Role of monocarboxylate transporters in prostate carcinoma

INSTITUTO DE CIENCIAS BIOMÉDICAS ABEL SALAZAR INSTITUTO DE CIENCIAS DA VIDA E CIENCIAS DA VIDA E SAUDE (ICVS) NELMA ISABEL AROUCA PÉRTEGA GOMES ROLE OF MONOCARBOXYLATE TRANSPORTERS IN PROSTATE CARCINOMA Tese de Candidatura ao grau de Doutor em Ciências (Especialidade em: Patologia e Genética Molecular), submetida ao Instituto de Ciências Biomédicas Abel Salazar da Universidade do Porto. Orientador – Maria de Fátima Monginho Baltazar Categoria – Professora Auxiliar Afiliação – Instituto de Ciências da Vida e Saúde, Braga, Portugal; Escola de Ciências da Saúde, Universidade do Minho, Braga, Portugal. Co-orientador – Carlos Alberto da Silva Lopes Categoria – Professor Catedrático Afiliação – Instituto de Ciências Biomédicas Abel Salazar da Universidade do Porto, Porto, Portugal. A tese de doutoramento aqui apresentada foi desenvolvida no âmbito de financiamento pela Fundação para a Ciência e Tecnologia (FCT) através de uma bolsa individual de doutoramento, com a referência SFRH/BD/61027/2009, e do projecto “Role of Monocarboxylate Transporters (MCTs) in the metabolism of prostate cancer”, com a referência PTDC/SAL-MET/113415/2009, no âmbito do Programa Operacional Temático Factores de Competitividade (COMPETE) do Quadro Comunitário de Apoio III e co-financiado pelo Fundo Comunitário Europeu FEDER. ACKNOWLEDGEMENTS I would like to thank my supervisor Prof. Fátima Baltazar, for giving me the opportunity to enter in this “Scientific World”. Thank you for introducing me to Cancer and Metabolism research field and for always giving me the opportunity/liberty to establish collaborations with other research groups during my PhD. This was extremely valuable during my PhD journey. To Professor Carlos Lopes as co-supervisor of this work and Director of the Pathology and Molecular Genetics PhD Programme for allowing me to join this Course, turning this journey into a real learning experience. A very special thanks to Dr. José Ramon Vizcaino for accepting me at the Pathology Department of Centro Hospitalar do Porto and to accept to be part of our Project. Thank you for your scientific supervision, optimism, patience, ideas and friendship. My thesis would not be the same without our frequent scientific/speculative talks. Thank you so much!!! A special thank to Professor David Neal and Lee Fryer at Cancer Research UK Cambridge Institute/University of Cambridge for allowing me to join their lab and develop my skills and scientific knowledge in such a high-quality research Institute. At a professional and personal level this was a huge experience for me. Thanks for still believing my work. I can just promise that I will always do my best to correspond your expectations. Thank you!! Many thanks to all that in one way or another have collaborated with me, receiving me in most cases at their labs. Professor Rui Henrique, Professor Carmen Jerónimo and Professor Alexandre Lobo da Cunha. I also would like to thank Elsa Oliveira and Carlos Gouveia, thank you all. A special thanks to Dra. Daniela Ribeiro and Dr. Valdemar Máximo, many thanks for believe in my ideas and for helping me to reach my aims!! I also want to thank all my friends inside and outside the lab: At ICVS I would like to thank Carina and Ricardo. Thanks for all the good moments inside and outside the lab, I am sure that we will be friends forever no matter what. Filipe Pinto and Isabel Valença my dear partners in party and work. It is indeed possible to establish good collaborative work and have fun at the same time. I think it is one of the best combinations ever. Thanks for that!! To “HOXAs” group, thank you girls for all the funny moments, lunch times, dinners, coffees and so on. Wish you all the best. My friends and collgues at CRI: Vinny, Mohammad, Charlie, Sarah, Jan, Karan, Helen, Steve, Harveer, Antonio for all their friendship and support and for allowing me to learn new things from your experience and for sharing with me expertise inside and outside the lab. A very special thanks to Sarah Jurmeister who was my every hour support in many different ways. Thank you for always be available to teach me something new and for your support and encouragement to be and to do better. Thank you for all your statistical support and refreshing ideas about how to look at my results. To all my friends out of the lab, especially Silvia, Bia, Liliana, Vânia e Dora, Aldina and Diana for all the good times, for their support, friendship and for accepting me as I am turning every moment that I was down into a good moment. For always encouraging me for the best, thank you my friends. To Julia and Jjonas for being the best housemates ever!!! Finally I would like to show all my gratitude to my family, namely my parents and brother, who always supported me. Thank you for helping me to deal with my frustrations and barriers along this journey. Thank you for raising me and for all the encouragement to be a better person giving me the freedom to do my choices and learn from them. I would like to dedicate this thesis to all of you!! We cannot do science alone!!! DIRETIVAS LEGAIS No cumprimento do disposto no Decreto de Lei nrº 230/2009, declara-se que a autora desta dissertação participou ativamente na execução do trabalho experimental que esteve na origem dos resultados apresentados bem como na redação dos respetivos manuscritos. Fazem parte desta tese os seguintes artigos publicados ou em preparação: Pértega-Gomes N, Vizcaíno JR, Miranda-Gonçalves V, Pinheiro C, Silva J, Pereira H, Monteiro P, Henrique RM, Reis RM, Lopes C, Baltazar F. “Monocarboxylate transporter 4 (MCT4) and CD147 overexpression is associated with poor prognosis in prostate cancer.” BMC Cancer. 2011 Jul 25;11:312. doi: 10.1186/1471-2407-11-312. (Citações ISI: 19) Pértega-Gomes N, Vizcaíno JR, Gouveia C, Jerónimo C, Henrique RM, Lopes C, Baltazar F. “Monocarboxylate transporter 2 (MCT2) as putative biomarker in prostate cancer.” Prostate. 2013 May;73(7):763-9. doi: 10.1002/pros.22620. Epub 2012 Nov 28. Pértega-Gomes N, Valença I, Vizcaíno JR, Ribeiro D, Baltazar F. “Localization of MCT2 at peroxisomes is associated with malignant transformation in prostate cancer” to be published in an international scientific periodical with referees. Pértega-Gomes N, Vizcaíno JR, Sousa S, Coelho R, Attig J, Jurmeister S, Oliveira E, Pereira L, Pinheiro C, Jerónimo C, Henrique RM, Lobo da Cunha A, Lopes C, Maximo V and Baltazar F. “Metabolic heterogeneity in prostate cancer is linked to disease progression and aggressiveness” to be published in an international scientific periodical with referees. Pértega-Gomes N, Vizcaíno JR,Gouveia C, Lopes C and Baltazar F. “A lactate shuttle system between tumour and stromal cells is associated with poor prognosis in prostate cancer” to be published in an international scientific periodical with referees. Pértega-Gomes N and Baltazar F. “Explotation of MCTs as potential therapeutic targets in prostate cancer” invited work to be published in Advances in Prostate Cancer Research and Treatment journal. TABLE OF CONTENTS Abbreviation List…………………………………………………………….…i Abstract …………………………………………………………………………iii Resumo………………………………………………………………………....v Aims and Thesis Layout……………………………………………………...vii Chapter 1. General Introduction………………………………………......3 1.1 Reprogramming energy metabolism as an emerging hallmark of cancer………………………………………………………………………3 1.1.1 The Warburg effect as the basis to explored altered cellular Metabolism in cancer…………………………………………………………4 1.1.2 A Metabolic switch that confers advantage for cancer cells…...6 1.1.2.1 Why cancer cells prefer aerobic glycoysis…………………...6 1.1.2.2 A deeper insight into hypoxia and Lactate export: Great contributors to malignancy………………………………………9 1.2 Monocarboxylate Transporters (MCTs) in the context of cellular metabolism…………………………………………………………….......13 1.2.1 The MCT family…………………………………………………….....14 1.2.1.1 MCT1……………………………………………………………......16 1.2.1.2 MCT2……………………………………………………………......17 1.2.1.3 MCT4………………………………………………………….…….18 1.2.1.4 Other MCT isoforms…………………………………………..….18 1.2.2 MCTs regulation………………………………………………………20 1.2.2.1 Transcriptional Level……………………………………………..20 1.2.2.2 Post-Transcriptional Level………………………………………22 1.2.2.3 Transporter Activity Level………………………….…………....23 1.2.2.4 Hormonal Regulation………………………………………….....25 1.2.3 MCTs inhibition………………………………………………….……25 1.2.3.1 The Classical MCT Inhibitor: CHC………………………..........26 1.2.3.2 Specific MCT Inhibitors…………………………………………..27 1.2.3.3 Other MCT inhibitors……………………………………………..27 iv represent promising therapeutic targets in different phases of neoplastic transformation and progression. v RESUMO O conceito de alterações metabólicas sofridas pelas células tumorais foi recentemente incluído nas principais alterações sofridas no cotexto de transformação maligna. Uma das principais características metabólicas das células tumorais consiste no seu elevado consumo de glucose e consequente produção de lactato. De modo a assegurar o rápido efluxo de lactato, a maioria das células malignas expressam elevados níveis de transportadores de monocarboxilatos (MCTs). A inibição destes transportadores tem sido apresentada como um bom alvo para quimioterapia e vários estudos in vitro demonstraram já o potencial desta abordagem, no entanto, no contexto do carcinoma da próstata o impacto da inibição MCTs assim como a sua regulação durante a progressão tumoral continua em grande parte desconhecida. Adicionalmente, ainda não está claro qual é a via metabólica predominante no cancro da próstata e, portanto, qual a que se apresenta como alvo mais apropriado para a terapia. O principal objetivo deste trabalho foi investigar a expressão e regulação dos MCTs durante a progressão do carcinoma da próstata e se estes transportadores poderiam representar alvos promissores no tratamento desta doença. A expressão de MCTs juntamente com outras proteínas relacionadas com o metabolismo foi estudada usando uma grande série de tecidos de próstata humana e o seu valor clínico-patológico foi avaliado. Estudos in vitro foram realizados a fim de compreender como variam as exigências metabólicas das células durante a progressão tumoral e de que forma estas mudanças poderiam representar alvos terapêuticos para a doença, dando especial ênfase aos MCTs neste contexto. Os resultados obtidos indicam que o carcinoma da próstata não é tão glicolítico como outros tipos de tumor e que o aumento da glicólise é principalmente encontrado num estadio mais avançado da doença. Foi observado que diferentes isoformas de MCTs estão diferencialmente expressas em diversas fases de progressão do carcinoma da próstata e potencialmente relacionados com diferentes vias metabólicas e portanto não restritas ao seu clássico papel de transportadores de lactato na membrana citoplasmática e que a inibição das diferentes isoformas em diferentes condições afeta a viabilidade e proliferação vi das células malignas. Em conclusão, os resultados apresentados nesta tese são pioneiros na compreensão das exigências metabólicas do carcinoma da próstata durante a progressão tumoral, contextualizando os MCTs como possíveis alvos terapêuticos em diferentes fases da transformação neoplásica e progressão maligna. vii AIMS AND THESIS LAYOUT Metabolic changes during malignant transformation have been noted for many years. Otto Warburg first reported that cancer cells preferentially rely on glycolysis for energy production, even in the presence of oxygen, leading to production of high levels of lactate. The crucial role of lactate efflux and exchange within the tumour microenvironment drew attention to monocarboxylate transporters (MCTs). MCTs have been pointed as promising targets in cancer therapy and described in a large variety of tumours, however, studies showing how these isoforms contribute to the acquisition of the malignant phenotype are scarce and regarding prostate cancer it is totally unknown. The main aim of this thesis was to characterize MCTs expression in prostate tissues, namely prostate cancer, as well as to give a contribution to understand the role of MCTs in prostate cancer maintenance and progression providing evidence for the exploitation of MCTs as potential targets for prostate cancer therapy. This thesis is organized in 7 related chapters, aiming to show the most important results obtained during the development of this scientific project. In Chapter 1 a general introduction to the Hallmarks of cancer with a special emphasis to altered cellular metabolism as a new hallmark of cancer and the “Warburg effect” as the basis to explore MCTs as suitable targets for cancer therapy is provided. Also, the current knowledge on prostate cancer metabolism is reviewed in this chapter. In Chapters 2-6 major findings about metabolic changes across prostate cancer malignant transformation as well as the value of using MCTs as targets for prostate cancer therapy is presented. These chapters include results already published or submitted for publication in international scientific periodicals with referees, giving a contribution to understand the role of MCTs in the context of prostate cancer. In Chapter 2 the expression of MCTs in prostate tissues and the clinico-pathological value of this expression is presented. Chapter 3 explores more deeply the role of MCT2 in prostate cancer metabolism. In Chapter 4 a panel of key metabolic-related proteins expression was studied in an even bigger casuistic of prostate tissues. In vitro assays were also performed showing vii important metabolic alterations across prostate cancer malignant transformation and how MCTs inhibition affects prostate cancer cells in different tumour environment condition. In Chapter 5 an effort to understand the metabolic alterations in the stroma that surrounds prostate tumours is presented. Finally, Chapter 6 presents an insight on MCTs regulation across prostate cancer progression and in Chapter 7, the main conclusions of Chapters 2-6 are summarized and some important future directions are suggested. Each of these chapters has their own bibliography section. Role of MCTs in Prostate Carcinoma Pértega-Gomes N. CHAPTER 1. GENERAL INTRODUCTION Role of MCTs in Prostate Carcinoma Pértega-Gomes N. Role of MCTs in Prostate Carcinoma Pértega-Gomes N. 3 CHAPTER 1. GENERAL INTRODUCTION 1.1 Reprogramming of energy metabolism as an emerging Hallmark of Cancer Advances in cancer research have generated a rich but also extremely complex knowledge revealing cancer as a disease that involves several dynamic changes. In order to organize the complexity of cancer, the biological capabilities acquired during the multistep development of human tumours and shared by all cancers were grouped and denominated as the “hallmarks of cancer”. Sustaining proliferative signaling, evading growth suppressors, resisting cell death, enabling replicative immortality, inducing angiogenesis, and activating invasion and metastasis were the traits established in order to create an organized principle that provides a logical framework to understand the diversity of neoplastic disease [1]. Lately, new hallmarks have emerged and reprogramming of energy metabolism was considered an emerging hallmark of cancer since it was recognized that the chronic and uncontrolled cell proliferation that represents the essence of neoplastic disease involves also adjustments of energy metabolism in order to fuel cell growth and division [2]. Figure 1 represents the last version of the “Hallmarks of cancer”. Figure 1. The Hallmarks of Cancer [2]. Role of MCTs in Prostate Carcinoma Pértega-Gomes N. 4 1.1.1 The Warburg effect as the basis to explore altered cellular metabolism in cancer Altered energy metabolism is being proved to be as widespread in cancer cells as many of the other cancer-associated traits that have been accepted as hallmarks of cancer. However just recently a major relevance has been given to cancer metabolism, the observation that tumour cells exhibit an altered metabolism when compared to normal cells was made almost one century ago by the Nobel Prize winner Otto Warburg who described it as the first tumour specific-alteration [3]. Warburg described that in contrast to non-malignant cells, many types of cancer cells prefer glycolysis as the ATP source, even in the presence of abundant oxygen. If under aerobic conditions, normal cells process glucose, first to pyruvate via glycolysis in the cytosol and then to carbon dioxide in the mitochondria, under anaerobic conditions, glycolysis is favored and few pyruvate is send to the oxygen-consuming mitochondria. Warburg first observed that cancer cells can reprogram their glucose metabolism, and thus their energy production, by limiting their energy metabolism largely to glycolysis, leading to a state that has been called ‘‘aerobic glycolysis’’ (Figure 2). Figure 2. Schematic representation of the differences between OXPHOS, anaerobic glycolysis and “aerobic glycolysis” (Warburg effect) [4]. Role of MCTs in Prostate Carcinoma Pértega-Gomes N. 5 Glycolysis, the first major pathway of cellular metabolism, occurs in the cytoplasm and is functional even in the absence of oxygen. While glycolysis is able to produce ATP at high rates, it is considered a low efficiency pathway because it produces only two ATP molecules per glucose. In order to compensate for the lower efficiency of ATP production obtained by glycolysis relative to mitochondrial oxidative phosphorylation, cancer cells markedly increased uptake and utilization of glucose in many human tumor types being the rate of entry higher than in normal cells [5-7]. This has proven useful to detect tumours and also monitor their treatment, as it is the basis for the clinical use of positron emission tomography (PET) that uses a radiolabeled analog of glucose (18F-fluorodeoxyglucose, FDG) as a reporter [8]. FDG is recognized as a substrate for glucose transport systems; thus, the rate of entry of this glucose derivate into cells is determined by the activity of glucose transport systems. Inside the cells, the accumulation of the radioactive glucose analogue serves as a read-out for the rate of glucose entry into cells. Since tumour cells exhibit enhanced glucose uptake compared to adjacent normal cells, PET is able to detect tumours and differentiate them from normal tissue [9, 10] (Figure 3). Figure 3. PET imaging with FDG of a patient with lymphoma. The mediastinal nodes (purple arrow) and supravincular nodes (green arrows) show high uptake of FDG, showing that tumours in these nodes have high levels of FDG uptake. The bladder (yellow arrow) also has high activity, because of excretion of the radionuclide [11]. Role of MCTs in Prostate Carcinoma Pértega-Gomes N. 12 accumulation of HIF1-alpha, which would lead in turn to a stimulation of the glycolytic pathway by regulating glycolytic enzymes, providing an important positive feedback in the context of cancer. Moreover, lactate was demonstrated to stimulate molecules involved in the process of cancer invasion and metastasis. CD44, VEGF and transforming growth factor (TGFB2) are some of them [35-39]. Besides glycolysis, it is important to recognize that there are other pathways that can also contribute to lactate production in solid tumours such as glutaminolysis and serinolysis (Figure 7) [40,41]. Figure 7. Overview of the metabolic pathways leading to lactate production in cells (continuous lines). The discontinuous arrows indicate lactate uptake and flow inside oxidative cancer cells. Besides being mainly an end-product, lactate exported might also be a substrate for neighbor cells, a phenomenon known as “cell-cell lactate shuttle”, in which the peripheral and oxygenated oxidative cells consume the lactate produced by the central and hypoxic glycolytic cells (Figure 8). Role of MCTs in Prostate Carcinoma Pértega-Gomes N. 13 Figure 8. Scheme representing intratumoural hypoxia and metabolic symbiosis. The crucial role of lactate efflux and exchange within the tumour microenvironment, drew attention to monocarboxylate transporters (MCTs) which transport monocarboxylates such as lactate across the membranes, and therefore play a central role in cellular metabolism and metabolic communication between tissues. 1.2 Monocarboxylate Transporters (MCTs) in the context of cellular metabolism. The transport of monocarboxylates across the plasma membrane was originally thought to be via non-ionic diffusion of the free acid, however, the demonstration that lactate and pyruvate transport into human erythrocytes could be strongly inhibited after treatment with chemicals, allowed the identification of a specific monocarboxylate transport mechanism. The monocarboxylate transport was then characterized extensively in different cell types and the observed characteristics led to the rationale for of the existence of a family of monocarboxylate transporters [42-43]. Role of MCTs in Prostate Carcinoma Pértega-Gomes N. 14 1.2.1 The MCT Family MCTs are encoded by the SLC16 gene family, which is conserved among species, including rat, mouse, chicken and others. The family is presently composed by 14 members, identified through screening of genomic and expressed sequence tag databases [43]. These proteins catalyse the transport of lactate with a proton, with no energy input involved in this process [42, 44]. It is predicted that the topology of MCTs consists of 12 transmembrane domains (TMDs) with the Nand Ctermini located in the cytoplasm, as illustrated in Figure 9 for MCT1. The TMDs are highly conserved among isoforms with the greatest sequence variations observed in the C-terminus and the large intracellular loop between TMDs 6 and 7, which has a range of 29-105 amino acid residues [45] . This observed variability is common to transporters with 12 TMDs and it is thought that these sequence variations are related to substrate specificity or regulation of transport activity [44, 45]. Theoretical predictions and experimental evidence indicate that none of the MCT family members is glycosylated [46-49]. To function, an MCT translocates a proton and a monocarboxylate through the plasma membrane by an ordered mechanism in which H+ binding is followed by monocarboxylate binding to the protonated transporter [49, 50]. Therefore, MCT activity is dependent on both besides substrate concentration and proton gradient between the extracellular and intracellular milieus. Figure 9. Proposed topology of MCT1. The model shown is that predicted from the primary sequence using hydropathy plots and subsequently confirmed by proteolytic cleavage and labelling experiments as described by Juel & Halestrap [44]. Role of MCTs in Prostate Carcinoma Pértega-Gomes N. 15 Lactate is indeed the monocarboxylate whose transport across the plasma membrane is quantitatively more important, however, MCTs are also important for the transport of many other metabolically important monocarboxylates such as pyruvate, the branched-chain oxoacids derived from leucine, valine and isoleucine, and the ketone bodies acetoacetate, β-hydroxybutyrate and acetate [45]. Consequently, MCTs have a central role in mammalian cell metabolism and are critical for the communication between cells as illustrated in Figure 10. Figure 10. Metabolic pathways involving monocarboxylate transport across the mitochondrial and plasma membranes [45]. Besides being a family of 14 members, only the first four (MCT1-MCT4) have been demonstrated experimentally to facilitate the proton-linked transport of metabolically important monocarboxylates [46-49]. Since MCT3 is a very specialized MCT, being limited to the retinal pigment and choroid plexus epithelia [50,51] this introduction will only focus on MCT1, MCT2 and MCT4 isoforms, whose function is responsible for the name of this family of transporters [52-56]. Role of MCTs in Prostate Carcinoma Pértega-Gomes N. 16 Figure 11 shows that MCT1-MCT4 are associated in the same cluster presenting high homology. Importantly, this cluster is yet sub-divided in two branches MCT1-2 and MCT3-4 which correlates with the transported substrate as well as the affinities by which those transport is performed. Figure 11. Human MCT family members’ phylogram, based on amino acid sequence. Boxes limited by dots represent the main clusters [57]. 1.2.1.1 MCT1 Human MCT1 gene, SLC16A1 is located on chromosome 1 (1p13.2-p12) and comprises 5 coding exons. MCT1 functional protein is composed by 500 amino acids and has a molecular weight of 53.958 Daltons [58]. Among the MCT family, MCT1 isoform is the most well studied and functionally characterized member, since it is the only MCT expressed in human erythrocytes. Although ubiquitously expressed, MCT1 is especially prominent in heart and red muscle, where it is up-regulated in response to increased work, suggesting a special role in lactic acid oxidation [59,60]. The wide pattern of expression observed for MCT1 may be explained by its substrate affinities that indicate that Role of MCTs in Prostate Carcinoma Pértega-Gomes N. 17 MCT1, may be involved in both uptake and efflux of monocarboxylates from cells. MCT1 transports a variety of substrates including short chain (C2-C5) unbranched aliphatic monocarboxylates such as acetate and propionate. Monocarboxylates with C2 or C3 substitutions (excluding aminoand amido-) are also transported or even preferred (e.g pyruvate, L-lactate, acetoacetate and βhydroxybutyrate) (3537c). The simplest monocarboxylate, formate, is a poor substrate whereas bicarbonate; dicarboxylates, tricarboxylates and sulphonates are not transported. Also MCT1 is stereoselective for lactate but not for β-hydroxybutyrate. MCT1 was also found in mitochondria [61,62] and peroxisomes [63], where it is believed to participate in a lactate oxidation complex to maintain organelle redox and proper functioning. 1.2.1.2 MCT2 MCT2 is encoded by the SLC16A7 gene located in chromosome 12 (12q13) and comprises 5 coding exons [64]. Although only one transcript is identified, there is evidence for alternatively spliced mRNA species in human and rat [51, 63], but no evidence of splice variants of the protein. This isoform shares approximately 50% sequence identity with MCT1, contains 478 amino acids and has a molecular weight of 52,186 Da. The fact that, when both MCT1 and MCT2 are expressed in the same tissue, the expression pattern is cell-specific, suggested a distinct functional role between these isoforms [64-68]. Subsequent expression of MCT2 in Xenopus oocytes revealed its unique biochemical feature of facilitating the proton-linked transport of especially pyruvate, with a considerable high affinity, supporting the previous evidence of an alternative biological role [69]. As so, MCT2 also catalysis the proton-linked transport of a range of monocarboxylates, but with a considerably higher affinity than MCT1. As a higher affinity transporter, MCT2 has also a more restricted expression, being adapted to perform the uptake of monocarboxylates into cells. As a result, MCT2 is found in tissues that use lactate as a respiratory fuel, like brain or cardiac and skeletal muscle, kidney and liver were lactate is the major gluconeogenic substrate Role of MCTs in Prostate Carcinoma Pértega-Gomes N. 18 [66,67,69,70]. As MCT1, MCT2 is also found in mitochondria [71,72] and peroxisomes [63]. 1.2.1.3 MCT4 MCT4 is encoded by the human SLC16A3 gene, which is located in chromosome 17 (17q25.3), comprises 5 exons and 3 transcripts, with different initiation sites but no difference in protein product has been identified. The protein is constituted by 465 amino acids, corresponding to a molecular weight of 49,469 Da. MCT4 demonstrates remarkable similarities with MCT1 and in contrast to MCT2 has a broader distribution. The principal difference between MCT4 and MCT1 isoforms lies on their specific localization and substrate affinities. MCT4 shows a much lower affinity for substrates than MCT1 and MCT2. Accordingly, MCT4 is predominantly expressed in glycolytic cells such as white muscle and white blood cells, suggesting that its physiological function is lactate efflux and in fact, the kinetic properties of MCT4 show that this isoform is adapted to the export of lactate. This led to the hypothesis that MCT4 might be of particular importance in cells that rely on high rates of glycolytic metabolism to meet their energy demands, producing high amounts of lactate that need to be rapidly exported, such as cancer cells [73]. 1.2.1.4 Other MCT isoforms Other MCT isoforms are being characterized in the last years. SLC18A2 (MCT8) gene has been described as a thyroid hormone transporter [74] and mutations in this gene have been associated with X linked severe mental retardation and neurological dysfunction [76-80]. MCT6 (SLC16A5) transports bumetanide, but neither L-lactic acid or L-tryptophan, in a pHand membrane potential-sensitive but in a non-proton gradient-dependent manner [81]. MCT9 (SLC16A9) polymorphism was found to be associated with altered serum uric acid [82] however MCT9 substrate is still unknown. Importantly, SLC16A12 (MCT12) has been identified as a possible biomarker for colon, prostate and breast carcinoma due to gene hypermethylation [83] however the substrate for MCT12 is still Role of MCTs in Prostate Carcinoma Pértega-Gomes N. 19 unknown, as for the remaining members of the family (MCT5, MCT7, MCT11, MCT13 and MCT14). Table 1 summarizes the characteristics of the human SLC16 family of transporters and Table 2 shows the substrate affinities for the various MCT isoforms in humans and rats. Table 1. The human SLC16 family of transporters [47]. Role of MCTs in Prostate Carcinoma Pértega-Gomes N. 20 Table 2. Comparision of substrate affinities for the various MCT isoforms in human and rat [47]. 1.2.2 MCT Regulation Although the regulatory mechanisms of MCT expression are far from being completely understood, evidence indicates that MCTs might be regulated at various points up to the functional protein. This includes both transcriptional and post-transcriptional level [84-87] that affects protein amounts as well as regulators of transporter activity, like chaperone proteins. Hormone regulation has also been described for MCTs, as well as regulation by signaling pathways like insulin-like growth factor receptor type I (IGF-IR) activation which upregulates MCT1 [88]. MCT1 expression is particularly more described than the other isoforms in different physiological and pathological conditions. Reports including MCT up-regulation in skeletal muscle in response to training [85, 89-96], downregulation after muscle Role of MCTs in Prostate Carcinoma Pértega-Gomes N. 21 denervation and inflammatory bowel diseases [97,98], changes in MCT expression during development [99-103] or substrate induced MCT1 up-regulation [86, 87, 104] are relatively frequent. However and importantly, regulatory mechanisms vary among MCTs isoforms, which allows induction of specific isoforms upon different stimuli, adapting cells to different energy demands. 1.2.2.1 Transcriptional Level The analysis of SLC16A1 5’-flanking region allowed the identification of putative binding site sequences for the transcription factors USF, nuclear factor-kappaB (NF-kB), activated protein 1 and 2 (AP1 and AP2) and stimulating protein-1 (Sp1). USF1 and USF2 have been described as potential repressor proteins for MCT1 [105] whereas NF-kB pathway has been involved in the butyrate-induced MCT1 up-regulation [106]. AP2 has been associated to protein kinase C (PKC)- dependent stimulation of the SLC16A1 promoter [107] . Lactate-induced increase in MCT1 has been linked to activation of NF-kB and nuclear factor erythroid 2 (NF-E2) pathways, as well as cAMP-response elementbinding protein (CREB) and NF-E2 related factor 2 (Nrf2) transcription factors, the last three elements possessing also putative transcription binding sites in the SLC16A1 5’-flanking region. The co-activators peroxisome proliferator-activated receptor gamma, co-activator 1 alpha (PGC-1a) [108] and peroxisome proliferator-activated receptor alpha (PPAR) [109,110] have been associated with MCT1, but not with MCT2 and MCT4 up-regulations. Promoter analyses of MCT1, MCT2 and MCT4 have shown that the SLC16A1 promoter contains two peroxisome proliferator-activated receptor response elements (PPRE) while SLC16A7 and SLC16A3 each contain one PPRE. In expression studies, SLC16A1 was also shown to be activated by c-myc and nmyc proto-oncogenes, while the pro-inflammatory cytokines and TNF-a, have also been implicated in the transcriptional control of MCT1, by down-regulating SLC16A1 transcription [111-113]. Role of MCTs in Prostate Carcinoma Pértega-Gomes N. 28 through the same transporters. Quercetin is also described as an inhibitor of the monocarboxylate transporter isoform 1 (MCT1). In previous studies, it was demonstrated that Quercetin acts as reversible noncovalent inhibitor of L-lactate transport by tumour cells or red blood cells [144, 145]. More recently, a study aiming to clarify the role of flavonoids in the modulation of MCT1-mediated transport of hydroxybutyrate in vitro and in vivo demonstrated that Quercetin (among others) is an effective inhibitor of MCT1-mediated transport [146]. Lonidamine is a derivative of indazole-3-carboxylic acid, which for a long time, has been known to inhibit glycolysis in cancer cells. Although this action was originally attributed to hexokinase inhibition [147, 148], further studies revealed that lonidamine inhibits lactate efflux from cancer cells through inhibition of MCT1 and MCT4 [149, 150]. Actually, despite a lack of knowledge of its precise mechanism of action, lonidamine has been effective in clinical trials against various tumours, especially as a sensitizer to other chemotherapies [151]. 1.3 Monocarboxylate Transporters in the Context of Cancer 1.3.1 MCTs in human cancer. What is described? Regarding cancer research, there are already several evidence for the upregulation of MCTs in several solid tumours, such as colorectal carcinomas [152], uterine cervix carcinomas [153], glioblastomas, breast carcinomas [154, 155] and lung tumours [156], pointing to an important role of this transporters in the maintenance of these malignancies. The first report on MCT expression in human tumor samples described a decrease of MCT1 expression in the colonic transition from normality to malignancy [157], which was further supported by a larger study [158]. In breast cancer, silencing of SLC16A1 by gene promoter hypermethylation was suggested in 4 of 20 breast cancer cases (20 %). In contrast, results from our group showed a significant increase of MCT1 cytoplasmic and plasma membrane expression in breast carcinoma, when comparing to normal breast epithelium [154]. MCT4 only showed a significant increase in tumor samples for cytoplasm expression, with no differences in plasma membrane expression. The literature is also controversial in lung cancer. A study Role of MCTs in Prostate Carcinoma Pértega-Gomes N. 29 by Koukourakis and collaborators, no expression of MCTs in normal lung was found, while expression of MCT1 was found in all tumors examined and both MCT2 and MCT4 were also expressed in cancer cells. This study also analyzed the possible metabolic cooperation between lung cancer cells and the tumourassociated stroma, however, tumour associated stroma expressed MCTs weakly [15]. In opposition, a recent study by our group showed that normal lung presents a high frequency of MCT expression and, in fact, MCT4 is less expressed in tumor samples than in normal epithelium. MCT expression has also been described in some gynecological tumors like cervical and ovarian cancer [159]. In cervical cancer, a significant increase in overall and plasma membrane expression of MCT1 and MCT4 was observed. In ovarian cancer, staining for MCT1 and MCT4 as well as their chaperone CD147 was not found in normal ovarian tissues and benign ovarian tissues, while around 80 % of epithelial ovarian primary and metastatic tumors showed expression of these proteins. MCT1 was significantly associated with low grade tumors, high FIGO stage, presence of residual tumor, lack of relapse and presence of ascites; MCT4 was significantly associated with high grade tumors, high FIGO stage, presence of residual tumor, relapse and presence of ascites. Importantly, MCT expression was associated with the expression of the multidrug resistance markers MDR1 and MRP2. In contrast to what was found in the previous types of tumours, neither MCT1 nor MCT4 were found to be up-regulated in gastric adenocarcinomas [160]. Actually, MCT4 expression was more frequently observed in normal gastric mucosa than in gastric cancer cells and even less frequently observed in lymph-node metastasis, indicating a progressive loss of this MCT isoform with disease progression. Table 3 shows an overview on MCT1 and MCT4 expression and the impact on prognosis in different tumour types. Role of MCTs in Prostate Carcinoma Pértega-Gomes N. 30 Table 3. Overview of MCT1 and MCT4 expression and impact on prognosis in different tumour types [57]. Role of MCTs in Prostate Carcinoma Pértega-Gomes N. 31 Overall, the data available in the literature support the hypothesis of a major role of MCTs in the emergence of the hyper-glycolytic and acid-resistant phenotypes, as adaptations to the hypoxic microenvironment. The up-regulation of MCTs in the plasma membrane of different type of tumors is an adaptive mechanism to allow continuous high glycolytic rates, by exporting the accumulating end-product, lactate, as well as to counteract acid-induced apoptosis or necrosis. However, it was clear that this might not be the case for all tumor types, therefore, in most cases there are no functional studies showing the dependence of the tumors on MCT expression and activity. As so, additional studies on MCT expression in other tumor types, confirmation of the results already published as well as additional functional studies are needed to deeply understand the role of MCTs in cancer maintenance and aggressiveness and exactly in which cases these transporters could be used for therapy. 1.3.2 MCTs as suitable targets for cancer therapy. What is known? Lately there was an increase in the exploitation of treatments that target tumour metabolism, being some of them already in clinical trial phase. Figure 8 shows actual and future therapeutic targets of tumour metabolism by targeting metabolic enzymes. Figure 13. Actual and future therapeutic targets (dashed lines) of tumour metabolism by targeting metabolic enzymes [161]. Role of MCTs in Prostate Carcinoma Pértega-Gomes N. 32 Several studies have already shown the in vitro and in vivo effect of targeting different MCTs isoforms in human cancers. It was demonstrated that MCT inhibition decreases intracellular pH [162], leads to cell death and, importantly, enhances cancer cell radiosensitivity [163]. Results for MCT4 show a decrease in cancer cell migration, by mechanisms that also involve interaction of MCT4 with β1-integrin [164]. In contrast, another study showed that silencing of MCT1 or MCT4 inhibited cancer cell invasion, but did not influence cell migration [165]. In vivo models have also been used, where administration of αcyano-4-hydroxycinnamic acid (CHC), retarded tumor growth, rendered tumor cells sensitive to radiation, induced tumor necrosis and decreased tumor invasion [166]. The importance of MCTs for in vivo tumor growth was confirmed by a more specific approach, where combined silencing of MCT1 and MCT4 or silencing of CD147 significantly reduced glycolytic flux and tumor growth. There are also other MCT inhibitors described which are either non-isoform specific (ARC155858 targets both MCT1 and MCT2 [167] or target other molecules besides MCTs (e.g., lonidamine primary target is hexokinase II)) [167,168]. However, these compounds have been little explored as lactate transport inhibitors in the cancer context. In sum, it seems clear that MCTs represent a suitable target for cancer therapy; however, the effect of this inhibition seems highly dependent on the cell type and the isoform that is being targeted. Figure 14 shows the model for therapeutic targeting of lactate-based metabolic symbiosis in tumours. It shows that MCTs inhibition would have a major effect on lactate transport, pH balance and tumour homeostasis, by compromising aerobic glycolysis and microenvironmental acidosis, as well as, the cell-cell lactate shuttle between aerobic and hypoxic cell populations. Role of MCTs in Prostate Carcinoma Pértega-Gomes N. 33 Figure 14. Model for therapeutic targeting of lactate-based metabolic symbiosis in tumours [118] Regarding prostate cancer, which is our main focus in this thesis, few studies exist reporting the effect of MCTs inhibition in prostate cancer cells. As so, it is important to invest in studies evaluating both blocking and the silencing of MCTs in prostate cancer in order to reach a conclusion about the effectiveness of this therapeutic approach. 1.4 Prostate Cancer 1.4.1 Epidemiology of the disease Prostate cancer is one of the most prevalent and incident cancers in the male population worldwide after lung cancer, and the fifth most common cancer overall. In Portugal, prostate cancer is the leading cancer among men with 5,140 estimated incident cases in 2008 [169, 170]. The patterns of incidence and mortality provide a number of interesting leads with peak incidences in the United States, Australia and the Scandinavian countries. Incidence rates Role of MCTs in Prostate Carcinoma Pértega-Gomes N. 34 in Asian countries are generally low, but in recent years have risen proportionately more than in western countries. Incidence rates increased dramatically through the early 1990s [171]. In the late 1970s and early 1980s there was a rise in incidence in part, due to increased detection with more frequent surgical treatment for benign prostatic hyperplasia (BPH), particularly, transurethral resection of the prostate (TURP) [172]. Between 1986 and 1992 there was another sharp rise in incidence largely due to increasing use of prostate specific antigen (PSA) [173]. A recently published study points out an increase of 26% in the incidence of PCa in the period between 1986 to 2005 in the United States, which means that more than a million additional men were diagnosed with prostate cancer. Besides PSA screening programmes, other aspects such as the introduction of better diagnostic tools like transrectal ultrasonography (TRUS) –guided biopsy may be partially responsible for the increased incidence of prostate cancer in developed countries. However, screening practice differences alone are unlikely to explain the nearly huge differences in prostate cancer risk between highand lowrisk populations [174, 175]. Despite prostate cancer's high morbidity, its etiology remains obscure, with the only established risk factors being increasing age, race, and a family history. Many putative risk factors, including hormones, dietary factors, obesity, physical inactivity, occupation, vasectomy, smoking, sexual factors, and genetic susceptibility, have been implicated, but the epidemiologic evidence is inconclusive. While it is not known whether the risk factors explaining the observed patterns are environmental, lifestyle, or genetic, it is likely that a complex interplay of these factors is associated with prostate cancer development. Concerning mortality, the rates are much less variable than PCa incidence, leading to a less accentuated difference between developed and developing countries. With the introduction of PSA a slight increase in mortality rates was observed but this increase was verified both in high and low risk countries [176]. The reasons for this time trends in mortality rates are still controversial but the introduction of the PSA screening was in certain way responsible for the Role of MCTs in Prostate Carcinoma Pértega-Gomes N. 35 increasing rates due to miscertification of cause of death among men diagnosed with latent tumours, however it may also play a role in decreasing rates because of an earlier diagnosis of most of the cases. Early detection in prostate cancer is crucial since only organ-confined disease is amenable to curative treatment whereas patients with advanced disease can only be palliated. Although there are some available tools frequently used for PCa detection, their performance is sub-optimal due to non-satisfactory sensitivity and specificity rates. In this context, new biomarkers are urgently needed, not only to early detection but also as ancillary tools for diagnosis, which is still based on histopathological evaluation of biopsy specimens. 1.4.2 Prostate Cancer Diagnosis Usually, men with PSA levels of 4.0 ng/mL or greater are candidates to perform a prostatic core needle biopsy [177]. This cut off value for the PSA test is still controversial because PSA level screening produces false-negative or falsepositive results that can lead men without disease to be submitted to unnecessary additional testing [177, 178]. Importantly, PSA levels screening are not acceptable diagnostic tools for PCa in routine clinical practice because they lack adequate sensitivity and specificity. Regardless the utility of diagnosis aid-tools, the only way to access diagnosis of prostate cancer is by histopathological analysis of prostatic tissue obtained from biopsy or prostatectomy specimens. It is crucial to perform a proper histopathological evaluation of the tissue collected from the patient in order to determine which are the best treatment options available for each patient because the combination of this evaluation results with other parameters, such as serum PSA levels, in nomograms do have prognostic impact [179]. Biopsy techniques have been improved in order to reduce morbidity and increase the rate and accuracy of detection in the first biopsy, although the optimal number of cores that must be collected is still a matter of debate [178]. Role of MCTs in Prostate Carcinoma Pértega-Gomes N. 36 1.4.2.1 Histophatological evaluation. The Gleason score system for prostate cancer grading The Gleason grading system was firstly described in 1996 and it is presently, with some modifications, the most commonly used method for prostate cancer grading. Because the Gleason score (GS) is directly correlated with PCa prognosis, its accurate evaluation is critical to the natural history of the disease or the risk of recurrence following radical prostatectomy or radiotherapy [180]. The system is based on the evaluation of the glandular architectural patterns of the tumour tissue, recognizing five different grades that range from good to poor grade of differentiation. Because prostate adenocarcinomas are heterogeneous, more than one of the five patterns defined by Gleason might be present in the same tumour. To incorporate this heterogeneity in tumour grading, the Gleason score (GS) was developed, resulting from the sum of two most predominant cancer pattern (grades) in a sample. GS varies from 2 (1+1) to 10 (5+5) (Figure 15). Figure 15. Updated Gleason score for histological grading of prostate tumours [181] . Pattern 1Closely packed small, uniform glands. Pattern 2More stroma between the glands that are round or oval with smooth ends and may minimally invade nonneoplastic tissue. Pattern 3Irregular size glands with angular shape and more infiltrative margins. Pattern 4Fused, cribiform or poorly defined glands. Pattern 5Only occasional gland formation. 1.4.2.2 Clinical and Pathological Staging Another aspect that helps to predict the progression of PCa is the staging which comprises information about the extension of the disease (local, regional or systemic) and it is widely classified using the UICC (International Union against Cancer) Tumour Node Metastasis (TNM) system (Table 4). In this classification, T represents the extent of the primary tumour, N the lymph node status and M to Role of MCTs in Prostate Carcinoma Pértega-Gomes N. 37 distant metastasis. The clinical stage is based on the evaluation of the patient by digital rectal exam (DRE), transrectal ultrasonography (TRUS), and, possibly, magnetic resonance imaging (MRI) [178,182,183] . Further information can be provided by biopsy histopathological evaluation and serum PSA levels [188]. Alternatively, pathological stage is determined after surgical removal of the prostate through adequate analysis of the prostatectomy specimen, and it predicts disease recurrence much more accurately [183]. There are several independent prognostic factors included in staging that are important to guide clinical decisions concerning the treatment options. These are extra-prostatic tumour invasion, seminal vesicles involvement, lymph node metastasis and distant metastasis. 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Role of MCTs in Prostate Carcinoma Pértega-Gomes N. 60 Role of MCTs in Prostate Carcinoma Pértega-Gomes N. 61 2.2.1 Monocarboxylate transporter 4 (MCT4) and CD147 overexpression is associated with poor prognosis in Prostate Cancer Role of MCTs in Prostate Carcinoma Pértega-Gomes N. 62 Role of MCTs in Prostate Carcinoma Pértega-Gomes N. 63 Role of MCTs in Prostate Carcinoma Pértega-Gomes N. 64 Role of MCTs in Prostate Carcinoma Pértega-Gomes N. 65 Role of MCTs in Prostate Carcinoma Pértega-Gomes N. 66 Role of MCTs in Prostate Carcinoma Pértega-Gomes N. 67 Role of MCTs in Prostate Carcinoma Pértega-Gomes N. 68 Role of MCTs in Prostate Carcinoma Pértega-Gomes N. 69 Role of MCTs in Prostate Carcinoma Pértega-Gomes N. 76 Role of MCTs in Prostate Carcinoma Pértega-Gomes N. 77 3.2.1 Monocarboxylate transporter 2 (MCT2) as a putative biomarker in prostate cancer. Role of MCTs in Prostate Carcinoma Pértega-Gomes N. 78 Role of MCTs in Prostate Carcinoma Pértega-Gomes N. 79 Role of MCTs in Prostate Carcinoma Pértega-Gomes N. 80 Role of MCTs in Prostate Carcinoma Pértega-Gomes N. 81 Role of MCTs in Prostate Carcinoma Pértega-Gomes N. 82 Role of MCTs in Prostate Carcinoma Pértega-Gomes N. 83 Role of MCTs in Prostate Carcinoma Pértega-Gomes N. 84 Role of MCTs in Prostate Carcinoma Pértega-Gomes N. 85 Role of MCTs in Prostate Carcinoma Pértega-Gomes N. 92 gluconeogenic substrate [8-13]. As MCT1, MCT2 is also found in mitochondria [12,13] and was also localized at peroxisomes in non-tumor liver fractions [14]. The presence of MCTs in mitochondria is justified by the need of a pyruvate carrier that plays a central role in carbohydrate and fat metabolism. In contrast, the presence of MCT1 and MCT2 in peroxisomes was justified as being involved in a lactate-pyruvate shuttle system present in the membrane of this organelle. This shuttle was suggested to be involved in the oxidation of NADH generated by βoxidation, being crucial for the maintenance of peroxisomal viability and consequent β-oxidation rates [14]. This was the only study regarding this matter and it was not done in the cancer context, as so, little is known about lactate shuttles as well as the involvement of peroxisomal alterations in cancer. In this work we aimed to unraveling MCT2 intracellular localization and expression across prostate malignant transformation using different models of disease progression in order to infer about the importance of this isoform in prostate cancer disease. Also, the other MCT isoforms where assessed in order to characterize in more detail their intracellular distribution. Material and methods Cell culture In this study we have used different prostate cell lines, PNT1A (non-malignant), 22Rv1 (localized tumor), LNCaP (lymph node metastasis) and PC3 (bone metastasis). Cells were seeded in RPMI-1640 (Gibco, Invitrogen, USA) supplemented with 10% of fetal bovine serum (FBS) (PAA Laboratories GmbH, Cölbe, Germany), 1% of antibiotic (penicillin/streptomycin) (PAA Laboratories GmbH, Cölbe, Germany) and incubated at 37⁰C in an atmosphere containing 5% CO2. Immunofluorescence and microscopy techniques Immunofluorescence was performed in order to identify the intracellular localization of MCT1, MCT2 (sc-14926, Santa Cruz Biotechnology, Santa Cruz, CA), MCT4 and its chaperones CD147 and gp70. Fluorophores TRITC (Jackson Role of MCTs in Prostate Carcinoma Pértega-Gomes N. 93 ImmunoResearch) and Alexa 488 (Invitrogen, Life Technologies, Grand Island, NY, USA) were used as secondary antibodies. Immunofluorescence was performed in cells seeded on glass cover slips that were fixed after 24h with 4 % paraformaldehyde in PBS, pH 7.4 for 20 min. Afterwards cells were permeabilized with 0.2 % Triton X-100 for 10 min, blocked with 1 % BSA solution for 10 min and incubated with primary (MCT2, catalase, PEX19 and Gp70) and secondary antibodies [TRITC (Jackson ImmunoResearch) and Alexa 488 (Invitrogen, Life Technologies, Grand Island, NY, USA)] for 1 h each. Between each step, cells were extensively rinsed 3 times with PBS, pH 7.4. Lastly, cells were stained with and mounted in slides using Mowiol 4-88 containing npropylgallate. Images were obtained using a Zeiss LSM 510 Meta Confocal setup (Carl Zeiss, Oberkochen, Germany) equipped with a plan-Apochromat 100×/1.4 oil objective. Western Blot Cells were lysed with specific lysis buffer (25 mM Tris-HCl, pH 8.0, 50 mM sodium chloride, 0.5% sodium deoxycholate, 0.5% Triton X-100 and a protease-inhibitor mix). To improve protein extraction, samples were passed 20 times through a 26gauge syringe needle and incubated on a rotary mixer at for 30 min at 4⁰C. After cleared by centrifugation (13300 rpm, 15 min) protein concentrations were determined by the Bradford assay (BioRad, Hercules, CA, USA). Blots were incubated with specific primary antibodies, MCT2, gp70, PEX14, Catalase, ACOX1, ACOX3 and PEX19. The antibodies were detected by a horseradish peroxidase-linked secondary antibody using an enhanced chemiluminescence system (GE Healthcare, Waukesha, WI, USA). Immunoprecipitation 22Rv1 cells were transfected with Pex19-YFP using Turbofect in vitro transfection kit (Fermentas, USA), according to the manufacturer’s instructions. For immunoprecipitation of Pex19-YFP we used GFP trap_M (Chromotek). Transfection with a plasmid containing GFP was used as negative control. After 48h of transfection cell pellets were incubated lysis buffer (10mM Tris-HCl, pH 7.5, 150mMNaCl, 0.5mM EDTA, 0.5%NP-40 and a protease-inhibitor mix). The lysate Role of MCTs in Prostate Carcinoma Pértega-Gomes N. 94 was cleared by centrifugation (13300 rpm, 15 min) and diluted with dilution buffer (10mM Tris-HCl, pH 7.5, 150mM NaCl, 0.5mM EDTA and a protease-inhibitor mix). Protein concentrations were determined by the Bradford assay (BioRad, Hercules, CA, USA). Ice-cold dilution buffer was used to equilibrate beads and cell lysates were incubated for 2 h at 4°C on a rotary mixer. Beads were washed 3 times with dilution buffer and resuspended in 3x SDS-sample buffer and boiled for 10 min to elute bound proteins. Immunoprecipitated samples were separated by running in a 12.5% SDS-polyacrylamide gel. Results MCT2 was detected in the peroxisomes of prostate cancer cells at different levels within disease progression The intracellular localization of MCT2 was accessed in different models of prostate cancer disease progression. As McClelland et al have shown a peroxisomal localization of MCT2 in non-tumor liver fractions, we firstly tested whether this protein would also be present in this organelle in prostate cancer cells. To that end, we have performed immunolocalization of MCT2 together with the peroxisomal marker catalase in different prostate cell lines: PNT1A (nonmalignant), 22Rv1 (localized tumor), LNCaP (lymph node metastasis) and PC3 (bone metastasis). Our results demonstrate that, while no co-localization was observed between MCT2 and the peroxisomal marker in PNT1A, this protein is localized at peroxisomes in all the malignant cell lines (Fig 1). We have, though, observed that the localization level varied across the different models. In 22Rv1 cells, MCT2 was mainly found in the peroxisomes with a minor portion spread throughout the cytoplasm as small aggregates (Fig1 d-f). However, the ratio of peroxisomal MCT2/cytoplasm MCT2 decreased with disease progression, culminating with almost no MCT2 in the peroxisomes in PC3 cells (Fig1 j-l). Surprisingly, the amount of MCT2 at peroxisomes appears to correlate with a change on the organelle’s morphology. In fact, in cells where no MCT2 was present in peroxisomes (PNT1A), this organelle exhibits a regular phenotype (Fig 1A: a-c). Curiously, in 22Rv1 cells (where MCT2 was mainly observed in Role of MCTs in Prostate Carcinoma Pértega-Gomes N. 95 peroxisomes) this organelle appears somewhat in clusters (Fig. 1A: d-f) LNCaP cells exhibit more distributed rounder peroxisomes with only a few clusters (Fig.1A: g-i). In PC3, the highly metastatic model, peroxisomes appear similar to the ones of the non-malignant cells PNT1A (Fig 1A: j-l). Strikingly, the expression level of MCT2 increases from non-tumor (PNT1A) to localized malignant cells (22Rv1), correlating to its change in localization from cytoplasmic to peroxisomal (Fig 1B). This increase in expression was also observed for Pex14 (a peroxisomal membrane protein) (Fig 1C), suggesting an increase in peroxisomal membrane surface/number that accompanies the malignant transformation. MCT2 travels to the peroxisomal membranes trough PEX19 The quick movement of monocarboxylates across the membranes is imperative for cellular metabolism. These proteins are thought to require chaperones such as CD147 in the case of MCT1 and MCT4, or gp70 in the case of MCT2, for appropriate expression and activity in the plasma membrane [15]. Since MCT2 was the only isoform found to localize in peroxisomes in prostate cancer cells, we aimed at unraveling its targeting mechanism to this organelle. A previous study was unable to find gp70 expression in human prostate samples that exhibited MCT2 expression [5]. However, our studies with confocal microscopy allowed us to observe some, although scarce, gp70 distributed in the cytoplasm without any co-localization with peroxisomal markers (Fig 2 a-l). Hence, the protein that behaves as chaperone for MCT2 transport to peroxisomes in prostate cells is still unknown. As Pex19 is the main protein responsible for the transport of peroxisomal membrane proteins to this organelle, we tested whether this protein could act as a chaperone for MCT2 in these cells. Being 22RV1 the cell line where MCT2 was mainly present at peroxisomes, it was the chosen model to study the possible interaction between MCT2 and PEX19. Immunoprecipitation experiments have shown, indeed, an interaction between MCT2 and PEX19 (Fig 3). These results suggest a highjack of the peroxisomal transport mechanism by the prostate cancer cells. Role of MCTs in Prostate Carcinoma Pértega-Gomes N. 96 The presence of MCT2 at peroxisomes seems to be related with an increase in the rates of peroxisomal β-oxidation McClelland et al observed a decrease in β-oxidation upon MCTs inhibition, suggesting that the presence of MCT2 at peroxisomes of non-malignant liver cells would be essential for the maintenance of peroxisomal viability and consequently β-oxidation rates. Hence, we hypothesized that the increase of MCT2 expression from non-malignant prostate cells (PNT1A) to localized prostate tumor cells (22RV1) could be related with an increase in peroxisomal β-oxidation. In fact, we observed an increase in the expression levels of ACOX1 and ACOX-3, central proteins in the β-oxidation pathway (Fig 4). These results interestingly suggest that, indeed, the increase in MCT2 expression levels as well as its presence at peroxisomes, are related to an increase in β-oxidation levels which may be crucial for malignant transformation. The distribution of other MCTs isoforms and CD147 at intracellular level suggests an additional role for these proteins in prostate cancer MCT1, MCT4 and CD147 expressions were also evaluated in this study. MCT1 showed a strong nuclear expression in PNT1a and 22RV1, decreasing for LNCaP and PC3, where this transporter was mainly found in the cytoplasm and plasma membrane (Fig.5). MCT4 however was shown to be present in the cytoplasm of the less tumorigenic cell lines 22RV1 and LNCaP in contrast to the highly metastatic PC3 where it was clearly present at the plasma membrane (Fig.6). CD147 was present in the plasma membrane, cytoplasm and nuclear envelope (Fig.7). Interestingly, some co-localization between MCTs 1 and 4 and peroxisomes, was also verified, however it was less evident than for MCT2. Discussion It is now widely accepted that the transport of lactate and pyruvate is mediated by a family of H+/monocarboxylate transporters (MCTs) known for their specificity to transport short chain monocarboxylates. They form an integral part of proposed Role of MCTs in Prostate Carcinoma Pértega-Gomes N. 97 shuttles that transport lactate between cells (cell–cell lactate shuttle) and within cells (intracellular lactate shuttle). A previous study of MCTs expression in prostate cancer drew attention to MCT2 that was shown to be clearly expressed in prostate malignant glands but at the cytoplasm, suggesting the involvement of this transporter in an organelle membrane [6], however there were no studies describing the existence of MCT2 in the organelles of prostate cancer cells. It is known, that once in the mitochondrial matrix, lactate and pyruvate undergo further breakdown making it difficult to infer their respective roles in the shuttling of reducing equivalents to the electron transport chain. Peroxisomes, on the other hand, lack the enzymes to catabolize monocarboxylates and although they are known to contain LDH [16], they are not known to contain any other glycolytic or any tricarboxylic acid cycle enzymes. One of the many functions of liver peroxisomes is the β-oxidation of long branched-chain fatty acids and for the maintenance of these reactions inside the peroxisome, a redox shuttle system exist across the peroxisomal membrane to reoxidize NADH. It is essential for the continuous functioning of peroxisomes that proper intra-organelle reduction– oxidation (redox) balance is maintained that ensures the reoxidation of NADH produced by β-oxidation and the continuous breakdown of fatty acids. Redox balance in many organelles is maintained in part by substrate shuttles. This would entail a shuttle system in peroxisomes that neither generates products nor consumes any substrates. McClelland et al proposed that a redox shuttle system that consists of a substrate cycle between lactate and pyruvate, as so, to play their role in peroxisomal redox balance, pyruvate and lactate must be transported across the peroxisomal membrane [14]. In the present study we describe for the first time that MCT2 is present in human prostate peroxisomes and a possible explanation for how these MCT2 ends up in the peroxisomal membrane was given by the evident interaction of MCT2 with PEX19 which might act as chaperone. The absence of glycolysis in this organelle strongly suggests that this shuttle serves to maintain redox balance and not glycolysis or some other metabolic function. Consequently, we point out the importance of peroxisomes in prostate cancer cells that as it was observed a clear Role of MCTs in Prostate Carcinoma Pértega-Gomes N. 98 change their morphology across prostate malignant transformation, which provides further evidence for the dynamic nature of the peroxisomes across disease progression. Importantly, the frequency of peroxisomal clusters and the colocalization with MCT2 that was mainly observed in the transition from the benign model (PNT1a) to the malignant model (22RV1), point to the involvement of these alterations in tumour initiation. Finally, the possibility of having MCT1 and MCT4 in the peroxisomal membrane, however less evident than for MCT2, corroborates the hypothesis of alternative roles for these transporters in metabolic pathways different from glycolysis in the present cells. However, further studies are needed to understand the role of these MCT isoforms in prostate peroxisomes. References 1. Comperat, E., J. Varinot, and J.R. Srigley, [Benign mimickers of the prostate cancer. Diagnostic challenges]. Ann Pathol, 2013. 33(4): p. 237-46. 2. Schmitz, W., R. Fingerhut, and E. Conzelmann, Purification and properties of an alphamethylacyl-CoA racemase from rat liver. Eur J Biochem, 1994. 222(2): p. 313-23. 3. Lloyd, M.D., et al., Alpha-methylacyl-CoA racemase--an 'obscure' metabolic enzyme takes centre stage. FEBS J, 2008. 275(6): p. 1089-102. 4. Halestrap, A.P., The SLC16 gene family - structure, role and regulation in health and disease. Mol Aspects Med, 2013. 34(2-3): p. 337-49. 5. Pertega-Gomes, N., et al., Monocarboxylate transporter 4 (MCT4) and CD147 overexpression is associated with poor prognosis in prostate cancer. BMC Cancer, 2011. 11: p. 312. 6. Pertega-Gomes, N., et al., Monocarboxylate transporter 2 (MCT2) as putative biomarker in prostate cancer. Prostate, 2013. 73(7): p. 763-9. 7. Lin, R.Y., et al., Human monocarboxylate transporter 2 (MCT2) is a high affinity pyruvate transporter. J Biol Chem, 1998. 273(44): p. 28959-65. 8. Pierre, K., et al., Cell-specific localization of monocarboxylate transporters, MCT1 and MCT2, in the adult mouse brain revealed by double immunohistochemical labeling and confocal microscopy. Neuroscience, 2000. 100(3): p. 617-27. 9. Pierre, K., P.J. Magistretti, and L. Pellerin, MCT2 is a major neuronal monocarboxylate transporter in the adult mouse brain. J Cereb Blood Flow Metab, 2002. 22(5): p. 586-95. 10. Debernardi, R., et al., Cell-specific expression pattern of monocarboxylate transporters in astrocytes and neurons observed in different mouse brain cortical cell cultures. J Neurosci Res, 2003. 73(2): p. 141-55. 11. Pellerin, L., et al., Cellular and subcellular distribution of monocarboxylate transporters in cultured brain cells and in the adult brain. J Neurosci Res, 2005. 79(1-2): p. 55-64. Role of MCTs in Prostate Carcinoma Pértega-Gomes N. 99 12. Yoshida, Y., et al., Negligible direct lactate oxidation in subsarcolemmal and intermyofibrillar mitochondria obtained from red and white rat skeletal muscle. J Physiol, 2007. 582(Pt 3): p. 1317-35. 13. Benton, C.R., et al., Monocarboxylate transporters in subsarcolemmal and intermyofibrillar mitochondria. Biochem Biophys Res Commun, 2004. 323(1): p. 249-53. 14. McClelland, G.B., et al., Peroxisomal membrane monocarboxylate transporters: evidence for a redox shuttle system? Biochem Biophys Res Commun, 2003. 304(1): p. 130-5. 15. Wilson, M.C., et al., Basigin (CD147) is the target for organomercurial inhibition of monocarboxylate transporter isoforms 1 and 4: the ancillary protein for the insensitive MCT2 is EMBIGIN (gp70). J Biol Chem, 2005. 280(29): p. 27213-21. 16. Baumgart, E., et al., L-lactate dehydrogenase A4and A3B isoforms are bona fide peroxisomal enzymes in rat liver. Evidence for involvement in intraperoxisomal NADH reoxidation. J Biol Chem, 1996. 271(7): p. 3846-55. Role of MCTs in Prostate Carcinoma Pértega-Gomes N. 100 Figures Figure 1 A) Immunohistochemical images of different human prostate cell lines PNT1a (Figure 1ac), 22RV1(Figure 1d-f), LNCaP (Figure 1g-i) and PC3 (Figure 1j-l). Superposition of signals from probes for MCT2 (green), and Catalase (red) shows clear colocalization (yellow in the peroxisomes of prostate cancer cells). Role of MCTs in Prostate Carcinoma Pértega-Gomes N. 101 PNT1a 22RV1 LNCaP PC3 MCT2 βTubulin Figure 1 B) Western blot analysis showing the levels of MCT2 in the different prostate cell line models. PNT1a 22RV1 LNCaP PC3 PEX14 Tubulin Figure 1 C) Western blot analysis, showing the expression levels of the peroxisomal protein PEX14 in the different in vitro models of prostate cancer progression (PNT1a, 22RV1, LNCaP and PC3). Role of MCTs in Prostate Carcinoma Pértega-Gomes N. 108 Role of MCTs in Prostate Carcinoma Pértega-Gomes N. 109 4.1 CHAPTER OVERVIEW We firstly described overexpression of MCT2 and MCT4 in PCa tissues when compared to the normal counterparts, however, the role of MCTs in PCa metabolism is still largely unknown. The presence of MCT4 in the cytoplasm of prostate cancer cells instead of plasma membrane together with the expression of MCT1 in both non-malignant and malignant glands, suggested that prostate cells may not firstly rely on glycolytic metabolism as the majority of tumours. This verification turned this work even more challenging in an attempt to contextualize the role of different MCTs isoforms across prostate malignant transformation and progression. As stated before, it remains unclear what is the primary energetic pathway in PCa and therefore which metabolic pathway represents the most appropriate target for metabolic inhibition in PCa. There are studies reporting the presence of hypoxia in PCa, pointing to the presence of a glycolytic phenotype, however other studies report that PCa is characterized by low rates of glycolysis and a low FDG activity on PET imaging. This fact raises the hypothesis that if glucose consumption is not elevated in PCa, alternative metabolic pathways must provide the energy needed for cancer cell proliferation and growth. Some evidence support a crucial role of fatty acid-related metabolism in the pathogenesis and progression of prostate malignancy. However, the reports are scarce with low number of clinical samples and with no information regarding the clinico-pathological significance of these alterations; as so, PCa metabolism is still largely unknown. This chapter includes results already submitted for publication, showing the expression pattern of several metabolic-related proteins involved in glycolytic and fatty acid oxidation pathways from benign prostate to metastatic PCa, investigating the prognostic impact of their expression. Also, using different in vitro models of disease progression we tested how the inhibition of these pathways affect the different models of prostate cancer cells in order to establish a rationale for metabolic phenotypes across prostate malignant transformation and progression. Overall, the results presented here indicate a switch from less aggressive tumor to glycolytic metabolism in the highly aggressive and metastatic prostate tumor Role of MCTs in Prostate Carcinoma Pértega-Gomes N. 110 and underscores the plasticity of bioenergetic pathways in prostate cancer cells which might have crucial implications at prognostic and therapeutic level. 4.2 PUBLISHED RESULTS The results presented in this chapter were: (i) Submitted for publication as an original article in an international peer reviewed journal Pértega-Gomes N, Vizcaíno JR, Sousa S, Coelho R, Attig J, Jurmeister S, Oliveira E, Pereira L, Pinheiro C, Jerónimo C, Henrique RM, Lobo da Cunha A, Lopes C, Maximo V and Baltazar F “Metabolic heterogeneity in prostate cancer is linked to disease progression and aggressiveness”. 2013. (ii) Invited for publication as an original article in an international peer reviewed journal Pértega-Gomes N. and Baltazar F. “MCTs as targets for prostate cancer therapy?” invited work to be published in Advances in Prostate Cancer Research and Treatment journal. 2014. (iii) Presented as oral communication in the following international scientific meeting Pértega-Gomes N, Vizcaíno JR, Lopes C and Baltazar F. “Tracing the metabolic profile of prostate cancer development” work presented at 23rd European Congress of Pathology at Helsinki, Finland. 2011. Pértega-Gomes N., Vizcaíno JR, Lopes C and Baltazar F. “Exploitation of metabolic alterations as importante prognostic, diagnostic and therapeutic tools in prostate cancer”. Work presented at 20th Meeting of the EAU Section of Urological Research (ESUR). 25-27 October 2012, Strasbourg, France. 2012. (iv) Presented as oral communication in the following national scientific meeting: Role of MCTs in Prostate Carcinoma Pértega-Gomes N. 111 Pértega-Gomes N, Lourenço T, Vizcaíno JR, Lopes C and Baltazar F. “Study of Metabolic profile of localized and metastatic tumour of the prostate. Monocarboxylate transporters as therapeutic targets” work presented at XVI Workshop on Oncological Urology, Carvoeiro, Portugal. 2011. Pértega-Gomes N, Lourenço T, Vizcaíno JR, Lopes C and Baltazar F. et al. “A metabolic switch could be involved in prostate cancer aggressiveness.”Work presented at the 12º Congress of the Portuguese Society of Oncology, Algarve, Portugal. 2011. (v) Presented as poster in the following international scientific meeting: Pértega-Gomes N, Lourenço T, Miranda-Gonçalves V, Vízcaíno JR, Oliveira E, Jerónimo C, Lopes C, Lobo da Cunha A. and Baltazar F. “Emerging roles for Monocarboxylate Transporters in prostate cancer metabolism”. at 2nd Symposium of the International Society for Proton Dynamics in Cancer in Nice, France. 2011. Miranda-Gonçalves V, Pértega-Gomes N, Lourenço T, Pinheiro C, Jerónimo C, Vizcaíno JR, Lopes C, and Baltazar F. “Prostate Cancer: exploitation of monocarboxylate transporters as potential therapeutic targets”. at the 39th Meeting of International Society of Oncology and Biomarkers in Florence, Italy. 2011. Pértega-Gomes N., Vizcaíno JR, Lourenço T, Lopes C. and Baltazar F “Exploiting metabolic alterations in prostate cancer diagnosis and prognosis” at XXI Porto Cancer Meeting, Porto. 2012. Pértega-Gomes N, Vizcaíno JR, Lourenço T, Lopes C. and Baltazar F “Tracing the metabolic profile of prostate cancer progression. Roles for monocarboxylate transporters (MCTs) in prostate cancer metabolism” at EACR Annual Congress, Barcelona. 2012. Pértega-Gomes N, Vizcaíno JR, Lourenço T, Lopes C. and Baltazar F. “Clinical impact of the metabolic phenotype of prostate cancer: Role of monocarboxylate transportes (MCTs).” at São Paulo Advanced School of Comparative Oncology. September 30th to October 6th. Águas de São Pedro-São Paulo, Brasil. 2012. Role of MCTs in Prostate Carcinoma Pértega-Gomes N. 112 Pértega-Gomes N, Vizcaíno JR, Lourenço T, Lopes C. and Baltazar F. “Metabolic phenotypes associated with distinct androgen-responsive conditions can be a valuable tool for diagnostic and therapeutic options in prostate cancer.” at A one day symposium with Carlos Caldas sponsored by EACR. 29TH October, Porto, Portugal. 2012. (vi) Were recognized with the the following prizes/awards: Pértega-Gomes N, Vizcaíno JR, Lopes C and Baltazar F “Tracing the metabolic profile of prostate cancer development”. Bursary Award given by the European Society of Pathology (ESP) to attend 23º Congress of the European Society of Pathology meeting in Helsinki, Finland. Pértega-Gomes N, Lourenço T., Vizcaíno JR, Lopes C and Baltazar F. “Study of Metabolic profile of localized and metastatic tumour of the prostate. Monocarboxylate transporters as therapeutic targets” 1st Prize (Janssen Award) and Honorable Mention given by for best poster and scientific communication in National Congress of Urology. Pértega-Gomes N, Lourenço T., Vizcaíno JR, Lopes C and Baltazar F. 2011. “A metabolic switch could be involved in prostate cancer aggressiveness. 1st Prize for best poster and scientific communication in 12º National Congress of Oncology. Pértega-Gomes N, Vizcaíno JR, Lourenço T, Lopes C. and Baltazar F. 2012. “Clinical impact of the metabolic phenotype of prostate cancer: Role of monocarboxylate transportes (MCTs).” Bursary award given by FAPESP - São Paulo Research Foundation to attend ESPCA-School of Comparative Oncology at Águas de São Pedro-São Paulo, Brasil, 2012. Role of MCTs in Prostate Carcinoma Pértega-Gomes N. 113 4.3.1 Metabolic heterogeneity in prostate cancer is linked to disease progression and aggressiveness Role of MCTs in Prostate Carcinoma Pértega-Gomes N. 114 Role of MCTs in Prostate Carcinoma Pértega-Gomes N. 115 Metabolic heterogeneity in prostate cancer is linked to disease progression and aggressiveness Running Title: Metabolic heterogeneity in PCa progression List of authors Nelma Pértega-Gomes1,2, José R. Vizcaíno3, Susana Sousa1,2, Ricardo Coelho4,5, Jan Attig6, Sarah Jurmeister7, Elsa Oliveira8, Luísa Pereira4, 9, Céline Pinheiro1,2, Carmen Jerónimo10,11,12, Rui M. Henrique11,12,13, Alexandre Lobo da Cunha8, Carlos Lopes3,12, Valdemar Maximo4,9and Fátima Baltazar1,2* 1Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Braga, Portugal 2ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal; 3Department of Pathology, Centro Hospitalar do Porto, Portugal; 4Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP), 4200-465 Porto, Portugal. 5Institute of Biomedical Sciences of Abel Salazar, University of Porto, 4050-313 Porto, Portugal. 6MRC Laboratory of Molecular Biology Francis Crick Avenue Cambridge Biomedical Campus Cambridge UK. 7Uro-oncology Research Group, Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge, UK. Role of MCTs in Prostate Carcinoma Pértega-Gomes N. 116 8Laboratory of Cell Biology, Institute of Biomedical Sciences Abel Salazar (ICBAS), University of Porto 9Department of Pathology and Oncology, Medical Faculty of the University of Porto, 4200-319 Porto, Portugal 10Cancer Epigenetics Group – Research Center, Portuguese Oncology InstitutePorto, Porto, Portugal; 11Department of Genetics, Portuguese Oncology Institute,Porto, Portugal 12Department of Pathology and Molecular Immunology, Institute of Biomedical Sciences Abel Salazar (ICBAS), University of Porto, Porto, Portugal 13Department of Pathology, Portuguese Oncology Institute-Porto, Porto, Portugal *Correspondence to: Fátima Baltazar, PhD, School of Health Sciences, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal. email:[email protected]. Tel: +351 253 60 48 28. Fax: +351 253 60 48 20 Conflict of interest statement The authors declare no conflicts of interest. Role of MCTs in Prostate Carcinoma Pértega-Gomes N. 117 Abstract Metabolic adaptation is now considered a new hallmark of cancer, in which cancer cells exhibit high rates of glucose consumption with consequent lactate production. However, the dominant energetic pathway as well as the clinical impact of altered cellular metabolism in prostate cancer is still largely unknown. In an attempt to understand the metabolic alterations during prostate cancer progression and how these alterations could represent suitable tools for prostate cancer at diagnostic, prognostic and therapeutic level, we studied the expression of several key metabolic-related proteins in 480 human prostate samples and using in vitro models we assessed metabolic changes associated with malignant transformation/progression and how their manipulation affects different models of prostate cancer cells. Also we provided an insight into the cell mitochondrial features and used publicly available genome-wide expression profiling datasets to assess the expression of the genes that codify metabolicrelated proteins during prostate cancer progression. This study provides evidence for increased glycolytic phenotype mainly in the advanced stages of prostate cancer, and its correlation with poor prognosis whereas the localized tumour showed more consistently upregulation of proteins involved in peroxisomal branched-chain fatty acid oxidation without any association with clinic-pathological data. Importantly, experiments using in vitro models showed that cell lines derived from distant metastasis are more glycolytic, sensitive to glycolytic inhibitors and with lower energy efficiency at the mitochondrial level. This is the most comprehensive study reporting the metabolic differences across prostate malignant transformation and correlating the metabolic heterogeneity of prostate cancer cells with unfavorable prognosis, showing that metabolic changes have a strong potential to be explored as diagnostic, prognostic and therapeutic tools in prostate cancer. Keywords: prostate cancer, cancer metabolism, monocarboxylate transporters, glycolytic metabolism, fatty acid oxidation, metabolic switch. Role of MCTs in Prostate Carcinoma Pértega-Gomes N. 124 there was a decrease in the survival of patients with tumours expressing CAIX, (data not shown). Different in vitro models of PCa progression exhibit different protein expression and glycolytic metabolism profiles Since the main differences observed across malignant progression in human samples were related to proteins involved in the glycolytic metabolism, in Figure 3 we show the expression profile of these proteins in different in vitro models of disease progression. Interestingly, MCT4 and HIF-1alpha were only proteins detected in DU145 and PC3 cell lines. Also, GLUT-1, LDHV and CAIX showed evident higher levels in PC3 cell line. Interestingly, the ICC results for the different cell line models show that MCT4 and GLUT-1 only appeared evidently expressed at the plasma membrane in the highly metastatic models, such as PC3, in contrast to the low tumorigenic cell line LNCaP (Figure 3B). Finally, a clear distinct metabolic behaviour between the low tumorigenic 22RV1 and LNCaP and the high tumorigenic PC3 and DU145 models was observed. PC3 and DU145 exhibited higher levels of glucose consumption (Figure 3C) and lactate production (Figure 3D) when compared to 22RV1 and LNCaP cells. Prostate cancer cells exhibited different sensitivities to inhibition of glycolysis and fatty acid oxidation Based in the idea that different models of disease progression exhibited a different metabolic behaviour, we aimed to study the effect of glycolysis and fatty acid oxidation inhibition in the different models. Oxamic acid (OA), an inhibitor of lactic dehydrogenase (LDH), was used to infer how the glycolytic metabolism in the different models is affected after inhibition of lactic acid production. Figure 4 shows the glucose consumption and lactate production of prostate cell lines after the treatment with OA. It is possible to observe that both parameters showed a significant decrease in 22RV1, PC3 and DU145 cell lines. In contrast, LNCaP cell line metabolism was less affected by OA treatment. Role of MCTs in Prostate Carcinoma Pértega-Gomes N. 125 The phenothiazine drug, thioridazine (TZ), described as a selective inhibitor of peroxisomal β-oxidation, was also used in our cell line models. As observed in Figure 5A, the IC50 values show that 22RV1 and LNCaP cell lines are more sensitive to TZ effect, especially at 72 h treatment. Accordingly, when we supplemented the culture media with phytanic acid (PA), a long branched chain fatty acid, to stimulate fatty acid oxidation, a decrease in PC3 and DU145 cell viability was observed, but not in 22RV1 and LNCaP that maintain their viability with increasing concentrations of PA, even after 48 hours of exposure (Figure 5B). Ultrastructural differences are evident among the different prostate cancer cell models So far, we have observed that differences at the expression levels of proteins involved in glycolytic and fatty acid metabolism as well as in cells metabolic behavior are evident across malignant transformation. As so, we also aimed to verify if such differences might be also reflected at the ultrastructural level of the cells. Figure 6 shows electron micrographs of LNCaP (Figure 6A), PC3 (Figure 6B) and two different human samples of prostate cancer (Figure 6C and 6D). It was possible to observe that either in LNCaP cells or the human samples (Figure 6A, 6C and 6D, respectively), cells are packed with lipid bodies. Also, in what concerns mitochondria morphology, these images clearly show differences in terms of subcellular features of localized prostate tumours and the low tumorigenic model LNCaP cell line in comparison with the cells derived from highly metastatic tumours (PC3). Analysis of the mitochondrial genome reflects a decrease in the efficiency of PC3 cell lines through increased expression of UCP2 protein A complete mtDNA sequencing of the cell lines LNCaP and PC3 (as two clearly distinct models of disease progression) was performed (Table 6). The results showed that the LNCaP haplotype can be affiliated in the European haplogroup H26c, displaying the coding private mutation in ND5 gene at position 13227, which is a synonymous polymorphism. The PC3 haplotype belongs to another European haplogroup, denominated U5a1c, and bears two non synonymous Role of MCTs in Prostate Carcinoma Pértega-Gomes N. 126 recent mutations: in gene ND4, at position 11120, leading to the F121L amino acid replacement, which has a MutPred pathogenicity score of 0.423; and in gene ND5, at position 13802, conducting to the amino acid replacement T489M, having a MutPred pathogenicity score of 0.6. We have inferred, on the basis of a large population dataset of mtDNA genomes, that the value of MutPred score dividing pathogenic mutations from tolerated mutations is around 0.7. The two nonsynonymous mutations in PC3 cell line have scores below this threshold, but we do not know yet if the mutations can contribute additively to a phenotype. As both PC3 mutations are located in proteins of the Complex I, if they have some functional impairment in the oxidative phosphorylation it will be upon this complex. Investigating Complex I activity in prostate cell lines we observed that in contrast with we were expecting, PC3 cells show increased Complex I activity (Figure 7A). However the increased Complex I activity in PC3 cells was not reflected in global cellular ATP levels (Figure 7B). These results suggest that the proton electrochemical gradient across the inner mitochondrial membrane (IMM) is being dissipated and not used for ATP production. In fact, investigating the expression levels of the mitochondrial uncoupling protein 2 (UCP2), we verified that it is significantly higher in PC3 cells, both using β-actin protein (p=0.02) as total protein loading, and the translocase of outer mitochondrial membrane 20 homolog (TOMM20) (p<0.01), as a mitochondrial protein loading indicating that PC3 cells exhibit few mitochondria and thus confirming our ultrastructural data (Figure 8A-D). Public genome-wide expression profiling datasets show association of metabolic-related proteins with disease progression By making use of publicly available genome-wide expression profiling datasets, we immediately observed that the mRNA levels of genes involved in the glycolytic pathway were higher in metastatic tumours compared to localized tumours (Figure 9A), whereas, the differences in the expression patterns observed for the genes involved in the fatty acid oxidation pathway were less striking (Figure 9B). Role of MCTs in Prostate Carcinoma Pértega-Gomes N. 127 Interestingly, looking to the genes that codify for lactate transporter proteins, only the mRNA levels of SLC16A3, the gene that codifies for MCT4, the isoform responsible for lactate extrusion were higher in metastatic compared to localized tumours (Figure 9E). Finally, the Glinsky dataset showed again SLC16A3 gene being associated with the time to biochemical recurrence after surgery (Figure 10). Discussion While numerous studies have investigated the involvement of altered cellular metabolism in various tumours, little is known about the metabolic alterations during prostate cancer progression. Costello et al. [25,26] described the transformation of a citrate-producing epithelial cell to a malignant citrate oxidizing, which must be an early event for malignancy and progression. Additionally, Shan Zha et al described the selective upregulation of peroxisomal branched chain fatty acid β-oxidation pathway in prostate cancer [17], which might represent a good source of acetylCoA for Krebs cycle, and later, Ranasinghe W. et al [27] linked hypoxia with aggressiveness and metastasis in prostate cancer. However, whether the expression of proteins involved in cellular energetic metabolism is altered during prostate malignant transformation and progression, as well as, the significance of their expression is largely unknown. In this study, we found a consistent overexpression of proteins involved in peroxisomal branched chain fatty acid oxidation in prostate cancer as well as in PIN lesions in contrast to benign glands, suggesting a possible etiological role of this pathway in malignant transformation. In the other hand, the expression of proteins involved in the glycolytic pathway showed association with reliable predictors of poor prognosis such as tumour stage (pT), Gleason score and biochemical recurrence, suggesting a role in disease progression. Accordingly, our in vitro studies showed that different models of disease progression exhibited different metabolic profiles. The cell lines derived from distant metastasis are more glycolytic and strongly express MCT4 and GLUT-1 Role of MCTs in Prostate Carcinoma Pértega-Gomes N. 128 at the plasma membrane, two major players in the glucose uptake and lactate extrusion essential for the proper function of the glycolytic pathway. Importantly, a change in the subcellular localization of these proteins from localized tumour to metastatic tumour was verified, with MCT4, GLUT1 and CAIX being present at the plasma membrane only in metastatic samples, suggesting an obvious link between the plasma membrane function of these proteins and the aggressive stage of the tumour. Using inhibitors of the different pathways we were able to support the idea that different metabolic pathways are active according to the disease stage of the tumour. Thus, targeting fatty acid oxidation seems to be more relevant in an early stage in contrast to the glycolytic pathway, which will produce more effects in an advanced stage of the disease. Additionally, differences were also evident at the organelle content level, between low and high tumorigenic models, which led us to investigate the idea of a less energetically efficient respiration of the highly tumorigenic models. In fact, we found an false increase in Complex I activity in PC3 cells masked by the overexpression of UCP2 protein, justifying a coupling defect in oxidative phosphorylation in these cells. Interestingly, a coupling defect in oxidative phosphorylation was also described in mitochondrion-rich (Hürthle or oncocytic) tumours of the thyroid [28-31]. Finally, publicity dataset supported our main findings showing that the expression of genes involved in fatty acid oxidation mostly changed from benign samples to localized tumour specimens, suggesting a clear role mainly in prostate malignant transformation rather than progression, whereas, genes involved in the glycolytic metabolism are aberrantly expressed mainly in the metastatic samples, being more compatible with a role in prostate cancer progression and aggressiveness, and drawing attention in this case for SLC16A3 gene that codifies MCT4 as a predictive marker of disease progression and aggressiveness. This data also indicates that prostate cancer does not fit in the traditional model of a metabolic switch to glycolysis from non malignant to malignant cells but instead this metabolic switch is more likely to Role of MCTs in Prostate Carcinoma Pértega-Gomes N. 129 be involved in the advanced stage of the disease and importantly prior to pathologic transformation, exhibiting a predictive value. To the best of our knowledge, this is the largest study investigating the role of key metabolic-related proteins in relation to outcome of radical treatment to localized prostate cancer, putting together a large variety of clinical samples and in vitro models, demonstrating the metabolic heterogeneity of prostate cancer and its significance as diagnostic, prognostic and therapeutic tool in prostate cancer disease. Acknowledgements NPG and SS received fellowships from the Portuguese Foundation for Science and Technology (FCT), refs. SFRH/BD/61027/2009 and PTDC/SAUMET/113415/2009 respectively. This work was supported by the FCT grant ref. PTDC/SAU-MET/113415/2009, under the scope of “Programa Operacional Tematico Factores de Competitividade” (COMPETE) of “Quadro Comunitario de Apoio III” and co-financed by Fundo Comunitario Europeu FEDER. This work was partially supported by the project (PIC/IC/83037/2007). Further funding was obtained from the project ‘Microenvironment, metabolism and cancer’ based at IPATIMUP and partially supported by Programa Operacional Regional do Norte (ON.2—O Novo Norte), under the Quadro de Referência Estratégico Nacional (QREN), and through the Fundo Europeu de Desenvolvimento Regional (FEDER). IPATIMUP and ICVS/3Bs are associate laboratories of the Portuguese Ministry of Science, Technology and Higher Education and is partially supported by the FCT. 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Details of the immunohistochemical procedure used to analyze the expression of the different proteins. Protein Antibody Company Antibody Dilution Positive Control Incubation Period Detection System MCT4 sc-50329 Santa Cruz Biotechnology 1:500 Colon tumor Overnight R.T.U. Vectastain Universal Elite ABC Kit, Vector, EUA MCT2 sc-50322 Santa Cruz Biotechnology 1:200 Muscle 2 hours Ultravision Detection System Anti-polyvalent, HRP, Labvision Corporation, Freemont, CA CD44 156-3C11 Serotec 1:1000 Colon tumor GLUT1 ab 15309 Abcam 1:2000 Head and neck tumor CAIX ab 15086 Abcam 1:2000 Stomach MCT1 sc-365501 Santa Cruz Biotechnology 1:500 Colon tumor Overnight R.T.U. Vectastain Universal Elite ABC Kit, Vector, EUA HIF-1α 610958 BD Biosciences 1:100 Glioblastoma GLUT12 ab 75441 Abcam 1:500 Rim Overnight Ultravision Detection System Anti-polyvalent, HRP, Labvision Corporation, Freemont, CA LDHV ab 53010 Abcam 1:1000 Colon tumor 2 hours HKII ab104836 Abcam 1:750 Colon tumor PDK1 ab110025 Abcam 1:500 Stomach CD147 sc-71038 Santa Cruz Biotechnology 1:400 Colon tumor AMACR 504R-16 Cell Marque 1:50 Kidney ACOX3 sc-135435 Santa Cruz Biotechnology 1:250 Liver DBP DBP antibody was a gift from Dr. Gabriele Moller from HelmholtzZentrum mÜnchen. Ready to use Kidney