Uncovering PRMT6 deregulation effect in prostate cancer

M 2014 UNCOVERING PRMT6 DEREGULATION EFFECT IN PROSTATE CANCER VÍTOR DIOGO ALMEIDA RIOS TESE DE MESTRADO APRESENTADA AO INSTITUTO DE CIÊNCIAS BIOMÉDICAS ABEL SALAZAR DA UNIVERSIDADE DO PORTO EM ONCOLOGIA MOLECULAR VITOR DIOGO ALMEIDA RIOS UNCOVERING PRMT6 DEREGULATION EFFECT IN PROSTATE CANCER Dissertação de mestrado apresentada ao Instituto de Ciências Biomédicas Abel Salazar da Universidade do Porto em Oncologia – Especialização Oncologia Molecular ORIENTADOR Professora Doutora Carmen de Lurdes Fonseca Jerónimo Professora Associada convidada com “Agregação” Departamento de Patologia e Imunologia Molecular Instituto de Ciências Biomédicas Abel Salazar Universidade do Porto Investigadora Auxiliar e coordenadora do Grupo de Epigenética e Biologia do Cancro – Centro de Investigação Instituto Português de Oncologia – Porto CO-ORIENTADOR Professor Doutor Rui Manuel Ferreira Henrique Professor Catedrático Departamento de Patologia e Imunologia Molecular Instituto de Ciências Biomédicas Abel Salazar Universidade do Porto Diretor do Serviço de Anatomia Patológica Investigador Sénior do Grupo de Epigenética e Biologia do Cancro – Centro de Investigação Instituto Português de Oncologia – Porto “You I advise not to work, But to fight. You I advise not to peace, But to victory. Let your work be a fight, Let your peace be a victory.” Friedrich Nietzsche, Thus Spoke Zarathustra, 1883 v AGRADECIMENTOS A realização desta tese de Mestrado representou para mim, o culminar de uma etapa importante na minha formação profissional. Há dois anos atrás, a minha ambição centrava-se em adquirir novos conhecimentos numa área do meu interesse e aplicá-los da melhor forma na realização desta tese. Volvidos dois anos, o apaziguamento que sinto mostra-me que esta etapa me enriqueceu muito para além da componente cognitiva e profissional. Desta forma, é legitimo que me sinta grato para com um leque de pessoas que me ajudaram, às quais quero endereçar o meu sincero agradecimento. À minha orientadora, Prof.ª Carmen Jerónimo, em primeiro lugar porque me mostrou que a lealdade é base essencial para respeitar e ser respeitado. Numa fase menos positiva para mim, ter acreditado no meu trabalho foi um gesto que me motivou até à última palavra desta tese. Penso que a cada contratempo que encontrei ao longo deste caminho, o desânimo que não conseguia esconder espelhava suficientemente bem a vontade que tinha em retribuir o voto de confiança em mim depositado. Nesses momentos menos conseguidos, o seu lado maternal foi essencial em cada conselho que me deu, em cada lição que me incutiu. A maneira singular como gere o Grupo de Epigenética e Biologia do Cancro ofereceu-me liberdade para seguir um determinado rumo de trabalho e para aprender com os erros cometidos. Ao meu Co-orientador, Prof. Rui Henrique, cujo vasto conhecimento, rigor científico e espírito crítico apenas são excedidos pela simplicidade e educação com que trata todos aqueles que o procuram. Talvez tenha sido abusivo da minha parte procurá-lo tantas vezes para discutir resultados e esclarecer dúvidas, mas teria sido ingénuo em desperdiçar a oportunidade de trabalhar e aprender, com alguém tão competente para partilhar o conhecimento como o Professor. A ambos, expresso o meu singelo agradecimento e considero-me um privilegiado por ter tido a oportunidade de trabalhar e aprender convosco. A este e a tantos outros níveis, são e serão no futuro, uma referência para mim. Ao Prof. Manuel Teixeira, Diretor do Serviço de Genética e do Centro de Investigação do Instituto Português de Oncologia do Porto, por me ter aceite no mesmo e me ter dado todas as condições necessárias para a elaboração deste trabalho. vi À Filipa, em primeiro lugar pela dura batalha que travou para que não a tratasse por “Professora Filipa”. Alcançada essa vitória, foi a pessoa que me acolheu no âmbito laboratorial quando eu ainda tinha um conhecimento muito superficial do que era estar num laboratório de investigação. Ensinaste-me tudo aquilo que necessitei para conseguir ser autónomo e orgulho-me de sentir que, neste sentido, tive os melhores ensinamentos possíveis. Se hoje me sinto metódico e organizado naquilo que faço, devo-o a ti. As risadas e os ombros que partilhámos neste período são a prova que fomos mais que companheiros de trabalho. Fomos fomentando uma boa amizade que, sem ser de sempre, espero que seja para sempre. À Inês, porque em boa hora apareceste nesta caminhada. Esse sorriso de orelha a orelha é a melhor recordação que posso guardar dos bons momentos partilhados. Foste um pilar essencial no rumo que este trabalho levou. Quando, numa fase decisiva, sofri um enorme revés, passível de colocar em causa a realização de um bom trabalho, tu foste absolutamente fantástica na forma como suportaste o meu mau humor, me motivaste e prontamente te disponibilizaste para me ajudar naquilo que precisasse. A forma empenhada e séria como discutiste os resultados obtidos e deste sugestões pertinentes, foram sem dúvida alguma, cruciais na etapa final e decisiva desta tese. Conciliar tudo isto com boa disposição constante, fez com que o tempo que trabalhámos juntos e facilitámos o difícil, seja uma imagem a guardar deste percurso. À Márcia, porque por motivos profissionais partilhámos cada dia deste percurso. Aprendemos a lidar com os nossos dias bons, os nossos dias maus e a tolerar a falta de sincronização entre os nossos dias bons e os nossos dias maus. Não foi fácil, nem tão pouco difícil. Foi uma experiência nossa. Cresci a cada desavença, a cada debate e a cada partilha de ideias. Por tudo isto, que os rumos que as nossas vidas levem, me façam sentir a tua ausência e que a ausência traga até mim a memória de cada riso incontrolado, soltado por razões que só nós conseguíamos perceber. Ao Pedro, à Joana e ao Diogo, por toda a partilha de ideias e ajuda que sempre me deram ao longo deste trabalho. Ao Pedro, porque te prestaste a ajudar quando precisei de ti e pelo rigor e espírito crítico que te caracterizam. À Joana porque sempre que te procurei e precisei de ti, foste simplesmente impecável. Ao Diogo porque bastavam uns minutos a falar contigo para a minha vii cultura geral sofrer um upgrade. Embora eu não saiba o que vou levar desta vida, espero conseguir levar a memória. Nela guardo todos os bons momentos que passámos, desde os lanches aos jantares e desde os jantares ao jogos no Dragão a sofrer pelo nosso Porto. Que no futuro nos possamos encontrar e que o reencontro seja nostálgico por aquilo que nos marcou, a boa disposição. Ao João, porque de uma forma profissional e particular foi discutindo comigo os resultados que fui obtendo, sugerindo sempre possíveis linhas de trabalho a seguir. Agradeço-te a partilha de ideias e de standards pois permitiram enriquecer o conteúdo desta tese. A todos os elementos do Grupo de Epigenética e Biologia do Cancro que conheci nestes dois anos. A vossa capacidade de conciliar o trabalho com a boa disposição é admirável e, sem qualquer sombra de dúvida, trabalhar assim tornase bem mais fácil. À Francisca, por todas as conversas científicas que tivemos e que me deixaram a pensar em teorias mirabolantes. Devo ainda um agradecimento especial às tuas aptidões para a alquimia, pois quase me mostraste que era possível tornar o preto, branco. A todos os meus colegas do Mestrado em Oncologia, pela boa experiência social que foi conhecer-vos, adquirir conhecimentos em conjunto e partilhar a ansiedade que antecedeu cada exame ou trabalho coletivo. Ao Jorge Sousa, porque chegar a casa e abstrair-me do trabalho nunca foi fácil. A cada tentativa de te explicar o que andava a fazer, estou certo que te enriqueci tanto cientificamente como tu a mim musicalmente, de cada vez que tive de ouvir o teu repertório para saxofone, desde a nobre música clássica à irreverente música contemporânea. A todos os elementos da minha equipa de futebol, assim como da adversária, que todas as segundas-feiras permitiram pausar o trabalho com uma agradável hora de competição saudável. Até nas derrotas foi uma honra partilhar as quatros linhas convosco. Que o nosso companheirismo continue a ser o nosso lema e o símbolo máximo da camisa que envergamos. À Técnica Maria do Amparo, que no decurso deste período me permitiu disponibilizar o tempo necessário para não comprometer a execução desta tese. xii (complexo MLL e a SMYD3) tenham aumentado significativamente. O silenciamento da PRMT6 associou-se significativamente com o aumento dos níveis de transcrito do p21, p27 e CD44, bem como com a diminuição da expressão da MMP-9. Relativamente às vias de sinalização celular, o silenciamento da PRMT6 conduziu a uma redução da via PI3K/AKT/mTOR e a um aumento da via dos receptores de androgénios, sugerindo um papel oncogénico desta enzima. De modo a confirmar que as alterações observadas são uma consequência direta da atividade da PRMT6 serão necessários estudos complementares. De igual modo, serão efetuados ensaios adicionais in vitro para esclarecer a utilidade terapêutica da re-expressão dos receptores de androgénios na linha celular PC-3. De facto, a restauração da expressão do AR em células PC-3 ShPRMT6, pode ser clinicamente relevante, uma vez que pode voltar a sensibilizar células neoplásicas andrógeno-insensitive a ADT. xiii SUMMARY Prostate cancer (PCa) is one of the most incident and prevalent cancers in men worldwide, and a leading cause of cancer-related morbidity and mortality. The limited knowledge about its biology hinders the development of new diagnostic and prognostic markers, able to improve management and therapeutic of this malignancy. Epigenetics plays an important role in prostate carcinogenesis and in fact, abnormal expression of histone modifier enzymes, such as histone methyltransferases (HMTs) and demethylases (HDMs), and its chromatin modifications are related to PCa. Notwithstanding, the specific role of deregulated activity/expression of several members of both HMTs and HDMs is still poorly understood. Previously, we have found in a small number of clinical samples that PRMT6 was overexpressed in PCa compared to normal prostate tissue samples (NPTs) and demonstrated a promising capacity to distinguish NPTs from PCa. Based on these previous results, the main goals of this work were to validate the overexpression of PRMT6 in PCa and further explore its putative oncogenic role in prostate carcinogenesis, using in vitro models. Using a large series of prostatic samples we found that PRMT6 was overexpressed in PCa, at both transcript and protein level. Intriguingly, PIN lesions displayed significantly higher PRMT6 expression levels, compared to PCa. Furthermore, PRMT6 mRNA levels are able to discriminate cancerous from noncancerous prostate tissues. Contrarily to LNCaP, stable PRMT6 knockdown in PC-3, attenuated the malignant phenotype, in which the increased apoptosis as well as the decreased viability levels, migration and invasion ability was observed. Moreover, at molecular level, PRMT6 silencing was associated with decreased H3R2me2a levels and increased MLL complex and SMYD3 expression, although no global H3K4me3 levels were found. The silencing of PRMT6 significantly associated with increased expression levels of p21, p27 and CD44, as well as decreased expression of MMP-9. Regarding signaling pathways, PRMT6 knockdown related with a reduction of the PI3K / AKT / mTOR pathway and an increase in AR, supporting an oncogenic role for this enzyme. xiv Nevertheless, to confirm the observed alterations as a direct consequence of PRMT6 activity further experiments are needed. Similarly, additional in vitro studies should be performed to elucidate the specific therapeutic utility of AR reexpression in PC-3 cell line. Indeed, restoration of AR expression in Sh-PRMT6 PC3 cells, might be of clinical relevance as it may re-sensitize androgen-insensitive neoplastic cells to ADT. xv TABLE OF CONTENTS INTRODUCTION 1! 1!PROSTATE 3! 1.1!PROSTATE ANATOMY AND HISTOLOGY 3! 1.2!NON-MALIGNANT DISORDERS OF PROSTATE 4! 1.3!PROSTATE CANCER 5! 1.3.1!Epidemiology of Prostate Cancer 6! 1.3.2!Risk Factors 7! 1.3.3!Current Guidelines for Prostate Cancer Diagnosis 8! 1.3.4!Prostate Cancer Grading System: Gleason Score 9! 1.3.5!Clinical and Pathological Staging 11! 1.3.6!Prostate Cancer Therapies 13! 2!EPIGENETICS 17! 2.1!DECIPHERING ITS GROWING RELEVANCE 17! 2.2!EPIGENETIC MECHANISMS OF GENE EXPRESSION CONTROL 18! 2.2.1!DNA Methylation 18! 2.2.2!Non-coding RNAs 19! 2.2.3!Histone Post-Translational Modifications 20! 3!HISTONE METHYLATION AND PROSTATE CANCER 23! 3.1!HISTONE METHYLATION: BASICS OF AN EXTENSIVE FIELD 23! 3.2!HISTONE METHYLATION: RELEVANCE IN PROSTATE CANCER 24! PRELIMINARY DATA 27! AIMS OF THE STUDY 31! MATERIALS AND METHODS 35! 1!CLINICAL SAMPLES: PATIENTS AND SAMPLE COLLECTION 37! 2!RNA ISOLATION 37! 3!PRMT6 GENE EXPRESSION IN CLINICAL SAMPLES 37! 4!IMMUNOHISTOCHEMISTRY 38! 5!CELL CULTURE 39! 6!PRMT6 KNOCKDOWN IN SELECTED PCA CELL LINES 40! 7!RNA EXTRATION FROM SILENCED CELL LINES 40! xvi 8!CDNA SYNTHESIS 40! 9!PRMT6 EXPRESSION ASSAY 41! 10!PROTEIN EXTRACTION AND QUANTIFICATION 41! 11!WESTERN BLOT ANALYSIS 42! 12!CELL VIABILITY ASSAY 43! 13!APOPTOSIS ASSAY 44! 14!MIGRATION ASSAY 45! 15!INVASION ASSAY 45! 16!STATISTICAL ANALYSIS 46! RESULTS 47! 1!PRMT6 GENE EXPRESSION IN A LARGE SERIES OF CLINICAL SAMPLES 49! 2!ASSOCIATION BETWEEN PRMT6 EXPRESSION LEVELS AND CLINICOPATHOLOGICAL FEATURES 49! 3!EVALUATION OF PRMT6 EXPRESSION AS DIAGNOSTIC BIOMARKER 50! 4!PRMT6 EXPRESSION OF MATCHED PIN AND PCA SAMPLES 51! 5!PRMT6 PROTEIN LEVELS IN A LARGE SERIES OF CLINICAL SAMPLES 52! 6!KNOCKDOWN OF PRMT6 IN PC-3 AND LNCAP CELL LINES 54! 7!IMPACT ON VIABILITY AND APOPTOSIS OF PRMT6 KNOCKDOWN IN PC-3 AND LNCAP CELL LINES 55! 8!EFFECT OF PRMT6 KNOCKDOWN IN MIGRATION AND INVASION CAPACITY OF PC-3 CELLS 56! 9!EFFECT OF PRMT6 KNOCKDOWN IN HISTONE PTMS PATTERNS IN PC-3 CELL LINE 57! 10!MOLECULAR PATHWAYS AND PUTATIVE TARGET GENES REGULATED BY PRMT6 59! DISCUSSION 61! CONCLUSION AND FUTURE PERSPECTIVES 69! REFERENCES 73! xvii FIGURE INDEX Figure 1: Anterior oblique view of the prostate and urethra. 4 Figure 2: Progression pathway for prostate cancer. 6 Figure 3: Estimated age-standardized incidence rates of PCa per 100,000, worldwide. 6 Figure 4: Incidence and mortality of different cancers in Europe and Portugal. 7 Figure 5: Modified Gleason grading system for PCa. 10 Figure 6: Models of progression to CRPC. 16 Figure 7: Altered DNA-methylation patterns in carcinogenesis. 19 Figure 8: Chromatin structure and histone PTMs. 21 Figure 9: Epigenetic writers, readers and erasers. 22 Figure 10: Major lysine methylation marks on the amino-termini of histones H3 and H4. 23 Figure 11: Histone-modifications map for a typical chromosome in normal and cancer cell. 25 Figure 12: Expression levels of PRMT6 in NPT and Tumor. 30 Figure 13: Principle of BioCoat Matrigel Invasion Chamber and subsequent analysis of invasion assay. 46 Figure 14: PRMT6 relative expression in prostate tissues. 49 Figure 15: Performance of PRMT6 mRNA expression as a biomarker for PCa. 50 Figure 16: Relative expression of PRMT6 in paired PIN and PCa tissue samples. 51 Figure 17: Illustrative images of PRMT6 immunostaining in NPT, PIN and PCa samples. 53 Figure 18: Distribution of PRMT6 immunoexpression (A) and of PRMT6 transcript levels (B) in a series of prostate tissue samples (NPT, PIN and PCa), grouped according to PRMT6 immunostaining. 53 Figure 19: (A) PRMT6 expression in PCa cell lines. (B) The efficiency of PRMT6 knockdown was confirmed at mRNA, using RT-qPCR (upper panel), and protein level, using Western-Blot (lower panel), in LNCaP and PC-3 cells. 54 55 xviii Figure 20: Impact of PRMT6 knockdown in cell viability and apoptosis of LNCaP and PC-3 cell lines. Figure 21: Impact of PRMT6 knockdown in migration and invasion properties of PC-3. 56 Figure 22: Reduction of asymmetrical dimethylation of arginine 2 of histone 3 after PRMT6 knockdown in PC-3 cell line. 57 Figure 23: Relative expression of methyltransferases that catalyze H3K4me3 in PC3 PRMT6-silenced cells. 58 Figure 24: H3K4me3 and MYC expression after PRMT6 knockdown in PC-3 cell line. 58 Figure 25: Western blot analysis of p21 and p27 expression in PC-3 cell line after PRMT6 knockdown. 59 Figure 26: Impact of PRMT6 knockdown in MMP-9 and CD44 transcript levels in PC-3 cell line. 59 Figure 27: Western blot analysis of AKT, pAKT and mTOR expression in PC-3 cell line. 60 Figure 28: Overexpression of AR in PC-3 PRMT6 silenced cells. 60 xix TABLE INDEX Table 1: The 2010 AJCC/UICC TNM staging classification for PCa. 12 Table 2: Clinical and pathological features of patients included in the screening of HMTs and HMDs expression in NPTs and PCa samples. 29 Table 3: Culture media conditions for PCa cell lines. 39 Table 4: Essential information about the primary antibodies used in this study. 43 Table 5: Number of cells plated for in vitro experiments in PCa cell lines used in this study. 44 Table 6: Clinical and pathological data of patients included in this study. 50 Table 7: Description of PRMT6 performance as diagnostic biomarker of prostate malignancy. 51 Table 8: Immunohistochemical expression of PRMT6 in a series of NPT, PIN lesions and primary PCa. 52 Table 9: Association between PRMT6 expression and prostate tissues. 52 Uncovering PRMT6 Deregulation Effect In Prostate Cancer 4 1.2 Non-malignant Disorders of Prostate Prostate has higher frequency and extent of non-malignant lesions than other organs [9], being more common in aged men [10]. Benign prostatic hyperplasia (BPH), prostatic intraepithelial neoplasia (PIN) and proliferative inflammatory atrophy (PIA) are among the most common pre-malignant lesions of this gland. BPH is a chronic disease affecting more than half of the male population over 50 years old [10]. Coexistence of BPH and prostate cancer (PCa) was first described in 1957 in a necropsy study, in which BPH was identified in 80% and 45% of cadavers with and without PCa, respectively [11]. Although BHP and PCa share several common pathophysiological mechanisms they exhibit important differences in terms of histology and localization. However, it is still not clear if BHP represents the first step in the pathway to PCa [11]. Figure 1: Anterior oblique view of the prostate and urethra. The prostate is divided into four zones: central zone (CZ), transition zone (TZ), peripheral zone (PZ) and anterior fibromuscular stroma (AFS). Adapted from [8]. Introduction 5 In 1987, PIN lesions were first described by Bostwick and Brawer [12] being those lesion classified into low-grade (LGPIN) and high-grade (HGPIN). Although, only HGPIN is widely accepted as a precursor lesion of PCa. This lesion is characterized by progressive phenotype and genotype abnormalities that are intermediate between benign prostatic epithelium and cancer [13, 14]. The frequency of HGPIN in prostate biopsies averages 9% and the severity and frequency of these lesions at postmortem examination are greatly increased in individuals with cancerous prostates comparing with noncancerous prostates [13, 15]. In addition to the similar cytologic features, HGPIN and PCa share genetic and molecular markers as well as other characteristics, such increased incidence with age and preferential occurrence at prostatic peripheral zone. These characteristics, combined with the fact that HGPIN precedes the onset of PCa in about 10 years, suggest that HGPIN represents an intermediate stage between benign epithelium and the invasive malignant carcinoma. Thus, HGPIN might have a clinical significance as a tool to identify patients at risk of malignancy [14]. PIA has been also suggested as a precursor of PCa and HGPIN. This lesion is described as a hyperproliferative entity associated with chronic inflammation and with distinct morphological appearance recognized as prostatic atrophy [16]. The hypothesis that PIA could be a potential precursor of other prostate lesions stems from the fact that this lesion usually occurs in the prostate peripheral zone and shares some molecular features found both in PIN and PCa [13]. Despite those findings, evidence to date is inconclusive as to whether PIA is a precursor of HGPIN and PCa [13]. 1.3 Prostate Cancer Adenocarcinoma of the prostate accounts for 95% of all malignancies in this gland [3]. PCa is an age-related disease, being very heterogeneous in terms of pathologic, biologic and clinical behavior, ranging from clinical indolent to highly aggressive neoplasia [6, 17]. This malignancy is multicentric and has no specific symptoms, which leads to a late diagnosis and compromises not only prognosis but also the attempts to develop target therapies [6]. Uncovering PRMT6 Deregulation Effect In Prostate Cancer 6 Histologically, PCa is characterized by loss of both basal cell layer and membrane. Remarkably, in HGPIN this cell layer is fragmented and in BPH, both are intact. This finding suggests the existence of a sequential pathway in PCa development (Figure 2) although the underlying mechanisms that lead to PCa still not fully understood [18]. 1.3.1 Epidemiology of Prostate Cancer Current epidemiological data indicates this neoplasm as a major health concern. In 2012, 1,111,689 new cases were diagnosed worldwide (approximately 15% of total newly diagnosed cancers in men), making it the second most commonly diagnosed cancer in male population, only outshined by lung cancer [19]. PCa incidence rates are very heterogeneous around globe, being estimated more than a 25-fold variation. In fact, about 70% of new diagnosed cases in 2012 occurred in more developed countries, being the higher incidences reported in United States, Australia and Nordic countries, whereas lower incidence rates are observed in Asia [19]. (Figure 3) Figure 2: Progression pathway for prostate cancer. Adapted from [3]. Figure 3: Estimated age-standardized incidence rates of PCa per 100,000, worldwide. Adapted from [19]. Introduction 7 Regarding mortality, a slight decrease in mortality rates were observed among developed countries. However, the mortality rates are much less variable than incidence, accounting only a 10-fold variation worldwide [19]. Restricting this analysis to Europe, PCa arises as the most incident cancer and the third cause of cancer-related mortality. In Portugal, PCa is also the most frequent cancer among men, with 6,622 newly diagnosed cases in 2012 (23,3% of overall cancers) and the third leading cause of cancer related death (11,1% of overall cancer related deaths) [19]. (Figure 4) 1.3.2 Risk Factors Although the etiology and pathogenesis of PCa is quite complex, there are three well-established and widely accepted risk factors [20]. Race: PCa remains as one of the most singular cancers regarding geographical distribution. Globally, African American men have a higher risk to develop PCa and tend to be diagnosed at younger ages and with more advanced stage, which reflects the higher mortality rates in this population, comparing to European American men [21]. Asian men have the lowest incidence rates of PCa but Asian males living in the United States exhibit a higher risk to develop this malignancy [22]. The exact and scientific explanation for these disparities is still unknown, however genetic, Figure 4: Incidence and mortality of different cancers in Europe and Portugal. Adapted from [19]. Uncovering PRMT6 Deregulation Effect In Prostate Cancer 8 environment, social or cultural factors also in addition to differences in the decision-making practice, access to medical care and preferred diagnostic tools, might play an important role in this unequal distribution of PCa around the globe [23, 24]. Family history: Men´s probability of developing PCa is higher within a family history of the disease is present. Currently, it is estimated that 10-15% of all PCa are associated with familial cases [23]. In fact, men with first-degree relatives (father or brother) harboring PCa have more than double of risk to develop this disease. Additionally, the number of members affected and the early-onset of disease increase even more the risk of developing PCa. This might suggest an involvement of a genetic predisposition, although other features like exposure to the same environmental factors should also be considered [22, 23]. Age: As a consequence of a clinical latent course, age is the most important recognized risk factor for PCa incidence [17, 23]. Malignancies of prostate gland are more frequent in older men, with a median age at diagnosis of 67 years. Approximately 85% of PCa cases are diagnosed after the age of 65 years old and in men older than 90 years old, the incidence is higher than 90% [22, 23, 25]. 1.3.3 Current Guidelines for Prostate Cancer Diagnosis Diagnosis of PCa is quite hampered by indolent behavior of this malignancy. Thus, improvement of detection tools to diagnose the disease when it is organ confined is the best approach. Serum concentration of prostatespecific antigen (PSA) and digital rectal examination (DRE) are generally used as a standard methodology to PCa screening and management [26]. DRE is capable to detect about 18% of all PCa, regardless of the PSA level [27]. PSA is a glycoprotein with protease activity produced by epithelial cells of prostate gland, which is secreted in the glands lumina and is released into the bloodstream in increased quantities when disruption of normal prostatic membrane structures occurs. This phenomenon arises not only in malignant lesions, but also in the context of inflammation, hyperplasia and urologic manipulation. Therefore, PSA is prostate specific, but not PCa specific. Additionally, PSA not only lacks sensitivity to detect an important number of PCa Introduction 9 cases, especially at early stages, but also increases the overdiagnosis and overtreatment of indolent tumors, demonstrated a limited value as a screening test [28]. According with international guidelines, men with inconclusive or suspicious DRE result and/or PSA value equal or higher than 4.0 ng/mL, must be considered for transrectal ultrasound (TRUS)-guided biopsy. Additionally, when a patient is considered for biopsy, clinicians should take into account factors such patients’ age, possible comorbidities and therapeutic consequences. It should be taken in consideration that both PSA and DRE are fallible, once there are PCa cases with normal DRE and/or lower levels of PSA [29-31]. Nevertheless, the combination of PSA test and DRE allowed for an important decrease in the number of cases with advanced disease at the diagnosis (about 70-80% of PCa are organ confined), which increases the diseasefree survival [4]. 1.3.4 Prostate Cancer Grading System: Gleason Score The Gleason score (GS) is the widespread accepted system for grading PCa. This system is based on the histological evaluation of glandular epithelial architecture pattern, excluding cytological aspects from the analysis. Given the PCa heterogeneity, it classifies the two most prevalent patterns in a 1 to 5 scale, with a decreasing grade of differentiation (1Most differentiated; 5Least differentiated) (Figure 5). Thus, the GS consists in the sum of the classification of the two most common patterns, ranging from 2 (1+1) to 10 (5+5) [4, 32]. In 2005, International Society of Urological Pathology (ISUP) published the last update of this classification, considering that GS of a prostate biopsy should include the Gleason grade of the prevalent carcinoma pattern and the highest grade, independently of its extent. The predominant pattern has a crucial role in prognostic evaluation, specifically in GS = 7 lesions, because the predominance of pattern 4 (Gleason 4+3) carries more than a 3-fold higher risk of PCa mortality than Gleason pattern 3+4 [33, 34]. Since 1966, when Donald Gleason firstly proposed this grading system, it is along with pathologic stage, a powerful tool for PCa prognostic assessment. Thus, GS is fundamental in predicting PCa natural history and the risk of recurrence after radical prostatectomy (RP) or radiotherapy, influencing treatment approach [30]. Uncovering PRMT6 Deregulation Effect In Prostate Cancer 10 Notwithstanding, inter-observer variability remains the principal pitfall of GS, although the updated system has proved to increase in 20% agreement between observers [32, 33]. Figure 5: Modified Gleason grading system for PCa. Pattern 1: Closely-packed, uniform, rounded to oval glands; Pattern 2: More loosely arranged glands with smooth ends that minimally invade non neoplastic tissue; Pattern 3: Irregular size and shape glands with more infiltrative margins; Pattern 4: Fused, cribriform or ill-defined glands; Pattern 5: Almost no glandular differentiation. Adapted from [34]. Introduction 11 1.3.5 Clinical and Pathological Staging PCa is an important cause of cancer morbidity and mortality worldwide and therefore, accurate staging is a crucial approach for prognosis assessment and treatment planning [35]. The most commonly used staging system for PCa was proposed by the American Joint Committee on Cancer (AJCC) and the Union for International Cancer Control (UICC): the TNM (Tumor Node Metastasis) system [36]. This staging system consists in the evaluation of primary tumor (T) extension, regional involved lymph nodes (N) and presence or absence of distant metastases (M) (Table 1) [37]. According with AJCC and UICC, TNM stage for PCa could be clinical (cTNM) or pathological (pTNM) [38]. Clinical stage provides information regarding cancer extension, before definitive treatment onset and is generally achieved by DRE, TRUS or magnetic resonance imaging (MRI), and serum PSA levels [36, 39]. Since pathological staging requires macro and microscopic evaluation of surgical specimen and dissected regional lymph nodes, it can only be determined after RP [4, 38, 40]. Consensually, pathological staging predicts recurrence with more accuracy than clinical stage and, it is also useful as a prognostic factor [40]. In this sense, clinicians developed monograms, combining independent prognostic factors such as capsule invasion, preoperative serum PSA levels, GS for the RP specimen, positive surgical margins, lymph node metastases, seminal vesicles involvement and distant metastases [4, 35, 41]. Uncovering PRMT6 Deregulation Effect In Prostate Cancer 12 Table 1: The 2010 AJCC/UICC TNM staging classification for PCa. Adapted from [36]. PRIMARY TUMOR (T) CLINICAL Tx Primary tumor cannot be assessed T0 No evidence of primary tumor T1 Clinically unapparent tumor neither palpable nor visible by imaging T1a Tumor incidental histologic finding in 5% or less of tumor resected T1b Tumor incidental histologic finding in more than 5% of tumor resected T1c Tumor identified by needle biopsy T2 Tumor confined within prostate gland T2a Tumor involves one half of one side or less T2b Tumor involves more than one half of one lobe but not both lobes T2c Tumor involves both lobes T3 Tumor extends through prostate capsule T3a Extracapsular extension (unilateral or bilateral) T3b Tumor invades seminal vesicle(s) T4 Tumor is fixed or invades adjacent structures other than seminal vesicles, such as: external sphincter, rectum, bladder, levator muscles, and/or pelvic wall PATHOLOGIC (PT)* pT2 Organ confined pT2a Unilateral, one half of one side or less pT2b Unilateral, involving more than one half of one lobe but not both lobes pT2c Bilateral disease pT3 Extraprostatic extension pT3a Extraprostatic extension or microscopic invasion of bladder neck pT3b Seminal vesicle invasion pT4 Invasion of rectum, levator muscles and/or pelvic wall REGIONAL LYMPH NODES (N) CLINICAL Nx Regional lymph nodes were not assessed N0 No regional lymph node metastasis N1 Metastasis in regional lymph node(s) PATHOLOGIC (PN) pNx Regional nodes not sampled pN0 No positive regional nodes pN1 Metastasis in regional node(s) DISTANT METASTASIS (M) M0 No distant metastasis M1 Distant metastasis M1a Non-regional lymph node(s) M1b Bone(s) M1c Other site(s) with or without bone disease *There is no pathologic pT1 classification Introduction 13 1.3.6 Prostate Cancer Therapies Unequivocally, PCa is associated with a substantial mortality and morbidity, but in fact, most men do die with, rather than of, their PCa. Furthermore, given the intrinsic heterogeneity of PCa, men with similar history (clinical stage, serum PSA levels and biopsy features) can have significantly different outcomes. Hereupon, the main challenge to physicians is to recognize the faction of men with aggressive local PCa and thus choose a suitable therapy approach for these, sparing the remainder of the morbidity associated to overtreatment [4]. Current therapeutic options for PCa include surgery, radiation, hormone therapy and chemotherapy. Treatment choice usually depends on disease and patient ´s features such as age, life expectancy, life quality, TNM stage, GS and preoperative serum PSA level [37]. Concerning clinically localized PCa, standard treatment options are active surveillance (AS), RP, external-beam radiotherapy and interstitial radiation therapy (brachytherapy). AS is suitable for patients with very-low risk of PCa progression. The requirements for belong to this group of patients are: T1-T2 TNM staging (organ confined PCa), GS ≤6 and serum PSA level < 10ng/mL. These patients are subjected to frequent PSA measurements and prostatic biopsies to detect disease progression [31]. RP remains the standard procedure for high-risk locally advanced PCa, showing survival benefits comparing with conservative methodologies. Patients with a biopsy GS ≤8, TNM staging ≤ T3a, PSA level < 20ng/mL and life expectancy of 10 or more years are indicated for this surgery. This procedure consists in the removal of whole prostate gland and seminal vesicles and has been optimized in order to decrease the associated morbidities, such as erectile dysfunction and urinary incontinence [31, 42]. Other therapeutic option for organ-confined PCa is radiotherapy since both external-beam radiotherapy and brachytherapy present rates of disease-free survival similar to that obtained by RP. External-beam radiotherapy is a noninvasive methodology with low morbidity rates suitable for patients without disseminated disease and which not tolerate RP or brachytherapy [42]. Regarding brachytherapy, this approach consists in the permanent ultrasound guided insertion of radioactive seeds, with the half-life of 60 days, directly in the Uncovering PRMT6 Deregulation Effect In Prostate Cancer 20 estimated that they regulate the expression of 50% of human genes [78]. Additionally, one miRNA can target many mRNAs, which in turn can be targeted by several miRNAs [75]. Thereby, due the scope of miRNA regulation, they play important functions in biological processes such as apoptosis, differentiation and proliferation [78, 79]. MiRNAs aberrant expression has been widely reported in several diseases, especially in cancer. The disruption of normal miRNA expression patterns is associated with genetic and epigenetic events [71, 74, 80]. A trend to a global miRNAs downregulation is observed in cancer, although upregulation also has been described [81]. Concerning miRNA role in carcinogenesis, depending on the neoplastic context and target genes, miRNAs can act as oncogenes (oncomiRs) or tumor-suppressors [82]. Hereupon, tumor-suppressors miRNAs decreased expression in cancer can be attributed to deficiencies in miRNA biogenesis leading to anomalous expression of their targets. In turn, oncomiRs overexpression and/or amplification promote a decreased expression of their miRNA-target tumor-suppressor genes, which could contribute to tumor induction [82]. 2.2.3 Histone Post-Translational Modifications In eukaryotic cells, chromatin is the macromolecular complex formed by DNA and histone proteins that contains the inherited material of cells [83]. The basic functional unit of chromatin, nucleosomes, contain 147 base pairs of DNA wrapped around a histone octamer, which is composed by one pair of histones H2A, H2B, H3 and H4, linked by H1 (Figure 8) [83-85]. A great portion of histone covalent post-translational modifications (PTMs) is established in histone tails [86]. PTMs define the chemical modifications of proteins that signal to other proteins, which in turn recognize the modification [87]. Additionally, chromatin is a dynamic structure that is divided in two major regions: heterochromatin, which is recognized as highly condensed regions that contain inactive genes; and euchromatin, which is relatively open and comprise transcriptional active genes [83]. There are several PTMs described so far such as methylation, acetylation, phosphorylation, ubiquitylation, sumoylation and deamination. Introduction 21 The epigenetic machinery responsible for the dynamics of PTMs can be divided into different groups based on their biological function: writers that lay down epigenetic marks in histones; readers which recognize them; and erasers that remove the PTMs (Figure 9) [88]. The distinct patterns of PTMs within the histone tails have been recognized as “histone code” and along with DNA methylation play a major role in gene activity regulation [83, 89] Regarding methylation and acetylation, the respective epigenetic writers comprise histone methyltransferases (HMTs) and histone acetyltransferases (HATs), while epigenetic erasers are composed by histone demethylases (HDMs) and histone deacetylases (HDACs) [88]. Histone acetylation has an important impact in gene transcriptional activity since it affects the electrostatic charge of the histones. Alterations in electrostatic charge of the histones influence not only the chromatin structure but also the binding affinity of histones to DNA. Indeed, the chromatin packaging controls the capacity of epigenetic readers to recognize histone PTMs in histone tails and thus, active or repress transcription [88, 90, 91]. Figure 8: Chromatin structure and histone PTMs. (a) Chromatin is made of repeating units of nucleosomes, composed by ≈ 147 base pairs of DNA wrapped around a histone octamer consisting in two copies each of the core histones H2A, H2B, H3 and H4. Linker H1 is positioned on top of the nucleosome core. (b) Histone PTMs on histone tails (N-terminal). Adapted from [85]. Uncovering PRMT6 Deregulation Effect In Prostate Cancer 22 Since histone PTMs are crucial for gene transcription regulation, they require a balance between the epigenetic machinery responsible by their dynamics. Disruption of this balanced is associated with pathological processes including cancer [92]. However, the plasticity nature of PTMs allowed the developing of “epigenetic drugs”, capable to target abnormal activity/expression of important chromatin-modifying enzymes [93, 94]. Figure 9: Epigenetic writers, readers and erasers. Adapted from [88]. Introduction 23 3 HISTONE METHYLATION AND PROSTATE CANCER 3.1 Histone Methylation: Basics of an Extensive Field Unlike acetylation, histone methylation does not alter the charge of the target residue, whereby is unlikely to be able to directly change chromatin structure. Methylation marks serves as binding sites for proteins that maintain nucleosomes together or signalizes for additional regulatory proteins such as binding domains [87]. Histone methylation occurs mainly in lysine residues (K), by lysine methyltransferases (KMTs), and in arginine residues (R) by arginine methyltransferases (PRMTs). Lysines can be mono- (me1), di- (me2) or trimethylated (me3), whereas arginines might be monoand symmetrically (me2s) or asymmetrically di-methylated (me2a) [93]. Transcriptional effect of histone methylation is relatively more complex than histone acetylation. Histone methylation can either lead to transcription repression or gene activation depending on the specific target residue, as well as the degree of methylation (Figure 10) [95]. Figure 10: Major lysine methylation marks on the amino-termini of histones H3 and H4. The embedded numbers refer to the methylated amino acid residue on each histone. The general function of each mono- , diand trimethylation state is depicted in dots of distinct colors, according with its function. Adapted from [95]. Uncovering PRMT6 Deregulation Effect In Prostate Cancer 24 In actively transcribed genes, there is an enrichment of H3K4me2 and H3K4me3, H3K36me3 and H3K79. Conversely, H3K9me3, H3K27me3 and H4K20me3 are associated with repressed genes [96]. More than 50 HMTs have been described, so far, and all of them use S-adenosylmethionine (SAM) as a cosubstrate to transfer methyl groups [87, 93]. Additionally, HMTs can be classified in three different groups: SET domain KMTs, non-SET domain KMTs and PRMTs [97]. Regarding PRMTs, they might be classified in three types: Type I, II and III generate monomethylarginine (MMA). Specifically, type I catalyzes the subsequent generation of asymmetric dimethylarginine (ADMA) and type II catalyzes symmetric dimethylarginine (SDMA) [98]. Histone methylation was considered for many years a stable and static histone modification until the discovery of the first KDM (Lysine-specific demethylase 1 - LSD1) in 2004 [93]. Since then, more than 30 HDMs were described and classified in two families: Lys-specific demethylases (LSD) and jumonji C (JMJC) histone demethylases [96]. This dynamic character of histone methylation requires an efficient balance between HMTs and HDMs in order to avoid alterations in normal cellular phenotype [92]. 3.2 Histone Methylation: Relevance in Prostate Cancer Aberrant histone modification profiles as well as the deregulated activity of correspondent enzymes are linked to cancer onset and progression, mainly by two mechanisms: alterations on normal gene expression of both oncogenes and TSGs, and genomic instability [93] (Figure 11). Concerning PCa, several alterations of normal histone modifications patterns have been associated with different clinical outcomes. While H3K4me1 and H3K4me2 are associated with increased risk of PCa recurrence [99, 100], methylation of H3K4 and H3K27 was correlated with tumor grade or recurrence [99, 101]. Additionally, levels of H4R3me2 and H3K4me2 allowed the distinction between two groups of low-grade PCa with different prognosis outcome (GS = 6 or GS < 6) [102]. Similarly to aberrant patterns of histone modifications, abnormal expression of some histone modifying-enzymes was also reported in PCa. Deregulation of some KMTs, such as Mixed-Lineage Leukemia 2 (MLL2), MixedLineage Leukemia 3 (MLL3), nuclear receptor binding SET domain protein 1 (NSD1), enhancer of zeste homolog 2 (EZH2) or SET and MYND domain containing Introduction 25 3 (SMYD3), were already described in PCa [99, 103]. EZH2 catalyzes repressive marks (H3K27me3 and at less extent H3K9me2) and was demonstrated to be a driver in prostatic carcinogenesis [102, 104-107]. EZH2 was found overexpressed in metastatic PCa being associated with intensification of tumoral phenotype [108, 109]. Moreover, repression of AR target genes was correlated with methylation of H3K9. Some studies showed that knockdown of its specific demethylases (LSD1, lysine-specific demethylase 3A – KDM3A; lysine-specific demethylase 4C – KDM4C) increased the levels of repressive marks on the regulatory regions of AR targeted genes, which in turn leads to their decreased expression [110, 111]. Additionally, LSD1 upregulation is associated with aggressive PCa, CRPC and high risk of disease relapse [112-114]. Therefore, a better understanding of the mechanisms underlying disruption of methylation marks in cancer is needed to develop new drugs and molecular strategies, capable to inhibit the imbalance between HMTs and HDMs, responsible for aberrant deposition of histone´s methylation marks. Figure 11: Histone-modifications map for a typical chromosome in normal and cancer cell. In normal cell, histone marks associated with active transcription are enriched in genomic regions including the promoters of tumor-suppressor genes, whereas DNA repeats and other heterochromatic regions are characterized by presence of repressive marks. In cancer cells, there are loss of the ‘active’ histone marks on tumor-suppressor gene promoters, and loss of repressive marks at subtelomeric DNA and other DNA repeats, leading to a more ‘relaxed’ chromatin conformation in these regions. Adapted from [69]. P RELIMINARY D ATA Preliminary Data 29 1 BRIEF CONTEXTUALIZATION The work presented in this Master Thesis arises from a previous PhD project developed in Cancer Epigenetics and Biology Group (GBEC), which aimed to clarify the role of HMTs and HDMs in PCa. Thereby, in order to identify what HMTs and HDMs displayed an abnormal expression pattern during prostatic carcinogenesis, TaqMan Array 96-Well Plates were designed to assess expression levels on 37 HMTs and 20 HDMs in normal prostate and PCa tissue samples by RT-qPCR. Five normal prostate tissues (NPTs) and ten PCa samples were selected to cover the full spectrum of prostate carcinomas considering the GS and pathological stage (relevant clinical and pathological data are depicted in Table 2). The mRNA levels of the genes studied were normalized to the betaglucuronidase (GUSB) reference gene and the median value of NPTs and PCa samples was chosen to calculate the fold variation in gene expression between groups, using the comparative Ct method. Table 2: Clinical and pathological features of patients included in the screening of HMTs and HMDs expression in NPTs and PCa samples. CLINICOPATHOLOGICAL FEATURES PCA NPT NUMBER OF PATIENTS, N 10 5 AGE (YEARS) Median (range) 59 (53 – 71) 61 (49 – 66) PSA LEVELS (NG/ML) Median (range) 12.3 (3.5 – 19.9) NA PATHOLOGICAL STAGE, N (%) pT2 4 (40.0) NA pT3a 2 (20.0) pT3b 4 (40.0) GLEASON SCORE, N (%) < 7 3 (30.0) NA ≥ 7 7 (70.0) NA, Not applicable Materials and Methods 37 1 CLINICAL SAMPLES: PATIENTS AND SAMPLE COLLECTION Samples of 195 primary tumors of prostate and 38 PIN lesions were prospectively collected from patients with clinically localized prostate adenocarcinoma, consecutively diagnosed and primarily treated with RP at the Portuguese Oncology Institute, Porto, Portugal. For control purposes, samples of 15 non-neoplastic prostate tissues normal prostate tissue (NPT) were obtained from the peripheral zone of prostates not harboring PCa, collected from cystoprostatectomy specimens of bladder cancer patients. All tissue specimens were immediately frozen after surgery and storage at -80ºC, following informed consent. Five-micron thick frozen sections were cut and stained for the identification of the areas of PCa (i.e., the index or dominant tumor) and normal tissue. Then, the tissue block was trimmed to maximize the yield of target cells (>70% of target cells). Histological slides from formalin-fixed, paraffin embedded (FFPE) tissue fragments were also obtained from the same surgical specimens and assessed for GS and TNM stage. Relevant clinical data were collected from the clinical records, and these studies were approved by the institutional review board [Comissão de Ética para a Saúde-(CES 295/2013)] of Portuguese Oncology Institute - Porto, Portugal. 2 RNA ISOLATION All tissue samples were suspended in TRIzol® reagent (Invitrogen, Carlsbad, CA, USA) and total RNA was purified using the PureLink™ _ RNA Mini Kit (Invitrogen) following manufacturer’s recommendations. The concentration, purity and integrity of RNA samples were determined on a Nanodrop ND-1000 (ThermoScientific, Wilmington, DE, USA) and agarose gel electrophoresis and then RNA samples were stored ate -80ºC. 3 PRMT6 GENE EXPRESSION IN CLINICAL SAMPLES PRMT6 mRNA levels were assessed in the previously described series of prostate tissue samples used in this study. Briefly, a total of 300ng was reverse Uncovering PRMT6 Deregulation Effect In Prostate Cancer 38 transcribed and amplified using TransPlex® Whole Transcriptome Amplification Kit (Sigma-Aldrich®, Schnelldorf, Germany) with subsequent purification using QIAquick PCR Purification Kit (QIAGEN, Germany), according to manufacturer’s instructions. Expression levels of PRMT6 and BGUS, a housekeeping gene used as endogenous control, were evaluated using TaqMan® Gene Expression Assay (Applied Biosystems®, Life TechnologiesTM, Foster City, CA, USA) (Hs_00250803_s1 and Hs_99999908_m1, respectively). To calculate the relative expression levels in each sample, the expression values of PRMT6 were normalized using the median of the reference gene (BGUS) to obtain a ratio (PRMT6/GUSB). Each plate included 2 negative controls and five sequential dilutions of a cDNA from human prostate RNA (Ambion®, Invitrogen, Carlsbad, CA, USA) to construct a standard curve for each plate. All experiments were run in triplicate. 4 IMMUNOHISTOCHEMISTRY Histological slides from FFPE tissue fragments were achieved from the same surgical specimens used in clinical sample series through the realization of 4μm thickness histological sections. Firstly, slides were deparaffinized in xylene (Sigma-Aldrich®, St. Louis, MO, USA) and then hydrated in a decreasing series of ethanol solutions (Merck, Darmstadt, Germany). Epitope retrieval was performed with ethylenediaminetetraacetic acid (EDTA) buffer (Thermo Scientific, Waltham, MA, USA) for 20 minutes, in a microwave at 700W. Endogenous peroxidase activity was neutralized for 20 minutes with 0.6% hydrogen peroxide (Merck). Protein detection was performed using the NovolinkTM Max Polymer Detection System (Leica Biosystems, Nussloch, Germany), according to manufacturer instructions. Slides were incubated overnight with a mouse monoclonal antibody specific for PRMT6 (sc-271744; Santa Cruz Biotechnology Inc., Santa Cruz, CA) in a 1:150 dilution at 4ºC, inside a humid chamber. Subsequent washing steps were performed with tris buffered saline with Tween® 20 (TBS-T) (Sigma-Aldrich®). Antigen-antibody binding reaction was revealed through the slides incubation for 7 minutes, in the dark, in a 0.05% (m/v) 3,3’-diaminobenzidine (DAB) solution (Sigma-Aldrich®) in phosphate-buffered saline (PBS) (Biochrom Ltd., Cambridge, United Kingdom) previously activated with a 0,1% hydrogen peroxide solution. Counterstaining of the slides were obtained with hematoxylin (Merck) for about 5 seconds and then slides were washed for 1 minute in a 0.25% ammonium Materials and Methods 39 solution (Merck). Lastly, the slides were dehydrated in an increasing series of ethanol content and diaphanized in xylene. After the coverslip was mounted, slides were dried. For positive control of the immunohistochemistry (IHC) reaction, FFPE tissue from a normal testis was also included. Slides were observed at the optical microscope and evaluated for PRMT6 immunoexpression by an experienced Uropathologist. Scoring criteria were adapted from a previous publication of our research group [116]: +1 for weak expression in ≤ 50% of cells; +2 for weak expression in > 50% of cells or moderate expression in ≤ 50% of cells; +3 for moderate expression in > 50% of cells or intense expression (which typically occurred in more than 50% of cells). 5 CELL CULTURE In present study, although five PCa cell lines (LNCaP, 22RV1, VCaP, PC-3 and DU145) were firstly screened for PRMT6 expression, only LNCaP and PC-3 were maintained in culture for further in vitro studies. Concerning these two PCa cell lines, LNCaP is a hormone-sensitive and PC-3 is a hormone refractory cell line. Both LNCaP and PC-3 were kindly provided by Prof. Ragnhild A. Lothe from the Department of Cancer Prevention at The Institute for Cancer Research at Oslo, Norway. Cell lines were grown using the recommended medium and correspondent supplements (Table 3) and 1% of Penicillin-Streptomycin (P-S) (GIBCO, Invitrogen, Carlsbad, CA, USA), in an humidifying chamber (37ºC and 5% CO2). TrypLE™ Express (GIBCO) dissociation reagent allowed subculture the selected cell lines to harvest them. Additionally, the cultured cell lines were routinely tested by a specific multiplex PCR for contamination by Mycoplasma spp. Table 3: Culture media conditions for PCa cell lines PCA CELL LINE GROWTH MEDIUM SUPPLEMENTS PC-3 RPMI-1640 + F-12 10% Fetal bovine serum (FBS) LNCAP RPMI-1640 Uncovering PRMT6 Deregulation Effect In Prostate Cancer 40 6 PRMT6 KNOCKDOWN IN SELECTED PCa CELL LINES PRMT6 knockdown was achieved through viral transduction in LNCaP and PC3 cell lines using MISSION® shRNA Lentiviral Transduction Particles (SHCLNV_TRCN0000299934 for LNCaP cell line and SHCLNV_TRCN0000299933 for PC-3 cell line; Sigma-Aldrich®) in the presence of polybrene (Santa Cruz Biotechnology Inc.) as described by the manufacturer. Moreover, control LNCaP and PC3 cells were generated using a MISSION® Non-Mammalian shRNA Control Transduction Particles (SHC002V; Sigma-Aldrich®). After transduction, stable clones with shRNA were selected with Puromycin dihydrochloride (cat. 631306, Clontech Laboratories Inc.) at a final concentration of 3μg/mL or 5μg/mL in LNCaP or PC3 cells, respectively. 7 RNA EXTRATION FROM SILENCED CELL LINES Cell culture flasks (75 cm3) at 100% confluence with LNCaP and PC-3 PRMT6 silenced cells were harvested with a dissociation reagent, TrypLE™ Express (GIBCO®) and centrifuged for 5 minutes at 1,200 rpm. Cell pellets were ressuspended in 1 mL of PBS (GIBCO®), and centrifuged for 5 minutes at 1,200 rpm. The supernatant was discarded and cell pellets were stored at -80oC. Total RNA from LNCaP and PC-3 PRMT6 silenced cells was extracted by TRIzol® Reagent (Invitrogen), according to manufacturer instructions. The pellets generated from total RNA extraction were dried and eluted in RNA storage solution (1 mM sodium citrate, pH 6.4) (Ambion®, Life TechnologiesTM, Foster City, CA, USA). The concentration, purity and integrity of RNA samples were determined as previously described and RNA samples were stored ate -80ºC. 8 CDNA SYNTHESIS In order to evaluate PRMT6 mRNA expression in LNCaP and PC-3 PRMT6 silenced cells, 1000ng of cDNA was synthesized from total extracted RNA. A cDNA synthesis was performed using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems®) according manufacturer instructions. Reverse transcription was performed in a Veriti® Thermal Cycler (Applied Biosystems®) and samples were then stored at -20ºC.! Materials and Methods 41 9 PRMT6 EXPRESSION ASSAY In order to confirm the efficiency of PRMT6 knockdown in transduced cell lines, PRMT6 mRNA levels were assessed by RT-qPCR. Briefly, cDNA products were diluted 10x in distilled water and reactions were carried out in 96-well plates using a 7500 Real-Time PCR system (Applied Biosystems®). For each well, 9 μL of cDNA product were mixed with 10 μL TaqMan® Universal PCR Master Mix (Applied Biosystems®) and 1 μL TaqMan® Gene Expression Assay, specific for PRMT6 (Hs_00250803_s1, Applied Biosystems®). Each sample was run in biological and experimental triplicate, and in every plate, two negative template controls were included. The analysis method used to ascertain PRMT6 knockdown in selected PCa cell lines was ΔΔCt method. LNCaP and PC-3 cells transduced with MISSION® NonMammalian shRNA Control Transduction Particles (Sigma-Aldrich®), from now on designated as LNCaP and PC-3 Sh-Scramble, respectively, were used to normalize results for LNCaP and PC-3 cells treated with MISSION® shRNA Lentiviral Transduction Particles specific for PRMT6, from now on designated LNCaP and PC-3 Sh-PRMT6. BGUS was used as a reference gene to normalize results obtained for the PRMT6 gene. 10 PROTEIN EXTRACTION AND QUANTIFICATION Protein was extracted from whole-cell lysates using the Kinexus Lysis Buffer with Lysis Buffer Cocktail (Kinexus Bioinformatics Corporation, Vancouver, British Columbia, Canada). Briefly, growth medium was removed from 75 cm3 cellculture flasks, and cells were washed with PBS to remove the residual culture medium. After PBS removal, 200 µL of Kinexus Lysis Buffer with Lysis Buffer Cocktail were added to each flask. Cells were scrapped with the help of a cell scrapper (Santa Cruz Biotechnology Inc.) to stimulate cell detachment and lysis, being then collected to a 1.5 mL tube. Cell lysate was sonicated in ice for 4 x 10 seconds cycles, with 10 seconds gap between each cycle. After that, tubes containing cell lysates were centrifuged for 30 minutes at maximum speed at 4ºC. Supernatant was carefully transferred to a new 1.5 mL tube. Uncovering PRMT6 Deregulation Effect In Prostate Cancer 42 Protein concentration was determined using BCA assay (Thermo Scientific, Waltham, MA, USA) according to manufacturer's information and extracted protein samples were stored at -80ºC. 11 WESTERN BLOT ANALYSIS PRMT6 gene silencing and alterations in the different protein levels were assessed by Western Blot analysis. Briefly, 30 µg of total protein was heated for 5 minutes at 95ºC to promote protein denaturation, centrifuged and finally loaded in a 10% sodium dodecyl sulfate polyacrylamide gel, for further electrophoresis (SDS-PAGE). Proteins were blotted onto 0.2 µm nitrocellulose or PVDF membranes (Bio-Rad Laboratories Inc., Hercules, CA, USA) and after their blocking membranes were incubated overnight at 4ºC with primary antibody (detailed information about primary antibodies in Table 4). The blots were developed with the ClarityTM Western ECL Substrate (Bio-Rad Laboratories Inc.) and AmershamTM Hyperfilm ECL (GETM Healthcare, Buckinghamshire, United Kingdom), to which membranes were exposed. To ascertain equal loading of protein, the membranes were stripped with Antibody Erasing Buffer (Komabiotech, Seoul, South Korea) and reprobed with a monoclonal mouse antibody against β-Actin (1:8000, Sigma-Aldrich®) or with a rabbit polyclonal antibody against Histone H3 (1:500, Abcam). Materials and Methods 43 Table 4: Essential information about the primary antibodies used in this study. PRIMARY ANTIBODY DILUTION HOST COMPANY ANTI-PRMT6 1:500 Rabbit pAb Novus Biologicals (Littleton, CO, USA) ANTIH3R2ME2A 1:500 Rabbit pAb Novus Biologicals ANTI-H3K4ME3 1:500 Rabbit pAb Abcam ANTI-AKT 1:500 Mouse mAb Santa Cruz Biotechnologies Inc. ANTI-PAKT 1:500 Mouse mAb Millipore (Billerica, MA, USA) ANTI-MTOR 1:1000 Rabbit mAb Cell Signaling Technology (Cell Signaling Technology, Inc., Danvers, MA, USA) ANTI-P21 1:500 Mouse mAb BD Biosciences (Franklin Lakes, NJ, USA) ANTI-P27 1:500 Mouse mAb Transduction Laboratories (BD Biosciences, Franklin Lakes, NJ, USA) ANTI-AR 1:250 Rabbit pAb Cell Signalling ANTI-PSA 1:6000 Rabbit mAb Abcam pAb: Polyclonal antibody; mAb: monoclonal antibody 12 CELL VIABILITY ASSAY The effect of PRMT6 on LNCaP and PC-3 cell lines viability was assessed by 3-(4, 5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium (MTT; Sigma-Aldrich®) assay. This assay consists on the cleavage of yellow-colored, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide, into a blue-colored formazan by the mitochondrial enzyme succinate-dehydrogenase. Briefly, the cells were seeded in 96-well plates (Sarstedt, Numbrecht, Germany) at different concentrations (Table 5), depending on cell line, in 200μL of complete medium and incubated in a humidified chamber at 37ºC and 5% CO2. The viability assay was performed after cells adhered to the plate (0 hours) and in subsequent days (24h, 48h and 72h). On each viability assay, cells were incubated with a solution of MTT (Sigma-Aldrich®) diluted in complete medium at 37ºC for 2h. Then, MTT solution was removed, formazan crystals were dissolved in Dimethyl sulfoxide Uncovering PRMT6 Deregulation Effect In Prostate Cancer 44 DMSO (Sigma-Aldrich®) and plates were shaken for 15 minutes for complete dissolution. An automated plate reader (FLUOstar Omega, BMG Labtech, Offenburg, Germany) at 540nm with a reference filter of 630nm allowed for colorimetric quantification. The optical density (OD) was directly proportional to the number of viable cells. Three biological independent experiments were performed with methodological triplicates for each experiment. Table 5: Number of cells plated for in vitro experiments in PCa cell lines used in this study. CELL LINE NUMBER OF CELLS PER WELL (96-WELL PLATES) PC-3 4000 LNCAP 8000 13 APOPTOSIS ASSAY Apoptotic levels were assessed using the APOPercentage™ kit (Biocolor Ltd., Newtownabbey, Northern Ireland, UK). Shortly, this assay consists in APOPercentage dye incorporation by cells undergoing apoptosis through phosphatidylserine transmembrane movements. Cells were grown in 96-well plates (Sarstedt) at different concentrations depending on cell line (Table 5), in complete medium and incubated in a humidified chamber at 37ºC and 5% CO2. 72h after cell adherence to the 96-well plate, the APOPercentage assay was performed according to manufacturer's instructions. Quantification of apoptosis was performed using a FLUOstar Omega microplate reader (BMG Labtech), by the OD measurement of the released dye at 550nm with a reference filter of 620 nm. To normalize the OD measured in the apoptotic test to the cell number, the OD of apoptosis assay was divided by the OD of the viability assay, also performed in 96-well plates. The results of the apoptosis assay on the silenced cells were expressed as the ratio of the values obtained for scramble cells (set as 100%). Three biological independent experiments were performed with methodological triplicates for each experiment. Materials and Methods 45 14 MIGRATION ASSAY The impact of PRMT6 silencing in migration ability of PC-3 cell line into a defect in a monolayer culture was determined using the wound-healing assay. Briefly, cells were grown (37ºC and 5% CO2 atmosphere) to full confluence in 24well plates, the medium was removed and scratches were performed using a 100µL tip. Cells were then washed with PBS and medium replaced. Scratch closure was analyzed under the inverted microscope and images were captured at different time points. Three biological independent experiments were performed with methodological quadruplicates for each experiment. 15 INVASION ASSAY Invasion capacity of PC-3 PRMT6 silenced cells was evaluated through Biocoat Matrigel Invasion Chambers (BD Biosciences) according to manufacturer’s protocol (Figure 13). Briefly, 2.5x104 cells in 500µL of serum-free medium were seeded in Matrigel inserts. Additionally, for each well plated with Matrigel inserts, the lower portion of insert was completed with 500µL of medium with serum as a chemoattractant. Then, plates were incubated in a humidified chamber at 37ºC and 5% CO2 for 24h. Non-invading cells were removed from the top of the insert and cells that migrated were fixed in methanol and stained with VECTASHIELD® Mounting Media containing DAPI (Vector Laboratories, Inc., Burlingame, CA, USA). Migrated cells were manually counted under a fluorescence microscope and results were displayed as percentages of invasion relative to scramble. Three biological independent experiments were performed with methodological duplicates for each experiment. Uncovering PRMT6 Deregulation Effect In Prostate Cancer 52 5 PRMT6 PROTEIN LEVELS IN A LARGE SERIES OF CLINICAL SAMPLES To assess PRMT6 protein expression in tissue samples, IHC was performed in a series of 248 FFPE prostate tissue samples (NPT=15; PIN=38; PCa=195) that perfectly matched with series of frozen tissues utilized for assessment of PRMT6 mRNA expression. Table 8 depicts the distribution of IHC scores in each group of samples and illustrative examples are presented in Figure 17. Table 8: Immunohistochemical expression of PRMT6 in a series of NPT, PIN lesions and primary PCa. CLINICAL SAMPLE GROUP SCORE +1 N (%) SCORE +2 N (%) SCORE +3 N (%) NPT 15 (100%) - - PIN 14 (37%) 15 (39%) 9 (24%) PCA 100 (51%) 63 (32%) 32 (16%) A significant increase of PRMT6 protein levels from NPTs to PIN lesions and PCa samples is apparent. Score distribution of PRMT6 immunostaining across the three groups of clinical samples is illustrated in Figure 18 - A. Paralleling PRMT6 mRNA expression results, the highest percentage of cases scored as +2 and +3 were found in PIN. A statistically significant association between PRMT6 transcript and protein levels was apparent (p < 0.001), indicating that, globally, PRMT6 protein levels follow the same trend as that of the transcript (Figure 18 – B). In pairwise comparisons, statistical significance was detected between scores +1 vs +2 and +1 vs +3 (p < 0.01 and p < 0.001, respectively). Additionally, a significant association was obtained between immunoexpression and prostate tissues. Table 9: Association between PRMT6 expression and prostate tissue. CLINICAL SAMPLE GROUP NORMAL EXPRESSION OVEREXPRESSION P NPT 15 (100%) - **** NEOPLASTIC PROSTATE TISSUE 114 (49%) 119 (51%) Neoplastic Prostate Tissue: PIN lesions and PCa samples; Normal Expression: +1; Overexpression: +2 and +3; ****p<0.0001 (Fisher´s Exact Test). Results 53 PIN PCa NPT 1+ 2+ 3+ Figure 17: Illustrative images of PRMT6 immunostaining in NPT, PIN and PCa samples. Figure 18: Distribution of PRMT6 immunoexpression (A) and of PRMT6 transcript levels (B) in a series of prostate tissue samples (NPT, PIN and PCa), grouped according to PRMT6 immunostaining. **p<0.01;***p<0.001;n.s. non significant (Mann-Whitney U-test). Uncovering PRMT6 Deregulation Effect In Prostate Cancer 54 6 KNOCKDOWN OF PRMT6 IN PC-3 AND LNCaP CELL LINES Firstly, PRMT6 expression levels were assessed by RT-qPCR in five PCa cell lines (LNCaP, 22RV1, VCaP, PC-3 and DU145) (Figure 19-A). All cell lines expressed PRMT6, although at variable levels. Then, the androgen responsive and the androgen refractory cell lines that expressed higher levels of PRMT6 (LNCaP and PC-3, respectively) were chosen for in vitro studies. Furthermore, effective PRMT6 knockdown was achieved in the selected cell lines and confirmed both at mRNA and protein level (Figure 19-B). A Figure 19: (A) PRMT6 expression in PCa cell lines. (B) The efficiency of PRMT6 knockdown was confirmed at mRNA, using RT-qPCR (upper panel), and protein level, using Western-Blot (lower panel), in LNCaP and PC-3 cells. *p<0.05 (MannWhitney U-test). Results 55 7 IMPACT ON VIABILITY AND APOPTOSIS OF PRMT6 KNOCKDOWN IN PC-3 AND LNCaP CELL LINES In LNCaP PRMT6-knocked down cell line, the MTT assay showed a 33% and 31% increase in cell viability at 24h and 48h, respectively. However, at 72h cell viability was similar to that of Sh-scramble (Figure 20-A). Concerning PC-3, a significant decrease in cell viability was observed after PRMT6 knockdown, both at 48h and 72h (10% and 24%, respectively) (Figure 20-B). Moreover, knockdown of PRMT6 led to an increase of apoptosis, in both LNCaP and PC-3 cell lines. Nevertheless, this increase was only statistically significant in PC-3 cells (Figure 20-C and D). Figure 20: Impact of PRMT6 knockdown in cell viability and apoptosis of LNCaP and PC-3 cell lines. (A) Cell viability in LNCaP and (B) cell viability in PC-3: quantification of cell viability by MTT assay in Sh-Scramble and Sh-PRMT6 at 0h, 24h, 48h and 72h. Quantification of apoptosis by APOPercentage in Sh-Scramble, Sh-PRMT6 LNCaP (C) and PC-3 (D) at 72h. *p<0.05 (Mann-Whitney U-test). Uncovering PRMT6 Deregulation Effect In Prostate Cancer 56 8 EFFECT OF PRMT6 KNOCKDOWN IN MIGRATION AND INVASION CAPACITY OF PC-3 CELLS Because PC-3 is widely recognized as a highly invasive PCa cell line, we evaluated the impact of PRMT6 knockdown in migration and invasion potential in this cell line. A significant decrease in migration ability was observed in PC-3 PRMT6-silenced cells (Figure 21-A). Furthermore, a decrease in invasion capacity of PC-3 PRMT6-silenced cells was also observed, although it did not reach statistical significance (Figure 21-B). Figure 21: Impact of PRMT6 knockdown in migration and invasion properties of PC-3. (A) Woundhealing scratch assay in PC-3 cell line. The upper panel shows the migration rate at 12h and 16h, and the lower panel displays the illustrative images at the beginning and endpoint of the assay. (B) Matrigel Invasion assay in PC-3. The upper panel shows the percentage of invasive cells at 24h and the lower panel displays illustrative images at endpoint of the assay (24h). ***p<0.001 (Mann-Whitney U-test). Results 57 9 EFFECT OF PRMT6 KNOCKDOWN IN HISTONE PTMs PATTERNS IN PC-3 CELL LINE As PRMT6 knockdown produced phenotypic alterations in silenced PC-3 cells, we ascertained alterations in histone marks established by this arginine methyltransferase. Therefore, western blot was performed to quantitate H3R2me2a. The observed reduction of this mark, established by PRMT6 [117], is illustrated in Figure 22. PRMT6 methyltransferase activity in arginine 2 of histone 3 counteracts the MLL complex activity on lysine 4 of the same histone, being mutually exclusive [117-119]. As PRMT6 was overexpressed in PCa samples, we evaluated the impact of this deregulation in MLL complex expression and activity. We had previously found that MLL family genes (MLL1-5) were downregulated in PCa [120]. Because the case series used in the present study is partially common to that of our previous publication, we interrogated the combined datasets concerning a putative association between PRMT6 and MLL complex expression. Although Spearman correlation analysis did not disclose an association between PRMT6 and MLL complex transcript levels in primary PCa tissues, PRMT6 knockdown was associated with MLL3-5 upregulation in PC-3 cells (Figure 23). The same trend was observed for SMYD3, another H3K4me3 methyltransferase (Figure 23). H3R2me2a H3 Figure 22: Reduction of asymmetrical dimethylation of arginine 2 of histone 3 after PRMT6 knockdown in PC-3 cell line. Uncovering PRMT6 Deregulation Effect In Prostate Cancer 58 Moreover, the impact of PRMT6 knockdown on MLL complex activity in vitro was tested through the evaluation of the differences in H3K4me3 expression, in PC-3 cells. However, no significant alterations in expression were apparent after PRMT6 knockdown (Figure 24 – A). Because PRMT6 activity is associated with a subset of transcriptionally inactive Hox genes and presumably also with some MYC-dependent genes [117], we evaluated MYC transcript levels before and after PRMT6 silencing. RT-qPCR analysis depicted a significant increase in MYC mRNA levels in PC-3 Sh-PRMT6 (Figure 24 - B). Figure 23: Relative expression of methyltransferases that catalyze H3K4me3 in PC3 PRMT6-silenced cells. (A-E) MLL complex genes are upregulated in PC3 Sh-PRMT6 cells, reaching statistical significance for MLL35. (F) SMYD3 is also significantly upregulated in PC3 knocked down cells. *p<0.05 (Mann-Whitney U-test). Figure 24: H3K4me3 and MYC expression after PRMT6 knockdown in PC-3 cell line. (A) Western blot analysis of H3K4me3 expression after PRMT6 knockdown in PC-3 cells. (B) MYC transcript levels in PC3 PRMT6 silenced cells. *p<0.05 (Mann-Whitney U-test). H3K4me3 H3 A Results 59 10 MOLECULAR PATHWAYS AND PUTATIVE TARGET GENES REGULATED BY PRMT6 To find genes regulated by PRMT6, we evaluated putative targets associated with cellular processes and pathways relevant in PCa. Firstly, cellular senescence was investigated, since this biological process has been previously associated with PRMT6 activity [121, 122]. Among molecules responsible for induction of cellular senescence, p21 and p27 play a major role [121-123]. In PC-3 Sh-PRMT6 cells, western blot analysis showed a considerable increase of p27 expression whilst p21 was only slightly increased (Figure 25). Considering the decrease in invasion and migration ability of PC-3 ShPRMT6 cells, we further evaluated the expression levels of genes involved in those cellular processes. We found that PRMT6 silencing was associated with matrix metalloproteinase 9 (MMP-9) downregulation and CD44 upregulation (Figure 26). Figure 26: Impact of PRMT6 knockdown in MMP-9 and CD44 transcript levels in PC-3 cell line. *p<0,05 (Mann-Whitney U-test). Figure 25: Western blot analysis of p21 and p27 expression in PC-3 cell line after PRMT6 knockdown. p27 p21 β-Actin Uncovering PRMT6 Deregulation Effect In Prostate Cancer 60 The PI3K/AKT/mTOR pathway, which is frequently deregulated in PCa was also investigated [124]. In PC-3 Sh-PRMT6 cells, AKT, phosphorylated AKT and mTOR protein expression was significantly decreased (Figure 27), suggesting PI3K/AKT/mTOR pathway downregulation due to PRMT6 silencing. Finally, the AR pathway, which is critically involved in PCa initiation and progression [125], was examined. In PC-3 cells, which have only residual expression of AR [126], PRMT6 knockdown led to AR upregulation, both at transcript and protein level (Figure 28). To confirm these results, expression of PSA (a well-known downstream target of AR) was assessed, and an impressive increase in PSA protein levels was apparent in PC-3 Sh-PRMT6 cells (Figure 28 - B). Figure 23: Overexpression of AR in PC-3 PRMT6 silenced cells. RT-qPCR analysis of AR transcript levels in PC-3 cell line (left panel). Western blot analysis of AR and PSA protein levels in PC-3 cell line. *p<0,05 (Mann-Whitney U-test). PSA AR β-Actin B mTOR AKT Figure 27: Western blot analysis of AKT, pAKT and mTOR expression in PC-3 cell line. β-Actin pAKT DISCUSSION C ONCLUSION AND F UTURE P ERSPECTIVES Conclusion and Future Perspectives 71 1 CONCLUSION AND FUTURE PERSPECTIVES In this study we confirmed, in a large series of prostate tissue samples, that PRMT6 was overexpressed in PCa, at both transcript and protein level. Intriguingly, PIN lesions displayed significantly higher PRMT6 expression levels, compared to PCa. Furthermore, PRMT6 mRNA levels are able to discriminate cancerous from non-cancerous prostate tissues. In stable PRMT6 knockdown models, attenuation of the malignant phenotype was consistently demonstrated in PC-3 but not in LNCaP cells, probably due to its different AR expression status. At molecular level, PRMT6 silencing was associated with decreased H3R2me2a levels and increased MLL complex and SMYD3 expression, although global H3K4me3 levels remained unchanged. Moreover, the expression of several key genes, involved in critical cellular pathways, was shown to be affected by PRMT6 downregulation, including p21, p27, MMP-9, CD44, AKT, phospho-AKT, mTOR and AR, globally supporting an oncogenic role for PRMT6. Finally, restoration of AR expression in Sh-PRMT6 PC-3 cells, might be of clinical relevance as it may re-sensitize androgen-insensitive neoplastic cells to ADT. The therapeutical potential of PRMT6 silencing/inhibition is probably the most exciting finding of this study and it is surely the one that more deserves further studies. Chromatin immunoprecipitation (ChIP) experiments are required to firmly establish a link between PRMT6 activity and AR expression (de- )regulation. Furthermore, the androgen sensitivity of Sh-PRMT6 PC-3 cells must be proven by exposure to di-hydro-testosterone, as well as through combined treatment with bicalutamide, to demonstrate the re-acquisition of sensitivity to ADT. Although results observed for Sh-PRMT6 LNCaP cells seem discouraging, the hypothesis that the paradoxical effect of PRMT6 downregulation in cell viability is due to increased AR expression must be tested. As LNCaP cells might be considered representative of androgen-sensitive PCa, which are those that predominate at diagnosis, the effects of PRMT6 inhibition/downregulation must be carefully investigated, to ascertain whether the combined therapeutic strategy Uncovering PRMT6 Deregulation Effect In Prostate Cancer 72 previously hypothesized for CRPC (PRMT6 inhibition and bicalutamide) might constitute a potentially harmful therapy for androgen-sensitive PCa. Importantly, in case the combined therapy proves useful in in vitro models, our study provides evidence that PRMT6 expression, assessed by IHC (a commonly used technique in most Pathology labs) might serve as a predictive marker of response, through the identification of PCa cases overexpressing PRMT6, which are those that are most likely to respond. R EFERENCES References 75 1 REFERENCES 1. Oesterling JE. The origin and development of benign prostatic hyperplasia. An age-dependent process. 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