Dissertations / Theses on the topic 'Prostate cancer; epigenetic modification'

Enhancer of zeste homolog 2 (EZH2) is the catalytic subunit of the polycomb repressive complex 2 and suppresses gene expression by catalyzing histone H3 methylation on lysine 27. EZH2 is overexpressed in metastatic prostate cancer and has been shown to promote cell proliferation and metastasis. Here we show that EZH2 also suppresses prostate cancer apoptosis by coordinating the epigenetic silencing of two pro-apoptotic microRNAs, miR-205 and miR-31. We previously reported that miR-205 is silenced in prostate cancer through promoter methylation. In this study, we found that EZH2 suppresses miR-31 expression by trimethylation of H3K27 on the miR-31 promoter. SiRNA knockdown of EZH2 increased miR-31 expression and decreased the anti-apoptotic protein E2F6 (a target of miR-31), resulting in the sensitization of prostate cancer cells to docetaxel-induced apoptosis and vice versa. We further demonstrated that miR-205 silencing is linked to miR-31 silencing through EZH2. Suppression of miR-205 caused an increase of EZH2 protein, which in turn inhibited miR-31 expression and vice versa. Thus, EZH2 integrates the epigenetic silencing of miR-205 and miR-31 to confer resistance to chemotherapy-induced apoptosis. Besides the histone modification by histone methyltransferases (HMTs) such as EZH2, histone deacetylases (HDACs) offer another mechanism to epigenetically regulate gene expressions in cancer. The class I selective inhibitor of HDACs, mocetinostat, has promising antitumor activities in both preclinical studies and the clinical trials. To understand how mocetinostat induces apoptosis in prostate cancer cells, we examined the effects of mocetinostat on miR-31. We found that miR-31 was significantly upregulated by mocetinostat in prostate cancer cells. E2F6 was decreased by mocetinostat treatment. Mocetinostat also increased the expression of pro-apoptotic protein Bad and activated caspase-3 and caspase-9. SiRNA iv knockdown of E2F6 sensitized cancer cells to mocetinostat-induced apoptosis. Importantly, we found the same results in the primary prostate cancer stem cells. Thus, activation of miR-31 and downregulation of E2F6 contribute to mocetinostat-induced apoptosis in prostate cancer. In summary, the epigenetic silencing of miR-31 confers a resistance mechanism for chemotherapy-induced apoptosis in prostate cancer cells. Using mocetinostat to activate miR-31 expression is a novel strategy to overcome resistance to apoptosis and improve response to therapy.

Inhibitor of DNA binding/differentiation protein 4 (ID4) is a dominant negative regulator of basic helix loop helix (bHLH) family of transcription factors. ID4 shares the homology of HLH domain with other ID proteins (ID1, ID2, and ID3) and lack the basic DNA binding region. Evidence suggested that unlike ID1, ID2 and ID3, ID4 acts as a tumor suppressor in prostate cancer by attenuating cell proliferation and promoting apoptosis. Consistent with these observations ID4 is epigenetically silenced in DU145 prostate cancer cell line. In this study we investigated whether ID4 is also epigenetically silenced in prostate cancer. We also examined association between ID4 promoter hyper-methylation and its expression in prostate cancer cell lines. ID4 protein expression was analyzed in human prostate adenocarcinoma samples by Immunohistochemistry (IHC). ID4 promoter methylation pattern on prostate cancer cell lines was examined by methylation specific PCR. In addition, we performed methylation specific PCR on the human prostate tissues and genomic DNA to correlate cell line studies with clinical studies. IHC demonstrated decreased ID4 protein expression in human prostate tissue samples, whereas higher nuclear ID4 expression was found in normal prostate tissues. ID4 methylation specific PCR (MSP) on prostate cancer cell lines, showed ID4 methylation in DU145, but not in LNCaP and C33 cells. C81 and PC3 cells showed partial methylation. Increased ID4 methylation in C81 as compared to LNCaP suggests its epigenetic silencing as cells acquire androgen independence. Tumors with ID4 promoter hyper-methylation showed distinct loss of ID4 expression. However, the underlying mechanism involved in epigenetic silencing of ID4 is currently unknown. We hypothesized that ID4 promoter methylation is initiated by an EZH2 dependent tri-methylation of histone 3 at lysine 27 (H3K27Me3). ID4 expressing (LNCaP) and non-expressing (DU145 and C81) prostate cancer cell lines were used to investigate EZH2, H3K27Me3 and DNMT1 enrichment on ID4 promoter by Chromatin immuno-precipitation (ChIP). Increased enrichment of EZH2, H3K27Me3 and DNMT1 in DU145 and C81 cell lines was compared to ID4 expressing LNCaP cell line. Knockdown of EZH2 in DU145 cell line led to re-expression of ID4 and decrease in enrichment of EZH2, H3K27Me3 and DNMT1 demonstrating that ID4 is regulated in an EZH2 dependent manner. ChIP on prostate cancer tissue specimens and cell lines suggested EZH2 occupancy and H3K27Me3 marks on the ID4 promoter. Collectively, our data indicate a PRC2 dependent mechanism in ID4 promoter silencing in prostate cancer through recruitment of EZH2 and a corresponding increase in H3K27Me3. Increased EZH2, but decreased ID4 expression in prostate cancer strongly supports this model.

Perturbation of androgen signalling drives progression of human prostate cancer (CaP) to castration-resistant prostate cancer (CRPC). Additionally, CaP is initiated and maintained by cancer stem cells (CSC)s which are analogous to normal prostate stem cells (SC)s. This study presents a qPCR assay to detect androgen receptor gene amplification (GAAR), which is the most common mechanism of castration resistance ( > 30%). Also, the epigenetic regulation and function of two SC-silenced genes with tumour-suppressive activity (Latexin (LXN) and Retinoic Acid Receptor Responder 1 (RARRES1)) were interrogated using micro-ChIP, transcriptional profiling and mass spectrometry. Traditionally, GAAR is detected using FISH which is labour-intensive and semi-quantitative, limiting clinical applicability. The mechanism of action of LXN or RARRES1 in CaP is unknown, and epigenetic regulation by DNA methylation has been ruled-out in primary CaP. The qPCR assay can detect GAAR in minor cell populations (~1%) within a heterogeneous sample and also quantifies X chromosome aneuploidy (XCA) - a predictor of poor-prognosis in CaP. GAAR and XCA were detected in near-patient xenografts derived from CRPC-tissue indicating that these abnormalities are present in cells capable of in vivo tumour-reconstitution. Micro-ChIP analysis of fractionated primary CaP cultures identified bivalent chromatin at LXN and RARRES1 promoters. Transcriptomic profiling failed to reveal significant changes in gene expression after transduction with LXN or RARRES1. However, an interactome for LXN and RARRES1 was successfully generated in PC3 cells. Additionally, confocal microscopy of mVenus-tagged LXN revealed a pan-cellular distribution which is reflected in the interactome. Screening for GAAR and XCA, using a high-throughput qPCR assay, could facilitate a targeted-medicine strategy in the treatment of CaP and CRPC. Further investigation of the LXN and RARRES1 interactomes may identify their mechanism(s) of action and the micro-ChIP assay could be used to identify epigenetic-inducers of LXN and RARRES1 which could provide a CSC-targeted strategy for CaP treatment.

Western studies have established that men from African descent are disproportionally affected by prostate cancer (PCa). Annual incidence rates in this population vary from 1.5 to 2 times when compared to their counterparts from other racial groups. They also record the worse outcomes in terms of prognosis. Additionally, with the rise of PCa in Subsaharan Africa, new cancer control policies and programs are increasingly demanded. Understanding therefore, factors that underpin racial inequality in distribution and especially why the disease preferentially niches in African males can help better address PCa in both Western and Subsaharan countries. There is also the potential to develop new therapeutic options. A genetic susceptibility was first hypothesized, however available data suggest that they only account for less than 20% of the cases. Current findings from epidemiological and molecular investigations suggest an important role of complex and dynamic environmental interactions involving the different levels of calcium regulation. Using a multi-method design, this research aims at developing an integrative mechanistic model of PCa. We argue that given the versatile and ubiquitous role calcium plays in nutrition, physical environment, and in key cellular processes, that mineral cation is central to prostate tumorigenesis and in shaping its populational distribution. Thus a tree-level investigation was conducted: (i) a critical analysis and synthesis of empirical evidence on calcium interactions with cancer mechanisms (ii) a population-wide prospective cohort study of calcium intake patterns in a group of Subsaharan males in Côte d’Ivoire, namely the African Prostate Cancer Study (APCS) (iii) a proteomics research investigating the responses of prostate cancer cell lines when exposed to a high affinity synthetic calcium binding peptide. This monograph describes the research methods, instruments design and validation and the preliminary findings of the ongoing research, portions of which have already been published, presented at two international cancer seminars or under review. Findings at this stage include: mechanistic models of prostate cancer differential distribution and outcomes, a novel calcium questionnaire specific to African diet, synthesis of a high affinity calcium-binding peptide (Peptide#1). New concepts and constructs related to prostatic carcinogenesis have been developed as well.

BORIS, a paralogue of the transcription factor CTCF, is a member of the cancer-testis antigen family. BORIS is normally present in the testes; however, it is aberrantly expressed in various tumours and cell lines. The main aim of this study was to investigate BORIS expression in prostate cell lines and tumours, and the importance of BORIS in the regulation of genes in prostate cells, in particular, the androgen receptor CAR) gene, associated with the development of more aggressive prostate tumours.

One important objective of prostate cancer (PCa) research is to understand the molecular basis underlying the progression of these cancers from an androgen dependent to an androgen independent state. Hypermethylation of the promoter CpG islands is associated with the transcriptional silencing of specific gene sets in each tumor type and subtype. Transcriptional silencing of antitumor genes via CpG island hypermethylation could be a mechanism mediating PCa progression from an androgen-dependent to an androgen-independent state. Hypermethylation associated gene silencing has been reported for a great number of genes in PCa with the exception of the genes that undergo methylation associated silencing specifically during cancer development to androgen independence. The first aim of this thesis is to identify novel glenes which undergo DNA hypermethylation associated gene silencing during the cancer progression. The androgen-dependent (AD, as defined as the inability of celill to proliferate in the absence of androgen) PCa cell line LNCaP gives rise to the androgen-independent (AI) subline LNCaPcs generated by maintaining LNCaP in medium with charcoal-stripped (CS) serum for over 30 passages. This LNCaP cell model was used to identify differentially methylated sequences between the two genomes using the Methylation-Sensitive Restriction Fingerprinting (MSRF) technique. One sequence identified is located in a 5' CpG island, which encompasses part of the promoter, exon 1, and part of intron 1, of the Peroxisomal Membrane Protein 24 KD (PMP24) gene. PMP24 is silenced in concert with the hypermethylation of its CpG island in AI LNCaPcsand PC-3 cell lines. The silencing is reactivated by the treatment with a DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (5AZAdC). PMP24 is specifically silenced in PCa cancer cell lines and shows potential antitumor properties. These results demonstrate the utility of MSRF in the identification of novel, differentially methylated DNA sequences in the genome and suggest that hypermethylation-mediated silencing of PMP24 is an epigenetic event involved in PCa progression to androgen independence. The next study investigated the molecular mechanism for DNA methylation associated gene silencing of PMP24 in AI LNCaPcs and PC-3 cell lines. We demonstrated that PMP24 transcription is repressed by the disruption of transcription factor binding to a critical cis-element by hypermethylation of its promoter CpG island. We found a CpG containing activator protein 2 (AP-2) cis-element in the intron 1 of PMP24 whose first CpG dinucleotidle is essential for the sequence-specific protein binding and the promoter activity of the gene. We presented first in cellulo evidence that the methylation of AP-2 cis-element alone but not the whole CpG island, using a newly developed methylated oligonucleotides treatment, is sufficient for the downregulation of PMP24. Our study is the first to report that the silencing mechanism for PMP24 in AI LNCaPcs and PC-3 is mediated by the complete methylation of a single GpG site of AP-2 cis-element in the intron 1 part of the CpG island, which interferes with transcription factor binding. Most interestingly, the promoter CpG island of PMP24 is hypermethylated in AD LNCaP cells with the incomplete methylation specifically at the AP-2 cis-element. The silencing of PMP24 in AD LNCaP cells was reactivated not by the 5AZAdC treatment but by the treatment with Trichostatin A (TSA), a histone deacetylase inhibitor. An alternative silencing mechanism for PMP24 other than the interference with transcription factor binding by methylation is therefore likely involved at this androgen-dependent stage. During the androgen ablation process, this mechanism is either evolved by the spread of methylation in the promoter CpG island or selected against, leading to the methylation-dominant silencing mechanism in the AI cells as seen in LNCaPcsand PC-3 cells. Taken together, this thesis emphasized the important role of DNA methylation in the progression of PCa into androgen independence. Particular respect should be paid to the specific CpG dinucleotides in cis-elements critical for the promoter activity, whose complete methylation could dominate the silencing mechanism which is independent of androgen. This thesis also pointed to the importance of monitoring the effects of cell culture on the methylation status of genes. Most importantly, this thesis raised the possibility that the silencing mechanisms for PMP24 could be different in AD LNCaP cells as compared to AI LNCaPcs and PC-3 cells. Either the evolution of such mechanism or the selectivity against it during the androgen ablation process would result in a methylation-dominant silencing mechanism of the genes such as PMP24 in AI cells and may contribute to the overall androgen independence of the cells.

Le suppresseur de tumeur DOK1 (downstream of tyrosine kinases1) est une protéine régulatrice de voies de signalisation impliquées dans des processus cellulaires tel que la prolifération, la migration et l'apoptose. Le rôle suppresseur de tumeur de DOK1 a été démontré dans des modèles animaux. Les souris knock-out pour DOK1 présentent une forte susceptibilité de développer des leucémies, des tumeurs malignes hématologiques, des adénocarcinomes pulmonaires, ainsi que des sarcomes histiocytaires agressifs. En outre, nous avons rapporté précédemment que le gène DOK1 peut être muté et son expression réprimée dans différentes tumeurs malignes humaines, telles que les lignées cellulaires de lymphome de Burkitt (BL) et la leucémie lymphoïde chronique (LLC). Cependant, les mécanismes de dérégulation de DOK1 restent inconnus, notamment dans les processus de carcinogenèse induite ou non par des oncovirus. Dans ce projet de thèse, nous avons d'abord caractérisé le promoteur de DOK1 et le rôle du facteur de transcription E2F1 comme le principal régulateur de l'expression de DOK1. Nous avons démontré pour la première fois la contribution de DOK1 dans la réponse cellulaire au stress par son rôle suppresseur de prolifération cellulaire et promoteur d'apoptose. Nous avons trouvé que l'expression du gène DOK1 est réprimée dans une variété de cancers humains, y compris le cancer de la tête et du cou, les lymphomes de Burkitt et les cancers du poumon. Cette répression est due à l'hyperméthylation aberrante de DOK1. Nous avons donc étudié les événements épigénétiques, qui sont souvent altérés dans les cancers, et leurs implications dans la répression de DOK1 dans les lignes cellulaire cancéreuses de la tête et du cou. Nous nous sommes par la suite intéressés aux mécanismes de dérégulation de DOK1 par le virus d'Epstein Barr dans le cadre de sa propriété oncogénique dans les lymphocytes B humains ainsi que dans les lignes cancéreuses du lymphome de Burkitt. Nos résultats apportent de nouvelles informations sur les mécanismes de régulation de l'expression de DOK1 dans la carcinogenèse induite ou non par des oncovirus, ce qui pourrait le définir comme un biomarqueur potentiel de cancer et comme une cible intéressante pour des thérapies épigénétiques
The newly identified tumor suppressor DOK1 (downstream of tyrosine kinases1) inhibits cell proliferation, negatively regulates MAP kinase activity, opposes leukemogenesis, and promotes cell spreading, motility, and apoptosis. DOK1 also plays a role in the regulation of immune cell activation, including B cells. The tumor suppressor role of DOK1 was demonstrated in animal models. DOK1 knockout mice show a high susceptibility to develop leukemia, hematological malignancies as well as lung adenocarcinomas and aggressive histiocytic sarcoma. In addition, we previously reported that the DOK1 gene can be mutated and its expression is down-regulated in human malignancies such as Burkitt’s lymphoma cell lines (BL) and chronic lymphocytic leukemia (CLL). However, very little is known about the mechanisms underlying DOK1 gene regulation and silencing in viral- and non viral-related tumorigenesis. In the present project, we first characterized the DOK1 promoter. We have shown the role of E2F1 transcription factor as the major regulator of DOK1 expression and how DOK1 plays a role in DNA stress response though opposing cell proliferation and promoting apoptosis. We demonstrated that DOK1 gene expression is repressed in a variety of human cancers, including head and neck, Burkitt’s lymphoma and lung cancers, as a result of aberrant hypermethylation. We investigated the link between the epigenetic events and DOK1 silencing in non viral head and neck cancer cell lines, and by Epstein Barr virus in relation to its oncogenic activity in human B cells and neoplasia such as Burkitt’s lymphoma. These data provide novel insights into the regulation of DOK1 in viral and non viral-related carcinogenesis, and could define it as a potential cancer biomarker and an attractive target for epigeneticbased therapy

Les protéines à " Methyl-CpG-binding domain " (MBD) jouent un rôle important dans l'interprétationde la méthylation de l'ADN conduisant à la répression transcriptionnelle via le recrutement decomplexes remodelant la chromatine. Dans les cancers, MBD2 jouerait un rôle essentiel dans la perted'expression des gènes hyperméthylés. Ainsi, MBD2 serait une cible potentielle pour rétablir, enpartie au moins, leur expression. Caractériser, à l'échelle du génome, la distribution de MBD2 et sesconséquences sur la répression transcriptionnelle au cours de la cancérogenèse est donc une étapeincontournable. (1) L'impact sur l'expression génique de l'inhibition de MBD2 par interférence àl'ARN, a été étudié en utilisant des puces, dans des cellules normales MRC5. La perte de MBD2n'induit pas de surexpression génique globale et la densité en CpG des promoteurs méthylés sembleêtre une composante importante dans la force de répression par MBD2. (2) Les profils de méthylationde l'ADN, de liaisons de MBD2 et de l'ARN polymérase II dans les cellules HeLa ont été analysés parChIP-on-chip avec des puces promoteurs. Ces mêmes approches couplées à l'analyse de l'acétylationdes histones H3 ont été réalisées dans un modèle cellulaire syngénique de progression tumoralemammaire humain. Dans les modèles étudiés, une forte proportion de gènes silencieux et méthylés estliée par MBD2. Les comparaisons entre cellules immortalisées et transformées ne montrent pas dechangements majeurs de la méthylation de l'ADN ou de la répression transcriptionnelle, par contreune redistribution de MBD2 parmi ces sites est observée, suggérant une redondance entre les protéinesliant l'ADN méthylé.

Prostate cancer is the second-leading cause of cancer death in Australian men. Current therapies for advanced prostate cancer are not curative and most patients eventually develop castrate resistant prostate cancer. Epigenetic modifications are heritable and reversible biochemical changes of the chromatin that regulate gene expression and are important in prostate tumourigenesis. There is also evidence that excess foetal nutrition is associated with increased risk of developing prostate cancer. Hence, the aims of this thesis were to determine the involvement of epigenetic modifications in: the early origin of prostate cancer, prostate cancer progression, as prognostic and therapeutic targets in prostate cancer. The first aim of this thesis was to use a rodent model to determine if a maternal high fat diet (MHFD) is associated with increased risk of prostate cancer in offspring. Offspring exposed to a MHFD had increased incidence of prostate abnormalities compared to offspring exposed to a maternal control diet. GSTP1 is hypermethylated and silenced in human prostate cancer and was decreased in these offspring prostates. The MHFD altered the male offspring prostates microRNA expression and provided insights of possible underlying mechanisms that support a link between MHFD and risk of prostate cancer in adult offspring. The second aim was to investigate if specific histone modifications H3K18Ac and H3K4diMe were prognostic markers for prostate cancer. High levels of H3K18Ac and H3K4diMe were associated with increased risk of prostate cancer relapse respectively. To further investigate the underlying mechanisms, epigenetic genes were mined in microarray data, and an epigenetic gene signature was identified which distinguished non-malignant from tumour prostate tissues in an independent prostate cancer cohort. To investigate if the DNA methyltransferase inhibitor (DNMTi) 5-aza-CdR was a potential treatment agent for prostate cancer, proliferation assays were performed in prostate cancer cells. A daily low-dose and prolonged 5-aza-CdR treatment regime was the most effective treatment in prostate cancer cells compared to high doses administered less frequently. Furthermore, GSTP1 DNA methylation and protein status were good indicators of DNMTis efficacy in vitro, where demethylation indicated growth suppression and protein re-expression indicated cell death induction. To investigate if the Kruppel-like-factor 6 (KLF6) prostate cancer susceptibility gene is epigenetically altered during prostate cancer progression, DNA methylation analyses were performed in human and mouse (TRAMP) prostate cancers. Our results suggest that DNA hypermethylation is not responsible for decreased KLF6 expression in human and TRAMP prostate cancers in our study. Collectively, the findings of this thesis further support the importance of epigenetic modifications in prostate tumourigenesis. We demonstrated the potential of using epigenetic modifications as prognostic markers, therapeutic targets and as a marker of treatment efficacy. Lastly, we provide evidence, for the first time, that MHFD is a risk factor for prostate cancer and that miRNAs are involved. This finding is important and suggests the potential of early prevention/ intervention of prostate cancer by targeting epigenetic modifications and diet intervention.
Thesis (Ph.D.) -- University of Adelaide, School of Medicine, 2010

ABSTRACT: Prostate cancer is a highly prevalent malignancy and a major cause of cancer-related morbidity and mortality. Radical prostatectomy technique remains the major treatment opbon for men with potenbal cure and life expectancy exceeding 10 years. In the very recent 2018 published follow-up of 29 years of SPG4 study (watchful waibng versus radical prostatectomy), men who did RP gained a mean of 2.9 years of life. One of the relevant issues about surgery is its influence in the oncologic prognosis of pabents, namely, the presence of posibve surgical margins and its impact in biochemical recurrence and subsequent treatments. For this reason, we present, in this thesis, a book chapter published in 2015 about the classical open technique with detailed technical specificabons. Nowadays, more and more main urological surgeries are thoroughly robobc. Pabents with biochemical relapse aoer radical prostatectomy are counseled to do salvage radiotherapy directed to prostabc loca and pelvic lymphabc chains, and hormonal therapy within 2 years. In the majority of cases, we do not know if the pabent actually has disease or where it is located. Also, we do not have any independent biomarker that can accurately indicate if the disease will be aggressive and we should treat, or if the tumor is indolent and the pabent can wait safely, without being submiwed to morbid adjuvant treatments. Convenbonally, prostate cancer prognosis evaluabon is mainly based on PSA, surgical margin status, lymph node status, pathological stage and Gleason score. Unfortunately, the prognosbc power of these is, indeed, insufficient. In 2011, we studied the Amadora (Lisbon) Cohort from Hospital Prof. Doutor Fernando Fonseca with 171 pabents, submiwed to retropubic radical prostatectomy between 2000 and 2005. The stabsbcal risk analysis concluded that the presence of PSM in RRP is strongly influenced by pathological stage ≥ pT3a. The presence of PSM and their number increase significantly the risk of BR compared to other factors. In the absence of PSM, the factors that seemed to be crucial and with greater impact on BR were inibal PSA > 10 ng/ml and pathological Gleason score ≥8. The conclusion, thereaoer, and in concordance with evidence, was that it is important to consider inibal PSA pathological Gleason and surgical margins status when making treatment decisions aoer radical prostatectomy. Nowadays, new tools of molecular nuclear imaging are available, namely, 68Ga-PSMAPET/CT, that have accuracy to detect and localize inibal and recurrent disease with very low levels of PSA (≥ 0,2 ng/ml). We are starbng to “see” and to localize prostate cancer. The cons of this nuclear test are probably the costs and the low availability that restricts it to a limited number of cases that can be done per day, especially if no direct access to the generator/reactor is possible. The evolubon of Magne]c Resonance Imaging with its mul]parametric tools, is allowing clinicians to actually visualize the disease, as well. The novel prostate cancer biomarkers, such as liquid biopsies, epigenebc and inflammabon-related biomarkers and its importance are discussed. In this context, we present two manuscripts. The first, an editorial, about a possible novel biomarker and therapeubc target, the Prostate Stem Cell Anbgen (PSCA). The second, a review arbcle about epigenebc and inflammatory biomarkers that is in peer-review in World Journal of Clinical Oncology (WJCO). We think and believe that the genesis and development of prostate cancer is linked to chronic inflamma]on and epigene]c modifica]ons. The new concepts of urinary microbiota and proliferabve inflammatory atrophy (PIA), a prostate cancer precursor, are explained. Owing to the limitabons of current clinical, serologic, and pathologic parameters in predicbng disease inibabon and progression, we sought to invesbgate the prognosbc value of epigenebc and inflammatory biomarkers by immunochemistry on TMA´s of 234 prostabc specimens from two disbnct cohorts: Hospital Professor Doutor Fernando Fonseca in Amadora, Lisbon and Insbtuto Português de Oncologia of Porto. We sought to assess in a retrospecbve way, several biomarkers, namely, prostabc epigenebc biomarkers not so known in prostabc se{ng: the histone modifiers HDAC1, HDAC4 and histone mark H3Ac, and the inflammatory biomarkers CXCR4, CXCR7, CXCL12, that could be representabve of aggressiveness of prostate cancer. We awempted to correlate the biomarkers expression levels with the clinical and pathological features and other variables such as disease-specific survival (DSS), disease-free survival (DFS) and overall survival (OS). Log-rank test and Cox regression model were used to idenbfy which biomarkers were independent predictors of prognosis. Complete informabon was available for 231 pabents and median follow-up bme was 13.7 years. A total of 16 (6.9%) pabents died from PCa and 89 (38,5%) pabents displayed biochemical relapse. Pathological stage (pTStage) and WHO Grade Groups strabfied pabents in respect to DSS (P < 0.0001 and P = 0.011, respecbvely) and to DFS (P=0.004 and P=0.027, respecbvely). Pabents with higher CXCR4 immunoexpression experienced significantly worse DSS compared to pabents with low expression (HR=1.016, 95% CI:1.002-1.031). The same occurred with CXCL12 (HR=0.546 95% CI:0.322-0.926) and H3Ac (HR=1.015, 95% CI: 1.001 - 1.029). No significant effect on DSS was found for the other biomarkers. In what concerns to DFS, the biomarker CXCR4 disclosed stabsbcally significant associabon with this variable. Pabents with higher expression of this biomarker were significantly more prone to experience disease recurrence (HR=1.003, 95% CI: 1.000-1.005). CXCR7 also corroborated stabsbcal evidence of associabon with DFS (HR=1.111, 95% CI:1.032-1.196). No significant effect on DFS was found for the other biomarkers. When adjusted to pTStage and WHO Grade Groups, the majority of the biomarkers lost stabsbcal significance, except pabents with higher immunoexpression of CXCR7. CXCR7 was the only biomarker which maintained independent impact on DFS (HR =1.119, 95% CI:1.032-1.214). No significant effect on OS was found for any other studied variable. From this analysis we concluded that high-level of CXCR7 expression is an independent predictor of poor prognosis aoer surgery and might provide important informabon for pabent management. To answer the quesbon posed by this thesis Which epigeneDc and inflammaDonrelated biomarkers can idenDfy clinically aggressive prostate cancer, we need to have stronger evidence but our results sustain that targebng biomarkers in blood and in urine are capable of early detecbon of PCa. The CXCL12-CXCR4/CXCR7 signaling axis may be a potenbal strategy. The same biomarkers would also be capable of doing pabent strabficabon and, eventually, be a treatment target. If detecbon of acbve inflammatory axis could be made earlier, earlier treatments could be implemented. This data come to meet the issue of immunotherapy in PCa. Prostate cancer seems to be an ideal model for therapeubc cancer vaccines, once prostate is an unessenbal organ with mulbple tumor-associated anbgens as potenbal targets. In general, PCa is an indolent disease that provides enough bme for the anbtumor immune response to be installed. A two phase approach of immune sbmulabon and immune modulabon seems to produce the best results. Many clinical trials are assessing immunotherapy combinabons and some of them in early stages of the disease. Examples are a STAT3 inhibitor trial (Phase I/II NCT01563302) and 2 trials with PSMA (Phase I NCT01723475 and Phase I NCT02991911). Although this thesis was based in a retrospecbve study, we think it was a relevant work, that opens doors to the understanding and evaluabon of prostate cancer behavior. Further invesbgabons must be pursed so that we can use these news tools and substanbally improve our pabent care in the near future. It will be a valuable task to learn how the intensity of CXCL12 and CXCR7 expression in prostate specimens correlates with its serum levels and, in turn, prospecbvely study how these levels relate to cancer aggressiveness and be able to bewer tailor pabent treatment based in a less invasive proceeding.

Prostate cancer is one of the most frequently diagnosed cancers and the second leading cause of cancer-related deaths in male population. While localized prostate cancer can be successfully treated with surgery or radiation therapy, the metastatic disease has no curable options. Metastasis can be developed as a result of failed therapy of localized cancer or present at initial diagnosis. As metastasis is the most common cause of prostate cancer-related death, developing novel approaches and improving the efficiency of existing therapies for the metastatic prostate cancer treatment will significantly improve patients’ survival.

Ubiquitin C-terminal hydrolase L1 (UCHL1) is a multifunctional protein primarily expressed in neuronal cells and involved in numerous cellular processes. UCHL1 has been linked with neurodegenerative diseases and a wide range of cancers but its specific role remains unknown. Previous UCHL1 knockdown studies have shown that UCHL1 controls the expression of pro- and anti-apoptotic genes as well as genes involved in cell cycle regulation but it is unknown how UCHL1 regulates these genes. We have shown that UCHL1 is cross-linked to DNA in DU145 but not in LNCaP or PC3 prostate cancer cells. Therefore, we hypothesized that UCHL1 regulates the expression of pro- or anti-apoptotic genes as well as the genes involved in the cell cycle through its interaction with DNA. By utilizing ChIP and ChIP-seq analyses it is possible to determine the UCHL1 target sequences on the genomic DNA. It was shown that UCHL1 is only expressed in DU145 but not in LNCaP, PC3 or C4-2 prostate cancer cell lines. Additionally, UCHL1 is expressed and cross-linked to DNA in HEK293T cells. It is believed that UCHL1 is silenced by upstream promoter methylation when it is not expressed. However, treatment with the epigenetic drugs 5-aza-2′-deoxycytidine and trichostatin A (TSA) did not result in induction of UCHL1 expression in LNCaP, PC3 or C4-2 prostate cancer cell lines. UCHL1 is also associated with p53. However, ChIP assay results have shown that UCHL1 and p53 do not bind to genomic DNA of upstream promoter regions CDKN1A and BAX genes. Additionally, through UCHL1 ChIP-seq analyses in DU145 and HEK293T cells, we discovered that UCHL1 co-localizes to the DNA with the shelterin complex shedding light on a new role of UCHL1 that has never been described before.
October 2014

Prostate cancer is the second most frequently diagnosed type of cancer and the fifth leading cause of cancer death in men worldwide. Prostate carcinogenesis is characterized by progressive alterations in genetic and epigenetic mechanisms that deregulate gene expression. The Six Transmembrane Epithelial Antigen of the Prostate 1 (STEAP1) gene encodes a protein with six transmembrane domains. In normal tissues, STEAP1 expression is very low but is overexpressed in several human cancers, mainly in prostate cancer. Some studies have indicated that STEAP1 overexpression seems to promote cell growth, suggesting that STEAP1 may act as an oncogene. Previous studies demonstrated that STEAP1 mRNA and protein have higher stability in LNCaP prostate cancer cell lines when compared with PNT1A non-neoplastic prostate cell lines, possibly due to post-transcriptional and post-translational modifications. However, these alterations do not justify the overexpression of STEAP1 in tumor cells, suggesting that other mechanisms may be involved. Therefore, the aim of this study was to explore the hypothesis that genetic and / or epigenetic alterations may be involved in overexpression of STEAP1. In order to evaluate genetic alterations in the STEAP1 gene sequence, the promoter region of STEAP1 in LNCaP and PNT1A cells was sequenced. To study the involvement of epigenetic mechanisms, the methylation patterns of STEAP1 in PNT1A and LNCaP cells were compared. In addition, the effect of treatment with DNA methyltransferases (DNMT) and histone deacetylases (HDAC) inhibitors on STEAP1 mRNA expression in PNT1A cells was evaluated. The sequence analysis of the promoter region of STEAP1 revealed some differences in both PNT1A and LNCaP cells when compared with the available genomic sequence. In silico analysis of the identified variants revealed several alterations in the transcription factors (TF) that can bind to each allelic variant including the binding of transcriptional activators to the altered allele of the variants. The analysis of the methylation pattern of STEAP1 gene in PNT1A and LNCaP cells showed differences in the promoter region near the transcription start site. The treatment with 5-Aza-2’-deoxycytidine (DNMT inhibitor) induced a slight increase in STEAP1 mRNA expression (3 fold-variation in comparison with control group, p<0.01) while the treatment with both 5-Aza-2’-deoxycytidine and TSA (HDAC inhibitor) induced a marked increase in STEAP1 mRNA expression (15 fold-variation relatively to control, p<0.001). The difference in the methylation pattern of STEAP1 between PNT1A and LNCaP cells, along with the increased STEAP1 mRNA expression in response to DNMT and HDAC inhibitors, indicates that STEAP1 gene expression seems to be regulated by epigenetic mechanisms.
O cancro da próstata é o segundo tipo de cancro mais frequentemente diagnosticado e a quinta principal causa de morte por cancro nos homens em todo o mundo. O desenvolvimento do cancro da próstata é caracterizado por alterações progressivas nos mecanismos genéticos e epigenéticos o que conduz a uma desregulação da expressão genética. O gene Six transmembrane epithelial antigen of the prostate 1 (STEAP1) codifica uma proteína com seis domínios transmembranares. Nos tecidos normais, a expressão do STEAP1 é muito baixa, no entanto é sobre-expresso em vários tipos de cancro nomeadamente no cancro da próstata. Vários estudos indicaram que a sobre-expressão do STEAP1 parece promover o crescimento celular, sugerindo que este pode actuar como um oncogene. Estudos anteriores demonstraram também que o mRNA e a proteína STEAP1 apresentam uma maior estabilidade em linhas celulares de cancro da próstata LNCaP quando comparado com as linhas celulares da próstata não-neoplásicas PNT1A. Esta diferença pode ser devida a modificações pós-transcricionais e/ou pós-translacionais. No entanto, estas alterações não justificam a sobre-expressão do STEAP1 em células tumorais, sugerindo assim o envolvimento de outros mecanismos de regulação. Portanto, o objectivo do presente trabalho foi explorar a hipótese de que alterações genéticas e/ou epigenéticas poderão estar envolvidas na sobre-expressão do STEAP1. A fim de avaliar a possível presença de alterações genéticas na sequência do gene STEAP1, foi sequenciada a região promotora do STEAP1 em células LNCaP e PNT1A. Para estudar o envolvimento de mecanismos epigenéticos, foram comparados os padrões de metilação do STEAP1 entre as linhas celulares PNT1A e LNCaP. Para além disso, foi ainda avaliado o efeito de um tratamento com inibidores das DNA metiltransferases (DNMT) e histonas desacetilases (HDAC) na expressão do gene STEAP1 em células PNT1A. A análise da sequência da região promotora do STEAP1 revelou algumas variantes tanto nas células LNCaP como PNT1A quando comparada com a sequência genómica disponível. A análise in silico das variantes mostrou diferenças nos fatores de transcrição que se podem ligar a cada variante alelica incluindo a ligação de activadores transcripcionais ao alelo alterado das variantes. A análise do padrão de metilação do STEAP1 entre células PNT1A e LNCaP mostrou diferenças na região promotora próxima do local de início da transcrição. O tratamento com 5-Aza-2’-deoxicitidina (inibidor das DNMT) induziu um ligeiro aumento na expressão do STEAP1 (três vezes em comparação com o grupo de controlo, p<0.01), enquanto que o tratamento com ambos os inibidores 5-Aza-2’-deoxicitidina e TSA (inibidor das HDAC) induziu um aumento acentuado na expressão do STEAP1 (quinze vezes relativamente ao grupo de controlo, p<0.001). A diferença no padrão de metilação do STEAP1 entre as células LNCaP e PNT1A, juntamente com o aumento da expressão do STEAP1 em resposta ao tratamento com os inibidores de HDACs e DNMTs, indica que a expressão génica do STEAP1 parece ser regulada por mecanismos epigenéticos.

by Cheung Tak Chi.
Thesis (M.Phil.)--Chinese University of Hong Kong, 1998.
Includes bibliographical references (leaves 117-123).
Abstract also in Chinese.
Acknowledgeements --- p.i
Abbreviations --- p.ii
Abstract --- p.v
List of Figures --- p.viii
List of Tables --- p.xiv
Contents --- p.xv
Contents
Chapter 1. --- Introduction --- p.1
Chapter 1.1 --- Epidemiological Risk Factors --- p.1
Chapter 1.1.1 --- Age --- p.1
Chapter 1.1.2 --- Race --- p.2
Chapter 1.1.3 --- Environmental or Migratory Factor --- p.2
Chapter 1.1.4 --- Diet --- p.2
Chapter 1.1.5 --- Genetics --- p.3
Chapter 1.2 --- Regulation of Normal Prostate Development and Function --- p.4
Chapter 1.3 --- Biochemistry and Development of Prostate Cancer --- p.6
Chapter 1.3.1 --- Androgen-Dependent Prostate Cancer --- p.6
Chapter 1.3.2 --- Androgen-Independent Prostate Cancer --- p.8
Chapter 1.4 --- Classification of Prostate Cancer --- p.9
Chapter 1.4.1 --- Stage A Prostate Cancer --- p.10
Chapter 1.4.2 --- Stage B Prostate Cancer --- p.10
Chapter 1.4.3 --- Stage C Prostate Cancer --- p.11
Chapter 1.4.4 --- Stage D Prostate Cancer --- p.11
Chapter 1.5 --- Methods for Early Detection of Prostate Cancer --- p.12
Chapter 1.6 --- Clinical Treatment of Prostate Cancer --- p.12
Chapter 1.6.1 --- Surgery --- p.12
Chapter 1.6.2 --- Radiotherapy --- p.13
Chapter 1.6.3 --- Chemotherapy --- p.13
Chapter 1.6.4 --- Hormonal Therapy --- p.13
Chapter 1.7 --- Objective --- p.14
Chapter 1.8 --- Estrogen and Its Related Compounds --- p.16
Chapter 1.8.1 --- 17β-Estradiol --- p.16
Chapter 1.8.2 --- Tamoxifen --- p.18
Chapter 1.8.3 --- Aromatase Inhibitor --- p.20
Chapter 1.9 --- Anticancer Drugs --- p.23
Chapter 1.9.1 --- Doxorubicin --- p.23
Chapter 1.9.2 --- cis-Platinum --- p.24
Chapter 1.10 --- Apoptotic Pathways --- p.25
Chapter 1.10.1 --- BCL-2 /BAD Pathway --- p.26
Chapter 1.10.2 --- FADD Pathway --- p.27
Chapter 1.10.3 --- CAS Pathway --- p.27
Chapter 2. --- Materials and Methods --- p.28
Chapter 2.1 --- Materials --- p.28
Chapter 2.2 --- Cell Lines --- p.32
Chapter 2.3 --- Preparation of Drugs --- p.32
Chapter 2.4 --- Drug Sensitivity Assay --- p.33
Chapter 2.5 --- Cell Cycle Analysis --- p.35
Chapter 2.6 --- DNA Fragmentation Assay --- p.36
Chapter 2.7 --- Annexin Binding Assay --- p.37
Chapter 2.8 --- Western Blot Analysis --- p.38
Chapter 2.9 --- Data Analysis --- p.41
Chapter 3. --- Results --- p.42
Chapter 3.1 --- Response of Human Androgen-Independent Prostate Cancer Cells to Doxorubicin and cis-Platinum --- p.42
Chapter 3.2 --- The Effect of 17p-Estradiol on the Growth and Anticancer Drug Sensitivity of Human Androgen-Independent Prostate Cancer Cells --- p.45
Chapter 3.2.1 --- 17β-Estradiol on Cell Growth --- p.45
Chapter 3.2.2 --- 17β-Estradiol on Anticancer Drug Sensitivity --- p.45
Chapter 3.2.3 --- 17β-Estradiol and Doxorubicin on Cell Cycle Progression --- p.51
Chapter 3.2.4 --- 17β-Estradiol and Doxorubicin Induced DNA Fragmentation --- p.57
Chapter 3.2.5 --- 17β-Estradiol and Doxorubicin on Annexin Staining --- p.59
Chapter 3.2.6 --- 17β-Estradiol and Doxorubicin on Apoptotic Protein Expression --- p.62
Chapter 3.3 --- The Effect of Tamoxifen on the Growth and Anticancer Drug Sensitivity of Human Androgen-Independent Prostate Cancer Cells --- p.64
Chapter 3.3.1 --- Tamoxifen on Cell Growth of Human --- p.65
Chapter 3.3.2 --- Tamoxifen on Anticancer Drug Sensitivity --- p.65
Chapter 3.3.3 --- Tamoxifen and Doxorubicin on Cell Cycle Progression --- p.71
Chapter 3.3.4 --- Tamoxifen and Doxorubicin Induced DNA Fragmentation --- p.76
Chapter 3.3.5 --- Tamoxifen and Doxorubicin on Annexin Staining --- p.78
Chapter 3.3.6 --- Tamoxifen and Doxorubicin on Apoptotic Protein Expression --- p.79
Chapter 3.4 --- The Effect of Aromatase Inhibtiors on the Growth and Anticancer Drug Sensitivity of Human Androgen-Independent Prostate Cancer Cells --- p.81
Chapter 3.4.1 --- Aromatase Inhibitors on Cell Growth --- p.81
Chapter 3.4.2 --- Aromatase Inhibitors on Anticancer Drug Sensitivity --- p.83
Chapter 3.4.3 --- 4-AcA and Doxorubicin on Cell Cycle Progression --- p.93
Chapter 3.4.4 --- 4-AcA and Doxorubicin Induced DNA Fragmentation --- p.99
Chapter 3.4.5 --- 4-AcA and Doxorubicin on Annexin Staining --- p.100
Chapter 3.4.6 --- 4-AcA and Doxorubicin on Apoptotic Protein Expression --- p.102
Chapter 4. --- Discussion --- p.105
Chapter 4.1 --- 17 β-Estradiol and Anticancer Drug Sensitivity --- p.106
Chapter 4.2 --- Tamoxifen and Anticancer Drug Sensitivity --- p.109
Chapter 4.3 --- Aromatase Inhibitors and Anticancer Drug Sensitivity --- p.112
Chapter 4.4 --- DU145 Cells vs PC3 Cells --- p.115
Chapter 5. --- Conclusion and Perspectives --- p.116
Chapter 6. --- References --- p.117

Aberrant promoter methylation is known to silence tumor-suppressor genes in prostate cancer. Using a quantitative real-time PCR assay(MethyLight), I determined promoter methylation levels of APC, RASSF1A, CYP26A1 and TBX15 in 219 radical prostatectomies diagnosed between 1998-2001, examined their correlation with clinicopathological follow-up data including Gleason Pattern(GP), Gleason Score(GS) and pathological stage, and explored their potential in predicting biochemical recurrence(BR) using univariate and multivariate analyses. I demonstrated that methylation status of all four genes could accurately differentiate normal from cancerous tissues. Quantitative methylation levels of APC and TBX15 correlated strongly with GP, GS, and pathological stage. Both APC and TBX15 methylation levels could significantly predict BR in univariate analysis(p-value=0.028 and 0.003, respectively). The methylation profiles of APC and TBX15 combined could discriminate patients into high, intermediate, and low risk groups of BR(p-value=0.005). My project demonstrated that quantitative increase in promoter methylation levels of APC and TBX15 were associated with PCa progression.

碩士
中山醫學大學
醫學研究所
102
Thapsigargin (TG) was isolated from the Mediterranean plant Thapsia garganica. The highly lipophilic characteristic of TG accounts for their excellent penetration of biological membranes. TG can induce ER Stress and increase intracellular calcium through inhibiting sarco-endoplasmic reticulum Ca2+-ATPases. It has been reported that TG induces apoptosis and autophagy. In our previous study, TG inhibits telomerase activity by decreasing hTERT expression in A549 cells. In this study, we investigated the effects of TG on cytotoxicity, cell senescence and epigenetic regulation of hTERT. In our previous studies, fungal immunomodulatory protein Ganoderma tsugae (FIP-gts) has anticancer effects. FIP-gts can inhibit telomerase activity via ER Stress/calcium pahway in wide-type EGFR A549 cells. In this study, we investigated the effects of FIP-gts on telomerase activity and hTERT expression in EGFR mutation lung cancer cells. On MTT assay, the cell viability was reduced by TG in A549, H1355 and H1299 cells and reduced by FIP-gts in A549, H1975 and HCC827 cells. However, FIP-gts did not inhibit cell survival in H1650 cells. On clonogenic assay, TG inhibited A549 cell colony formation. Several autophagy inhibitors, 3-methyladenine (3-MA), chloroquine (CQ), and bafilomycin A1 (BafA1) were used to clarify the role of autophagy in TG-induced cell death. However, only 3-MA, an autophagy initiation inhibitor, enhanced the TG-induced cell-killing effect. The flow cytometry analysis was performed to detect the TG-induced ROS and senescence. Different from most of the genes, promoter hypermethylation turns on the hTERT expression. The TRAP, RT-PCR and Real Time PCR were used to analyze telomerase activity and hTERT expression in lung cancer cells treated with TG or FIP-gts. The western blot assay was performed to detected hTERT, TRF1 and TRF2 expression in A549 cells treated TG with or without 5-Azadc. The results demonstrated that 5-Azadc did not affect the TG-inhibiting telomerase activity and hTERT expression. DNA methyltransferase(DNMTs) enzyme are plays an important role that methylated genomic DNA. On RT-PCR and western blot assay, TG and 5-Azadc co-treatment downregulated the expressions of DNMT1 and DNMT3b, but did not alter the DNMT3a in A549 cells. To study the methylation patterns in more detail, bisulfite sequencing analysis confirm the hTERT promoter region (-196 form +46) in TG-treated cells. TG did not induce the methylation of site-specific CpGs on the hTERT promoter. The reporter assay was used to investigate of effect of transcription factors on hTERT promoter activity regulation. Thapsigargin inhibited the transcriptional activities of hTERT promoter (-548, -212, -196 and -155). Our results suggested that TG induces cell death via inhibited hTERT and telomerase activity. FIP-gts can inhibit cell survival and telomerase activity in EGFR mutation lung cancer cells.

Dissertação de mestrado em Genética Molecular
Prostate cancer (PCa) is the most common noncutaneous malignancy in men and the major cause of cancer-related morbidity and mortality worldwide. Due to genetic and epigenetic deregulations, prostate cancer is characteristically asymptomatic in early stages. Deeper understanding of this mechanisms strength the development of new and improved diagnostic and prognostic tools and, therefore, better treatment strategies. The shuffle of canonical histones, an epigenetic mechanism, is highly conserved among species and expression alterations of these histones variants, such as macroH2A1, are related to cancer development. H2AFY gene codifies two isoforms of the H2A histone variant macroH2A1: macroH2A1.1 and macroH2A1.2. MacroH2A1.1 inhibits cell proliferation and cell migration, whilst macroH2A1.2 has opposite functions. To date, there were studies of this histone variant in several cancer types, but none in PCa. Thus, our aim was to assess whether macroH2A1 is implicated in prostate carcinogenesis. In a large series of prostate samples from Portuguese Oncology Institute-Porto, we found that macroH2A1.1 transcript levels were downregulated in high-grade prostatic intraepithelial neoplasia (PIN) and primary PCa compared to normal prostatic tissues. Moreover, QKI, a splicing regulator that induces macroH2A1.1 expression, presented similar results. Compared with clinicopathological data, macroH2A1.1 and QKI expression were associated with Gleason Score and PSA blood levels. Both transcripts were able to discriminate cancerous from noncancerous prostate tissues. MacroH2A1.1 in vitro overexpression in a PCa Cell line decreased cell viability. Thus, macroH2A1.1 seems to play a critical role in PCa development.
O cancro da próstata é, mundialmente, a neoplasia não-cutânea mais comum do sexo masculino e a maior causa de morbilidade e mortalidade associada ao cancro. Com alterações genéticas e epigenéticas, o cancro da próstata é, inicialmente, assintomático. Uma melhor compreensão sobre estes mecanismos oferece o desenvolvimento de novas e aperfeiçoadas análises diagnósticas e, posteriormente, uma melhor aplicação de tratamentos. A substituição das histonas canónicas, um mecanismo epigenético, encontra-se conservada ao longo da evolução. Alterações da expressão dessas variantes de histonas, como a macroH2A1, correlacionam-se com o desenvolvimento de cancro. O gene H2AFY codifica duas isoformas da variante macroH2A1, da família H2A: macroH2A1.1 e macroH2A1.2. Enquanto a macroH2A1.1 inibe a proliferação e a migração celular, a macroH2A1.2 tem consequências opostas. Até hoje, há registos desta variante de histona em diversos estudos de cancro, embora nenhum em cancro da próstata. Com base no que foi descrito, esta tese tem como principal objectivo determinar se a variante macroH2A1 está associada com o desenvolvimento do carcinoma da próstata. Utilizando uma longa série de amostras de próstata do Instituto Português de Oncologia – Porto, descobrimos que os níveis de transcrito da macroH2A1.1 se encontravam mais baixos em neoplasias intraepiteliais prostáticas (PIN) de alto grau e tecidos primários de cancro da próstata, quando comparados com tecidos nãoneoplásicos de próstata. Adicionalmente, o QKI, um regulador de splicing que induz a expressão da macroH2A1.1, demonstrou resultados semelhantes. Comparando com os dados clinico patológicos, a expressão dos genes macroH2A1.1 e QKI estão associados com o Gleason Score e níveis de PSA no sangue. Ambos os transcritos também discriminam significativamente tecidos primários de cancro da próstata de tecidos não neoplásicos. A sobreexpressão de macroH2A1.1 numa linha de cancro da próstata diminuiu a viabilidade celular. Assim, a macroH2A1.1 parece desempenhar um papel relevante no desenvolvimento de cancro da próstata.

Relatório de Estágio do Mestrado Integrado em Ciências Farmacêuticas apresentado à Faculdade de Farmácia
Epigenetics comprises the study modifications in gene expression patterns that do not alter the primary DNA sequence, involving the reversible chemical modification of DNA, RNA and histones. Epigenetic changes are regulated by sets of enzymes that add or remove specific epigenetic markers and alter the expression of associated genes, producing an epigenetic code. Chromatin has several independent epigenetic mechanisms involved in modifying its structure, among which DNA methylation, post-translational modification of histones and non-coding RNAs. DNA methylation is an important regulator of gene expression and comprises the transfer of a methyl group to the fifth position carbon of the cytosine carbon ring, giving rise to 5-methylcytosine (5mC), through the DNMTs family of enzymes. One of the most relevant and most studied post-translational modifications is histone acetylation, which influences chromatin structure. The acetylation of histones consists of the addition of acetyl groups from Acetyl-Coenzyme A (acetyl-coA), to histone core and tails catalyzed by the histone acetyltransferase (HAT) family of enzymes. Epigenetics is influenced by externa factors, such as diet and metabolism, and, contrary to genetic abnormalities, epigenetic changes have a reversible character and can thus recover the function of the affected genes. A new trend in te molecular mechansims of disease is the identification of epigenetic patterns with specific diseases, such as the hypermethylation of DNA in cancer. Thus, understanding the impact that epigenetic mechanisms have on human diseases is crucial to succeeding in reversing or stabilizing the worsening of these diseases, namely cancer. Based on this knowledge, the great potential of epigenetic therapies and the development of drugs capable of modulating the epigenetic profile emerges. Among the drugs that act on the epigenetic machinery, the histone deacetylases inhibitors (HDACi), histone methyltransferases inhibitors (HMTi), acetyltransferases (HATi), demethylases (HDMi) and, finally, DNA methyltransferases inhibitors (DNMTi). Furthermore, resistance to chemotherapy is a limiting factor in anti-tumor therapies and, therefore, the combination of epigenetic drugs with classical anti-tumor therapies increases the probability of treatment success. Epigenetics is based on a promising therapeutic approach and with numerous application opportunities in different areas of clinical practice. In this work a general introduction to epigenetics and epigenetic mechanisms is followed by a more deeper revision concerning the use of epigenetic drugs in the case of cancer.
A epigenética compreende o estudo das modificações do DNA, RNA e de histonas com impacto na expressão genética que não alteram a sequência primária do DNA. As mudanças epigenéticas consistem, regra geral, na adição e remoção de grupos químicos (marcadores epigenéticos) de um modo regulado por famílias de enzimas que ao adicionar e remover os marcadores epigenéticos específicos alteram a expressão dos genes associados, produzindo um código epigenético. A cromatina apresenta vários mecanismos epigenéticos independentes envolvidos na modificação da sua estrutura, dos quais se destaca, por serem os mais bem estudados, a metilação do DNA, as modificações pós-translacionais de histonas e RNAs não codificantes. De facto, a metilação do DNA é um importante regulador da expressão genética e compreende a transferência de um grupo metilo para o carbono da quinta posição do anel de carbono de citosina, originando a 5-metilcitosina (5mC), por intermédio da família de enzimas, metiltransferases, DNMTs. Uma das modificações pós-translacionais mais relevante e mais estudada é a acetilação de histonas, que tem influência na estrutura da cromatina. A acetilação de histonas consiste na adição de grupos acetilo da Acetil-Coenzima A (acetil-coA) às caudas e “core” das histonas pela família de enzimas acetiltransferases das histonas (HAT). As modificações epigenéticas têm a particularidade de ser afetadas pelo ambiente, metabolismo e pela dieta e, contrariamente às anormalidades genéticas, as modificações epigenéticas têm um caráter reversível, benéfico ou não, podendo desta forma recuperar-se a função/expressão dos genes afetados. Um dos desenvolvimentos mais recentes no âmbito da saúde , diz respeito à caracterização de padrões de modificações epigenéticas associadas a doenças específicas, como por exemplo a hipermetilação do DNA no caso do cancro. Assim, a compreensão do impacto que os mecanismos epigenéticos têm nas doenças humanas, é crucial para conseguir reverter ou estabilizar o agravamento destas doenças, nomeadamente do cancro. Com base neste conhecimento baseia-se o grande potencial das terapias epigenéticas e o desenvolvimento de fármacos capazes de modular o perfil epigenético. Dentro dos fármacos que atuam na maquinaria epigenética citam-se os inibidores das histonas desacetilases (iHDAC), inibidores das histonas metiltransferases (iHMTs), das acetiltransferases (iHATs), das desmetilases (iHDMs) e, por fim, os inibidores das DNA metiltransferases (iDNMTs).Ademais, a resistência à quimioterapia é um fator limitante nas terapias antitumorais e desta forma, a combinação de fármacos epigenéticos com as terapias clássicas anti-tumorais, aumentam a probabilidade de êxito do tratamento. A epigenética assenta numa abordagem terapêutica promissora e com inúmeras oportunidades de aplicações em diversas áreas da prática clínica. Neste trabalho farei, inicialmente, uma abordagem genérica da epigenética para, depois, e dada a novidade e impacto na saúde humana, concentrar a atenção em mecanismos relacionados com fármacos epigenéticos.

Lung cancer is the third most prevalent cancer in the world; however it is the leading cause of cancer related deaths worldwide. Non-small cell lung cancer (NSCLC) accounts for ~85% of the lung cancer cases. The current strategies to treat NSCLC patients with frequent causal genetic mutations is through targeted therapeutics. Approximately 10-35% of NSCLC patient tumors have activated mutations in the Epidermal Growth Factor Receptor (EGFR) resulting in uncontrolled cellular proliferation. The standard-of care for such patients is EGFR-Tyrosine Kinase Inhibitors (EGFR-TKIs), a class of targeted therapeutics that specifically inhibit EGFR activity. One such EGFR-TKI used in this study is erlotinib. Following erlotinib treatment, tumors rapidly regress at first; however, over 50% of patients develop erlotinib resistance within a year post treatment. Development of resistance remains to be the major challenge in treatment of NSCLC using EGFR-TKIs such as erlotinib.

In approximately 60% of cases, acquired erlotinib resistance in patients is attributed to a secondary mutation in EGFR, whereas in about 20% of cases, activation of alternative signaling pathways is the reported mechanism. For the remaining 15-20% of cases the mechanism of resistance remains unknown. Therefore, it can be speculated that the common methods used to identify genetic mutations in tumors post erlotinib treatment, such as histologic analysis and genetic screening may fail to identify alterations in epigenetic mediators of erlotinib resistance, also including microRNAs (miRNAs). MiRNAs are short non-coding RNAs that post-transcriptionally negatively regulate their target transcripts. Hence, in this study two comprehensive screens were simultaneously conducted in erlotinib sensitive cells: 1) a genome-wide knock-out screen, conducted with the hypothesis that loss of function of certain genes drive erlotinib resistance, 2) a miRNA overexpression screen, conducted with the hypothesis that certain miRNAs drive the development of erlotinib resistance when overexpressed. The overreaching goal of the study was to identify novel drivers of erlotinib resistance such as microRNAs or other epigenetic factors in NSCLC.

The findings of this study led to the identification of a tumor suppressive protein and an epigenetic regulator, SUV420H2 (KMT5C) that has never been reported to be involved in erlotinib resistance. On the other hand, the miRNA overexpression screen identified five miRNAs that contribute to erlotinib resistance that were extensively analyzed using multiple bioinformatic tools. It was predicted that the miRNAs mediate erlotinib resistance via multiple pathways, owing to the ability of each miRNA to target multiple transcripts via partial complementarity. Importantly, a correlation between the two screens was identified clearly supporting the use of two simultaneous screens as a reliable technique to determine highly significant miRNA-target interactions. Overall, the findings from this study suggest that epigenetic factors, such as histone modifiers and miRNAs function as critical mediators of erlotinib resistance, possibly belonging to the 15-20% of NSCLC cases with unidentified mechanisms involved in erlotinib resistance.

Indiana University-Purdue University Indianapolis (IUPUI)
Given the numerous benefits of a healthy diet and exercise for cancer survivors, there has been an increase in the number of lifestyle intervention trials for this population in recent years. However, the extent to which adherence to a diet and exercise intervention predicts health-related outcomes among cancer survivors is currently unknown. To address this question, data from the Reach out to ENhancE Wellness in Older Cancer Survivors (RENEW) diet and exercise intervention trial were analyzed. RENEW was a yearlong telephone and mailed print intervention for 641 older (>65 years of age), overweight (body mass index: 25.0-39.9), long-term (>5 years post-diagnosis) survivors of colorectal, breast, and prostate cancer. Participants were randomized to the diet and exercise intervention or a delayed-intervention control condition. The RENEW telephone counseling sessions were based on determinants of behavior derived from Social Cognitive Theory (SCT) (e.g., building social support, enhancing self-efficacy). These factors have been hypothesized to improve health behaviors, which in turn should improve health outcomes. Thus, drawing on SCT and prior diet and exercise research with cancer survivors, I hypothesized that telephone counseling session attendance would be indirectly related to health-related outcomes (i.e., physical function, basic and advanced lower extremity function, mental health, and body mass index) through intervention-period strength and endurance exercise and dietary behavior (i.e., fruit and vegetable intake, saturated fat intake). The proposed model showed good fit to the data; however, not all of the hypothesized relationships were supported. Specifically, increased telephone counseling session attendance was related to engagement in all of the health behaviors over the intervention period. In turn, (a) increased endurance exercise was related to improvement in all of the health-related outcomes with the exception of mental health; (b) increased strength exercise was solely related to improved mental health; (c) increased fruit and vegetable intake was only related to improved basic lower extremity function; and (d) saturated fat intake was not related to any of the health-related outcomes. Taken together, these findings suggest that SCT determinants of behavior and the importance of session attendance should continue to be emphasized in diet and exercise interventions. Continued exploration of the relationship between adherence to a diet and exercise intervention and health-related outcomes will inform the development of more cost-effective and efficacious interventions for cancer and other medical populations.