Caloric restriction (CR) extends lifespan from yeast to mammals. In budding yeast, inhibition of the conserved TOR and/or PKA pathways has been shown to mediate lifespan extension by CR partly through the activation of stress response. However, how the stress response is regulated at the systems level is poorly understood. In this study, by using fluorescent reporters whose expression is dependent on the transcription factors Msn2/4 and Gis1, two separate screenings were conducted to reveal novel regulators of the stress response induced by starvation. A 'focused' screening on the 272 'signalling' mutants revealed that, apart from the previously identified Rim15, Yak1 and Mck1 kinases, the SNF1/AMPK complex, the cell wall integrity (CWI) pathway and a number of cell cycle regulators are necessary to elicit appropriate stress response. The chronological lifespan (CLS) of these signalling mutants correlates well with the amount of accumulated storage carbohydrates but poorly with transition-phase cell cycle status. Subsequent analyses reveal that the levels of intracellular reactive oxygen species are controlled by Rim15, Yak1 and Mck1. Furthermore, CLS extension enabled by tor1 deletion is dependent on the above three kinases. These data suggest that the signalling pathways (SNF1 and CWI) and the kinases downstream of TOR/PKA (Rim15, Yak1 and Mck1) coordinate the metabolic reprogramming (to accumulate storage carbohydrates) and the activation of anti-oxidant defence systems (to control ROS levels) to extend chronological lifespan. A 'genome-wide' screening of a haploid deletion library indicates that less than 10% of the non-essential genes are implicated in the regulation of starvation-induced stress response. Gene ontology analysis suggests that they can be grouped into major clusters including mitochondrial function, r-RNA processing, DNA damage and repair, transcription from RNA polymerase and cell cycle regulation. Further phenotypic assays confirm the previous observation that CLS extension is mostly correlated with the accumulation of storage carbohydrates. Compromised expression of stress response reporters is confirmed by FACS in a variety of mitochondrial mutants, suggesting that mitochondrial respiration also plays a key role in the activation of stress response. Put together, the above findings indicate that stress response and metabolic reprogramming induced by glucose starvation are coordinated by multiple signalling pathways and the activation of mitochondrial respiration is essential to both cellular processes and to CLS extension.

Hexavalent Chromium (Cr(VI) induces malignant cell transformation in normal bronchial epithelial (BEAS-2B) cells. Cr(VI)-transformed cells exhibit increased level of antioxidants, are resistant to apoptosis, and are tumorigenic. RNAseq analysis in Cr(VI)-transformed cells showed that expression of transcripts associated with mitochondrial oxidative phosphorylation is reduced, and the expression of transcripts associated with pentose phosphate pathway, glycolysis, and glutaminolysis are increased. Sirtuin-3 (SIRT3) regulates mitochondrial adaptive response to stress, such as metabolic reprogramming and antioxidant defense mechanisms. SIRT3 was upregulated and it positively regulated mitochondrial oxidative phosphorylation in Cr(VI)-transformed cells. Our results suggests that SIRT3 plays an important role in mitophagy deficiency of Cr(VI)-transformed cells. Furthermore, SIRT3 knockdown suppressed cell proliferation and tumorigenesis of Cr(VI)-transformed cells. Nrf2 is a transcription factor that regulates oxidative stress response. This study investigated the role of Nrf2 in regulating metabolic reprogramming in Cr(VI)-transformed cells. We observed that in Cr(VI)-transformed cells p-AMPKthr172 was increased, when compared to normal BEAS-2B cells. Additionally, Nrf2 knockdown reduced p-AMPKthr172. Our results suggest that Nrf2 regulated glycolytic shift via AMPK regulation of PFK1/PFK2 pathway. Furthermore, our results showed that Nrf2 constitutive activation in Cr(VI-transformed cells increased cell proliferation and tumorigenesis. Overall this dissertation demonstrated that Cr(VI)-transformed cells undergo metabolic reprogramming. We demonstrated that Nrf2 constitutive activation plays decisive role on metabolic reprogramming induction, and SIRT3 activation contributing to increased cancer cell proliferation and tumorigenesis.

Skeletal muscle can perceive cellular energy status and substrate availability and demonstrates remarkable plasticity in response to environmental changes. Nonetheless, how skeletal muscle and its resident stem cells (satellite cells; SCs) sense and respond to nutrient flux remains largely undefined. The dynamic post-translational modification O-GlcNAcylation has been shown to serve as a cellular nutrient sensor in a wide range of cells and tissues, yet its role in skeletal muscle and SCs remains unexplored. Here, we ablated skeletal muscle O-GlcNAc transferase (OGT), and thus O-GlcNAcylation, and found the knockout mice exhibited enhanced glucose uptake, insulin sensitivity, and resistance to high-fat diet induced obesity. Additionally, mKO mice had a 3-fold increase in circulating levels of interleukin-15 (IL-15), a potent anti-obesity cytokine, potentially through epigenetic regulation of Il15 by OGT. To further investigate if there was a causal relationship between OGT ablation and the lean phenotype, we generated muscle specific OGT and interleukin-15 receptor alpha (IL-15ra) double knockout mice (mDKO). As a result, mDKO mice had blunted IL-15 secretion and minimal protection against HFD-induced obesity. Together, these data indicate the skeletal muscle OGT-IL15 axis plays an essential role in the maintenance of skeletal muscle and whole-body metabolic homeostasis. As satellite cells (SCs) play an indispensable role in postnatal muscle growth and adult regenerative myogenesis, we investigated the role of O-GlcNAcylation in SC function. To this end, we conditionally ablated OGT in SCs (cKO) and found cKO mice had impaired SC proliferation, in vivo cycling properties, population stability, metabolic regulation, and adult regenerative myogenesis. Together these findings show that SCs require O-GlcNAcylation, presumably to gauge nutritional signals, for proper function and metabolic homeostasis. Another critical yet often neglected player in myogenesis are mitochondria. Traditionally depicted as a power plant in cells, mitochondria are critical for numerous nonconventional, energy-independent cellular process. To investigate the role of both mitochondrial energy production and alternative mitochondrial functions in myogenic regulation, we ablated ATP synthase subunit beta (ATP5b) and ubiquinol-cytochrome c reductase (UQCRFS1) in C2C12 myoblasts to disrupt mitochondrial ATP production and mitochondrial membrane potential, respectively. Ablation of UQCRFS1, but not ATP5b, impaired myoblast proliferation, although lack of either gene compromised myoblast fusion. Interestingly, addition of the potent myogenic stimulator IGF-1 rescued ATP5b fusion but could not override UQCRFS1 knockout effects on proliferation or differentiation. These data demonstrate mitochondrial ATP production is not the "metabolic switch" that governs myogenic progression but rather an alternative mitochondrial function. In summary, skeletal muscle and their resident stem cell population (SCs) both use O-GlcNAcylation, feasibly to sense and respond to nutritional cues, for the maintenance of metabolic homeostasis and normal physiology. A deeper understand of both muscle and SC metabolic regulation may provide therapeutic targets to improve global metabolism and muscle growth. / Doctor of Philosophy / Skeletal muscle is responsible for approximately 20% of basal energy expenditure and 70-90% of insulin-mediated glucose disposal, and as such changes in skeletal muscle metabolism and insulin sensitivity have profound impacts on whole body metabolism. Skeletal muscle is a plastic tissue that can perceive nutrient availability, which permits metabolic adaptations to environmental changes. Deletion of the nutrient sensing pathway O-GlcNAcylation in skeletal muscle (mKO) protected mice from high-fat diet induced obesity and ameliorates whole-body insulin sensitivity. Skeletal muscle can secrete myokines to elicit endocrine effects on other tissues in the body, and as such, we proposed perturbation of this nutrient sensing pathway in skeletal muscle alters myokine secretion to elicit responses in other metabolically active tissues to support its energy requirements. Indeed, circulating levels of interleukin-15, a potent anti-obesity myokine, increased 3-fold in mKO mice. To determine the contribution of IL-15 to the mKO phenotype, we used a genetic approach to blunt IL-15 secretion from skeletal muscle (mDKO), which partially negated the lean mKO phenotype. Our findings show the ability of skeletal muscle to "sense" changes in nutrients through O-GlcNAcylation is necessary for proper muscle and whole-body metabolism. Moreover, this nutrient sensing mechanism is also important for proper muscle stem cell function, also known as satellite cells (SCs). Loss of O-GlcNAcylation in SCs impairs their ability to regenerate muscle after injury, which can be attributed to a reduced capacity to proliferate and an inability to maintain a healthy SC population. Interestingly, SCs lacking O-GlcNAcylation have a greater mitochondrial content. Using a myoblast cell line, we investigated the contribution of mitochondria to myogenesis, the formation of muscle, and found mitochondrial energy production is dispensable in the myogenic process. Our studies show skeletal muscle and SCs rely on highly integrated signaling cascades that sense and respond to intrinsic metabolic changes and extrinsic nutritional cues to function properly.

Estudos recentes relataram oncogenes induzindo a reprogramação metabólica no câncer. Essa reprogramação é fundamental para que as células cancerosas tenham nutrientes e biomoléculas suficiente para manter sua alta taxa proliferativa. A mitocôndria tem um papel central no metabolismo energético da célula e alterações no seu genoma, tanto em relação a mutações como em número de cópias, já foram bastante observados em vários tipos tumorais. Além disso, deficiência no fator de transcrição mitocondrial A (TFAM), fundamental para a transcrição e estabilidade do mtDNA, já foi associada com o crescimento tumoral. Diante disso, nosso estudo teve como objetivo avaliar o papel da instabilidade do genoma mitocondrial no metabolismo energético e crescimento do melanoma. Para isso, nós medimos a instabilidade do mtDNA utilizando como parâmetros: o acúmulo de mutações no mtDNA, alterações no mtDNAcn e a expressão do TFAM. O impacto da instabilidade do mtDNA foi avaliado em três modelos diferentes de melanoma: um modelo in vitro de linhagens celulares, dados de expressão gênica de tumores de melanoma metastático proveniente do TCGA e um modelo murino induzível de melanoma (BrafV600E/Ptennull), adicionado a um background alternativo de deficiência para o TFAM/mtDNAcn. Esse modelo murino também nos permitiu avaliar a deficiência do TFAM limitada a células tumorais (Tfamflox) e tanto em células tumorais, como no seu microambiente (Tfam+/-). Nas análises in vitro, nós observamos correlações positivas entre o mtDNAcn e a expressão do TFAM com a taxa de consumo de glicose e produção de ATP, indicando um impacto desses parâmetros na bioenergética celular. Análises de expressão gênica, utilizando tanto as linhagens de melanoma como tumores de melanoma metastático, nos sugeriram que o TFAM regula genes indutores de angiogênese, a resposta imunológica humoral e vias metabólicas de aminoácidos. Nas análises in vivo, nós observamos um aumento dos tumores em camundongos Tfam+/-, indicando que a deficiência de TFAM/mtDNAcn em células tumorais e no seu microambiente induz a tumorigênese, o que confirma os dados de expressão gênica encontrados com linhagens e tecido de melanoma. Além disso, análises de metabolômica e transcriptômica combinadas nos sugeriram que as células de melanoma com deficiência no TFAM/mtDNAcn são mais dependentes do metabolismo de glutamina. Diante disso, nós concluímos que a deficiência do TFAM/mtDNAcn tem um papel importante no crescimento do melanoma, induzindo a expressão de genes pro-tumorigênicos e aumentando o consumo da glutamina para suprir as necessidades proliferativas das células cancerosas. Esses dados são relevantes e podem nos ajudar a entender melhor o papel da mitocondrial na progressão do melanoma. / Recent studies have shown many oncogenes triggering metabolic reprogramming in cancer. The metabolic switch in cancer cells is necessary to supply the high demand for nutrients and biomolecules for proliferative cells. In this context, mitochondria play a central role in the energetic metabolism of the cell and changes in its genome, such as an increased load of mutations and alterations in mtDNA content, have been reported in several cancers. In addition, deficiency in the Mitochondrial Transcription Factor A (TFAM), responsible for transcription and maintenance of mtDNA stability, was previously associated with tumor growth. Based on that, our goal was to evaluate the impact of the mitochondrial genome instability in the energetic metabolism and melanoma growth. mtDNA instability was inferred measuring mtDNA mutations load and content, as well as TFAM expression. Its impact was evaluated in three different melanoma models: an in vitro model using melanoma cell lines, gene expression data from metastatic melanoma tumors, publicly available at TCGA, and an inducible murine model of melanoma (BRAFV600E/PTENnull), crossed onto different TFAMdeficient backgrounds. The murine model also provides us a tractable model to examine the consequences of mtDNA instability limited to cancer cells (Tfamflox) and in both cancer cells and tumor microenvironment (Tfam+/-). In vitro analysis showed us a positive correlation between mtDNA copy number (mtDNAcn) and TFAM expression with glucose consumption and ATP production, pointing an impact of these parameters in cellular bioenergetics. Further gene expression analysis, using both cell lines and metastatic melanoma data, suggested that TFAM could regulate the expression of angiogenesis genes, humoral immunity and amino acid metabolism. In vivo analysis confirmed the gene expression data, and revealed a higher melanoma growth in Tfam+/-. Also, combined metabolomics and transcriptomics data suggested that TFAM/mtDNAcn deficient melanoma cells rely mostly on glutamine metabolism to supply their energetic requirements. In conclusion, these data indicate that TFAM/mtDNAcn influences melanoma growth by triggering pro-tumorigenic signals and inducing metabolic reprogramming towards glutamine metabolism. These results are relevant and might help us understand how mitochondria affect melanoma progression.

Estudos recentes relataram oncogenes induzindo a reprogramação metabólica no câncer. Essa reprogramação é fundamental para que as células cancerosas tenham nutrientes e biomoléculas suficiente para manter sua alta taxa proliferativa. A mitocôndria tem um papel central no metabolismo energético da célula e alterações no seu genoma, tanto em relação a mutações como em número de cópias, já foram bastante observados em vários tipos tumorais. Além disso, deficiência no fator de transcrição mitocondrial A (TFAM), fundamental para a transcrição e estabilidade do mtDNA, já foi associada com o crescimento tumoral. Diante disso, nosso estudo teve como objetivo avaliar o papel da instabilidade do genoma mitocondrial no metabolismo energético e crescimento do melanoma. Para isso, nós medimos a instabilidade do mtDNA utilizando como parâmetros: o acúmulo de mutações no mtDNA, alterações no mtDNAcn e a expressão do TFAM. O impacto da instabilidade do mtDNA foi avaliado em três modelos diferentes de melanoma: um modelo in vitro de linhagens celulares, dados de expressão gênica de tumores de melanoma metastático proveniente do TCGA e um modelo murino induzível de melanoma (BrafV600E/Ptennull), adicionado a um background alternativo de deficiência para o TFAM/mtDNAcn. Esse modelo murino também nos permitiu avaliar a deficiência do TFAM limitada a células tumorais (Tfamflox) e tanto em células tumorais, como no seu microambiente (Tfam+/-). Nas análises in vitro, nós observamos correlações positivas entre o mtDNAcn e a expressão do TFAM com a taxa de consumo de glicose e produção de ATP, indicando um impacto desses parâmetros na bioenergética celular. Análises de expressão gênica, utilizando tanto as linhagens de melanoma como tumores de melanoma metastático, nos sugeriram que o TFAM regula genes indutores de angiogênese, a resposta imunológica humoral e vias metabólicas de aminoácidos. Nas análises in vivo, nós observamos um aumento dos tumores em camundongos Tfam+/-, indicando que a deficiência de TFAM/mtDNAcn em células tumorais e no seu microambiente induz a tumorigênese, o que confirma os dados de expressão gênica encontrados com linhagens e tecido de melanoma. Além disso, análises de metabolômica e transcriptômica combinadas nos sugeriram que as células de melanoma com deficiência no TFAM/mtDNAcn são mais dependentes do metabolismo de glutamina. Diante disso, nós concluímos que a deficiência do TFAM/mtDNAcn tem um papel importante no crescimento do melanoma, induzindo a expressão de genes pro-tumorigênicos e aumentando o consumo da glutamina para suprir as necessidades proliferativas das células cancerosas. Esses dados são relevantes e podem nos ajudar a entender melhor o papel da mitocondrial na progressão do melanoma. / Recent studies have shown many oncogenes triggering metabolic reprogramming in cancer. The metabolic switch in cancer cells is necessary to supply the high demand for nutrients and biomolecules for proliferative cells. In this context, mitochondria play a central role in the energetic metabolism of the cell and changes in its genome, such as an increased load of mutations and alterations in mtDNA content, have been reported in several cancers. In addition, deficiency in the Mitochondrial Transcription Factor A (TFAM), responsible for transcription and maintenance of mtDNA stability, was previously associated with tumor growth. Based on that, our goal was to evaluate the impact of the mitochondrial genome instability in the energetic metabolism and melanoma growth. mtDNA instability was inferred measuring mtDNA mutations load and content, as well as TFAM expression. Its impact was evaluated in three different melanoma models: an in vitro model using melanoma cell lines, gene expression data from metastatic melanoma tumors, publicly available at TCGA, and an inducible murine model of melanoma (BRAFV600E/PTENnull), crossed onto different TFAMdeficient backgrounds. The murine model also provides us a tractable model to examine the consequences of mtDNA instability limited to cancer cells (Tfamflox) and in both cancer cells and tumor microenvironment (Tfam+/-). In vitro analysis showed us a positive correlation between mtDNA copy number (mtDNAcn) and TFAM expression with glucose consumption and ATP production, pointing an impact of these parameters in cellular bioenergetics. Further gene expression analysis, using both cell lines and metastatic melanoma data, suggested that TFAM could regulate the expression of angiogenesis genes, humoral immunity and amino acid metabolism. In vivo analysis confirmed the gene expression data, and revealed a higher melanoma growth in Tfam+/-. Also, combined metabolomics and transcriptomics data suggested that TFAM/mtDNAcn deficient melanoma cells rely mostly on glutamine metabolism to supply their energetic requirements. In conclusion, these data indicate that TFAM/mtDNAcn influences melanoma growth by triggering pro-tumorigenic signals and inducing metabolic reprogramming towards glutamine metabolism. These results are relevant and might help us understand how mitochondria affect melanoma progression.

Apolipoprotéines L est une famille nouvellement caractérisée en humain sans une fonction patho- physiologique définitive. Ces protéines sont classiquement considérées être impliquées dans le transport et métabolisme des lipides, principalement due à l'association de son premier membre de la famille sécrétée l’apolipoprotéine L1 aux particules des lipoprotéines de haute densité. Néanmoins, le reste des membres sont des protéines intracellulaires (absence de domaine de peptide signal). Apolipoprotéine L1 a été initialement identifiée comme l'élément clé du facteur trypanolytique dans le sérum humain. L'exploration de la séquence des différents apolipoprotéines L a révélé un domaine distinct «B cell lymphoma-2 homology domain 3» ayant des similitudes structurelles et fonctionnelles avec le domaine B cell lymphoma-2 homology domain 3 des protéines de la famille B cell lymphoma-2. Ainsi la découverte de ce domaine peut contribuer à la compréhension de la fonction et rôle des apoLs dans différents mécanismes et processus tels que la mort cellulaire programmée, la prolifération cellulaire, le métabolisme cellulaire .Notre étude visait à caractériser les fonctions de patho- physiologique du premier membre de la famille «apolipoprotéine L1 ». L’expression de l’apolipoprotéine L1 ARNm, à partir de 48 carcinomes papillaires de la thyroïde, a été évaluée par des études à haut débit et normalisée à un pool de tissus normal de la thyroïde. Une confirmation de PCR en temps réel valide ainsi la surexpression d’apoL1 dans 91,67 % des cas testés. Le niveau élevé de l’apolipoprotéine L1 ARNm est en corrélation avec une expression protéique élevée dans les échantillons histologiques (70%), et détermine que les cellules folliculaires de la thyroïde dans la zone de la tumeur sont les cellules principales responsables de l’expression spécifique de l’apolipoprotéine L1. Nous avons étudié l'expression apolipoprotéine L1 dans le modèle de cancer pour approfondir notre compréhension des relations reliant cette expression distincte dans le cancer papillaire de la thyroïde et son rôle et fonction concernant le métabolisme du cancer (de reprogrammation métabolique :effet Warburg).7En outre, la localisation de l’apolipoprotéine L1 dans la mitochondrie des cellules cancéreuses de la thyroïde ainsi que dans la mitochondrie de levure, a été le point de départ de la recherche dans ce nouveau modèle, il nous a permis de révéler et d'introduire de nouvelles hypothèses pour expliquer l'effet inhibiteur de l’apolipoprotéine L1 en fonction des conditions métabolique variantes et l’effet pléotropiques de l’apolipoprotéine L1 sur la levure (dommages des mitochondries et vacuoles). Dans ce manuscrit, nous avons décrit nos efforts à mettre en évidence la spécificité d'expression de l’apolipoprotéine L1 dans le cancer papillaire thyroïdien notamment au niveau de la transcription ainsi que la localisation mitochondriale et l'interférence probable avec les voies métaboliques. / Option Biologie moléculaire du Doctorat en Sciences / info:eu-repo/semantics/nonPublished

<p>Breast cancer is the predominant cancer diagnosed among women, and the second most deadly cancer. The vast majority of cancer-related deaths is caused by the metastatic spread of cancer from the primary tumor to a distant site in the body. Therefore, new strategies which minimize breast cancer metastasis are imperative to improve patient survival. Cancer cells which acquire anchorage independence, or the ability to survive without extracellular matrix attachment, and metabolic flexibility have increased potential to metastasize. In the present studies, the ability to survive detachment and subsequent metabolic changes were determined in human Harvey-<i>ras</i> transformed MCF10A-<i>ras</i> breast cancer cells. Detachment resulted in reduced viability in a time-dependent manner with the lowest cell viability observed at forty hours. In addition, decreased cell viability was observed in both glutamine and glucose depleted detached conditions, suggesting a dependence on both nutrients for detached survival. Compared to attached cells, detached cells had reduced total pool sizes of pyruvate, lactate, α-ketoglutarate, fumarate, malate, alanine, serine, and glutamate, suggesting the metabolic stress which occurs under detached conditions. However, intracellular citrate and aspartate pools were unchanged, demonstrating a preference to maintain these pools in detached conditions. Compared to attached cells, detached cells had suppressed glutamine metabolism, as determined by decreased glutamine flux into the TCA cycle and reduced mRNA abundance of glutamine metabolizing enzymes. Further, detached glucose anaplerosis through pyruvate dehydrogenase activity was decreased, while pyruvate carboxylase (PC) expression and activity were increased. A switch in metabolism was observed away from glutamine anaplerosis to a preferential utilization of PC activity to replenish the TCA cycle, determined by reduced PC mRNA abundance in detached cells treated with a cell-permeable analog of α-ketoglutarate, the downstream metabolite of glutamine which enters the TCA cycle. These results suggest that detached cells elevate PC to increase flux of carbons into the TCA cycle when glutamine metabolism is reduced. </p> <p>Vitamin D is recognized for its role in preventing breast cancer progression, and recent studies suggest that regulation of energy metabolism may contribute to its anticancer effects. Vitamin D primarily acts on target tissue through its most active metabolite, 1α,25-dihydroxyvitamin D (1,25(OH)₂D). The present work investigated 1,25(OH)₂D’s effects on viability of detached cells through regulation of energy metabolism. Treatment of MCF10A-<i>ras</i> cells with 1,25(OH)₂D resulted in decreased viability of detached cells. While 1,25(OH)₂D treatment did not affect many of the glucose metabolism outcomes measured, including intracellular pyruvate and lactate pool sizes, glucose flux to pyruvate and lactate, and mRNA abundance of enzymes involved in glucose metabolism, 1,25(OH)₂D treatment reduced detached PC expression and glucose flux through PC. A reduction in glutamine metabolism was observed with 1,25(OH)₂D treatment, although no 1,25(OH)₂D target genes were identified. Further, PC depletion by shRNA decreased cell viability in detached conditions with no additional effect with 1,25(OH)₂D treatment. Moreover, PC overexpression resulted in increased detached cell viability and inhibited 1,25(OH)₂D’s negative effects on viability. These results suggest that 1,25(OH)₂D reduces detached cell viability through regulation of PC. Collectively this work identifies a key metabolic adaptation where detached cells increase PC expression and activity to compensate for reduced glutamine metabolism and that 1,25(OH)₂D may be utilized to reverse this effect and decrease detached cell viability. These results contribute to an increased understanding of metastatic processes and the regulation of these processes by vitamin D, which may be effective in preventing metastasis and improve breast cancer patient survival.</p>

No description available.

Metabolic reprogramming has been recognised as a hallmark in solid tumours. Malignant modification of the tumour’s bioenergetics provides energy for tumour growth and progression. Otto Warburg first reported these metabolic and biochemical changes in 1927. In prostate cancer (PCa) epithelial cells, the tumour metabolism also changes during development and progress. These alterations are partly driven by the androgen receptor, the key regulator in PCa development, progress, and survival. In contrast to other epithelial cells of different entities, glycolytic metabolism in prostate cells sustains physiological citrate secretion in the normal prostatic epithelium. In the early stages of PCa, citrate is utilised to power oxidative phosphorylation and fuel lipogenesis, enabling tumour growth and progression. In advanced and incurable castration-resistant PCa, a metabolic shift towards choline, amino acid, and glycolytic metabolism fueling tumour growth and progression has been described. Therefore, even if the metabolic changes are not fully understood, the altered metabolism during tumour progression may provide opportunities for novel therapeutic strategies, especially in advanced PCa stages. This review focuses on the main differences in PCa’s metabolism during tumourigenesis and progression highlighting glutamine’s role in PCa.

O Adenocarcinoma ductal pancreático (ADP) é o tumor pancreático mais comum e apresenta um dos piores prognósticos. A primeira alteração crítica que desencadeia o processo de progressão tumoral, é a ativação desregulada do gene KRAS, na qual está presente em 90% dos casos. Várias iniciativas terapêuticas buscaram como alvo direto a atividade da oncoproteína RAS, sem no entanto, obter resultados satisfatórios. Desta forma, a investigação de moléculas efetoras downstream às vias reguladas por RAS, poderiam resultar em estratégias mais eficazes. Dentre estas moléculas efetoras estão as MAPKs, que modulam diversos processos celulares essenciais para o desenvolvimento tumoral, onde a cascata RAS/RAF/MEK/ERK representa uma importante via canônica de transdução de sinais. A transdução de sinais desta via pode ser favorecida por proteínas conhecidas como proteínas de arcabouço, como SHOC2, funcionando como uma plataforma para ligação de RAS-RAF-1 e consequentemente potencializando sua ligação. KRAS, têm sido associado à regulação de vias metabólicas importantes, como a glicólise que interferem diretamente na capacidade de proliferação e sobrevivência celular, para o estabelecimento e manutenção da biologia tumoral. Assim, o objetivo deste trabalho foi investigar o papel da proteína SHOC2 na indução do estresse oxidativo e capacidade de sobrevivência de linhagens celulares de ADP. Foram realizados os ensaios de morte celular por apoptose, avaliação da capacidade clonogênica e quantificação dos níveis de glutationa e quantificação da produção de espécies reativas de oxigênio. As linhagens celulares MIA PaCa2 e PANC-1 apresentaram uma redução significativa da capacidade de formação de colônias. A taxa de apoptose induzida pelo tratamento com Gemcitabina não diferiu entre as linhagens modificadas para silenciar a função de SHOC2. No ensaio da quantificação dos níveis de glutationa e na produção de espécies reativas de oxigênio, os resultados não foram concordantes com o esperado. Para análise dos níveis proteicos de p-ERK1/2, podemos observar uma redução na sua expressão, mesmo se mostrando de maneira sutil. Os resultados sugerem que pode haver alguma relação entre o silenciamento de SHOC2, estresse oxidativo e sobrevivência, porém existem outras vias alternativas modulando este processo. / Pancreatic ductal adenocarcinoma (PDAC) is the most common pancreatic tumor and has one of the worst prognoses. The first critical change that triggers the process of tumor progression is the dysregulated activation of the KRAS gene, in which it is present in 90% of cases. Several therapeutic initiatives aimed directly at the activity of the RAS oncoprotein, without, however, obtaining satisfactory results. Thus, investigating downstream effector molecules on RAS-regulated pathways could result in more effective strategies. Among these effector molecules are MAPKs, which modulate several cellular processes essential for tumor development, where the RAS / RAF / MEK / ERK cascade represents an important canonical pathway for signal transduction. Signal transduction of this pathway may be favored by proteins known as scaffold proteins, such as SHOC2, serving as a platform for RAS-RAF-1 binding and hence potentiating its interaction. KRAS, have been associated with the regulation of important metabolic pathways, such as glycolysis, for the establishment and maintenance of tumor biology. Thus, the objective of this work was to investigate the role of SHOC2 in the induction of oxidative stress and survival capacity of ADP cell lines. Cell death assays were performed by apoptosis, and quantification of glutathione levels and the production of reactive oxygen species were performed. MIA PaCa2 and PANC-1 cell lines showed a reduction in colony formation capacity. Gemcitabine-mediated cell death by apoptosis has not been induced after SHOC2 knockdown. Also, the measurement of reactive oxygen species and quantification of glutathione levels did not reveal any change mediated by SHOC2. The analysis of ERK1 / 2 activation has shown a discrete reduction in its expression. The results suggest that there may be some relationship between SHOC2 silencing, oxidative stress and survival, but there are other alternative pathways modulating this process which needs claryfication.