Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis. The aggressiveness and therapeutic recalcitrance of this malignancy has been attributed to multiple factors including the influence of an active desmoplastic stroma. How the stromal microenvironment of PDAC contributes to the fatal nature of this disease is not well defined. In the analysis of clinical specimens, we observed diverse expression of the hypoxic marker carbonic anhydrase IX and the lactate transporter MCT4 in the stromal compartment. These stromal features were associated with the epithelial to mesenchymal phenotype in PDAC tumor cells, and with shorter patient survival. Cultured cancer- associated fibroblasts (CAFs) derived from primary PDAC exhibited a high basal level of hypoxia inducible factor 1a (HIF1 alpha) that was both required and sufficient to modulate the expression of MCT4. This event was associated with increased transcription and protein synthesis of HIF1a in CAFs relative to PDAC cell lines, while surprisingly the protein turnover rate was equivalent. CAFs utilized glucose predominantly for glycolytic intermediates, whereas glutamine was the preferred metabolite for the TCA cycle. Unlike PDAC cell lines, CAFs were resistant to glucose withdrawal but sensitive to glutamine depletion. Consistent with the lack of reliance on glucose, CAFs could survive the acute depletion of MCT4. In co-culture and xenograft studies CAFs stimulated the invasive potential and metastatic spread of PDAC cell lines through a mechanism dependent on HIF1a and MCT4. Together, these data indicate that stromal metabolic features influence PDAC tumor cells to promote invasiveness and metastatic potential and associate with poor outcome in patients with PDAC.

OVCA is the most lethal gynecological cancer, due primarily to late diagnosis and chemoresistance (Canada, 2014; Society, 2014b). CDDP resistance is a major hurdle to successful therapy (MayoClinic, 2014). The mechanism of chemoresistance is multi-factorial including defects in apoptotic pathway and key tumor suppressor as well as dysregulation of metabolism (Borst et al., 2000; Galluzzi et al., 2012a; Siddik, 2003). Elevated aerobic glycolysis is a major source for fulfilling high energy demand of cancer, but the role of metabolic reprogramming and its regulatory mechanism in OVCA cells remain unknown. p53 is a key tumor suppressor involved in apoptosis and frequent defect of p53 (> 80%) exist in epithelial OVCA. HKII is a key metabolic enzyme involved in the first step of glycolysis and its frequent presence in the mitochondria (80% >) has been reported in multiple cancers. We demonstrate here that CDDP-induced, p53-mediated HKII down-regulation and mitochondrial p53-HKII interaction are determinants of chemosensitivity in OVCA. CDDP decreased HKII (mRNA abundance, protein level), altered its cellular localization and glycolysis in p53-wt chemosensitive OVCA cells, a response loss or attenuated in p53 deficient cells. HKII depletion sensitized chemoresistant cells to CDDP -induced apoptosis in a p53- dependent manner. In addition, p53 binds to HKII and facilitates its nuclear localization. Mechanistically, our data suggest that CDDP-activated p53 (phosphorylated p53; P-p53 Ser15) interacts with HKII in the nucleus for its regulation. Upon entry to the nucleus, P-p53(Ser15) transcriptionally regulates HKII by promoter binding, contributing to the regulation of HKII and aerobic glycolysis, eliciting apoptosis in chemosensitive OVCA cells. Conversely, this response is compromised in p53 defect chemoresistant cells. Using proximity ligation assay (PLA) in human OVCA cell lines and primary tumor cells and tumor sections from OVCA patients, we have demonstrated that nuclear HKII-P-p53(Ser15) intracellular trafficking is associated with chemosensitivity in vitro and in vivo. Furthermore, the nuclear HKII-P-p53(Ser15) interaction may be useful as a biomarker for chemosensitivity in multiple epithelial subtypes of OVCA.

Tumor metabolism is a highly dysregulated process that is identified as a unique target for therapy. Current philosophy proposes that tumor metabolism is a plastic and flexible process which sustains proliferative and survival advantages. Tumors employ an anaerobic glycolytic pathway resulting in the overproduction of lactate. Additional thinking suggests that the conversion of pyruvate to lactate regenerates the NAD+ pool in the cell, maintaining a sustainable oxidative environment. Regardless of the reasons for lactate overproduction, its excretion and build up in the microenvironment results in acidic tumor microenvironments. Tumor acidosis has been measured with several different methods, but consistently averages from pH 6.6 to 7.0. Tumor acidity can thus be measured as a biomarker for tumor metabolism. This work examines the commonly explored energy pathways available to the cancer cell and a non-invasive MRI method to measure the efficacy of the tumor metabolism targeting agent. Appendix A is an introduction to tumor metabolism pathways and the large list of candidate therapies in interfering with energy production. Glucose, fatty acid, and glutamine metabolisms are all discussed along with PI3K/AKT/mTOR and HIF growth signals and ion transport. Magnetic resonance imaging and positron emission tomography are examined as imaging methods for non-invasively interrogating tumor acidosis. Appendix B presents the findings in a study where tumor metabolism was targeted with an mTOR inhibitor, where tumor growth rate was initially decreased and accompanied by an early, acute increase in tumor extracellular pH with acidoCEST MRI. Chapter 2 discusses the combination of a lactate dehydrogenase inhibitor in conjunction with doxorubicin in a breast cancer model. Tumor extracellular pH was shown to increase when measured with acidoCEST MRI, and an increase in cell death was measured. Chapter 4 discusses the studies and experimental designs that can be done in the near future.

O câncer de mama ocupa o primeiro lugar em mortalidade dentre todos os tipos de câncer. O subtipo triplo-negativo (triple-negative breast cancer - TNBC) representa 15-20% de todos os tipos de câncer de mama com alta prevalência em mulheres pré-menopausa e destaca-se pelo seu grande tamanho tumoral e agressividade no estabelecimento de metástases, com impacto direto na redução da sobrevida dos pacientes. Apesar das evidências sobre os efeitos anti-tumorigênicos do exercício físico, tanto na prevenção como durante a carcinogênese, é comum que pacientes alterem sua rotina após o diagnóstico de câncer, frequentemente reduzindo as atividades físicas durante e após o tratamento. Em adição, os mecanismos pelos quais o exercício físico exerce papel anti-tumoral são pouco compreendidos. O objetivo deste estudo foi avaliar os efeitos do exercício físico aeróbico moderado em modelo experimental de câncer de mama de tipo triplo-negativo, com ênfase na modulação do metabolismo energético tumoral. Foram utilizados camundongos fêmeas BALB/c em desenho experimental de 12 semanas, cuja inoculação de 1x104 células 4T1 foi realizada após 8 semanas de treinamento. Após protocolo de exercício aeróbico moderado em esteira, foram realizadas análises do metabolismo mitocondrial tumoral, composição lipídica e expressão de genes relacionados à bioenergética e proliferação celular. Os resultados mostraram que o exercício aeróbico moderado reduziu 54,5% do volume e 42% da massa tumoral de animais que foram treinados antes e após a inoculação tumoral. Animais treinados apresentaram fosforilação oxidativa mais próxima ao seu limite máximo respiratório e menor respiração mitocondrial no tecido tumoral quando comparados ao grupo sedentário. O treinamento ocasionou redução no conteúdo de ácido fosfatídico e fosfatidilcolina. Enquanto a análise de expressão relativa de mRNA demonstrou aumento na expressão de genes relacionados à via metabólica glicolítica, como Hif1a, Glut-1, HKII, Ldha e Pdk, além dos supressores tumorais p53 e Lats2. Nossos resultados sugerem que a redução na velocidade de crescimento tumoral proporcionada pelo exercício físico aeróbico de carga moderada seja devida, pelo menos em parte, à modulação do metabolismo energético tumoral. Em conjunto, os dados do nosso estudo abrem novas perspectivas para a identificação de vias metabólicas sensíveis ao exercício físico, permitindo o melhor o entendimento de seus efeitos antitumorigênicos / Breast cancer ranks first in mortality among all types of cancer. The triple-negative breast cancer (TNBC) accounts for 15-20% of all types of breast cancer with a high prevalence in premenopausal women and is notable for its large tumor size and aggressiveness in the establishment of metastasis, with a direct impact on the reduction of patients\' survival. Altough evidence highlight the anti-tumorigenic effects of physical exercise both on the prevention as well as during carcinogenesis, patients commonly change their routine after cancer diagnostic, usually reducing physical activity during and after treatment. Moreover, the mechanisms underlying the anti-tumor role of physical exercise remain poorly understood. The objective of this study was to evaluate the effects of moderate aerobic physical exercise in an experimental model of triple-negative breast cancer, with emphasis on the modulation of tumor energy metabolism. Female BALB / c mice were used in a 12-week experimental design, whose inoculation of 1x104 4T1 cells was performed after 8 weeks of training. After the protocol of moderate aerobic exercise was carried out on the treadmill, analyzes of mitochondrial tumor metabolism, lipid content and qPCR of genes related to bioenergetics and tumorigenic process were performed. The results showed that moderate aerobic exercise reduced 54.5% of the volume and 42% the tumor mass of animals trained before and after tumor inoculation. Trained animals showed oxidative phosphorylation closest to the maximum respiratory limit and lower mitochondrial respiration in tumor tissue when compared to the sedentary group. The training resulted in a reduction in the content of phosphatidic acid and phosphatidylcholine. In the trained group, relative mRNA quantification analysis showed increased expression of genes related to the glycolytic metabolic pathway, such as Hif1a, Glut-1, HKII, Ldha, and Pdk, as well as of the tumor suppressors p53 and Lats2. Our results suggest that the reduction in tumor growth velocity provided by moderate-intensity aerobic physical exercise is due, at least in part, to the modulation of tumor energy metabolism. Together, data from our study open new perspectives for the identification of metabolic pathways sensitive to exercise, allowing better understanding of its anti-tumorigenic effects

<p>Pathophysiologic lactate accumulation is characteristic of solid tumors and has been associated with metastases and poor overall survival in cancer patients. In recent years, there has been a resurgence of interest in tumor lactate metabolism. In the past, our group has shown that lactate can be used as a fuel in some cancer cell lines; however, survival responses to exogenous lactate alone are not well-described. We hypothesized that lactate utilization and cellular responses to exogenous lactate were varied and dynamic, dependent upon factors such as lactate concentration, duration of lactate exposure, and of expression of the lactate transporter, monocarboxylate transporter 1 (MCT1). We hypothesized that pharmacological inhibition of MCT1 with a small molecule, competitive MCT1 inhibitor, &#945;-cyano-4-hydroxycinnamic acid (CHC), could elicit cancer cell death in high lactate conditions typical of that seen in breast cancer. </p><p>My work focused on defining: 1. Lactate levels in locally advanced breast cancer (LABC); 2. Lactate uptake and catabolism in a variety of cancer cell lines; 3. The effect of exogenous lactate on cancer cell survival; 4. Whether the lactate-transporters, MCT1 and MCT4 can be used as markers of cycling hypoxia. </p><p>Lactate levels in LABC biopsies were assessed ex vivo by bioluminescence. NMR techniques were employed extensively to determine metabolites generated from 13C-labeled lactate. Cell viability in response to extracellular lactate ( ± glucose and ± CHC) was measured with Annexin V / 7-AAD staining to assess acute survival responses and clonogenic assays to evaluate long-term colony forming ability after lactate treatment. MCT1 and MCT4 protein expression was evaluated in cancer cell lines with Western blots after exposure to chronic or cycling hypoxia. Immunofluorescence was employed to assess MCT1 and MCT4 expression in head and neck cancer biopsies, and the expression patterns of the transporters were correlated to areas of hypoxia, as indicated by hypoxia marker EF5. </p><p>Lactate concentrations in LABC biopsied ranged from 0 - 12.3 µmol/g of tissue. The LABC dataset was too small to derive statistical power to test if lactate accumulation in LABC biopsies was associated with poor patient outcome or other clinical parameters of known prognostic significance. All cell lines tested (normal and cancer) showed uptake and metabolism of labeled lactate, with dominant generation of alanine and glutamate; however, relative rates and the diversity of metabolites generated was different among cell lines. MCF7 cells showed greater overall lactate uptake (mean = 18mM) over five days than MDA-MB-231 cells (mean = 5.5mM). CHC treatment effectively prevented lactate uptake in cancer cells when lactate concentrations were &#8804;20mM. </p><p>Cell survival was dependent upon lactate concentration and glucose availability. Acute responses to exogenous lactate did not reflect the long-term consequences of lactate exposure. Acutely, HMEC and R3230Ac cells were tolerant of all lactate concentrations tested (0-40mM) regardless of presence or absence of glucose. MCF7 and MDA-MB-231 cells were tolerant of lactate within the concentration ranges seen in biopsies. Cytotoxicity was seen after 24 hr incubation with 40mM lactate (-glucose), but this concentration is three times higher than any measurement made in human biopsies of LABC. Similarly, HMEC and MCF7 cells showed significantly decreased colony formation in response to 40mM exogenous lactate (+ glucose) while R3230Ac and MDA-MB-231 cells showed no impairment in colony-forming abilities with any lactate concentration (+ glucose). 5mM CHC significantly increased cell death responses independent of lactate treatment, indicating off-target effects at high concentrations. </p><p>MCT1 was found to be expressed in a majority of the cell lines tested, except for MDA-MB-231 cells. Cancer cells exposed to exogenous lactate showed upregulation of MCT1 but not MCT4. Chronic hypoxia resulted in an increase in protein expression of MCT4 but a decrease in MCT1 expression in cancer cell lines. The time course of regulation of protein levels of each transporter suggested the possibility of expression of both transporters during cycling hypoxia. When cancer cells were exposed to cycling hypoxia, both transporters showed upregulation. In head and neck tumor biopsies, MCT1 expression was significantly positively correlated to aerobic tumor regions and inversely correlated to hypoxic tumor regions. </p><p>Cancer cell responses to exogenous lactate were not uniform. Some cell lines demonstrated a lactate-tolerant and/or a lactate-consuming phenotype while other cell lines demonstrated lactate-intolerant and/or non-lactate-consuming phenotype. My work indicates that exogenous lactate was well-tolerated at clinically relevant concentrations , especially in the presence of glucose. Evidence of glutamate metabolism from lactate indicated that exogenous lactate partially progresses through the TCA cycle, suggesting that lactate may be utilized for fuel. The cell death elicited from 5mM CHC treatment was not dependent upon presence of lactate, indicating that manipulation of lactate metabolism may not be the best option for targeting cancer metabolism. When attempting to manipulate lactate metabolism in tumors, microenvironmental factors, such as hypoxia and glucose, must be taken into account in order to ensure a predictable and favorable outcome. Together, these results illustrate the importance of characterizing tumor metabolism before therapeutic intervention.</p> / Dissertation

Malgré les avancées récentes, le cancer reste la première cause de mortalité en France avec ~ 150000 décès recensés par an (INCa 2012). Notamment, le cancer broncho-pulmonaire est l’un des plus agressifs, avec 29100 décès en 2011. La survie à cinq ans de seulement 10% des patients atteints des cancers du poumon non à petites cellules (NSCLC, 80% des cancers bronchiques), pose un problème d’ordre scientifique, médical et de santé publique. Il est communément admis qu’une sous-population de la tumeur acquiert en raison d’une instabilité génétique de nouvelles propriétés agressives nécessaires à sa dissémination, sa prolifération et une plus grande résistance aux chimiothérapies. Une meilleure compréhension de ces propriétés tumorales constitue un enjeu majeur pour prévenir à terme cette progression maligne. Nous avons axé notre recherche sur la macro-autophagie, un processus catabolique lysosomal essentiel à l’homéostasie cellulaire et sur un de ses substrats SQSTM1 (séquestosome ou p62). Au moment où j’ai initié ma thèse, une relation étroite entre l’autophagie, SQSTM1 et la progression tumorale venait d’être mise en lumière. Plusieurs membres de la machinerie autophagique sont fréquemment mutés/déletés dans les cancers. De façon paradoxale, l’autophagie peut également permettre la survie des cellules tumorales face à l’hypoxie, la carence nutritionnelle ou les chimiothérapies. / Despite recent advances, cancer remains the leading cause of death in France with~150,000 annual deaths (INCA, 2012). Notably, lung cancer is one of the most aggressive, with 29 100 deaths in 2011. The five-year survival is only 10 % of patients with lung cancer non-small cell (NSCLC, 80% of lung cancers), so it’s a medical/scientific challenge, and public health problem. Due to genetic instability, it’s commonly accepted that a subpopulation of tumor acquires new aggressive properties for its dissemination, its proliferation and greater resistance to chemotherapy. A better understanding of these tumor properties is a major issue to prevent this malignant progression. We focused our research on macro-autophagy, a lysosomal catabolic process essential for cellular homeostasis, and alos on one of its substrates SQSTM1 (Sequestosome or p62). When I started my PhD, a close relationship between autophagy, SQSTM1 and tumor progression had been highlighted. Several members of the autophagic machinery are frequently mutated/deleted in cancers. Paradoxically, autophagy can also allow the survival of tumor cells under hypoxia, nutritional deficiency or chemotherapy. How could we understand these conflicting functions, considering that the only autophagy substrates known are long half-lives proteins, damaged organelles and scaffold SQSTM1? Thus, SQSTM1 is essential for RasV12 driven oncogenesis (A Duran et al, Cancer Cell 2008).

Orientador: Sandra Martha Gomes Dias / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-23T00:13:51Z (GMT). No. of bitstreams: 1 Gomes_EmersonRodrigoMachi_M.pdf: 4424058 bytes, checksum: 4547741a163b53b4836c32f103096cd3 (MD5) Previous issue date: 2013 / Resumo: Células tumorais apresentam uma autonomia metabólica aumentada em comparação a células não-transformadas, incorporando nutrientes e metabolizando-os através de vias que suportam o seu crescimento e proliferação. O foco deste trabalho foi a enzima glutaminase, a qual processa glutamina em glutamato para posterior produção de alfa-cetoglutarato pela enzima glutamato desidrogenase, reabastecendo o ciclo do TCA e suportando seu funcionamento e geração de metabólitos essenciais para a síntese de macromoléculas. O gene GLS1 codifica para as isoformas glutaminase kidney-type (KGA) e glutaminase C (GAC). Estas proteínas apresentam outros domínios além do catalítico, e, no caso da KGA, repetições do tipo ankirin, sabidamente envolvidas em contatos proteínas-proteínas. Os objetivos deste projeto foram de encontrar parceiros de interação para a glutaminase kidney-type (KGA) e avaliar o impacto desta interação para o metabolismo tumoral. Um candidato inicialmente avaliado, a Aldolase A, não foi confirmado como parceiro de interação. Outro candidato, a BNIP-H, apesar de ter sido mostrado interagir com a KGA em células nervosas, não mostrou indícios de interação com a KGA em linhagem de células de câncer de mama. Por fim, estudos de duplo-híbrido em levedura revelaram o receptor nuclear PPAR? (Peroxisome proliferator-activated receptor gamma) como forte candidato a parceiro de interação. Realizou-se um mapeamento dos domínios responsáveis pela interação entre estas duas proteínas, também por duplo híbrido, tendo sido identificado o domínio LBD da proteína PPAR? como envolvido na interação. Mesmo estudos realizados com fragmento da KGA, apesar de incompletos, mostraram que a interação não ocorre pelo domínio carboxi-terminal da enzima. Ensaios de anisotropia de fluorescência com as proteínas KGA e PPAR? purificadas indicaram que a interação é favorecida pela presença do produto da reação glutaminolítica, glutamato, e apresenta um Kd de 4,6 ± 0,5 ?M. Microscopia confocal de imunofluorescência mostrou que ambas as proteínas se co-localizam no citoplasma, mas não no núcleo. Mais se verificou que em células HEK 293T a presença de KGA diminui a capacidade de transativação de PPAR? induzida pelo ativador roziglitazona, enquanto que celular PC3 com superexpressão de KGA apresentam níveis diminuídos de expressão da proteína ACADL, alvo do PPAR?. Da mesma maneira, ensaios in vitro de atividade da KGA mostraram que a presença de PPAR? inibe a atividade da glutaminase. Nossos resultados mostram a interação in vitro entre as proteínas KGA-PPAR? e a potencial influência funcional que uma proteína exerce sobre a outra. Dado a participação de ambas as proteínas no processo tumoral, especula-se que esta interação possa ter impacto no desenvolvimento do câncer / Abstract: Tumor cells have an increased metabolic autonomy compared to non-transformed cells, metabolizing nutrients and incorporating them through pathways that support cell growth and proliferation. The focus of this study was the glutaminase enzyme, which processes glutamine to glutamate for subsequent production of alpha-ketoglutarate, by the glutamate dehydrogenase enzyme, replenishing TCA cycle and bearing its function and the generation of metabolites essential for the synthesis of macromolecules. The gene GLS1 codes for the isoforms kidney-type glutaminase (KGA) and glutaminase C (GAC). These proteins exhibit other domains besides the catalytic, and in the case of KGA, ankirin repeats, known to be involved in protein-protein contacts. The goal of this project was to investigate potential interacting partners of KGA and contextualize the interaction within the metabolic demands of tumor cells. A candidate initially evaluated, the Aldolase A, was not confirmed as a partner of interaction. Another candidate, the BNIP-H, despite having been shown to interact with the KGA in nervous cells, showed no evidence of interaction with KGA in one tested breast cancer cell lines. Finally, yeast two-hybrid studies revealed the nuclear receptor peroxisome proliferator-activated receptor gamma (PPAR?) as a strong interaction partner candidate. We mapped the domains responsible for the interaction between these two proteins, also by two-hybrid and identified the LBD domain of PPAR? as involved in the interaction. The same studies with KGA fragments, although incomplete, showed that the interaction did not involve the carboxy-terminal domain of the enzyme. KGA and PPAR? proteins were expressed in E. coli, purified and their interaction was analyzed by pull-down, fluorescence anisotropy, electrophoresis under native conditions, gel filtration chromatography and crosslinking. The assays indicated that the interaction is favored by the presence of the reaction product glutamate and has a Kd of 4,6 ± 0,5 ?M and disfavored by phosphate. Immunogold labeling followed by transmission electron microscopy of SKBR3 cells revealed a curious nuclear staining pattern probably heterochromatic of KGA. Immunofluorescence confocal microscopy showed that both proteins co-localize in the cytoplasm but not in the nucleus. Moreover, it was found that in HEK 293T cells, the presence of KGA decreases PPAR? ability of inducing transactivation of a reporter gene, while PC3 cell overexpressing KGA have low levels of protein expression ACADL target this receptor. Likewise, activity in vitro assays in the presence of KGA showed that PPAR? receptor inhibits the glutaminase activity. Our results demonstrate the in vitro interaction between proteins KGA-PPAR? and the potential functional influence that these proteins exerts on each other. Given the involvement of both proteins in the tumor growth, it is speculated that this interaction may have impact on the development of cancer / Mestrado / Clinica Medica / Mestre em Ciências

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Selenium is a micronutrient that is used by the selenocysteine biosynthesis pathway to produce the amino acid selenocysteine, which is required in selenoproteins. Many of the 25 human selenoproteins, such as glutathione peroxidases and thioredoxin reductases, play important roles in maintaining cellular redox homeostasis. In this study we characterize how this metabolic pathway is upregulated in cancer cells and how this increase in activity creates a unique vulnerability. We have outlined the evidence and underlying mechanisms for how many metabolites normally produced in cells are highly toxic, and we describe this concept as illustrated in selenocysteine metabolism. My thesis explores how SEPHS2, an enzyme in the selenocysteine biosynthesis pathway, is essential for survival of cancer, but not normal cells. SEPHS2 is required in cancer cells to detoxify selenide, an intermediate that is formed during selenocysteine biosynthesis. Breast and other cancer cells are selenophilic, owing to a secondary function of the cystine/glutamate antiporter SLC7A11 that promotes selenium uptake and selenocysteine biosynthesis, which, by allowing production of selenoproteins such as GPX4, protects cells against ferroptosis. However, this activity also becomes a liability for cancer cells because selenide is poisonous and must be processed by SEPHS2. These results show that SEPHS2 is a cancer specific target and indicates the therapeutic potential of SEPHS2 inhibition in the treatment of cancer. Collectively, this thesis identifies SEPHS2 as a targetable vulnerability of cancer cells, defines the role of selenium metabolism in cancer, and outlines a roadmap for future studies regarding toxic metabolites and cancer.