. The pulse sequence acquires a stack-of-spirals at two spin echoes after a single excitation pulse and encodes the kz-dimension in an interleaved manner to enhance robustness to B0 inhomogeneity. Spectral-spatial pulses are used to acquire dynamic 3D images from selected hyperpolarized 13C-labeled metabolites. A nominal spatial/temporal resolution of 1.25 x 1.25 x 2.5 $mm^3$ x 2 s was achieved in tumour images of hyperpolarized [1-13C]pyruvate and [1-13C]lactate acquired in vivo. An advanced sequence is also described in this thesis in a later study to acquire higher resolution images with isotropic voxels (1.25 x 1.25 x 1.25 $mm^3$) at no cost of temporal resolution. EPI is a sequence widely used in hyperpolarized 13C MRI because images can be acquired rapidly with limited RF exposure. However, EPI suffers from Nyquist ghosting, which is normally corrected for by acquiring a reference scan. In this thesis a workflow for hyperpolarized 13C EPI is proposed that requires no reference scan and, therefore, that does not sacrifice a time point in the dynamic monitoring of tissue metabolism. To date, most of the hyperpolarized MRI on metabolism are based on 13C imaging, while 1H is a better imaging target for its 4 times higher gyromagnetic ratio and hence 16 times signal. In this thesis the world’s first dynamic 1H imaging in vivo of hyperpolarized [1-13C]lactate is presented, via a novel double-dual-spin-echo INEPT sequence that transfers the hyperpolarization from 13C to 1H, achieving a spatial resolution of 1.25 x 1.25 $mm^2$.

Cancer cells exhibit an abnormal metabolic phenotype characterized by glycolysis and lactate fermentation in the presence of oxygen, a phenomenon known as the Warburg effect. This dysregulated metabolism plays an important role in every aspect of cancer progression, from tumorigenesis to invasion and metastasis. The Warburg effect is a common phenotype shared by most, if not all, cancer types. It is especially prominent in metastatic tumors, which are notoriously resistant to treatment and responsible for the majority of cancer-related deaths. Thus, metabolic therapies which target the Warburg effect could offer novel therapeutic options for most cancer patients, including those with aggressive or late-stage cancers. The ketogenic diet is a high fat, low carbohydrate diet that induces a physiological state of nutritional ketosis - decreased blood glucose and elevated blood ketones. It has been investigated as a cancer therapy for its potential to exploit the Warburg effect by restricting glucose availability to glycolysis-dependent tumors, and has been reported to slow cancer progression in some animal models as well as in anecdotal reports and small clinical studies in humans. Interestingly, there is some evidence that the elevation in blood ketones induced by the ketogenic diet contributes to its anti-cancer effects, suggesting that ketone supplementation could possibly inhibit cancer progression on its own. Rapid growth outstrips a tumor's ability to adequately perfuse its tissue, creating regions of tumor hypoxia which exacerbate the Warburg effect and promote a malignant phenotype. Hyperbaric oxygen therapy is the administration of 100% oxygen at elevated barometric pressure. It supersaturates the blood with oxygen, increasing its diffusion distance into the tissues, and can therefore be used to increase intratumoral pO2 and reverse tumor hypoxia. Here we present evidence that the ketogenic diet, ketone supplementation, and hyperbaric oxygen therapy work individually and in combination to slow progression and extend survival in the VM-M3 model of metastatic cancer. This study strongly suggests that these cost effective, non-toxic metabolic therapies should be further evaluated in animal and human studies to determine their potential clinical use.

No description available.

The purpose of this thesis was to characterize cancer metabolism in vitro using epithelial ovarian cancer as a model on an untargeted, systems-level, basis with particular attention paid to the difference between cancer stem cell metabolism and cancer cell metabolism. Two-dimensional gas chromatography coupled to mass spectrometry was used to measure the metabolite profiles of the ovarian cancer and cancer stem cell lines under normal baseline conditions and also under chemotherapeutic and environmental perturbations. These two cell lines exhibited significant metabolic differences under normal baseline conditions and results demonstrated that metabolism in the ovarian cancer stem cell line was distinct from that of more differentiated isogenic cancer cells, showing similarities to stem cell metabolism that suggest the potential importance of metabolism for the cancer stem cell phenotype. Glucose deprivation, hypoxia, and ischemia all perturbed ovarian cancer and cancer stem cell metabolism, but not in the same ways between the cell types. Chemotherapeutic treatment with docetaxel caused metabolic changes mostly in amino acid and carbohydrate metabolism in ovarian cancer cells, while ovarian cancer stem cell metabolism was not affected by docetaxel. Overall, these metabolic differences between the two cell types will deepen our understanding of the metabolic changes occurring within the in vivo tumor and will help drive development of cancer stem cell targeted therapeutics.

Metabolic reprogramming in cancer cells provides energy and important metabolites required to sustain tumour proliferation. Hypoxia represents a hostile environment that can encourage these transformations and other adaptive responses that contribute to poor prognosis and resistance to radiation and chemotherapy. Hypoxic signatures associated with worse prognosis were previously derived in different cancer types, and led to the selection of two candidates with potential metabolic implications, namely the mir210-putuative target iron-sulfur scaffold protein ISCU and glycogen phosphorylase (PYGL). Firstly, it was verified that the hypoxia-induced miR-210 targets ISCU. Iron-sulfur clusters represent cofactors for key enzymes involved in Krebs cycle and electron transport chain. Downregulation of ISCU was associated with the induction of reactive oxygen species (ROS) and reduced mitochondrial complex I and aconitase activity, caused a shift to glycolysis in normoxia and enhanced cell survival. This indicates that the induction of a single microRNA, miR-210, can mediate a new mechanism of adaptation to hypoxia, by regulating mitochondrial function via iron-sulfur cluster metabolism and free radical generation. Secondly, it was found that changes in PYGL expression reflect a characteristic upregulation of glycogen metabolism in hypoxia in both tumour xenografts and in cancer cell lines. More specifically, hypoxia stimulates glycogen accumulation and its utilisation, as well as the concurrent upregulation of several glycogen-metabolizing enzymes such as glycogen synthase (GYS1) and PYGL. PYGL depletion led to glycogen accumulation in hypoxic cells, increased intracellular levels of ROS, and a reduction in proliferation due to a p53-dependent induction of senescence. Furthermore, depletion of PYGL was associated with markedly impaired tumorigenesis in vivo. Finally, metabolic analyses indicated that glycogen degradation by PYGL is important for the optimal functioning of the pentose phosphate pathway. Collectively, this study shows the contribution of two important pathways to the metabolic adaptations induced by hypoxia.

<p>Proper balancing of the activities of metabolic pathways to meet the challenge of providing necessary products for biosynthetic and energy demands of the cell is a key requirement for maintaining cell viability and allowing for cell proliferation. Cell metabolism has been found to play a crucial role in numerous cell settings, including in the cells of the immune system, where a successful immune response requires rapid proliferation and successful clearance of dangerous pathogens followed by resolution of the immune response. Additionally, it is now well known that cell metabolism is markedly altered from normal cells in the setting of cancer, where tumor cells rapidly and persistently proliferate. In both settings, alterations to the metabolic profile of the cells play important roles in promoting cell proliferation and survival.</p><p>It has long been known that many types of tumor cells and actively proliferating immune cells adopt a metabolic phenotype of aerobic glycolysis, whereby the cell, even under normoxic conditions, imports large amounts of glucose and fluxes it through the glycolytic pathway and produces lactate. However, the metabolic programs utilized by various immune cell subsets have only recently begun to be explored in detail, and the metabolic features and pathways influencing cell metabolism in tumor cells in vivo have not been studied in detail. The work presented here examines the role of metabolism in regulating the function of an important subset of the immune system, the regulatory T cell (Treg) and the role and regulation of metabolism in the context of malignant T cell acute lymphoblastic leukemia (T-ALL). We show that Treg cells, in order to properly function to suppress auto-inflammatory disease, adopt a metabolic program that is characterized by oxidative metabolism and active suppression of anabolic signaling and metabolic pathways. We found that the transcription factor FoxP3, which is highly expressed in Treg cells, drives this phenotype. Perturbing the metabolic phenotype of Treg cells by enforcing increased glycolysis or driving proliferation and anabolic signaling through inflammatory signaling pathways results in a reduction in suppressive function of Tregs. </p><p>In our studies focused on the metabolism of T-ALL, we observed that while T-ALL cells use and require aerobic glycolysis, the glycolytic metabolism of T-ALL is restrained compared to that of an antigen activated T cell. The metabolism of T-ALL is instead balanced, with mitochondrial metabolism also being increased. We observed that the pro-anabolic growth mTORC1 signaling pathway was limited in primary T-ALL cells as a result of AMPK pathway activity. AMPK pathway signaling was elevated as a result of oncogene induced metabolic stress. AMPK played a key role in the regulation of T-ALL cell metabolism, as genetic deletion of AMPK in an in vivo murine model of T-ALL resulted in increased glycolysis and anabolic metabolism, yet paradoxically increased cell death and increased mouse survival time. AMPK acts to promote mitochondrial oxidative metabolism in T-ALL through the regulation of Complex I activity, and loss of AMPK reduced mitochondrial oxidative metabolism and resulted in increased metabolic stress. Confirming a role for mitochondrial metabolism in T-ALL, we observed that the direct pharmacological inhibition of Complex I also resulted in a rapid loss of T-ALL cell viability in vitro and in vivo. Taken together, this work establishes an important role for AMPK to both balance the metabolic pathways utilized by T-ALL to allow for cell proliferation and to also promote tumor cell viability by controlling metabolic stress.</p><p>Overall, this work demonstrates the importance of the proper coupling of metabolic pathway activity with the function needs of particular types of immune cells. We show that Treg cells, which mainly act to keep immune responses well regulated, adopt a metabolic program where glycolytic metabolism is actively repressed, while oxidative metabolism is promoted. In the setting of malignant T-ALL cells, metabolic activity is surprisingly balanced, with both glycolysis and mitochondrial oxidative metabolism being utilized. In both cases, altering the metabolic balance towards glycolytic metabolism results in negative outcomes for the cell, with decreased Treg functionality and increased metabolic stress in T-ALL. In both cases, this work has generated a new understanding of how metabolism couples to immune cell function, and may allow for selective targeting of immune cell subsets by the specific targeting of metabolic pathways.</p> / Dissertation

Thesis advisor: Thomas Seyfried / Lipids, in all their forms from structural components of the membranes (phosphoglycerides, glycolglycerolipids) to signaling molecules (IP3, DAG, prostaglandins, etc.,) post-translational modification of proteins (palmitoylated, farnesylated, prenylated, and GPI anchoring) play an essential role in cancer cell survival, proliferation, and metastasis. Alteration in structural lipids can impair transport, and signaling cascades. Abnormalities in lipids, such as cardiolipin (Ptd2Gro), impair mitochondrial function, bioenergetics, and could play a role in precipitatting the high incidence of spontaneous tumors in VM/Dk mice. This thesis explores the role of glucose and glutamine in their incorporation into lipids in the VM-M3 murine glioblastoma cell line as well as the inheritance of brain cardiolipin fatty acids abnormalities in VM/Dk mice. I used labeled [14C]-U-D-glucose and [14C]-U-L-glutamine to examine the profile of de novo lipid biosynthesis in the VM-M3 cell line. The major lipids synthesized included phosphatidylcholine (PtdCho), phosphatidylethanolamine (EtnGpl), phosphatidylinositol (PtdIns), phosphatidylserine (PtdSer), sphingomyelin (CerPCho), bis(monoacylglycero)phosphate (BMP) / phosphatidic acid (PtdOH), cholesterol (C), Ptd2Gro, and the gangliosides. The data show that the incorporation of labeled glucose and glutamine into synthesized lipids was dependent on the type of growth environment, and that the VM-M3 glioblastoma cells could acquire lipids, especially cholesterol, from the external environment for growth and proliferation. In addition, this thesis also explores and evaluates the abnormality of Ptd2Gro fatty acid composition in VM mice in comparison to B6 mice. Although previously reported, I confirmed the finding in the abnormal cardiolipin fatty acid composition in the VM mice. The abnormal brain cardiolipin fatty acid composition was found to be inherited as an autosomal dominant trait in reciprocal B6 x VM F1 hybrids for both male and female. Impaired cognitive awareness under hypoxia observed for the VM mice and reciprocal F1 hybrids is associated with abnormalities in neural lipid composition. / Thesis (PhD) — Boston College, 2014. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Toxoplasma gondii is an obligate intracellular parasite, that is able to infect any nucleated cell. An important global pathogen, T. gondii can cycle between primary and secondary hosts, thus enabling widespread penetrance. Within its intracellular niche – a membrane-bound parasitophorous vacuole – T. gondii is nevertheless able to subvert a variety of host cell processes to allow its continued survival and replication. This includes modulation of host signalling processes as well as the scavenging of nutrient macromolecules. In recent years, microRNAs have emerged as important regulators of cellular processes including inflammation, tumorigenesis and metabolism, as well as development. It has become increasingly clear that this species of non-coding RNA is of great importance in ‘fine tuning’ many cellular responses. I hypothesise in this work that host cell miRNAs may be yet another means by which T. gondii manipulates its host upon infection. Using high-throughput-sequencing, I examine host cell transcriptional responses to infection both at the mRNA and microRNA level, using two strains of T. gondii at a variety of Multiplicities of Infection over a time course of 43 hours. Through these transcriptional analyses I identify a number of dysregulated pathways common in tumorigenesis, and contemplate the hypothesis that T. gondii may be behaving as an ‘intracellular tumour’, subverting host cell metabolic processes to mimic a long-known feature of cancer metabolism – that of aerobic glycolysis (the Warburg effect) – in order to satisfy its own energetic and metabolic needs.