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 Candida u a ao g au de Dou o em Ciências (Especialidade em: Pa ologia e Gené ica Molecula ), subme ida ao Ins i u o de Ciências Biomédicas Abel Salaza da Uni e sidade do Po o. O ien ado – Ma ia de Fá ima Monginho Bal aza Ca ego ia – P o esso a Auxilia A iliação – Ins i u o de Ciências da Vida e Saúde, B aga, Po ugal; Escola de Ciências da Saúde, Uni e sidade do Minho, B aga, Po ugal. Co-o ien ado – Ca los Albe o da Sil a Lopes Ca ego ia – P o esso Ca ed á ico A iliação – Ins i u o de Ciências Biomédicas Abel Salaza da Uni e sidade do Po o, Po o, Po ugal. A ese de dou o amen o aqui ap esen ada oi desen ol ida no âmbi o de inanciamen o pela Fundação pa a a Ciência e Tecnologia (FCT) a a és de uma bolsa indi idual de dou o amen o, com a e e ência SFRH/BD/61027/2009, e do p ojec o “Role o Monoca boxyla e T anspo e s (MCTs) in he me abolism o p os a e cance ”, com a e e ência PTDC/SAL-MET/113415/2009, no âmbi o do P og ama Ope acional Temá ico Fac o es de Compe i i idade (COMPETE) do Quad o Comuni á io de Apoio III e co- inanciado pelo Fundo Comuni á io Eu opeu FEDER. ACKNOWLEDGEMENTS I would like o hank my supe iso P o . Fá ima Bal aza , o gi ing me he oppo uni y o en e in his “Scien i ic Wo ld”. Thank you o in oducing me o Cance and Me abolism esea ch ield and o always gi ing me he oppo uni y/libe y o es ablish collabo a ions wi h o he esea ch g oups du ing my PhD. This was ex emely aluable du ing my PhD jou ney. To P o esso Ca los Lopes as co-supe iso o his wo k and Di ec o o he Pa hology and Molecula Gene ics PhD P og amme o allowing me o join his Cou se, u ning his jou ney in o a eal lea ning expe ience. A e y special hanks o D . José Ramon Vizcaino o accep ing me a he Pa hology Depa men o Cen o Hospi ala do Po o and o accep o be pa o ou P ojec . Thank you o you scien i ic supe ision, op imism, pa ience, ideas and iendship. My hesis would no be he same wi hou ou equen scien i ic/specula i e alks. Thank you so much!!! A special hank o P o esso Da id Neal and Lee F ye a Cance Resea ch UK Camb idge Ins i u e/Uni e si y o Camb idge o allowing me o join hei lab and de elop my skills and scien i ic knowledge in such a high-quali y esea ch Ins i u e. A a p o essional and pe sonal le el his was a huge expe ience o me. Thanks o s ill belie ing my wo k. I can jus p omise ha I will always do my bes o co espond you expec a ions. Thank you!! Many hanks o all ha in one way o ano he ha e collabo a ed wi h me, ecei ing me in mos cases a hei labs. P o esso Rui Hen ique, P o esso Ca men Je ónimo and P o esso Alexand e Lobo da Cunha. I also would like o hank Elsa Oli ei a and Ca los Gou eia, hank you all. A special hanks o D a. Daniela Ribei o and D . Valdema Máximo, many hanks o belie e in my ideas and o helping me o each my aims!! I also wan o hank all my iends inside and ou side he lab: A ICVS I would like o hank Ca ina and Rica do. Thanks o all he good momen s inside and ou side he lab, I am su e ha we will be iends o e e no ma e wha . Filipe Pin o and Isabel Valença my dea pa ne s in pa y and wo k. I is indeed possible o es ablish good collabo a i e wo k and ha e un a he same ime. I hink i is one o he bes combina ions e e . Thanks o ha !! To “HOXAs” g oup, hank you gi ls o all he unny momen s, lunch imes, dinne s, co ees and so on. Wish you all he bes . My iends and collgues a CRI: Vinny, Mohammad, Cha lie, Sa ah, Jan, Ka an, Helen, S e e, Ha ee , An onio o all hei iendship and suppo and o allowing me o lea n new hings om you expe ience and o sha ing wi h me expe ise inside and ou side he lab. A e y special hanks o Sa ah Ju meis e who was my e e y hou suppo in many di e en ways. Thank you o always be a ailable o each me some hing new and o you suppo and encou agemen o be and o do be e . Thank you o all you s a is ical suppo and e eshing ideas abou how o look a my esul s. To all my iends ou o he lab, especially Sil ia, Bia, Liliana, Vânia e Do a, Aldina and Diana o all he good imes, o hei suppo , iendship and o accep ing me as I am u ning e e y momen ha I was down in o a good momen . Fo always encou aging me o he bes , hank you my iends. To Julia and Jjonas o being he bes housema es e e !!! Finally I would like o show all my g a i ude o my amily, namely my pa en s and b o he , who always suppo ed me. Thank you o helping me o deal wi h my us a ions and ba ie s along his jou ney. Thank you o aising me and o all he encou agemen o be a be e pe son gi ing me he eedom o do my choices and lea n om hem. I would like o dedica e his hesis o all o you!! We canno do science alone!!! DIRETIVAS LEGAIS No cump imen o do dispos o no Dec e o de Lei n º 230/2009, decla a-se que a au o a des a disse ação pa icipou a i amen e na execução do abalho expe imen al que es e e na o igem dos esul ados ap esen ados bem como na edação dos espe i os manusc i os. Fazem pa e des a ese os seguin es a igos publicados ou em p epa ação: Pé ega-Gomes N, Vizcaíno JR, Mi anda-Gonçal es V, Pinhei o C, Sil a J, Pe ei a H, Mon ei o P, Hen ique RM, Reis RM, Lopes C, Bal aza F. “Monoca boxyla e anspo e 4 (MCT4) and CD147 o e exp ession is associa ed wi h poo p ognosis in p os a e cance .” BMC Cance . 2011 Jul 25;11:312. doi: 10.1186/1471-2407-11-312. (Ci ações ISI: 19) Pé ega-Gomes N, Vizcaíno JR, Gou eia C, Je ónimo C, Hen ique RM, Lopes C, Bal aza F. “Monoca boxyla e anspo e 2 (MCT2) as pu a i e bioma ke in p os a e cance .” P os a e. 2013 May;73(7):763-9. doi: 10.1002/p os.22620. Epub 2012 No 28. Pé ega-Gomes N, Valença I, Vizcaíno JR, Ribei o D, Bal aza F. “Localiza ion o MCT2 a pe oxisomes is associa ed wi h malignan ans o ma ion in p os a e cance ” o be published in an in e na ional scien i ic pe iodical wi h e e ees. Pé ega-Gomes N, Vizcaíno JR, Sousa S, Coelho R, A ig J, Ju meis e S, Oli ei a E, Pe ei a L, Pinhei o C, Je ónimo C, Hen ique RM, Lobo da Cunha A, Lopes C, Maximo V and Bal aza F. “Me abolic he e ogenei y in p os a e cance is linked o disease p og ession and agg essi eness” o be published in an in e na ional scien i ic pe iodical wi h e e ees. Pé ega-Gomes N, Vizcaíno JR,Gou eia C, Lopes C and Bal aza F. “A lac a e shu le sys em be ween umou and s omal cells is associa ed wi h poo p ognosis in p os a e cance ” o be published in an in e na ional scien i ic pe iodical wi h e e ees. Pé ega-Gomes N and Bal aza F. “Explo a ion o MCTs as po en ial he apeu ic a ge s in p os a e cance ” in i ed wo k o be published in Ad ances in P os a e Cance Resea ch and T ea men jou nal. TABLE OF CONTENTS Abb e ia ion Lis …………………………………………………………….…i Abs ac …………………………………………………………………………iii Resumo……………………………………………………………………….... Aims and Thesis Layou ……………………………………………………... ii Chap e 1. Gene al In oduc ion………………………………………......3 1.1 Rep og amming ene gy me abolism as an eme ging hallma k o cance ………………………………………………………………………3 1.1.1 The Wa bu g e ec as he basis o explo ed al e ed cellula Me abolism in cance …………………………………………………………4 1.1.2 A Me abolic swi ch ha con e s ad an age o cance cells…...6 1.1.2.1 Why cance cells p e e ae obic glycoysis…………………...6 1.1.2.2 A deepe insigh in o hypoxia and Lac a e expo : G ea con ibu o s o malignancy………………………………………9 1.2 Monoca boxyla e T anspo e s (MCTs) in he con ex o cellula me abolism…………………………………………………………….......13 1.2.1 The MCT amily…………………………………………………….....14 1.2.1.1 MCT1……………………………………………………………......16 1.2.1.2 MCT2……………………………………………………………......17 1.2.1.3 MCT4………………………………………………………….…….18 1.2.1.4 O he MCT iso o ms…………………………………………..….18 1.2.2 MCTs egula ion………………………………………………………20 1.2.2.1 T ansc ip ional Le el……………………………………………..20 1.2.2.2 Pos -T ansc ip ional Le el………………………………………22 1.2.2.3 T anspo e Ac i i y Le el………………………….…………....23 1.2.2.4 Ho monal Regula ion………………………………………….....25 1.2.3 MCTs inhibi ion………………………………………………….……25 1.2.3.1 The Classical MCT Inhibi o : CHC………………………..........26 1.2.3.2 Speci ic MCT Inhibi o s…………………………………………..27 1.2.3.3 O he MCT inhibi o s……………………………………………..27 i ep esen p omising he apeu ic a ge s in di e en phases o neoplas ic ans o ma ion and p og ession. RESUMO O concei o de al e ações me abólicas so idas pelas células umo ais oi ecen emen e incluído nas p incipais al e ações so idas no co ex o de ans o mação maligna. Uma das p incipais ca ac e ís icas me abólicas das células umo ais consis e no seu ele ado consumo de glucose e consequen e p odução de lac a o. De modo a assegu a o ápido e luxo de lac a o, a maio ia das células malignas exp essam ele ados ní eis de anspo ado es de monoca boxila os (MCTs). A inibição des es anspo ado es em sido ap esen ada como um bom al o pa a quimio e apia e á ios es udos in i o demons a am já o po encial des a abo dagem, no en an o, no con ex o do ca cinoma da p ós a a o impac o da inibição MCTs assim como a sua egulação du an e a p og essão umo al con inua em g ande pa e desconhecida. Adicionalmen e, ainda não es á cla o qual é a ia me abólica p edominan e no canc o da p ós a a e, po an o, qual a que se ap esen a como al o mais ap op iado pa a a e apia. O p incipal obje i o des e abalho oi in es iga a exp essão e egulação dos MCTs du an e a p og essão do ca cinoma da p ós a a e se es es anspo ado es pode iam ep esen a al os p omisso es no a amen o des a doença. A exp essão de MCTs jun amen e com ou as p o eínas elacionadas com o me abolismo oi es udada usando uma g ande sé ie de ecidos de p ós a a humana e o seu alo clínico-pa ológico oi a aliado. Es udos in i o o am ealizados a im de comp eende como a iam as exigências me abólicas das células du an e a p og essão umo al e de que o ma es as mudanças pode iam ep esen a al os e apêu icos pa a a doença, dando especial ên ase aos MCTs nes e con ex o. Os esul ados ob idos indicam que o ca cinoma da p ós a a não é ão glicolí ico como ou os ipos de umo e que o aumen o da glicólise é p incipalmen e encon ado num es adio mais a ançado da doença. Foi obse ado que di e en es iso o mas de MCTs es ão di e encialmen e exp essas em di e sas ases de p og essão do ca cinoma da p ós a a e po encialmen e elacionados com di e en es ias me abólicas e po an o não es i as ao seu clássico papel de anspo ado es de lac a o na memb ana ci oplasmá ica e que a inibição das di e en es iso o mas em di e en es condições a e a a iabilidade e p oli e ação i das células malignas. Em conclusão, os esul ados ap esen ados nes a ese são pionei os na comp eensão das exigências me abólicas do ca cinoma da p ós a a du an e a p og essão umo al, con ex ualizando os MCTs como possí eis al os e apêu icos em di e en es ases da ans o mação neoplásica e p og essão maligna. ii AIMS AND THESIS LAYOUT Me abolic changes du ing malignan ans o ma ion ha e been no ed o many yea s. O o Wa bu g i s epo ed ha cance cells p e e en ially ely on glycolysis o ene gy p oduc ion, e en in he p esence o oxygen, leading o p oduc ion o high le els o lac a e. The c ucial ole o lac a e e lux and exchange wi hin he umou mic oen i onmen d ew a en ion o monoca boxyla e anspo e s (MCTs). MCTs ha e been poin ed as p omising a ge s in cance he apy and desc ibed in a la ge a ie y o umou s, howe e , s udies showing how hese iso o ms con ibu e o he acquisi ion o he malignan pheno ype a e sca ce and ega ding p os a e cance i is o ally unknown. The main aim o his hesis was o cha ac e ize MCTs exp ession in p os a e issues, namely p os a e cance , as well as o gi e a con ibu ion o unde s and he ole o MCTs in p os a e cance main enance and p og ession p o iding e idence o he exploi a ion o MCTs as po en ial a ge s o p os a e cance he apy. This hesis is o ganized in 7 ela ed chap e s, aiming o show he mos impo an esul s ob ained du ing he de elopmen o his scien i ic p ojec . In Chap e 1 a gene al in oduc ion o he Hallma ks o cance wi h a special emphasis o al e ed cellula me abolism as a new hallma k o cance and he “Wa bu g e ec ” as he basis o explo e MCTs as sui able a ge s o cance he apy is p o ided. Also, he cu en knowledge on p os a e cance me abolism is e iewed in his chap e . In Chap e s 2-6 majo indings abou me abolic changes ac oss p os a e cance malignan ans o ma ion as well as he alue o using MCTs as a ge s o p os a e cance he apy is p esen ed. These chap e s include esul s al eady published o submi ed o publica ion in in e na ional scien i ic pe iodicals wi h e e ees, gi ing a con ibu ion o unde s and he ole o MCTs in he con ex o p os a e cance . In Chap e 2 he exp ession o MCTs in p os a e issues and he clinico-pa hological alue o his exp ession is p esen ed. Chap e 3 explo es mo e deeply he ole o MCT2 in p os a e cance me abolism. In Chap e 4 a panel o key me abolic- ela ed p o eins exp ession was s udied in an e en bigge casuis ic o p os a e issues. In i o assays we e also pe o med showing ii impo an me abolic al e a ions ac oss p os a e cance malignan ans o ma ion and how MCTs inhibi ion a ec s p os a e cance cells in di e en umou en i onmen condi ion. In Chap e 5 an e o o unde s and he me abolic al e a ions in he s oma ha su ounds p os a e umou s is p esen ed. Finally, Chap e 6 p esen s an insigh on MCTs egula ion ac oss p os a e cance p og ession and in Chap e 7, he main conclusions o Chap e s 2-6 a e summa ized and some impo an u u e di ec ions a e sugges ed. Each o hese chap e s has hei own bibliog aphy sec ion. Role o MCTs in P os a e Ca cinoma Pé ega-Gomes N. CHAPTER 1. GENERAL INTRODUCTION Role o MCTs in P os a e Ca cinoma Pé ega-Gomes N. Role o MCTs in P os a e Ca cinoma Pé ega-Gomes N. 3 CHAPTER 1. GENERAL INTRODUCTION 1.1 Rep og amming o ene gy me abolism as an eme ging Hallma k o Cance Ad ances in cance esea ch ha e gene a ed a ich bu also ex emely complex knowledge e ealing cance as a disease ha in ol es se e al dynamic changes. In o de o o ganize he complexi y o cance , he biological capabili ies acqui ed du ing he mul is ep de elopmen o human umou s and sha ed by all cance s we e g ouped and denomina ed as he “hallma ks o cance ”. Sus aining p oli e a i e signaling, e ading g ow h supp esso s, esis ing cell dea h, enabling eplica i e immo ali y, inducing angiogenesis, and ac i a ing in asion and me as asis we e he ai s es ablished in o de o c ea e an o ganized p inciple ha p o ides a logical amewo k o unde s and he di e si y o neoplas ic disease [1]. La ely, new hallma ks ha e eme ged and ep og amming o ene gy me abolism was conside ed an eme ging hallma k o cance since i was ecognized ha he ch onic and uncon olled cell p oli e a ion ha ep esen s he essence o neoplas ic disease in ol es also adjus men s o ene gy me abolism in o de o uel cell g ow h and di ision [2]. Figu e 1 ep esen s he las e sion o he “Hallma ks o cance ”. Figu e 1. The Hallma ks o Cance [2]. Role o MCTs in P os a e Ca cinoma Pé ega-Gomes N. 4 1.1.1 The Wa bu g e ec as he basis o explo e al e ed cellula me abolism in cance Al e ed ene gy me abolism is being p o ed o be as widesp ead in cance cells as many o he o he cance -associa ed ai s ha ha e been accep ed as hallma ks o cance . Howe e jus ecen ly a majo ele ance has been gi en o cance me abolism, he obse a ion ha umou cells exhibi an al e ed me abolism when compa ed o no mal cells was made almos one cen u y ago by he Nobel P ize winne O o Wa bu g who desc ibed i as he i s umou speci ic-al e a ion [3]. Wa bu g desc ibed ha in con as o non-malignan cells, many ypes o cance cells p e e glycolysis as he ATP sou ce, e en in he p esence o abundan oxygen. I unde ae obic condi ions, no mal cells p ocess glucose, fi s o py u a e ia glycolysis in he cy osol and hen o ca bon dioxide in he mi ochond ia, unde anae obic condi ions, glycolysis is a o ed and ew py u a e is send o he oxygen-consuming mi ochond ia. Wa bu g fi s obse ed ha cance cells can ep og am hei glucose me abolism, and hus hei ene gy p oduc ion, by limi ing hei ene gy me abolism la gely o glycolysis, leading o a s a e ha has been called ‘‘ae obic glycolysis’’ (Figu e 2). Figu e 2. Schema ic ep esen a ion o he di e ences be ween OXPHOS, anae obic glycolysis and “ae obic glycolysis” (Wa bu g e ec ) [4]. Role o MCTs in P os a e Ca cinoma Pé ega-Gomes N. 5 Glycolysis, he i s majo pa hway o cellula me abolism, occu s in he cy oplasm and is unc ional e en in he absence o oxygen. While glycolysis is able o p oduce ATP a high a es, i is conside ed a low e iciency pa hway because i p oduces only wo ATP molecules pe glucose. In o de o compensa e o he lowe e ficiency o ATP p oduc ion ob ained by glycolysis ela i e o mi ochond ial oxida i e phospho yla ion, cance cells ma kedly inc eased up ake and u iliza ion o glucose in many human umo ypes being he a e o en y highe han in no mal cells [5-7]. This has p o en use ul o de ec umou s and also moni o hei ea men , as i is he basis o he clinical use o posi on emission omog aphy (PET) ha uses a adiolabeled analog o glucose (18F-fluo odeoxyglucose, FDG) as a epo e [8]. FDG is ecognized as a subs a e o glucose anspo sys ems; hus, he a e o en y o his glucose de i a e in o cells is de e mined by he ac i i y o glucose anspo sys ems. Inside he cells, he accumula ion o he adioac i e glucose analogue se es as a ead-ou o he a e o glucose en y in o cells. Since umou cells exhibi enhanced glucose up ake compa ed o adjacen no mal cells, PET is able o de ec umou s and di e en ia e hem om no mal issue [9, 10] (Figu e 3). Figu e 3. PET imaging wi h FDG o a pa ien wi h lymphoma. The medias inal nodes (pu ple a ow) and sup a incula nodes (g een a ows) show high up ake o FDG, showing ha umou s in hese nodes ha e high le els o FDG up ake. The bladde (yellow a ow) also has high ac i i y, because o exc e ion o he adionuclide [11]. Role o MCTs in P os a e Ca cinoma Pé ega-Gomes N. 12 accumula ion o HIF1-alpha, which would lead in u n o a s imula ion o he glycoly ic pa hway by egula ing glycoly ic enzymes, p o iding an impo an posi i e eedback in he con ex o cance . Mo eo e , lac a e was demons a ed o s imula e molecules in ol ed in he p ocess o cance in asion and me as asis. CD44, VEGF and ans o ming g ow h ac o (TGFB2) a e some o hem [35-39]. Besides glycolysis, i is impo an o ecognize ha he e a e o he pa hways ha can also con ibu e o lac a e p oduc ion in solid umou s such as glu aminolysis and se inolysis (Figu e 7) [40,41]. Figu e 7. O e iew o he me abolic pa hways leading o lac a e p oduc ion in cells (con inuous lines). The discon inuous a ows indica e lac a e up ake and low inside oxida i e cance cells. Besides being mainly an end-p oduc , lac a e expo ed migh also be a subs a e o neighbo cells, a phenomenon known as “cell-cell lac a e shu le”, in which he pe iphe al and oxygena ed oxida i e cells consume he lac a e p oduced by he cen al and hypoxic glycoly ic cells (Figu e 8). Role o MCTs in P os a e Ca cinoma Pé ega-Gomes N. 13 Figu e 8. Scheme ep esen ing in a umou al hypoxia and me abolic symbiosis. The c ucial ole o lac a e e lux and exchange wi hin he umou mic oen i onmen , d ew a en ion o monoca boxyla e anspo e s (MCTs) which anspo monoca boxyla es such as lac a e ac oss he memb anes, and he e o e play a cen al ole in cellula me abolism and me abolic communica ion be ween issues. 1.2 Monoca boxyla e T anspo e s (MCTs) in he con ex o cellula me abolism. The anspo o monoca boxyla es ac oss he plasma memb ane was o iginally hough o be ia non-ionic di usion o he ee acid, howe e , he demons a ion ha lac a e and py u a e anspo in o human e y h ocy es could be s ongly inhibi ed a e ea men wi h chemicals, allowed he iden i ica ion o a speci ic monoca boxyla e anspo mechanism. The monoca boxyla e anspo was hen cha ac e ized ex ensi ely in di e en cell ypes and he obse ed cha ac e is ics led o he a ionale o o he exis ence o a amily o monoca boxyla e anspo e s [42-43]. Role o MCTs in P os a e Ca cinoma Pé ega-Gomes N. 14 1.2.1 The MCT Family MCTs a e encoded by he SLC16 gene amily, which is conse ed among species, including a , mouse, chicken and o he s. The amily is p esen ly composed by 14 membe s, iden i ied h ough sc eening o genomic and exp essed sequence ag da abases [43]. These p o eins ca alyse he anspo o lac a e wi h a p o on, wi h no ene gy inpu in ol ed in his p ocess [42, 44]. I is p edic ed ha he opology o MCTs consis s o 12 ansmemb ane domains (TMDs) wi h he N- and C- e mini loca ed in he cy oplasm, as illus a ed in Figu e 9 o MCT1. The TMDs a e highly conse ed among iso o ms wi h he g ea es sequence a ia ions obse ed in he C- e minus and he la ge in acellula loop be ween TMDs 6 and 7, which has a ange o 29-105 amino acid esidues [45] . This obse ed a iabili y is common o anspo e s wi h 12 TMDs and i is hough ha hese sequence a ia ions a e ela ed o subs a e speci ici y o egula ion o anspo ac i i y [44, 45]. Theo e ical p edic ions and expe imen al e idence indica e ha none o he MCT amily membe s is glycosyla ed [46-49]. To unc ion, an MCT ansloca es a p o on and a monoca boxyla e h ough he plasma memb ane by an o de ed mechanism in which H+ binding is ollowed by monoca boxyla e binding o he p o ona ed anspo e [49, 50]. The e o e, MCT ac i i y is dependen on bo h besides subs a e concen a ion and p o on g adien be ween he ex acellula and in acellula milieus. Figu e 9. P oposed opology o MCT1. The model shown is ha p edic ed om he p ima y sequence using hyd opa hy plo s and subsequen ly con i med by p o eoly ic clea age and labelling expe imen s as desc ibed by Juel & Hales ap [44]. Role o MCTs in P os a e Ca cinoma Pé ega-Gomes N. 15 Lac a e is indeed he monoca boxyla e whose anspo ac oss he plasma memb ane is quan i a i ely mo e impo an , howe e , MCTs a e also impo an o he anspo o many o he me abolically impo an monoca boxyla es such as py u a e, he b anched-chain oxoacids de i ed om leucine, aline and isoleucine, and he ke one bodies ace oace a e, β-hyd oxybu y a e and ace a e [45]. Consequen ly, MCTs ha e a cen al ole in mammalian cell me abolism and a e c i ical o he communica ion be ween cells as illus a ed in Figu e 10. Figu e 10. Me abolic pa hways in ol ing monoca boxyla e anspo ac oss he mi ochond ial and plasma memb anes [45]. Besides being a amily o 14 membe s, only he i s ou (MCT1-MCT4) ha e been demons a ed expe imen ally o acili a e he p o on-linked anspo o me abolically impo an monoca boxyla es [46-49]. Since MCT3 is a e y specialized MCT, being limi ed o he e inal pigmen and cho oid plexus epi helia [50,51] his in oduc ion will only ocus on MCT1, MCT2 and MCT4 iso o ms, whose unc ion is esponsible o he name o his amily o anspo e s [52-56]. Role o MCTs in P os a e Ca cinoma Pé ega-Gomes N. 16 Figu e 11 shows ha MCT1-MCT4 a e associa ed in he same clus e p esen ing high homology. Impo an ly, his clus e is ye sub-di ided in wo b anches MCT1-2 and MCT3-4 which co ela es wi h he anspo ed subs a e as well as he a ini ies by which hose anspo is pe o med. Figu e 11. Human MCT amily membe s’ phylog am, based on amino acid sequence. Boxes limi ed by do s ep esen he main clus e s [57]. 1.2.1.1 MCT1 Human MCT1 gene, SLC16A1 is loca ed on ch omosome 1 (1p13.2-p12) and comp ises 5 coding exons. MCT1 unc ional p o ein is composed by 500 amino acids and has a molecula weigh o 53.958 Dal ons [58]. Among he MCT amily, MCT1 iso o m is he mos well s udied and unc ionally cha ac e ized membe , since i is he only MCT exp essed in human e y h ocy es. Al hough ubiqui ously exp essed, MCT1 is especially p ominen in hea and ed muscle, whe e i is up- egula ed in esponse o inc eased wo k, sugges ing a special ole in lac ic acid oxida ion [59,60]. The wide pa e n o exp ession obse ed o MCT1 may be explained by i s subs a e a ini ies ha indica e ha Role o MCTs in P os a e Ca cinoma Pé ega-Gomes N. 17 MCT1, may be in ol ed in bo h up ake and e lux o monoca boxyla es om cells. MCT1 anspo s a a ie y o subs a es including sho chain (C2-C5) unb anched alipha ic monoca boxyla es such as ace a e and p opiona e. Monoca boxyla es wi h C2 o C3 subs i u ions (excluding amino- and amido-) a e also anspo ed o e en p e e ed (e.g py u a e, L-lac a e, ace oace a e and β- hyd oxybu y a e) (35- 37c). The simples monoca boxyla e, o ma e, is a poo subs a e whe eas bica bona e; dica boxyla es, ica boxyla es and sulphona es a e no anspo ed. Also MCT1 is s e eoselec i e o lac a e bu no o β-hyd oxybu y a e. MCT1 was also ound in mi ochond ia [61,62] and pe oxisomes [63], whe e i is belie ed o pa icipa e in a lac a e oxida ion complex o main ain o ganelle edox and p ope unc ioning. 1.2.1.2 MCT2 MCT2 is encoded by he SLC16A7 gene loca ed in ch omosome 12 (12q13) and comp ises 5 coding exons [64]. Al hough only one ansc ip is iden i ied, he e is e idence o al e na i ely spliced mRNA species in human and a [51, 63], bu no e idence o splice a ian s o he p o ein. This iso o m sha es app oxima ely 50% sequence iden i y wi h MCT1, con ains 478 amino acids and has a molecula weigh o 52,186 Da. The ac ha , when bo h MCT1 and MCT2 a e exp essed in he same issue, he exp ession pa e n is cell-speci ic, sugges ed a dis inc unc ional ole be ween hese iso o ms [64-68]. Subsequen exp ession o MCT2 in Xenopus oocy es e ealed i s unique biochemical ea u e o acili a ing he p o on-linked anspo o especially py u a e, wi h a conside able high a ini y, suppo ing he p e ious e idence o an al e na i e biological ole [69]. As so, MCT2 also ca alysis he p o on-linked anspo o a ange o monoca boxyla es, bu wi h a conside ably highe a ini y han MCT1. As a highe a ini y anspo e , MCT2 has also a mo e es ic ed exp ession, being adap ed o pe o m he up ake o monoca boxyla es in o cells. As a esul , MCT2 is ound in issues ha use lac a e as a espi a o y uel, like b ain o ca diac and skele al muscle, kidney and li e we e lac a e is he majo gluconeogenic subs a e Role o MCTs in P os a e Ca cinoma Pé ega-Gomes N. 18 [66,67,69,70]. As MCT1, MCT2 is also ound in mi ochond ia [71,72] and pe oxisomes [63]. 1.2.1.3 MCT4 MCT4 is encoded by he human SLC16A3 gene, which is loca ed in ch omosome 17 (17q25.3), comp ises 5 exons and 3 ansc ip s, wi h di e en ini ia ion si es bu no di e ence in p o ein p oduc has been iden i ied. The p o ein is cons i u ed by 465 amino acids, co esponding o a molecula weigh o 49,469 Da. MCT4 demons a es ema kable simila i ies wi h MCT1 and in con as o MCT2 has a b oade dis ibu ion. The p incipal di e ence be ween MCT4 and MCT1 iso o ms lies on hei speci ic localiza ion and subs a e a ini ies. MCT4 shows a much lowe a ini y o subs a es han MCT1 and MCT2. Acco dingly, MCT4 is p edominan ly exp essed in glycoly ic cells such as whi e muscle and whi e blood cells, sugges ing ha i s physiological unc ion is lac a e e lux and in ac , he kine ic p ope ies o MCT4 show ha his iso o m is adap ed o he expo o lac a e. This led o he hypo hesis ha MCT4 migh be o pa icula impo ance in cells ha ely on high a es o glycoly ic me abolism o mee hei ene gy demands, p oducing high amoun s o lac a e ha need o be apidly expo ed, such as cance cells [73]. 1.2.1.4 O he MCT iso o ms O he MCT iso o ms a e being cha ac e ized in he las yea s. SLC18A2 (MCT8) gene has been desc ibed as a hy oid ho mone anspo e [74] and mu a ions in his gene ha e been associa ed wi h X linked se e e men al e a da ion and neu ological dys unc ion [76-80]. MCT6 (SLC16A5) anspo s bume anide, bu nei he L-lac ic acid o L- yp ophan, in a pH- and memb ane po en ial-sensi i e bu in a non-p o on g adien -dependen manne [81]. MCT9 (SLC16A9) polymo phism was ound o be associa ed wi h al e ed se um u ic acid [82] howe e MCT9 subs a e is s ill unknown. Impo an ly, SLC16A12 (MCT12) has been iden i ied as a possible bioma ke o colon, p os a e and b eas ca cinoma due o gene hype me hyla ion [83] howe e he subs a e o MCT12 is s ill Role o MCTs in P os a e Ca cinoma Pé ega-Gomes N. 19 unknown, as o he emaining membe s o he amily (MCT5, MCT7, MCT11, MCT13 and MCT14). Table 1 summa izes he cha ac e is ics o he human SLC16 amily o anspo e s and Table 2 shows he subs a e a ini ies o he a ious MCT iso o ms in humans and a s. Table 1. The human SLC16 amily o anspo e s [47]. Role o MCTs in P os a e Ca cinoma Pé ega-Gomes N. 20 Table 2. Compa ision o subs a e a ini ies o he a ious MCT iso o ms in human and a [47]. 1.2.2 MCT Regula ion Al hough he egula o y mechanisms o MCT exp ession a e a om being comple ely unde s ood, e idence indica es ha MCTs migh be egula ed a a ious poin s up o he unc ional p o ein. This includes bo h ansc ip ional and pos - ansc ip ional le el [84-87] ha a ec s p o ein amoun s as well as egula o s o anspo e ac i i y, like chape one p o eins. Ho mone egula ion has also been desc ibed o MCTs, as well as egula ion by signaling pa hways like insulin-like g ow h ac o ecep o ype I (IGF-IR) ac i a ion which up- egula es MCT1 [88]. MCT1 exp ession is pa icula ly mo e desc ibed han he o he iso o ms in di e en physiological and pa hological condi ions. Repo s including MCT up- egula ion in skele al muscle in esponse o aining [85, 89-96], down egula ion a e muscle Role o MCTs in P os a e Ca cinoma Pé ega-Gomes N. 21 dene a ion and in lamma o y bowel diseases [97,98], changes in MCT exp ession du ing de elopmen [99-103] o subs a e induced MCT1 up- egula ion [86, 87, 104] a e ela i ely equen . Howe e and impo an ly, egula o y mechanisms a y among MCTs iso o ms, which allows induc ion o speci ic iso o ms upon di e en s imuli, adap ing cells o di e en ene gy demands. 1.2.2.1 T ansc ip ional Le el The analysis o SLC16A1 5’- lanking egion allowed he iden i ica ion o pu a i e binding si e sequences o he ansc ip ion ac o s USF, nuclea ac o -kappaB (NF-kB), ac i a ed p o ein 1 and 2 (AP1 and AP2) and s imula ing p o ein-1 (Sp1). USF1 and USF2 ha e been desc ibed as po en ial ep esso p o eins o MCT1 [105] whe eas NF-kB pa hway has been in ol ed in he bu y a e-induced MCT1 up- egula ion [106]. AP2 has been associa ed o p o ein kinase C (PKC)- dependen s imula ion o he SLC16A1 p omo e [107] . Lac a e-induced inc ease in MCT1 has been linked o ac i a ion o NF-kB and nuclea ac o e y h oid 2 (NF-E2) pa hways, as well as cAMP- esponse elemen - binding p o ein (CREB) and NF-E2 ela ed ac o 2 (N 2) ansc ip ion ac o s, he las h ee elemen s possessing also pu a i e ansc ip ion binding si es in he SLC16A1 5’- lanking egion. The co-ac i a o s pe oxisome p oli e a o -ac i a ed ecep o gamma, co-ac i a o 1 alpha (PGC-1a) [108] and pe oxisome p oli e a o -ac i a ed ecep o alpha (PPAR) [109,110] ha e been associa ed wi h MCT1, bu no wi h MCT2 and MCT4 up- egula ions. P omo e analyses o MCT1, MCT2 and MCT4 ha e shown ha he SLC16A1 p omo e con ains wo pe oxisome p oli e a o -ac i a ed ecep o esponse elemen s (PPRE) while SLC16A7 and SLC16A3 each con ain one PPRE. In exp ession s udies, SLC16A1 was also shown o be ac i a ed by c-myc and n- myc p o o-oncogenes, while he p o-in lamma o y cy okines and TNF-a, ha e also been implica ed in he ansc ip ional con ol o MCT1, by down- egula ing SLC16A1 ansc ip ion [111-113]. Role o MCTs in P os a e Ca cinoma Pé ega-Gomes N. 28 h ough he same anspo e s. Que ce in is also desc ibed as an inhibi o o he monoca boxyla e anspo e iso o m 1 (MCT1). In p e ious s udies, i was demons a ed ha Que ce in ac s as e e sible nonco alen inhibi o o L-lac a e anspo by umou cells o ed blood cells [144, 145]. Mo e ecen ly, a s udy aiming o cla i y he ole o la onoids in he modula ion o MCT1-media ed anspo o hyd oxybu y a e in i o and in i o demons a ed ha Que ce in (among o he s) is an e ec i e inhibi o o MCT1-media ed anspo [146]. Lonidamine is a de i a i e o indazole-3-ca boxylic acid, which o a long ime, has been known o inhibi glycolysis in cance cells. Al hough his ac ion was o iginally a ibu ed o hexokinase inhibi ion [147, 148], u he s udies e ealed ha lonidamine inhibi s lac a e e lux om cance cells h ough inhibi ion o MCT1 and MCT4 [149, 150]. Ac ually, despi e a lack o knowledge o i s p ecise mechanism o ac ion, lonidamine has been e ec i e in clinical ials agains a ious umou s, especially as a sensi ize o o he chemo he apies [151]. 1.3 Monoca boxyla e T anspo e s in he Con ex o Cance 1.3.1 MCTs in human cance . Wha is desc ibed? Rega ding cance esea ch, he e a e al eady se e al e idence o he up egula ion o MCTs in se e al solid umou s, such as colo ec al ca cinomas [152], u e ine ce ix ca cinomas [153], glioblas omas, b eas ca cinomas [154, 155] and lung umou s [156], poin ing o an impo an ole o his anspo e s in he main enance o hese malignancies. The i s epo on MCT exp ession in human umo samples desc ibed a dec ease o MCT1 exp ession in he colonic ansi ion om no mali y o malignancy [157], which was u he suppo ed by a la ge s udy [158]. In b eas cance , silencing o SLC16A1 by gene p omo e hype me hyla ion was sugges ed in 4 o 20 b eas cance cases (20 %). In con as , esul s om ou g oup showed a signi ican inc ease o MCT1 cy oplasmic and plasma memb ane exp ession in b eas ca cinoma, when compa ing o no mal b eas epi helium [154]. MCT4 only showed a signi ican inc ease in umo samples o cy oplasm exp ession, wi h no di e ences in plasma memb ane exp ession. The li e a u e is also con o e sial in lung cance . A s udy Role o MCTs in P os a e Ca cinoma Pé ega-Gomes N. 29 by Koukou akis and collabo a o s, no exp ession o MCTs in no mal lung was ound, while exp ession o MCT1 was ound in all umo s examined and bo h MCT2 and MCT4 we e also exp essed in cance cells. This s udy also analyzed he possible me abolic coope a ion be ween lung cance cells and he umou - associa ed s oma, howe e , umou associa ed s oma exp essed MCTs weakly [15]. In opposi ion, a ecen s udy by ou g oup showed ha no mal lung p esen s a high equency o MCT exp ession and, in ac , MCT4 is less exp essed in umo samples han in no mal epi helium. MCT exp ession has also been desc ibed in some gynecological umo s like ce ical and o a ian cance [159]. In ce ical cance , a signi ican inc ease in o e all and plasma memb ane exp ession o MCT1 and MCT4 was obse ed. In o a ian cance , s aining o MCT1 and MCT4 as well as hei chape one CD147 was no ound in no mal o a ian issues and benign o a ian issues, while a ound 80 % o epi helial o a ian p ima y and me as a ic umo s showed exp ession o hese p o eins. MCT1 was signi ican ly associa ed wi h low g ade umo s, high FIGO s age, p esence o esidual umo , lack o elapse and p esence o asci es; MCT4 was signi ican ly associa ed wi h high g ade umo s, high FIGO s age, p esence o esidual umo , elapse and p esence o asci es. Impo an ly, MCT exp ession was associa ed wi h he exp ession o he mul id ug esis ance ma ke s MDR1 and MRP2. In con as o wha was ound in he p e ious ypes o umou s, nei he MCT1 no MCT4 we e ound o be up- egula ed in gas ic adenoca cinomas [160]. Ac ually, MCT4 exp ession was mo e equen ly obse ed in no mal gas ic mucosa han in gas ic cance cells and e en less equen ly obse ed in lymph-node me as asis, indica ing a p og essi e loss o his MCT iso o m wi h disease p og ession. Table 3 shows an o e iew on MCT1 and MCT4 exp ession and he impac on p ognosis in di e en umou ypes. Role o MCTs in P os a e Ca cinoma Pé ega-Gomes N. 30 Table 3. O e iew o MCT1 and MCT4 exp ession and impac on p ognosis in di e en umou ypes [57]. Role o MCTs in P os a e Ca cinoma Pé ega-Gomes N. 31 O e all, he da a a ailable in he li e a u e suppo he hypo hesis o a majo ole o MCTs in he eme gence o he hype -glycoly ic and acid- esis an pheno ypes, as adap a ions o he hypoxic mic oen i onmen . The up- egula ion o MCTs in he plasma memb ane o di e en ype o umo s is an adap i e mechanism o allow con inuous high glycoly ic a es, by expo ing he accumula ing end-p oduc , lac a e, as well as o coun e ac acid-induced apop osis o nec osis. Howe e , i was clea ha his migh no be he case o all umo ypes, he e o e, in mos cases he e a e no unc ional s udies showing he dependence o he umo s on MCT exp ession and ac i i y. As so, addi ional s udies on MCT exp ession in o he umo ypes, con i ma ion o he esul s al eady published as well as addi ional unc ional s udies a e needed o deeply unde s and he ole o MCTs in cance main enance and agg essi eness and exac ly in which cases hese anspo e s could be used o he apy. 1.3.2 MCTs as sui able a ge s o cance he apy. Wha is known? La ely he e was an inc ease in he exploi a ion o ea men s ha a ge umou me abolism, being some o hem al eady in clinical ial phase. Figu e 8 shows ac ual and u u e he apeu ic a ge s o umou me abolism by a ge ing me abolic enzymes. Figu e 13. Ac ual and u u e he apeu ic a ge s (dashed lines) o umou me abolism by a ge ing me abolic enzymes [161]. Role o MCTs in P os a e Ca cinoma Pé ega-Gomes N. 32 Se e al s udies ha e al eady shown he in i o and in i o e ec o a ge ing di e en MCTs iso o ms in human cance s. I was demons a ed ha MCT inhibi ion dec eases in acellula pH [162], leads o cell dea h and, impo an ly, enhances cance cell adiosensi i i y [163]. Resul s o MCT4 show a dec ease in cance cell mig a ion, by mechanisms ha also in ol e in e ac ion o MCT4 wi h β1-in eg in [164]. In con as , ano he s udy showed ha silencing o MCT1 o MCT4 inhibi ed cance cell in asion, bu did no in luence cell mig a ion [165]. In i o models ha e also been used, whe e adminis a ion o α- cyano-4-hyd oxycinnamic acid (CHC), e a ded umo g ow h, ende ed umo cells sensi i e o adia ion, induced umo nec osis and dec eased umo in asion [166]. The impo ance o MCTs o in i o umo g ow h was con i med by a mo e speci ic app oach, whe e combined silencing o MCT1 and MCT4 o silencing o CD147 signi ican ly educed glycoly ic lux and umo g ow h. The e a e also o he MCT inhibi o s desc ibed which a e ei he non-iso o m speci ic (ARC155858 a ge s bo h MCT1 and MCT2 [167] o a ge o he molecules besides MCTs (e.g., lonidamine p ima y a ge is hexokinase II)) [167,168]. Howe e , hese compounds ha e been li le explo ed as lac a e anspo inhibi o s in he cance con ex . In sum, i seems clea ha MCTs ep esen a sui able a ge o cance he apy; howe e , he e ec o his inhibi ion seems highly dependen on he cell ype and he iso o m ha is being a ge ed. Figu e 14 shows he model o he apeu ic a ge ing o lac a e-based me abolic symbiosis in umou s. I shows ha MCTs inhibi ion would ha e a majo e ec on lac a e anspo , pH balance and umou homeos asis, by comp omising ae obic glycolysis and mic oen i onmen al acidosis, as well as, he cell-cell lac a e shu le be ween ae obic and hypoxic cell popula ions. Role o MCTs in P os a e Ca cinoma Pé ega-Gomes N. 33 Figu e 14. Model o he apeu ic a ge ing o lac a e-based me abolic symbiosis in umou s [118] Rega ding p os a e cance , which is ou main ocus in his hesis, ew s udies exis epo ing he e ec o MCTs inhibi ion in p os a e cance cells. As so, i is impo an o in es in s udies e alua ing bo h blocking and he silencing o MCTs in p os a e cance in o de o each a conclusion abou he e ec i eness o his he apeu ic app oach. 1.4 P os a e Cance 1.4.1 Epidemiology o he disease P os a e cance is one o he mos p e alen and inciden cance s in he male popula ion wo ldwide a e lung cance , and he i h mos common cance o e all. In Po ugal, p os a e cance is he leading cance among men wi h 5,140 es ima ed inciden cases in 2008 [169, 170]. The pa e ns o incidence and mo ali y p o ide a numbe o in e es ing leads wi h peak incidences in he Uni ed S a es, Aus alia and he Scandina ian coun ies. Incidence a es Role o MCTs in P os a e Ca cinoma Pé ega-Gomes N. 34 in Asian coun ies a e gene ally low, bu in ecen yea s ha e isen p opo iona ely mo e han in wes e n coun ies. Incidence a es inc eased d ama ically h ough he ea ly 1990s [171]. In he la e 1970s and ea ly 1980s he e was a ise in incidence in pa , due o inc eased de ec ion wi h mo e equen su gical ea men o benign p os a ic hype plasia (BPH), pa icula ly, ansu e h al esec ion o he p os a e (TURP) [172]. Be ween 1986 and 1992 he e was ano he sha p ise in incidence la gely due o inc easing use o p os a e speci ic an igen (PSA) [173]. A ecen ly published s udy poin s ou an inc ease o 26% in he incidence o PCa in he pe iod be ween 1986 o 2005 in he Uni ed S a es, which means ha mo e han a million addi ional men we e diagnosed wi h p os a e cance . Besides PSA sc eening p og ammes, o he aspec s such as he in oduc ion o be e diagnos ic ools like ans ec al ul asonog aphy (TRUS) –guided biopsy may be pa ially esponsible o he inc eased incidence o p os a e cance in de eloped coun ies. Howe e , sc eening p ac ice di e ences alone a e unlikely o explain he nea ly huge di e ences in p os a e cance isk be ween high- and low- isk popula ions [174, 175]. Despi e p os a e cance 's high mo bidi y, i s e iology emains obscu e, wi h he only es ablished isk ac o s being inc easing age, ace, and a amily his o y. Many pu a i e isk ac o s, including ho mones, die a y ac o s, obesi y, physical inac i i y, occupa ion, asec omy, smoking, sexual ac o s, and gene ic suscep ibili y, ha e been implica ed, bu he epidemiologic e idence is inconclusi e. While i is no known whe he he isk ac o s explaining he obse ed pa e ns a e en i onmen al, li es yle, o gene ic, i is likely ha a complex in e play o hese ac o s is associa ed wi h p os a e cance de elopmen . Conce ning mo ali y, he a es a e much less a iable han PCa incidence, leading o a less accen ua ed di e ence be ween de eloped and de eloping coun ies. Wi h he in oduc ion o PSA a sligh inc ease in mo ali y a es was obse ed bu his inc ease was e i ied bo h in high and low isk coun ies [176]. The easons o his ime ends in mo ali y a es a e s ill con o e sial bu he in oduc ion o he PSA sc eening was in ce ain way esponsible o he Role o MCTs in P os a e Ca cinoma Pé ega-Gomes N. 35 inc easing a es due o misce i ica ion o cause o dea h among men diagnosed wi h la en umou s, howe e i may also play a ole in dec easing a es because o an ea lie diagnosis o mos o he cases. Ea ly de ec ion in p os a e cance is c ucial since only o gan-con ined disease is amenable o cu a i e ea men whe eas pa ien s wi h ad anced disease can only be pallia ed. Al hough he e a e some a ailable ools equen ly used o PCa de ec ion, hei pe o mance is sub-op imal due o non-sa is ac o y sensi i i y and speci ici y a es. In his con ex , new bioma ke s a e u gen ly needed, no only o ea ly de ec ion bu also as ancilla y ools o diagnosis, which is s ill based on his opa hological e alua ion o biopsy specimens. 1.4.2 P os a e Cance Diagnosis Usually, men wi h PSA le els o 4.0 ng/mL o g ea e a e candida es o pe o m a p os a ic co e needle biopsy [177]. This cu o alue o he PSA es is s ill con o e sial because PSA le el sc eening p oduces alse-nega i e o alse- posi i e esul s ha can lead men wi hou disease o be submi ed o unnecessa y addi ional es ing [177, 178]. Impo an ly, PSA le els sc eening a e no accep able diagnos ic ools o PCa in ou ine clinical p ac ice because hey lack adequa e sensi i i y and speci ici y. Rega dless he u ili y o diagnosis aid- ools, he only way o access diagnosis o p os a e cance is by his opa hological analysis o p os a ic issue ob ained om biopsy o p os a ec omy specimens. I is c ucial o pe o m a p ope his opa hological e alua ion o he issue collec ed om he pa ien in o de o de e mine which a e he bes ea men op ions a ailable o each pa ien because he combina ion o his e alua ion esul s wi h o he pa ame e s, such as se um PSA le els, in nomog ams do ha e p ognos ic impac [179]. Biopsy echniques ha e been imp o ed in o de o educe mo bidi y and inc ease he a e and accu acy o de ec ion in he i s biopsy, al hough he op imal numbe o co es ha mus be collec ed is s ill a ma e o deba e [178]. Role o MCTs in P os a e Ca cinoma Pé ega-Gomes N. 36 1.4.2.1 His opha ological e alua ion. The Gleason sco e sys em o p os a e cance g ading The Gleason g ading sys em was i s ly desc ibed in 1996 and i is p esen ly, wi h some modi ica ions, he mos commonly used me hod o p os a e cance g ading. Because he Gleason sco e (GS) is di ec ly co ela ed wi h PCa p ognosis, i s accu a e e alua ion is c i ical o he na u al his o y o he disease o he isk o ecu ence ollowing adical p os a ec omy o adio he apy [180]. The sys em is based on he e alua ion o he glandula a chi ec u al pa e ns o he umou issue, ecognizing i e di e en g ades ha ange om good o poo g ade o di e en ia ion. Because p os a e adenoca cinomas a e he e ogeneous, mo e han one o he i e pa e ns de ined by Gleason migh be p esen in he same umou . To inco po a e his he e ogenei y in umou g ading, he Gleason sco e (GS) was de eloped, esul ing om he sum o wo mos p edominan cance pa e n (g ades) in a sample. GS a ies om 2 (1+1) o 10 (5+5) (Figu e 15). Figu e 15. Upda ed Gleason sco e o his ological g ading o p os a e umou s [181] . Pa e n 1- Closely packed small, uni o m glands. Pa e n 2- Mo e s oma be ween he glands ha a e ound o o al wi h smoo h ends and may minimally in ade non- neoplas ic issue. Pa e n 3- I egula size glands wi h angula shape and mo e in il a i e ma gins. Pa e n 4- Fused, c ibi o m o poo ly de ined glands. Pa e n 5- Only occasional gland o ma ion. 1.4.2.2 Clinical and Pa hological S aging Ano he aspec ha helps o p edic he p og ession o PCa is he s aging which comp ises in o ma ion abou he ex ension o he disease (local, egional o sys emic) and i is widely classi ied using he UICC (In e na ional Union agains Cance ) Tumou Node Me as asis (TNM) sys em (Table 4). In his classi ica ion, T ep esen s he ex en o he p ima y umou , N he lymph node s a us and M o Role o MCTs in P os a e Ca cinoma Pé ega-Gomes N. 37 dis an me as asis. The clinical s age is based on he e alua ion o he pa ien by digi al ec al exam (DRE), ans ec al ul asonog aphy (TRUS), and, possibly, magne ic esonance imaging (MRI) [178,182,183] . Fu he in o ma ion can be p o ided by biopsy his opa hological e alua ion and se um PSA le els [188]. Al e na i ely, pa hological s age is de e mined a e su gical emo al o he p os a e h ough adequa e analysis o he p os a ec omy specimen, and i p edic s disease ecu ence much mo e accu a ely [183]. The e a e se e al independen p ognos ic ac o s included in s aging ha a e impo an o guide clinical decisions conce ning he ea men op ions. These a e ex a-p os a ic umou in asion, seminal esicles in ol emen , lymph node me as asis and dis an me as asis. 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Nanni, C., e al., 11C/ 18F-choline PET o 11C/18F-ace a e PET in p os a e cance : may a choice be ecommended? Eu J Nucl Med Mol Imaging, 2007. 34(10): p. 1704-5. Role o MCTs in P os a e Ca cinoma Pé ega-Gomes N. 60 Role o MCTs in P os a e Ca cinoma Pé ega-Gomes N. 61 2.2.1 Monoca boxyla e anspo e 4 (MCT4) and CD147 o e exp ession is associa ed wi h poo p ognosis in P os a e Cance Role o MCTs in P os a e Ca cinoma Pé ega-Gomes N. 62 Role o MCTs in P os a e Ca cinoma Pé ega-Gomes N. 63 Role o MCTs in P os a e Ca cinoma Pé ega-Gomes N. 64 Role o MCTs in P os a e Ca cinoma Pé ega-Gomes N. 65 Role o MCTs in P os a e Ca cinoma Pé ega-Gomes N. 66 Role o MCTs in P os a e Ca cinoma Pé ega-Gomes N. 67 Role o MCTs in P os a e Ca cinoma Pé ega-Gomes N. 68 Role o MCTs in P os a e Ca cinoma Pé ega-Gomes N. 69 Role o MCTs in P os a e Ca cinoma Pé ega-Gomes N. 76 Role o MCTs in P os a e Ca cinoma Pé ega-Gomes N. 77 3.2.1 Monoca boxyla e anspo e 2 (MCT2) as a pu a i e bioma ke in p os a e cance . Role o MCTs in P os a e Ca cinoma Pé ega-Gomes N. 78 Role o MCTs in P os a e Ca cinoma Pé ega-Gomes N. 79 Role o MCTs in P os a e Ca cinoma Pé ega-Gomes N. 80 Role o MCTs in P os a e Ca cinoma Pé ega-Gomes N. 81 Role o MCTs in P os a e Ca cinoma Pé ega-Gomes N. 82 Role o MCTs in P os a e Ca cinoma Pé ega-Gomes N. 83 Role o MCTs in P os a e Ca cinoma Pé ega-Gomes N. 84 Role o MCTs in P os a e Ca cinoma Pé ega-Gomes N. 85 Role o MCTs in P os a e Ca cinoma Pé ega-Gomes N. 92 gluconeogenic subs a e [8-13]. As MCT1, MCT2 is also ound in mi ochond ia [12,13] and was also localized a pe oxisomes in non- umo li e ac ions [14]. The p esence o MCTs in mi ochond ia is jus i ied by he need o a py u a e ca ie ha plays a cen al ole in ca bohyd a e and a me abolism. In con as , he p esence o MCT1 and MCT2 in pe oxisomes was jus i ied as being in ol ed in a lac a e-py u a e shu le sys em p esen in he memb ane o his o ganelle. This shu le was sugges ed o be in ol ed in he oxida ion o NADH gene a ed by β- oxida ion, being c ucial o he main enance o pe oxisomal iabili y and consequen β-oxida ion a es [14]. This was he only s udy ega ding his ma e and i was no done in he cance con ex , as so, li le is known abou lac a e shu les as well as he in ol emen o pe oxisomal al e a ions in cance . In his wo k we aimed o un a eling MCT2 in acellula localiza ion and exp ession ac oss p os a e malignan ans o ma ion using di e en models o disease p og ession in o de o in e abou he impo ance o his iso o m in p os a e cance disease. Also, he o he MCT iso o ms whe e assessed in o de o cha ac e ize in mo e de ail hei in acellula dis ibu ion. Ma e ial and me hods Cell cul u e In his s udy we ha e used di e en p os a e cell lines, PNT1A (non-malignan ), 22R 1 (localized umo ), LNCaP (lymph node me as asis) and PC3 (bone me as asis). Cells we e seeded in RPMI-1640 (Gibco, In i ogen, USA) supplemen ed wi h 10% o e al bo ine se um (FBS) (PAA Labo a o ies GmbH, Cölbe, Ge many), 1% o an ibio ic (penicillin/s ep omycin) (PAA Labo a o ies GmbH, Cölbe, Ge many) and incuba ed a 37⁰C in an a mosphe e con aining 5% CO2. Immuno luo escence and mic oscopy echniques Immuno luo escence was pe o med in o de o iden i y he in acellula localiza ion o MCT1, MCT2 (sc-14926, San a C uz Bio echnology, San a C uz, CA), MCT4 and i s chape ones CD147 and gp70. Fluo opho es TRITC (Jackson Role o MCTs in P os a e Ca cinoma Pé ega-Gomes N. 93 ImmunoResea ch) and Alexa 488 (In i ogen, Li e Technologies, G and Island, NY, USA) we e used as seconda y an ibodies. Immuno luo escence was pe o med in cells seeded on glass co e slips ha we e ixed a e 24h wi h 4 % pa a o maldehyde in PBS, pH 7.4 o 20 min. A e wa ds cells we e pe meabilized wi h 0.2 % T i on X-100 o 10 min, blocked wi h 1 % BSA solu ion o 10 min and incuba ed wi h p ima y (MCT2, ca alase, PEX19 and Gp70) and seconda y an ibodies [TRITC (Jackson ImmunoResea ch) and Alexa 488 (In i ogen, Li e Technologies, G and Island, NY, USA)] o 1 h each. Be ween each s ep, cells we e ex ensi ely insed 3 imes wi h PBS, pH 7.4. Las ly, cells we e s ained wi h and moun ed in slides using Mowiol 4-88 con aining n- p opylgalla e. Images we e ob ained using a Zeiss LSM 510 Me a Con ocal se up (Ca l Zeiss, Obe kochen, Ge many) equipped wi h a plan-Apoch oma 100×/1.4 oil objec i e. Wes e n Blo Cells we e lysed wi h speci ic lysis bu e (25 mM T is-HCl, pH 8.0, 50 mM sodium chlo ide, 0.5% sodium deoxychola e, 0.5% T i on X-100 and a p o ease-inhibi o mix). To imp o e p o ein ex ac ion, samples we e passed 20 imes h ough a 26- gauge sy inge needle and incuba ed on a o a y mixe a o 30 min a 4⁰C. A e clea ed by cen i uga ion (13300 pm, 15 min) p o ein concen a ions we e de e mined by he B ad o d assay (BioRad, He cules, CA, USA). Blo s we e incuba ed wi h speci ic p ima y an ibodies, MCT2, gp70, PEX14, Ca alase, ACOX1, ACOX3 and PEX19. The an ibodies we e de ec ed by a ho se adish pe oxidase-linked seconda y an ibody using an enhanced chemiluminescence sys em (GE Heal hca e, Waukesha, WI, USA). Immunop ecipi a ion 22R 1 cells we e ans ec ed wi h Pex19-YFP using Tu bo ec in i o ans ec ion ki (Fe men as, USA), acco ding o he manu ac u e ’s ins uc ions. Fo immunop ecipi a ion o Pex19-YFP we used GFP ap_M (Ch omo ek). T ans ec ion wi h a plasmid con aining GFP was used as nega i e con ol. A e 48h o ans ec ion cell pelle s we e incuba ed lysis bu e (10mM T is-HCl, pH 7.5, 150mMNaCl, 0.5mM EDTA, 0.5%NP-40 and a p o ease-inhibi o mix). The lysa e Role o MCTs in P os a e Ca cinoma Pé ega-Gomes N. 94 was clea ed by cen i uga ion (13300 pm, 15 min) and dilu ed wi h dilu ion bu e (10mM T is-HCl, pH 7.5, 150mM NaCl, 0.5mM EDTA and a p o ease-inhibi o mix). P o ein concen a ions we e de e mined by he B ad o d assay (BioRad, He cules, CA, USA). Ice-cold dilu ion bu e was used o equilib a e beads and cell lysa es we e incuba ed o 2 h a 4°C on a o a y mixe . Beads we e washed 3 imes wi h dilu ion bu e and esuspended in 3x SDS-sample bu e and boiled o 10 min o elu e bound p o eins. Immunop ecipi a ed samples we e sepa a ed by unning in a 12.5% SDS-polyac ylamide gel. Resul s MCT2 was de ec ed in he pe oxisomes o p os a e cance cells a di e en le els wi hin disease p og ession The in acellula localiza ion o MCT2 was accessed in di e en models o p os a e cance disease p og ession. As McClelland e al ha e shown a pe oxisomal localiza ion o MCT2 in non- umo li e ac ions, we i s ly es ed whe he his p o ein would also be p esen in his o ganelle in p os a e cance cells. To ha end, we ha e pe o med immunolocaliza ion o MCT2 oge he wi h he pe oxisomal ma ke ca alase in di e en p os a e cell lines: PNT1A (non- malignan ), 22R 1 (localized umo ), LNCaP (lymph node me as asis) and PC3 (bone me as asis). Ou esul s demons a e ha , while no co-localiza ion was obse ed be ween MCT2 and he pe oxisomal ma ke in PNT1A, his p o ein is localized a pe oxisomes in all he malignan cell lines (Fig 1). We ha e, hough, obse ed ha he localiza ion le el a ied ac oss he di e en models. In 22R 1 cells, MCT2 was mainly ound in he pe oxisomes wi h a mino po ion sp ead h oughou he cy oplasm as small agg ega es (Fig1 d- ). Howe e , he a io o pe oxisomal MCT2/cy oplasm MCT2 dec eased wi h disease p og ession, culmina ing wi h almos no MCT2 in he pe oxisomes in PC3 cells (Fig1 j-l). Su p isingly, he amoun o MCT2 a pe oxisomes appea s o co ela e wi h a change on he o ganelle’s mo phology. In ac , in cells whe e no MCT2 was p esen in pe oxisomes (PNT1A), his o ganelle exhibi s a egula pheno ype (Fig 1A: a-c). Cu iously, in 22R 1 cells (whe e MCT2 was mainly obse ed in Role o MCTs in P os a e Ca cinoma Pé ega-Gomes N. 95 pe oxisomes) his o ganelle appea s somewha in clus e s (Fig. 1A: d- ) LNCaP cells exhibi mo e dis ibu ed ounde pe oxisomes wi h only a ew clus e s (Fig.1A: g-i). In PC3, he highly me as a ic model, pe oxisomes appea simila o he ones o he non-malignan cells PNT1A (Fig 1A: j-l). S ikingly, he exp ession le el o MCT2 inc eases om non- umo (PNT1A) o localized malignan cells (22R 1), co ela ing o i s change in localiza ion om cy oplasmic o pe oxisomal (Fig 1B). This inc ease in exp ession was also obse ed o Pex14 (a pe oxisomal memb ane p o ein) (Fig 1C), sugges ing an inc ease in pe oxisomal memb ane su ace/numbe ha accompanies he malignan ans o ma ion. MCT2 a els o he pe oxisomal memb anes ough PEX19 The quick mo emen o monoca boxyla es ac oss he memb anes is impe a i e o cellula me abolism. These p o eins a e hough o equi e chape ones such as CD147 in he case o MCT1 and MCT4, o gp70 in he case o MCT2, o app op ia e exp ession and ac i i y in he plasma memb ane [15]. Since MCT2 was he only iso o m ound o localize in pe oxisomes in p os a e cance cells, we aimed a un a eling i s a ge ing mechanism o his o ganelle. A p e ious s udy was unable o ind gp70 exp ession in human p os a e samples ha exhibi ed MCT2 exp ession [5]. Howe e , ou s udies wi h con ocal mic oscopy allowed us o obse e some, al hough sca ce, gp70 dis ibu ed in he cy oplasm wi hou any co-localiza ion wi h pe oxisomal ma ke s (Fig 2 a-l). Hence, he p o ein ha beha es as chape one o MCT2 anspo o pe oxisomes in p os a e cells is s ill unknown. As Pex19 is he main p o ein esponsible o he anspo o pe oxisomal memb ane p o eins o his o ganelle, we es ed whe he his p o ein could ac as a chape one o MCT2 in hese cells. Being 22RV1 he cell line whe e MCT2 was mainly p esen a pe oxisomes, i was he chosen model o s udy he possible in e ac ion be ween MCT2 and PEX19. Immunop ecipi a ion expe imen s ha e shown, indeed, an in e ac ion be ween MCT2 and PEX19 (Fig 3). These esul s sugges a highjack o he pe oxisomal anspo mechanism by he p os a e cance cells. Role o MCTs in P os a e Ca cinoma Pé ega-Gomes N. 96 The p esence o MCT2 a pe oxisomes seems o be ela ed wi h an inc ease in he a es o pe oxisomal β-oxida ion McClelland e al obse ed a dec ease in β-oxida ion upon MCTs inhibi ion, sugges ing ha he p esence o MCT2 a pe oxisomes o non-malignan li e cells would be essen ial o he main enance o pe oxisomal iabili y and consequen ly β-oxida ion a es. Hence, we hypo hesized ha he inc ease o MCT2 exp ession om non-malignan p os a e cells (PNT1A) o localized p os a e umo cells (22RV1) could be ela ed wi h an inc ease in pe oxisomal β-oxida ion. In ac , we obse ed an inc ease in he exp ession le els o ACOX1 and ACOX-3, cen al p o eins in he β-oxida ion pa hway (Fig 4). These esul s in e es ingly sugges ha , indeed, he inc ease in MCT2 exp ession le els as well as i s p esence a pe oxisomes, a e ela ed o an inc ease in β-oxida ion le els which may be c ucial o malignan ans o ma ion. The dis ibu ion o o he MCTs iso o ms and CD147 a in acellula le el sugges s an addi ional ole o hese p o eins in p os a e cance MCT1, MCT4 and CD147 exp essions we e also e alua ed in his s udy. MCT1 showed a s ong nuclea exp ession in PNT1a and 22RV1, dec easing o LNCaP and PC3, whe e his anspo e was mainly ound in he cy oplasm and plasma memb ane (Fig.5). MCT4 howe e was shown o be p esen in he cy oplasm o he less umo igenic cell lines 22RV1 and LNCaP in con as o he highly me as a ic PC3 whe e i was clea ly p esen a he plasma memb ane (Fig.6). CD147 was p esen in he plasma memb ane, cy oplasm and nuclea en elope (Fig.7). In e es ingly, some co-localiza ion be ween MCTs 1 and 4 and pe oxisomes, was also e i ied, howe e i was less e iden han o MCT2. Discussion I is now widely accep ed ha he anspo o lac a e and py u a e is media ed by a amily o H+/monoca boxyla e anspo e s (MCTs) known o hei speci ici y o anspo sho chain monoca boxyla es. They o m an in eg al pa o p oposed Role o MCTs in P os a e Ca cinoma Pé ega-Gomes N. 97 shu les ha anspo lac a e be ween cells (cell–cell lac a e shu le) and wi hin cells (in acellula lac a e shu le). A p e ious s udy o MCTs exp ession in p os a e cance d ew a en ion o MCT2 ha was shown o be clea ly exp essed in p os a e malignan glands bu a he cy oplasm, sugges ing he in ol emen o his anspo e in an o ganelle memb ane [6], howe e he e we e no s udies desc ibing he exis ence o MCT2 in he o ganelles o p os a e cance cells. I is known, ha once in he mi ochond ial ma ix, lac a e and py u a e unde go u he b eakdown making i di icul o in e hei espec i e oles in he shu ling o educing equi alen s o he elec on anspo chain. Pe oxisomes, on he o he hand, lack he enzymes o ca abolize monoca boxyla es and al hough hey a e known o con ain LDH [16], hey a e no known o con ain any o he glycoly ic o any ica boxylic acid cycle enzymes. One o he many unc ions o li e pe oxisomes is he β-oxida ion o long b anched-chain a y acids and o he main enance o hese eac ions inside he pe oxisome, a edox shu le sys em exis ac oss he pe oxisomal memb ane o eoxidize NADH. I is essen ial o he con inuous unc ioning o pe oxisomes ha p ope in a-o ganelle educ ion– oxida ion ( edox) balance is main ained ha ensu es he eoxida ion o NADH p oduced by β-oxida ion and he con inuous b eakdown o a y acids. Redox balance in many o ganelles is main ained in pa by subs a e shu les. This would en ail a shu le sys em in pe oxisomes ha nei he gene a es p oduc s no consumes any subs a es. McClelland e al p oposed ha a edox shu le sys em ha consis s o a subs a e cycle be ween lac a e and py u a e, as so, o play hei ole in pe oxisomal edox balance, py u a e and lac a e mus be anspo ed ac oss he pe oxisomal memb ane [14]. In he p esen s udy we desc ibe o he i s ime ha MCT2 is p esen in human p os a e pe oxisomes and a possible explana ion o how hese MCT2 ends up in he pe oxisomal memb ane was gi en by he e iden in e ac ion o MCT2 wi h PEX19 which migh ac as chape one. The absence o glycolysis in his o ganelle s ongly sugges s ha his shu le se es o main ain edox balance and no glycolysis o some o he me abolic unc ion. Consequen ly, we poin ou he impo ance o pe oxisomes in p os a e cance cells ha as i was obse ed a clea Role o MCTs in P os a e Ca cinoma Pé ega-Gomes N. 98 change hei mo phology ac oss p os a e malignan ans o ma ion, which p o ides u he e idence o he dynamic na u e o he pe oxisomes ac oss disease p og ession. Impo an ly, he equency o pe oxisomal clus e s and he co- localiza ion wi h MCT2 ha was mainly obse ed in he ansi ion om he benign model (PNT1a) o he malignan model (22RV1), poin o he in ol emen o hese al e a ions in umou ini ia ion. Finally, he possibili y o ha ing MCT1 and MCT4 in he pe oxisomal memb ane, howe e less e iden han o MCT2, co obo a es he hypo hesis o al e na i e oles o hese anspo e s in me abolic pa hways di e en om glycolysis in he p esen cells. Howe e , u he s udies a e needed o unde s and he ole o hese MCT iso o ms in p os a e pe oxisomes. Re e ences 1. Compe a , E., J. Va ino , and J.R. S igley, [Benign mimicke s o he p os a e cance . Diagnos ic challenges]. Ann Pa hol, 2013. 33(4): p. 237-46. 2. Schmi z, W., R. Finge hu , and E. Conzelmann, Pu i ica ion and p ope ies o an alpha- me hylacyl-CoA acemase om a li e . Eu J Biochem, 1994. 222(2): p. 313-23. 3. Lloyd, M.D., e al., Alpha-me hylacyl-CoA acemase--an 'obscu e' me abolic enzyme akes cen e s age. FEBS J, 2008. 275(6): p. 1089-102. 4. Hales ap, A.P., The SLC16 gene amily - s uc u e, ole and egula ion in heal h and disease. Mol Aspec s Med, 2013. 34(2-3): p. 337-49. 5. Pe ega-Gomes, N., e al., Monoca boxyla e anspo e 4 (MCT4) and CD147 o e exp ession is associa ed wi h poo p ognosis in p os a e cance . BMC Cance , 2011. 11: p. 312. 6. Pe ega-Gomes, N., e al., Monoca boxyla e anspo e 2 (MCT2) as pu a i e bioma ke in p os a e cance . P os a e, 2013. 73(7): p. 763-9. 7. Lin, R.Y., e al., Human monoca boxyla e anspo e 2 (MCT2) is a high a ini y py u a e anspo e . J Biol Chem, 1998. 273(44): p. 28959-65. 8. Pie e, K., e al., Cell-speci ic localiza ion o monoca boxyla e anspo e s, MCT1 and MCT2, in he adul mouse b ain e ealed by double immunohis ochemical labeling and con ocal mic oscopy. Neu oscience, 2000. 100(3): p. 617-27. 9. Pie e, K., P.J. Magis e i, and L. Pelle in, MCT2 is a majo neu onal monoca boxyla e anspo e in he adul mouse b ain. J Ce eb Blood Flow Me ab, 2002. 22(5): p. 586-95. 10. Debe na di, R., e al., Cell-speci ic exp ession pa e n o monoca boxyla e anspo e s in as ocy es and neu ons obse ed in di e en mouse b ain co ical cell cul u es. J Neu osci Res, 2003. 73(2): p. 141-55. 11. Pelle in, L., e al., Cellula and subcellula dis ibu ion o monoca boxyla e anspo e s in cul u ed b ain cells and in he adul b ain. J Neu osci Res, 2005. 79(1-2): p. 55-64. Role o MCTs in P os a e Ca cinoma Pé ega-Gomes N. 99 12. Yoshida, Y., e al., Negligible di ec lac a e oxida ion in subsa colemmal and in e myo ib illa mi ochond ia ob ained om ed and whi e a skele al muscle. J Physiol, 2007. 582(P 3): p. 1317-35. 13. Ben on, C.R., e al., Monoca boxyla e anspo e s in subsa colemmal and in e myo ib illa mi ochond ia. Biochem Biophys Res Commun, 2004. 323(1): p. 249-53. 14. McClelland, G.B., e al., Pe oxisomal memb ane monoca boxyla e anspo e s: e idence o a edox shu le sys em? Biochem Biophys Res Commun, 2003. 304(1): p. 130-5. 15. Wilson, M.C., e al., Basigin (CD147) is he a ge o o ganome cu ial inhibi ion o monoca boxyla e anspo e iso o ms 1 and 4: he ancilla y p o ein o he insensi i e MCT2 is EMBIGIN (gp70). J Biol Chem, 2005. 280(29): p. 27213-21. 16. Baumga , E., e al., L-lac a e dehyd ogenase A4- and A3B iso o ms a e bona ide pe oxisomal enzymes in a li e . E idence o in ol emen in in ape oxisomal NADH eoxida ion. J Biol Chem, 1996. 271(7): p. 3846-55. Role o MCTs in P os a e Ca cinoma Pé ega-Gomes N. 100 Figu es Figu e 1 A) Immunohis ochemical images o di e en human p os a e cell lines PNT1a (Figu e 1a- c), 22RV1(Figu e 1d- ), LNCaP (Figu e 1g-i) and PC3 (Figu e 1j-l). Supe posi ion o signals om p obes o MCT2 (g een), and Ca alase ( ed) shows clea colocaliza ion (yellow in he pe oxisomes o p os a e cance cells). Role o MCTs in P os a e Ca cinoma Pé ega-Gomes N. 101 PNT1a 22RV1 LNCaP PC3 MCT2 βTubulin Figu e 1 B) Wes e n blo analysis showing he le els o MCT2 in he di e en p os a e cell line models. PNT1a 22RV1 LNCaP PC3 PEX14 Tubulin Figu e 1 C) Wes e n blo analysis, showing he exp ession le els o he pe oxisomal p o ein PEX- 14 in he di e en in i o models o p os a e cance p og ession (PNT1a, 22RV1, LNCaP and PC3). Role o MCTs in P os a e Ca cinoma Pé ega-Gomes N. 108 Role o MCTs in P os a e Ca cinoma Pé ega-Gomes N. 109 4.1 CHAPTER OVERVIEW We i s ly desc ibed o e exp ession o MCT2 and MCT4 in PCa issues when compa ed o he no mal coun e pa s, howe e , he ole o MCTs in PCa me abolism is s ill la gely unknown. The p esence o MCT4 in he cy oplasm o p os a e cance cells ins ead o plasma memb ane oge he wi h he exp ession o MCT1 in bo h non-malignan and malignan glands, sugges ed ha p os a e cells may no i s ly ely on glycoly ic me abolism as he majo i y o umou s. This e i ica ion u ned his wo k e en mo e challenging in an a emp o con ex ualize he ole o di e en MCTs iso o ms ac oss p os a e malignan ans o ma ion and p og ession. As s a ed be o e, i emains unclea wha is he p ima y ene ge ic pa hway in PCa and he e o e which me abolic pa hway ep esen s he mos app op ia e a ge o me abolic inhibi ion in PCa. The e a e s udies epo ing he p esence o hypoxia in PCa, poin ing o he p esence o a glycoly ic pheno ype, howe e o he s udies epo ha PCa is cha ac e ized by low a es o glycolysis and a low FDG ac i i y on PET imaging. This ac aises he hypo hesis ha i glucose consump ion is no ele a ed in PCa, al e na i e me abolic pa hways mus p o ide he ene gy needed o cance cell p oli e a ion and g ow h. Some e idence suppo a c ucial ole o a y acid- ela ed me abolism in he pa hogenesis and p og ession o p os a e malignancy. Howe e , he epo s a e sca ce wi h low numbe o clinical samples and wi h no in o ma ion ega ding he clinico-pa hological signi icance o hese al e a ions; as so, PCa me abolism is s ill la gely unknown. This chap e includes esul s al eady submi ed o publica ion, showing he exp ession pa e n o se e al me abolic- ela ed p o eins in ol ed in glycoly ic and a y acid oxida ion pa hways om benign p os a e o me as a ic PCa, in es iga ing he p ognos ic impac o hei exp ession. Also, using di e en in i o models o disease p og ession we es ed how he inhibi ion o hese pa hways a ec he di e en models o p os a e cance cells in o de o es ablish a a ionale o me abolic pheno ypes ac oss p os a e malignan ans o ma ion and p og ession. O e all, he esul s p esen ed he e indica e a swi ch om less agg essi e umo o glycoly ic me abolism in he highly agg essi e and me as a ic p os a e umo Role o MCTs in P os a e Ca cinoma Pé ega-Gomes N. 110 and unde sco es he plas ici y o bioene ge ic pa hways in p os a e cance cells which migh ha e c ucial implica ions a p ognos ic and he apeu ic le el. 4.2 PUBLISHED RESULTS The esul s p esen ed in his chap e we e: (i) Submi ed o publica ion as an o iginal a icle in an in e na ional pee e iewed jou nal Pé ega-Gomes N, Vizcaíno JR, Sousa S, Coelho R, A ig J, Ju meis e S, Oli ei a E, Pe ei a L, Pinhei o C, Je ónimo C, Hen ique RM, Lobo da Cunha A, Lopes C, Maximo V and Bal aza F “Me abolic he e ogenei y in p os a e cance is linked o disease p og ession and agg essi eness”. 2013. (ii) In i ed o publica ion as an o iginal a icle in an in e na ional pee e iewed jou nal Pé ega-Gomes N. and Bal aza F. “MCTs as a ge s o p os a e cance he apy?” in i ed wo k o be published in Ad ances in P os a e Cance Resea ch and T ea men jou nal. 2014. (iii) P esen ed as o al communica ion in he ollowing in e na ional scien i ic mee ing Pé ega-Gomes N, Vizcaíno JR, Lopes C and Bal aza F. “T acing he me abolic p o ile o p os a e cance de elopmen ” wo k p esen ed a 23 d Eu opean Cong ess o Pa hology a Helsinki, Finland. 2011. Pé ega-Gomes N., Vizcaíno JR, Lopes C and Bal aza F. “Exploi a ion o me abolic al e a ions as impo an e p ognos ic, diagnos ic and he apeu ic ools in p os a e cance ”. Wo k p esen ed a 20 h Mee ing o he EAU Sec ion o U ological Resea ch (ESUR). 25-27 Oc obe 2012, S asbou g, F ance. 2012. (i ) P esen ed as o al communica ion in he ollowing na ional scien i ic mee ing: Role o MCTs in P os a e Ca cinoma Pé ega-Gomes N. 111 Pé ega-Gomes N, Lou enço T, Vizcaíno JR, Lopes C and Bal aza F. “S udy o Me abolic p o ile o localized and me as a ic umou o he p os a e. Monoca boxyla e anspo e s as he apeu ic a ge s” wo k p esen ed a XVI Wo kshop on Oncological U ology, Ca oei o, Po ugal. 2011. Pé ega-Gomes N, Lou enço T, Vizcaíno JR, Lopes C and Bal aza F. e al. “A me abolic swi ch could be in ol ed in p os a e cance agg essi eness.”Wo k p esen ed a he 12º Cong ess o he Po uguese Socie y o Oncology, Alga e, Po ugal. 2011. ( ) P esen ed as pos e in he ollowing in e na ional scien i ic mee ing: Pé ega-Gomes N, Lou enço T, Mi anda-Gonçal es V, Vízcaíno JR, Oli ei a E, Je ónimo C, Lopes C, Lobo da Cunha A. and Bal aza F. “Eme ging oles o Monoca boxyla e T anspo e s in p os a e cance me abolism”. a 2nd Symposium o he In e na ional Socie y o P o on Dynamics in Cance in Nice, F ance. 2011. Mi anda-Gonçal es V, Pé ega-Gomes N, Lou enço T, Pinhei o C, Je ónimo C, Vizcaíno JR, Lopes C, and Bal aza F. “P os a e Cance : exploi a ion o monoca boxyla e anspo e s as po en ial he apeu ic a ge s”. a he 39 h Mee ing o In e na ional Socie y o Oncology and Bioma ke s in Flo ence, I aly. 2011. Pé ega-Gomes N., Vizcaíno JR, Lou enço T, Lopes C. and Bal aza F “Exploi ing me abolic al e a ions in p os a e cance diagnosis and p ognosis” a XXI Po o Cance Mee ing, Po o. 2012. Pé ega-Gomes N, Vizcaíno JR, Lou enço T, Lopes C. and Bal aza F “T acing he me abolic p o ile o p os a e cance p og ession. Roles o monoca boxyla e anspo e s (MCTs) in p os a e cance me abolism” a EACR Annual Cong ess, Ba celona. 2012. Pé ega-Gomes N, Vizcaíno JR, Lou enço T, Lopes C. and Bal aza F. “Clinical impac o he me abolic pheno ype o p os a e cance : Role o monoca boxyla e anspo es (MCTs).” a São Paulo Ad anced School o Compa a i e Oncology. Sep embe 30 h o Oc obe 6 h. Águas de São Ped o-São Paulo, B asil. 2012. Role o MCTs in P os a e Ca cinoma Pé ega-Gomes N. 112 Pé ega-Gomes N, Vizcaíno JR, Lou enço T, Lopes C. and Bal aza F. “Me abolic pheno ypes associa ed wi h dis inc and ogen- esponsi e condi ions can be a aluable ool o diagnos ic and he apeu ic op ions in p os a e cance .” a A one day symposium wi h Ca los Caldas sponso ed by EACR. 29TH Oc obe , Po o, Po ugal. 2012. ( i) We e ecognized wi h he he ollowing p izes/awa ds: Pé ega-Gomes N, Vizcaíno JR, Lopes C and Bal aza F “T acing he me abolic p o ile o p os a e cance de elopmen ”. Bu sa y Awa d gi en by he Eu opean Socie y o Pa hology (ESP) o a end 23º Cong ess o he Eu opean Socie y o Pa hology mee ing in Helsinki, Finland. Pé ega-Gomes N, Lou enço T., Vizcaíno JR, Lopes C and Bal aza F. “S udy o Me abolic p o ile o localized and me as a ic umou o he p os a e. Monoca boxyla e anspo e s as he apeu ic a ge s” 1s P ize (Janssen Awa d) and Hono able Men ion gi en by o bes pos e and scien i ic communica ion in Na ional Cong ess o U ology. Pé ega-Gomes N, Lou enço T., Vizcaíno JR, Lopes C and Bal aza F. 2011. “A me abolic swi ch could be in ol ed in p os a e cance agg essi eness. 1s P ize o bes pos e and scien i ic communica ion in 12º Na ional Cong ess o Oncology. Pé ega-Gomes N, Vizcaíno JR, Lou enço T, Lopes C. and Bal aza F. 2012. “Clinical impac o he me abolic pheno ype o p os a e cance : Role o monoca boxyla e anspo es (MCTs).” Bu sa y awa d gi en by FAPESP - São Paulo Resea ch Founda ion o a end ESPCA-School o Compa a i e Oncology a Águas de São Ped o-São Paulo, B asil, 2012. Role o MCTs in P os a e Ca cinoma Pé ega-Gomes N. 113 4.3.1 Me abolic he e ogenei y in p os a e cance is linked o disease p og ession and agg essi eness Role o MCTs in P os a e Ca cinoma Pé ega-Gomes N. 114 Role o MCTs in P os a e Ca cinoma Pé ega-Gomes N. 115 Me abolic he e ogenei y in p os a e cance is linked o disease p og ession and agg essi eness Running Ti le: Me abolic he e ogenei y in PCa p og ession Lis o au ho s Nelma Pé ega-Gomes1,2, José R. Vizcaíno3, Susana Sousa1,2, Rica do Coelho4,5, Jan A ig6, Sa ah Ju meis e 7, Elsa Oli ei a8, Luísa Pe ei a4, 9, Céline Pinhei o1,2, Ca men Je ónimo10,11,12, Rui M. Hen ique11,12,13, Alexand e Lobo da Cunha8, Ca los Lopes3,12, Valdema Maximo4,9and Fá ima Bal aza 1,2* 1Li e and Heal h Sciences Resea ch Ins i u e (ICVS), School o Heal h Sciences, Uni e si y o Minho, B aga, Po ugal 2ICVS/3B’s - PT Go e nmen Associa e Labo a o y, B aga/Guima ães, Po ugal; 3Depa men o Pa hology, Cen o Hospi ala do Po o, Po ugal; 4Ins i u e o Molecula Pa hology and Immunology o he Uni e si y o Po o (IPATIMUP), 4200-465 Po o, Po ugal. 5Ins i u e o Biomedical Sciences o Abel Salaza , Uni e si y o Po o, 4050-313 Po o, Po ugal. 6MRC Labo a o y o Molecula Biology F ancis C ick A enue Camb idge Biomedical Campus Camb idge UK. 7U o-oncology Resea ch G oup, Cance Resea ch UK Camb idge Ins i u e, Uni e si y o Camb idge, Camb idge, UK. Role o MCTs in P os a e Ca cinoma Pé ega-Gomes N. 116 8Labo a o y o Cell Biology, Ins i u e o Biomedical Sciences Abel Salaza (ICBAS), Uni e si y o Po o 9Depa men o Pa hology and Oncology, Medical Facul y o he Uni e si y o Po o, 4200-319 Po o, Po ugal 10Cance Epigene ics G oup – Resea ch Cen e , Po uguese Oncology Ins i u e- Po o, Po o, Po ugal; 11Depa men o Gene ics, Po uguese Oncology Ins i u e,Po o, Po ugal 12Depa men o Pa hology and Molecula Immunology, Ins i u e o Biomedical Sciences Abel Salaza (ICBAS), Uni e si y o Po o, Po o, Po ugal 13Depa men o Pa hology, Po uguese Oncology Ins i u e-Po o, Po o, Po ugal *Co espondence o: Fá ima Bal aza , PhD, School o Heal h Sciences, Uni e si y o Minho, Campus de Gual a , 4710-057 B aga, Po ugal. email:[email p o ec ed]. Tel: +351 253 60 48 28. Fax: +351 253 60 48 20 Con lic o in e es s a emen The au ho s decla e no con lic s o in e es . Role o MCTs in P os a e Ca cinoma Pé ega-Gomes N. 117 Abs ac Me abolic adap a ion is now conside ed a new hallma k o cance , in which cance cells exhibi high a es o glucose consump ion wi h consequen lac a e p oduc ion. Howe e , he dominan ene ge ic pa hway as well as he clinical impac o al e ed cellula me abolism in p os a e cance is s ill la gely unknown. In an a emp o unde s and he me abolic al e a ions du ing p os a e cance p og ession and how hese al e a ions could ep esen sui able ools o p os a e cance a diagnos ic, p ognos ic and he apeu ic le el, we s udied he exp ession o se e al key me abolic- ela ed p o eins in 480 human p os a e samples and using in i o models we assessed me abolic changes associa ed wi h malignan ans o ma ion/p og ession and how hei manipula ion a ec s di e en models o p os a e cance cells. Also we p o ided an insigh in o he cell mi ochond ial ea u es and used publicly a ailable genome-wide exp ession p o iling da ase s o assess he exp ession o he genes ha codi y me abolic- ela ed p o eins du ing p os a e cance p og ession. This s udy p o ides e idence o inc eased glycoly ic pheno ype mainly in he ad anced s ages o p os a e cance , and i s co ela ion wi h poo p ognosis whe eas he localized umou showed mo e consis en ly up egula ion o p o eins in ol ed in pe oxisomal b anched-chain a y acid oxida ion wi hou any associa ion wi h clinic-pa hological da a. Impo an ly, expe imen s using in i o models showed ha cell lines de i ed om dis an me as asis a e mo e glycoly ic, sensi i e o glycoly ic inhibi o s and wi h lowe ene gy e iciency a he mi ochond ial le el. This is he mos comp ehensi e s udy epo ing he me abolic di e ences ac oss p os a e malignan ans o ma ion and co ela ing he me abolic he e ogenei y o p os a e cance cells wi h un a o able p ognosis, showing ha me abolic changes ha e a s ong po en ial o be explo ed as diagnos ic, p ognos ic and he apeu ic ools in p os a e cance . Keywo ds: p os a e cance , cance me abolism, monoca boxyla e anspo e s, glycoly ic me abolism, a y acid oxida ion, me abolic swi ch. Role o MCTs in P os a e Ca cinoma Pé ega-Gomes N. 124 he e was a dec ease in he su i al o pa ien s wi h umou s exp essing CAIX, (da a no shown). Di e en in i o models o PCa p og ession exhibi di e en p o ein exp ession and glycoly ic me abolism p o iles Since he main di e ences obse ed ac oss malignan p og ession in human samples we e ela ed o p o eins in ol ed in he glycoly ic me abolism, in Figu e 3 we show he exp ession p o ile o hese p o eins in di e en in i o models o disease p og ession. In e es ingly, MCT4 and HIF-1alpha we e only p o eins de ec ed in DU145 and PC3 cell lines. Also, GLUT-1, LDHV and CAIX showed e iden highe le els in PC3 cell line. In e es ingly, he ICC esul s o he di e en cell line models show ha MCT4 and GLUT-1 only appea ed e iden ly exp essed a he plasma memb ane in he highly me as a ic models, such as PC3, in con as o he low umo igenic cell line LNCaP (Figu e 3B). Finally, a clea dis inc me abolic beha iou be ween he low umo igenic 22RV1 and LNCaP and he high umo igenic PC3 and DU145 models was obse ed. PC3 and DU145 exhibi ed highe le els o glucose consump ion (Figu e 3C) and lac a e p oduc ion (Figu e 3D) when compa ed o 22RV1 and LNCaP cells. P os a e cance cells exhibi ed di e en sensi i i ies o inhibi ion o glycolysis and a y acid oxida ion Based in he idea ha di e en models o disease p og ession exhibi ed a di e en me abolic beha iou , we aimed o s udy he e ec o glycolysis and a y acid oxida ion inhibi ion in he di e en models. Oxamic acid (OA), an inhibi o o lac ic dehyd ogenase (LDH), was used o in e how he glycoly ic me abolism in he di e en models is a ec ed a e inhibi ion o lac ic acid p oduc ion. Figu e 4 shows he glucose consump ion and lac a e p oduc ion o p os a e cell lines a e he ea men wi h OA. I is possible o obse e ha bo h pa ame e s showed a signi ican dec ease in 22RV1, PC3 and DU145 cell lines. In con as , LNCaP cell line me abolism was less a ec ed by OA ea men . Role o MCTs in P os a e Ca cinoma Pé ega-Gomes N. 125 The pheno hiazine d ug, hio idazine (TZ), desc ibed as a selec i e inhibi o o pe oxisomal β-oxida ion, was also used in ou cell line models. As obse ed in Figu e 5A, he IC50 alues show ha 22RV1 and LNCaP cell lines a e mo e sensi i e o TZ e ec , especially a 72 h ea men . Acco dingly, when we supplemen ed he cul u e media wi h phy anic acid (PA), a long b anched chain a y acid, o s imula e a y acid oxida ion, a dec ease in PC3 and DU145 cell iabili y was obse ed, bu no in 22RV1 and LNCaP ha main ain hei iabili y wi h inc easing concen a ions o PA, e en a e 48 hou s o exposu e (Figu e 5B). Ul as uc u al di e ences a e e iden among he di e en p os a e cance cell models So a , we ha e obse ed ha di e ences a he exp ession le els o p o eins in ol ed in glycoly ic and a y acid me abolism as well as in cells me abolic beha io a e e iden ac oss malignan ans o ma ion. As so, we also aimed o e i y i such di e ences migh be also e lec ed a he ul as uc u al le el o he cells. Figu e 6 shows elec on mic og aphs o LNCaP (Figu e 6A), PC3 (Figu e 6B) and wo di e en human samples o p os a e cance (Figu e 6C and 6D). I was possible o obse e ha ei he in LNCaP cells o he human samples (Figu e 6A, 6C and 6D, espec i ely), cells a e packed wi h lipid bodies. Also, in wha conce ns mi ochond ia mo phology, hese images clea ly show di e ences in e ms o subcellula ea u es o localized p os a e umou s and he low umo igenic model LNCaP cell line in compa ison wi h he cells de i ed om highly me as a ic umou s (PC3). Analysis o he mi ochond ial genome e lec s a dec ease in he e iciency o PC3 cell lines h ough inc eased exp ession o UCP2 p o ein A comple e m DNA sequencing o he cell lines LNCaP and PC3 (as wo clea ly dis inc models o disease p og ession) was pe o med (Table 6). The esul s showed ha he LNCaP haplo ype can be a ilia ed in he Eu opean haplog oup H26c, displaying he coding p i a e mu a ion in ND5 gene a posi ion 13227, which is a synonymous polymo phism. The PC3 haplo ype belongs o ano he Eu opean haplog oup, denomina ed U5a1c, and bea s wo non synonymous Role o MCTs in P os a e Ca cinoma Pé ega-Gomes N. 126 ecen mu a ions: in gene ND4, a posi ion 11120, leading o he F121L amino acid eplacemen , which has a Mu P ed pa hogenici y sco e o 0.423; and in gene ND5, a posi ion 13802, conduc ing o he amino acid eplacemen T489M, ha ing a Mu P ed pa hogenici y sco e o 0.6. We ha e in e ed, on he basis o a la ge popula ion da ase o m DNA genomes, ha he alue o Mu P ed sco e di iding pa hogenic mu a ions om ole a ed mu a ions is a ound 0.7. The wo nonsynonymous mu a ions in PC3 cell line ha e sco es below his h eshold, bu we do no know ye i he mu a ions can con ibu e addi i ely o a pheno ype. As bo h PC3 mu a ions a e loca ed in p o eins o he Complex I, i hey ha e some unc ional impai men in he oxida i e phospho yla ion i will be upon his complex. In es iga ing Complex I ac i i y in p os a e cell lines we obse ed ha in con as wi h we we e expec ing, PC3 cells show inc eased Complex I ac i i y (Figu e 7A). Howe e he inc eased Complex I ac i i y in PC3 cells was no e lec ed in global cellula ATP le els (Figu e 7B). These esul s sugges ha he p o on elec ochemical g adien ac oss he inne mi ochond ial memb ane (IMM) is being dissipa ed and no used o ATP p oduc ion. In ac , in es iga ing he exp ession le els o he mi ochond ial uncoupling p o ein 2 (UCP2), we e i ied ha i is signi ican ly highe in PC3 cells, bo h using β-ac in p o ein (p=0.02) as o al p o ein loading, and he anslocase o ou e mi ochond ial memb ane 20 homolog (TOMM20) (p<0.01), as a mi ochond ial p o ein loading indica ing ha PC3 cells exhibi ew mi ochond ia and hus con i ming ou ul as uc u al da a (Figu e 8A-D). Public genome-wide exp ession p o iling da ase s show associa ion o me abolic- ela ed p o eins wi h disease p og ession By making use o publicly a ailable genome-wide exp ession p o iling da ase s, we immedia ely obse ed ha he mRNA le els o genes in ol ed in he glycoly ic pa hway we e highe in me as a ic umou s compa ed o localized umou s (Figu e 9A), whe eas, he di e ences in he exp ession pa e ns obse ed o he genes in ol ed in he a y acid oxida ion pa hway we e less s iking (Figu e 9B). Role o MCTs in P os a e Ca cinoma Pé ega-Gomes N. 127 In e es ingly, looking o he genes ha codi y o lac a e anspo e p o eins, only he mRNA le els o SLC16A3, he gene ha codi ies o MCT4, he iso o m esponsible o lac a e ex usion we e highe in me as a ic compa ed o localized umou s (Figu e 9E). Finally, he Glinsky da ase showed again SLC16A3 gene being associa ed wi h he ime o biochemical ecu ence a e su ge y (Figu e 10). Discussion While nume ous s udies ha e in es iga ed he in ol emen o al e ed cellula me abolism in a ious umou s, li le is known abou he me abolic al e a ions du ing p os a e cance p og ession. Cos ello e al. [25,26] desc ibed he ans o ma ion o a ci a e-p oducing epi helial cell o a malignan ci a e oxidizing, which mus be an ea ly e en o malignancy and p og ession. Addi ionally, Shan Zha e al desc ibed he selec i e up egula ion o pe oxisomal b anched chain a y acid β-oxida ion pa hway in p os a e cance [17], which migh ep esen a good sou ce o ace yl- CoA o K ebs cycle, and la e , Ranasinghe W. e al [27] linked hypoxia wi h agg essi eness and me as asis in p os a e cance . Howe e , whe he he exp ession o p o eins in ol ed in cellula ene ge ic me abolism is al e ed du ing p os a e malignan ans o ma ion and p og ession, as well as, he signi icance o hei exp ession is la gely unknown. In his s udy, we ound a consis en o e exp ession o p o eins in ol ed in pe oxisomal b anched chain a y acid oxida ion in p os a e cance as well as in PIN lesions in con as o benign glands, sugges ing a possible e iological ole o his pa hway in malignan ans o ma ion. In he o he hand, he exp ession o p o eins in ol ed in he glycoly ic pa hway showed associa ion wi h eliable p edic o s o poo p ognosis such as umou s age (pT), Gleason sco e and biochemical ecu ence, sugges ing a ole in disease p og ession. Acco dingly, ou in i o s udies showed ha di e en models o disease p og ession exhibi ed di e en me abolic p o iles. The cell lines de i ed om dis an me as asis a e mo e glycoly ic and s ongly exp ess MCT4 and GLUT-1 Role o MCTs in P os a e Ca cinoma Pé ega-Gomes N. 128 a he plasma memb ane, wo majo playe s in he glucose up ake and lac a e ex usion essen ial o he p ope unc ion o he glycoly ic pa hway. Impo an ly, a change in he subcellula localiza ion o hese p o eins om localized umou o me as a ic umou was e i ied, wi h MCT4, GLUT1 and CAIX being p esen a he plasma memb ane only in me as a ic samples, sugges ing an ob ious link be ween he plasma memb ane unc ion o hese p o eins and he agg essi e s age o he umou . Using inhibi o s o he di e en pa hways we we e able o suppo he idea ha di e en me abolic pa hways a e ac i e acco ding o he disease s age o he umou . Thus, a ge ing a y acid oxida ion seems o be mo e ele an in an ea ly s age in con as o he glycoly ic pa hway, which will p oduce mo e e ec s in an ad anced s age o he disease. Addi ionally, di e ences we e also e iden a he o ganelle con en le el, be ween low and high umo igenic models, which led us o in es iga e he idea o a less ene ge ically e icien espi a ion o he highly umo igenic models. In ac , we ound an alse inc ease in Complex I ac i i y in PC3 cells masked by he o e exp ession o UCP2 p o ein, jus i ying a coupling de ec in oxida i e phospho yla ion in hese cells. In e es ingly, a coupling de ec in oxida i e phospho yla ion was also desc ibed in mi ochond ion- ich (Hü hle o oncocy ic) umou s o he hy oid [28-31]. Finally, publici y da ase suppo ed ou main indings showing ha he exp ession o genes in ol ed in a y acid oxida ion mos ly changed om benign samples o localized umou specimens, sugges ing a clea ole mainly in p os a e malignan ans o ma ion a he han p og ession, whe eas, genes in ol ed in he glycoly ic me abolism a e abe an ly exp essed mainly in he me as a ic samples, being mo e compa ible wi h a ole in p os a e cance p og ession and agg essi eness, and d awing a en ion in his case o SLC16A3 gene ha codi ies MCT4 as a p edic i e ma ke o disease p og ession and agg essi eness. This da a also indica es ha p os a e cance does no i in he adi ional model o a me abolic swi ch o glycolysis om non malignan o malignan cells bu ins ead his me abolic swi ch is mo e likely o Role o MCTs in P os a e Ca cinoma Pé ega-Gomes N. 129 be in ol ed in he ad anced s age o he disease and impo an ly p io o pa hologic ans o ma ion, exhibi ing a p edic i e alue. To he bes o ou knowledge, his is he la ges s udy in es iga ing he ole o key me abolic- ela ed p o eins in ela ion o ou come o adical ea men o localized p os a e cance , pu ing oge he a la ge a ie y o clinical samples and in i o models, demons a ing he me abolic he e ogenei y o p os a e cance and i s signi icance as diagnos ic, p ognos ic and he apeu ic ool in p os a e cance disease. Acknowledgemen s NPG and SS ecei ed ellowships om he Po uguese Founda ion o Science and Technology (FCT), e s. SFRH/BD/61027/2009 and PTDC/SAU- MET/113415/2009 espec i ely. This wo k was suppo ed by he FCT g an e . PTDC/SAU-MET/113415/2009, unde he scope o “P og ama Ope acional Tema ico Fac o es de Compe i i idade” (COMPETE) o “Quad o Comuni a io de Apoio III” and co- inanced by Fundo Comuni a io Eu opeu FEDER. This wo k was pa ially suppo ed by he p ojec (PIC/IC/83037/2007). Fu he unding was ob ained om he p ojec ‘Mic oen i onmen , me abolism and cance ’ based a IPATIMUP and pa ially suppo ed by P og ama Ope acional Regional do No e (ON.2—O No o No e), unde he Quad o de Re e ência Es a égico Nacional (QREN), and h ough he Fundo Eu opeu de Desen ol imen o Regional (FEDER). IPATIMUP and ICVS/3Bs a e associa e labo a o ies o he Po uguese Minis y o Science, Technology and Highe Educa ion and is pa ially suppo ed by he FCT. Role o MCTs in P os a e Ca cinoma Pé ega-Gomes N. 130 S a emen o au ho con ibu ions All au ho s mee he c i e ia o au ho ship in ha hey ha e pa icipa ed in he concep ion, execu ion o in e p e a ion o a leas pa o he publica ion in hei ield o expe ise. 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Am J Pa hol, 2002. 160(5): p. 1857-65. 30. Sa agne , F., e al., De ec i e mi ochond ial ATP syn hesis in oxyphilic hy oid umo s. J Clin Endoc inol Me ab, 2001. 86(10): p. 4920-5. 31. Gaspa e, G., e al., Dis up i e mi ochond ial DNA mu a ions in complex I subuni s a e ma ke s o oncocy ic pheno ype in hy oid umo s. P oc Na l Acad Sci U S A, 2007. 104(21): p. 9001-6. Role o MCTs in P os a e Ca cinoma Pé ega-Gomes N. 133 Tables Table 1. De ails o he immunohis ochemical p ocedu e used o analyze he exp ession o he di e en p o eins. P o ein An ibody Company An ibody Dilu ion Posi i e Con ol Incuba ion Pe iod De ec ion Sys em MCT4 sc-50329 San a C uz Bio echnology 1:500 Colon umo O e nigh R.T.U. Vec as ain Uni e sal Eli e ABC Ki , Vec o , EUA MCT2 sc-50322 San a C uz Bio echnology 1:200 Muscle 2 hou s Ul a ision De ec ion Sys em An i-poly alen , HRP, Lab ision Co po a ion, F eemon , CA CD44 156-3C11 Se o ec 1:1000 Colon umo GLUT1 ab 15309 Abcam 1:2000 Head and neck umo CAIX ab 15086 Abcam 1:2000 S omach MCT1 sc-365501 San a C uz Bio echnology 1:500 Colon umo O e nigh R.T.U. Vec as ain Uni e sal Eli e ABC Ki , Vec o , EUA HIF-1α 610958 BD Biosciences 1:100 Glioblas oma GLUT12 ab 75441 Abcam 1:500 Rim O e nigh Ul a ision De ec ion Sys em An i-poly alen , HRP, Lab ision Co po a ion, F eemon , CA LDHV ab 53010 Abcam 1:1000 Colon umo 2 hou s HKII ab104836 Abcam 1:750 Colon umo PDK1 ab110025 Abcam 1:500 S omach CD147 sc-71038 San a C uz Bio echnology 1:400 Colon umo AMACR 504R-16 Cell Ma que 1:50 Kidney ACOX3 sc-135435 San a C uz Bio echnology 1:250 Li e DBP DBP an ibody was a gi om D . Gab iele Molle om Helmhol zZen um mÜnchen. Ready o use Kidney