O carcinoma de células renais (CCR) é o tumor mais agressivo que afeta o rim de pessoas adultas. O CCR é uma doença heterogênea, com diferentes alterações moleculares e variados patrões histológicos e clínicos que apresentam evolução diferente. Atualmente apenas variáveis anatomopatológicas clássicas são utilizadas para determinar o prognóstico dos pacientes. Utilizando uma plataforma de microarranjos de DNA, nosso grupo identificou em um trabalho anterior um conjunto de genes que se encontram diferencialmente expressos em tumores de rim. Neste estudo, nove candidatos foram selecionados para avaliação como marcadores de prognóstico no CCR. Foi confirmada a alteração na expressão dos genes ARNTL, ACTN4 e EPAS1 (p < 0,05) em amostras tumorais de CCR através de PCR em tempo real. Adicionalmente, foi observada a alteração da expressão dos genes ARNTL, EPAS1 e CASP7 em linhagens celulares imortalizadas derivadas de tumores renais, recapitulando por tanto, as alterações observadas nos tumores obtidos de pacientes. Posteriormente investigamos o padrão de expressão proteica destes candidatos por imunohistoquímica utilizando microarranjos de tecidos. Foi detectada a diminuição significativa (p < 0,05) da expressão das proteínas ACTN4, ARNTL, CASP7 e EPAS1 em tumores de pacientes com CCR relativamente ao tecido renal não tumoral. Além disso, foi possível determinar valores de imunomarcação capazes de estratificar pacientes com CCR em diferentes grupos de risco quanto à sobrevida câncer-específica, que adicionalmente apresentaram associação significativa com parâmetros anatomopatológicos utilizados na clínica. As imunomarcações de ACTN4, ARNTL, e EPAS1 se mostraram parâmetros independentes de prognóstico de sobrevida dos pacientes. A imunomarcação de CASP7 foi capaz de identificar subgrupos de pacientes com pior prognóstico dentro de um conjunto de pacientes de baixo risco em função do estadio clinico, além de identificar pacientes com menor risco de morte pelo câncer entre aqueles apresentaram recorrência em até 5 após a cirurgia. O conjunto de resultados obtidos aponta para um novo conjunto de biomarcadores moleculares com potencial relevância para auxiliar no prognóstico de pacientes com carcinoma de células renais. / The renal cell carcinoma (RCC) is the most aggressive tumor that affects the kidney in adult people. The RCC is a heterogeneous disease, with many different molecular alterations and varied histological and clinical patterns with different outcome. Currently, only classic anatomopathological variables are used to determine patients\' prognosis. Using a DNA microarray platform, our group identified in a previous work a set of genes differentially expressed in renal tumors. In this study, nine candidates were selected for evaluation as prognostic biomarkers in RCC. Alteration of the gene expression in RCC tumor samples was confirmed for ARNTL, ACTN4 and EPAS1 (p < 0.05) by real time PCR. Additionally, gene expression changes of ARNTL, EPAS1 and CASP7 were also observed in immortalized cell lines derived from renal tumors, recapitulating the expression changes detected in the patients\' tumors. Next, we used tissue microarrays to investigate the protein expression of the selected candidates by immunohistochemistry. Expression of the proteins ACTN4, ARNTL, CASP7 and EPAS1 was detected as significantly downregulated (p < 0.05) in patients´ tumors relative to non-tumor renal tissue. Furthermore, immunostaining patterns of the selected candidates were able to stratify patients with RCC in different risk groups according to cancer-specific survival, which also showed significant associations with anatomopathological parameters used in the clinics. ACTN4, ARNTL and EPAS1 immunostaining resulted as independent prognostic parameters of patient survival. CASP7 immunostaining was able to identify subgroups of patients with worse prognosis in a set of low risk patients as determined by their clinical stage, and also identified patients with lower risk of death from cancer amongst patients that relapsed within 5 years after surgery. Overall, these results point to a new set of molecular biomarkers with potential relevance to help in the prognosis of patients with renal cell carcinoma.

No description available.

In this study, we investigated the mechanisms by which PBRM1 functions as a tumor suppressor in clear cell renal cell carcinoma. PBRM1, also known as BAF180 or Polybromo, is a member of the PBAF SWI/SNF chromatin remodeling complex. Cancer sequencing studies have revealed that SWI/SNF components are widely mutated in cancer. PBRM1 is recurrently mutated in various human malignancies, but it has a particularly high mutation rate in clear cell renal cell carcinoma: ~40% of clear cell renal cell carcinomas have a PBRM1 mutation, making it the second most highly mutated gene in clear cell renal cell carcinoma behind VHL. Although many recent studies have looked at how other SWI/SNF components function in cancer control, relatively little is known about the tumor suppressive mechanisms of PBRM1 in clear cell renal cell carcinoma. To investigate PBRM1 function, we manipulated its expression in clear cell renal cell carcinoma cell lines. In cell lines with intact PBRM1, we stably knocked down its expression using shRNA. In a cell line with mutant PBRM1, we stably restored expression of the wild-type protein. We found that PBRM1 deficiency significantly enhanced the growth properties of cells, but only when the cells were grown under stressful conditions, such as reduced serum or a 3-D culture environment. To investigate genes and pathways influenced by PBRM1 that may confer this growth advantage, we compared gene expression differences in the clear cell renal cell carcinoma cell lines and murine embryonic fibroblasts with or without PBRM1. We found that PBRM1 regulated numerous cancer-related genes and pathways. One gene, ALDH1A1, was consistently upregulated with PBRM1 deficiency across our cell lines. Further expression analysis using two different clear cell renal cell carcinoma primary tumor datasets revealed that PBRM1 mutation in primary tumors was also associated with higher ALDH1A1 levels. ALDH1A1, or aldehyde dehydrogenase 1, is part of the retinoic acid metabolic pathway and irreversibly converts retinaldehyde to retinoic acid. It functions in hematopoietic stem cell development, white versus brown fat programming, and insulin signaling. Numerous studies have also identified ALDH1A1 as a marker of tumor-initiating cells, also known as cancer stem cells. Not much is known about the regulation of ALDH1A1 expression in cancer, and it has not previously been linked to PBRM1 or SWI/SNF. We confirmed that stable knockdown of PBRM1 in clear cell renal cell carcinoma cell lines resulted in higher ALDH1A1 mRNA and protein expression, and also higher ALDH1-class enzyme activity. Alternatively, re-expression of wild-type PBRM1, but not cancer-associated mutant PBRM1, lowered ALDH1A1 expression and activity in the PBRM1-mutant line. Additionally, inhibiting ALDH1A1 or knocking it down in the context of PBRM1 deficiency reduced anchorage-independent growth, while over-expressing ALDH1A1 in the PBRM1-normal setting increased tumorsphere-forming capacity. These results suggest that ALDH1A1 is not only a marker of tumor-initiating cells, but can also increase the tumorigenic potential of cells. Based on our gene expression analysis, we additionally explored PBRM1 regulation of the EGFR and IFN pathways. PBRM1 decreased total EGFR protein levels and dampened downstream signaling. These changes had functional consequences, as PBRM1 deficiency led to faster growth in response to EGF stimulation. However, it did not create a setting of oncogenic addiction, as PBRM1 deficient cells were also more resistant to EGFR inhibition. Alternatively, PBRM1 deficiency reduced basal and IFNα-induced levels of IFI27, a pro-apoptotic interferon response gene, and made cells more resistant to growth inhibition by IFNα. PBRM1 mutations in cancer would thus be expected to have wide-ranging effects on a cell, and the targeting of any one specific downstream pathway might have limited efficacy. Finally, we investigated the molecular mechanisms of how PBRM1 deficiency could alter transcription, keeping in mind that PBRM1 is one subunit of the larger PBAF complex. In our clear cell renal cell carcinoma cell lines, we found that mRNA and protein levels of another PBAF-specific subunit, ARID2, increased with PBRM1 deficiency. PBRM1 mutation in primary tumors was also associated with significantly higher ARID2 expression. Immunoprecipitation and glycerol gradient fractionation experiments suggested that more ARID2 may associate with the SWI/SNF components BRG1 and SNF5 after PBRM1 knockdown. ARID2 ChIP-seq analysis revealed that this remnant PBAF-like complex was bound to fewer locations in the genome, and its binding locations were broadly redistributed. Both gained and lost ARID2 binding were associated with differential gene expression, of both upregulated and downregulated genes, indicating that the genomic context influences whether PBAF-binding is activating or repressive. Interestingly, we also found that ARID2 was required for some of the pro-tumorigenic changes associated with PBRM1 deficiency, such as upregulation of ALDH1A1 and EGFR levels, but not others, such as decreased IFI27 levels, implying alternative modes of transcriptional regulation. In total, this study implicates PBRM1 in the regulation of numerous cancer-related genes and pathways in clear cell renal cell carcinoma. PBRM1 mutation would alter the genomic binding of a residual PBAF-like complex containing ARID2, leading to transcriptional changes that promote tumor formation and growth. A better understanding of this oncogenic mechanism may reveal novel therapeutic opportunities.

In 2018, it is estimated that 55,440 Americans will be diagnosed with pancreatic cancer and this figure is expected to continue to rise with increased life expectancy. Despite some measurable progress over the past few decades, pancreatic cancer remains one of the most lethal malignancies with five-year survival rate of 8.7%. Novel therapies, and their timely translation to the clinic, are urgently needed. As part of an effort to identify and characterize novel therapeutic strategies for pancreatic ductal adenocarcinoma, we began a study of the role of Bmi1 in tumor maintenance and progression. While Bednar and colleagues showed that Bmi1 is critical for the development of pancreatic cancer, and that its pancreas-specific deletion impairs PanIN formation, we were interested in assessing its function in established tumors. During the course of this work, we acquired a novel compound, PTC596, developed by PTC Therapeutics as a post-translational inhibitor of BMI1. Treatment with PTC596 leads to hyperphosphorylated BMI1, and this modification is associated a loss of protein activity. We planned to study this compound, in vitro and in vivo, as a complement to genetic perturbations of Bmi1. Initial characterizations of the effects of PTC596 on human and murine-derived pancreatic cancer cell lines revealed a potent anti-proliferative effect, accompanied by BMI1 hyperphosphorylation, and followed by polyploidy and cell death after prolonged treatment. Further analysis showed a clear G2/M arrest and elevated levels of phospho-histone H3. Bmi1 is known to play a role the cell cycle, but its inhibition in pancreatic cancer cell lines has been shown to induce G1 arrest. We decided to further explore the mechanism of PTC596’s antiproliferative effects by carrying out RNA sequencing on Aspc1 cells treated with PTC596. We found that 8 of the ten most down-regulated genes were members of the tubulin family and began to study this compound’s effect on microtubules. Compelling results from a cell-free tubulin polymerization assay support inhibition of tubulin polymerization as the mechanism of action for PTC596. These data are further supported by evidence that PTC596 increases the fraction of free-tubulin in treated cells, as well as dramatically alters the cell’s microtubule network. Given our laboratory’s interest in identifying novel therapies for pancreatic cancer, and the fact that PTC596 has already begun clinical trials, we continued to characterize this compound in vivo. We found PTC596 to have properties favorable for in vivo administration. PTC596 is orally available, has a plasma half-life of approximately 22 hours following oral administration, and accumulates in tumor tissue where it has an expected pharmacodynamic effect. Furthermore, it is well tolerated in vivo in combination with gemcitabine. We carried out a four-arm intervention study in tumor-bearing KPC mice, examining PTC596 alone and in combination with gemcitabine. We found that PTC596 synergizes with gemcitabine to significantly reduce tumor growth rates and provide a 3-fold extension of survival as compared to vehicle. These findings are, to our knowledge, the first evidence of in vivo synergy between a microtubule-destabilizing agent and gemcitabine for the treatment of pancreatic cancer. Importantly, this study identifies an alternative mechanism for PTC596 and implicates its efficacy in a novel treatment regimen for pancreatic ductal adenocarcinoma.

腎細胞癌是一種成人惡性腫瘤，治療效果不理想且常發生腫瘤轉移。目前對腎細胞癌的研究主要集中於鑒定並驗證可用於癌癥早期診斷和預後判斷的新型潛在生物標誌物。擬遺傳學變化尤其是啟動子CpG二核苷酸甲基化所導致的抑癌基因功能喪失已被廣泛認為是腫瘤發生的一個主要機理。迄今為止，已有許多抑癌基因在腎細胞癌中被報道出現啟動子甲基化。這些發現為腎癌發生的分子機制及潛在生物標誌物提供了新的思路。本課題旨在探索ZNF382和BCL6B這兩個鋅指蛋白抑癌基因在腎細胞癌中的啟動子甲基化情況，及其與腫瘤抑制有關的生物學功能和可能的分子機制。 / 鋅指蛋白轉錄抑制子ZNF382已在多種癌癥中被報道為功能性抑癌基因， 且常伴隨有啟動子甲基化導致的基因沈默，但其在腎細胞癌中尚未被報道。我們發現ZNF382在腎癌細胞系中由於啟動子CpG甲基化而致表達下調或基因沈默，並且其表達下調或沈默可被去甲基化藥物逆轉，在正常細胞系中則觀察不到這一現象。腎癌原發腫瘤組織中也廣泛檢測到ZNF382異常甲基化。在ZNF382沈默的腎細胞癌細胞系中，外源表達的ZNF382顯著地抑制了腫瘤細胞集落形成和細胞遷移，並且誘導細胞發生雕亡。而且，我們發現ZNF382在腎癌細胞系中可抑制多種致瘤基因和幹細胞標誌基因的表達。因此，本研究證明ZNF382通過抑制下遊致癌基因和幹細胞標誌基因的表達從而發揮抑制腫瘤的功能，並且其在腎細胞癌中常因啟動子高度甲基化而導致基因失活。 / 另一個鋅指蛋白基因BCL6B（ZNF62）已被證實可通過招募組蛋白去乙酰化酶抑制靶基因的轉錄，但其在腎細胞癌中的表達情況和生物學功能尚不清楚。我們發現，BCL6B基因在正常腎組織和正常細胞系中穩定表達， 但在腎癌細胞系中由於啟動子甲基化其表達下調或沈默。去甲基化藥物可以重新激活BCL6B的表達，同時伴隨其啟動子的去甲基化。BCL6B甲基化在腎癌原發腫瘤組織中也被頻繁檢測到。在腎癌細胞系中，外源表達BCL6B顯著抑制了腫瘤細胞集落形成和細胞遷移，並且誘導腫瘤細胞雕亡。我們進一步發現，BCL6B作為功能性轉錄抑制子在腎癌細胞系中抑制多種致癌基因和幹細胞標誌基因的表達。這些結果表明BCL6B是腎細胞癌的一個抑癌基因且其在腎癌中常被甲基化。 / 綜上所述，本課題從擬遺傳學和生物學功能兩個方面分別鑒定了腎癌中的兩個鋅指蛋白抑癌基因，ZNF382 和BCL6B。此研究可以幫助更好地了解腎癌發生的分子機理，並且為發展新的腎癌標誌物提供了更多思路。 / Renal cell carcinoma (RCC) is a malignant cancer in adults, often with poor outcome and frequent metastasis. Recent studies on this disease focus on the identification and verification of novel potential biomarkers for early detection and prognostic prediction of cancer. Epigenetic alterations, especially promoter CpG methylation, leading to the loss of tumor suppressor gene (TSG) function have been widely recognized as a major cause for tumor pathogenesis. To date, a number of TSGs with aberrant promoter methylation have been reported in RCC, which provides new insights into the molecular mechanism of renal cancer and the potential as biomarkers. The aim of this study is to characterize promoter methylation of two zinc finger tumor suppressors, ZNF382 and BCL6B, their biological functions and underlying molecular mechanisms in RCC. / Transcription repressor ZNF382 (zinc finger protein 382) was reported as a functional TSG with frequent inactivation by promoter methylation in multiple carcinomas, but not studied in RCC yet. I found that ZNF382 was silenced or downregulated in RCC cell lines due to promoter CpG methylation which could be reversed by pharmacologic demethylation treatment, but not in normal renal cell lines. Aberrant methylation of ZNF382 was also frequently detected in the RCC primary tumors. Ectopic expression of ZNF382 in the silenced RCC cells strongly inhibited their clonogenicity and migration, as well as promoted cell apoptosis. Moreover, I found that ZNF382 repressed the expression of multiple oncogenes and stem cell markers in RCC cells. Therefore, my results demonstrate ZNF382 exerts the tumor suppressive function through repressing the downstream target oncogenes and stem cell markers, and is often epigenetically inactivated by promoter methylation in RCC. / Another zinc finger protein, B cell CLL/lymphoma 6 member B (BCL6B, ZNF62) has been identified to repress transcription of its target genes by recruiting histone deacetylases, but its expression and biological function in RCC remain largely unknown. BCL6B was readily expressed in normal kidney tissue and renal cell line. BCL6B was silenced or downregulated by promoter CpG methylation in RCC cell lines. Pharmacologic demethylation reactivated BCL6B expression along with concomitant promoter demethylation. BCL6B methylation was also frequently detected in RCC primary tumors. Ectopic expression of BCL6B in RCC cells significantly inhibited tumor clonogenicity and migration of RCC cells, and induced tumor cell apoptosis. We further found that BCL6B as functional repressor suppressed the expression of multiple oncogenes and stem cell markers. These data indicated BCL6B was a functional tumor suppressor frequently methylated in RCC. / In summary, my study identified two zinc finger tumor suppressors, ZNF382 and BCL6B, in RCC from both epigenetical and functional aspects. This work may contribute to a better understanding of the molecular mechanisms of renal cancer pathogenesis and also give more clues to the discovery of novel biomarkers for RCC. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Rong, Rong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 110-128). / Abstracts also in Chinese. / Abstract in English --- p.i / Abstract in Chinese --- p.iii / Acknowledgements --- p.v / Table of Content --- p.vi / List of abbreviations --- p.xi / List of Figures --- p.xv / List of Tables --- p.xvii / List of Publications --- p.xviii / Chapter Chapter 1 --- Literature Reviews --- p.1 / Chapter 1.1 --- Molecular basis of cancer --- p.1 / Chapter 1.1.1 --- Oncogenes and TSGs --- p.2 / Chapter 1.1.2 --- Cancer genetics --- p.3 / Chapter 1.1.3 --- Cancer epigenetics --- p.4 / Chapter 1.1.4 --- DNA methylation --- p.4 / Chapter 1.1.4.1 --- Mechanism of DNA methylation --- p.5 / Chapter 1.1.4.2 --- DNA methylation and gene transcription --- p.6 / Chapter 1.1.4.3 --- Types of DNA methylation in human cancers --- p.7 / Chapter 1.1.4.3.1 --- Hypomethylation in cancer genome --- p.8 / Chapter 1.1.4.3.2 --- Hypermethylation of TSGs in cancer --- p.8 / Chapter 1.1.5 --- The link between cancer genetics and cancer epigenetics --- p.9 / Chapter 1.2 --- Renal cell carcinoma (RCC) --- p.10 / Chapter 1.2.1 --- Epidemiology of RCC --- p.10 / Chapter 1.2.2 --- Histopathology of RCC --- p.14 / Chapter 1.2.3 --- Genetic and epigenetic alterations in RCC --- p.16 / Chapter 1.2.3.1 --- Genetic alterations --- p.17 / Chapter 1.2.3.2 --- Epigenetic alterations --- p.22 / Chapter 1.2.3.2.1 --- Aberrant DNA hypermethylation in RCC --- p.22 / Chapter 1.2.3.2.2 --- Histone and chromatin regulations in RCC --- p.24 / Chapter 1.2.4 --- Signaling pathways associated with RCC --- p.25 / Chapter 1.2.4.1 --- VHL/HIF signaling in RCC --- p.26 / Chapter 1.2.4.2 --- PI3K/AKT/mTOR signaling in RCC --- p.27 / Chapter 1.2.4.3 --- Wnt/β-catenin signaling in RCC --- p.28 / Chapter 1.2.4.4 --- HGF/MET signaling in RCC --- p.31 / Chapter 1.3 --- Transcription factor family of zinc finger proteins --- p.32 / Chapter 1.3.1 --- Zinc Finger Protein 382 (ZNF382) --- p.33 / Chapter 1.3.2 --- B cell CLL/lymphoma 6, member B (BCL6B) --- p.34 / Chapter Chapter 2 --- Aim of Study --- p.36 / Chapter 2.1 --- Identify two zinc finger repressors as TSGs for RCC --- p.37 / Chapter 2.2 --- Study their tumor suppressor roles in RCC --- p.37 / Chapter 2.3 --- Explore the mechanisms of their tumor suppressor function --- p.38 / Chapter Chapter 3 --- Materials and Methods --- p.39 / Chapter 3.1 --- Cell lines and tissue samples --- p.39 / Chapter 3.1.1 --- Cell lines, tumors and normal tissue samples --- p.39 / Chapter 3.1.2 --- Maintenance of cell lines --- p.39 / Chapter 3.1.3 --- Drug treatment of cell lines --- p.40 / Chapter 3.1.4 --- Total RNA extraction --- p.40 / Chapter 3.1.5 --- Genomic DNA extraction --- p.41 / Chapter 3.2 --- General techniques --- p.42 / Chapter 3.2.1 --- Agarose gel electrophoresis --- p.42 / Chapter 3.2.2 --- TA cloning --- p.43 / Chapter 3.2.3 --- Transformation of cloning vectors into E. coli competent cells --- p.43 / Chapter 3.2.4 --- Plasmid DNA extraction --- p.44 / Chapter 3.2.4.1 --- Mini-prep of plasmid DNA --- p.44 / Chapter 3.2.4.2 --- Midi-prep of plasmid DNA --- p.45 / Chapter 3.2.5 --- Measurement of DNA and RNA concentrations --- p.45 / Chapter 3.2.6 --- Preparation of reagents and medium --- p.46 / Chapter 3.2.6.1 --- Reagents for agarose gel electrophoresis --- p.46 / Chapter 3.2.6.2 --- Reagents for mini-prep of plasmid DNA --- p.46 / Chapter 3.2.6.3 --- LB medium and LB plates --- p.46 / Chapter 3.3 --- Semi-quantitative Reverse transcription (RT)-PCR --- p.47 / Chapter 3.3.1 --- Reverse Transcription --- p.47 / Chapter 3.3.2 --- Semi-quantitative PCR --- p.48 / Chapter 3.3.2.1 --- Primer design --- p.48 / Chapter 3.3.2.2 --- PCR reaction --- p.49 / Chapter 3.4 --- Real-time PCR --- p.49 / Chapter 3.5 --- Methylation analysis --- p.50 / Chapter 3.5.1 --- Bisulfite treatment of genomic DNA --- p.50 / Chapter 3.5.2 --- Bioinformatical analysis of CpG island --- p.51 / Chapter 3.5.3 --- Methylation-specific PCR (MSP) --- p.51 / Chapter 3.5.3.1 --- Primers design --- p.51 / Chapter 3.5.3.2 --- PCR reaction --- p.53 / Chapter 3.5.4 --- Bisulfite genomic sequencing (BGS) --- p.53 / Chapter 3.5.4.1 --- Primers design --- p.53 / Chapter 3.5.4.2 --- PCR amplification and TA-cloning --- p.54 / Chapter 3.6 --- Construction of expression plasmids for studied genes --- p.54 / Chapter 3.6.1 --- Construction of the ZNF382-expressing vector --- p.54 / Chapter 3.6.2 --- Construction of the BCL6B-expressing vector --- p.55 / Chapter 3.7 --- Functional Study --- p.56 / Chapter 3.7.1 --- Colony formation assay on monolayer culture --- p.56 / Chapter 3.7.2 --- Wound healing assay --- p.57 / Chapter 3.7.3 --- TUNEL assay --- p.58 / Chapter 3.8 --- Western blot --- p.58 / Chapter 3.9 --- Statistical analysis --- p.58 / Chapter Chapter 4 --- Results --- p.60 / Chapter 4.1 --- Epigenetic and Functional study of ZNF382 in RCC --- p.60 / Chapter 4.1.1 --- Expression profiling of ZNF382 in human adult tissues --- p.60 / Chapter 4.1.2 --- Expression profiling of ZNF382 in RCC cell lines --- p.61 / Chapter 4.1.3 --- Dense promoter CpG methylation of ZNF382 correlated with its reduced expression in RCC --- p.62 / Chapter 4.1.4 --- Restoration of ZNF382 expression by pharmacologic demethylation --- p.65 / Chapter 4.1.5 --- Frequent methylation of ZNF382 in RCC primary tumors --- p.67 / Chapter 4.1.6 --- Functional study of ZNF382 in RCC --- p.68 / Chapter 4.1.6.1 --- Ectopic expression of ZNF382 inhibits clonogencity of RCC cells --- p.68 / Chapter 4.1.6.2 --- Ectopic expression of ZNF382 inhibits migration of RCC cells --- p.71 / Chapter 4.1.7 --- ZNF382 induces apoptosis of RCC cells --- p.72 / Chapter 4.1.8 --- ZNF382 represses the expression of multiple oncogenes and stem cell markers in RCC --- p.73 / Chapter 4.1.9 --- Discussion --- p.76 / Chapter 4.2 --- Epigenetic and Functional study of BCL6B in RCC --- p.82 / Chapter 4.2.1 --- Expression profiling of BCL6B in human adult tissues --- p.82 / Chapter 4.2.2 --- Expression profiling of BCL6B in RCC cell lines --- p.83 / Chapter 4.2.3 --- Correlation of the methylation status of ZNF382 promoter CpG island with its aborted expression in RCC --- p.84 / Chapter 4.2.4 --- Restoration of BCL6B expression by pharmacological demethylation --- p.87 / Chapter 4.2.5 --- Frequent BCL6B methylation in RCC primary tumors --- p.88 / Chapter 4.2.6 --- Functional study of BCL6B in RCC --- p.90 / Chapter 4.2.6.1 --- Ectopic expression of BCL6B inhibits clonogencity of RCC cells --- p.90 / Chapter 4.2.6.2 --- Ectopic expression of ZNF382 inhibits migration of RCC cells --- p.92 / Chapter 4.2.7 --- BCL6B induces apoptosis of RCC cells --- p.93 / Chapter 4.2.8 --- BCL6B represses the expression of multiple oncogenes and stem cell markers in RCC --- p.94 / Chapter 4.2.9 --- Discussion --- p.97 / Chapter Chapter 5 --- General discussion --- p.103 / Chapter Chapter 6 --- Summary --- p.106 / Chapter Chapter 7 --- Future Study --- p.108 / Chapter 7.1 --- Identification of key responsive elements in gene promoter --- p.108 / Chapter 7.2 --- Study of genetic alterations leading to gene inactivation --- p.109 / Chapter 7.3 --- Elucidation of the transcription-repressor activity in RCC --- p.109 / Reference List --- p.110

by Tong Hung Man Joanna. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 150-172). / Abstract --- p.i / Acknowledgments --- p.iii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter Chapter 2 --- Literature Review --- p.5 / Chapter 1. --- Overview of renal cell carcinoma --- p.6 / Chapter 1.1 --- Epidemiology --- p.6 / Chapter 1.2 --- Etiology --- p.6 / Chapter 1.3 --- Clinical features --- p.7 / Chapter 1.4 --- Pathology --- p.9 / Chapter 2. --- The biological cell cycle --- p.15 / Chapter 2.1 --- Cell Cycle --- p.15 / Chapter 2.2 --- Cell cycle control --- p.18 / Chapter 3. --- Overview of DNA ploidy and the relationship with the biological behavior of renal cell carcinoma --- p.18 / Chapter 3.1 --- Overview of DNA ploidy --- p.18 / Chapter 3.2 --- Intratumoral heterogeneity --- p.20 / Chapter 3.3 --- Controversial prognostic value of DNA ploidy analysis in RCC --- p.21 / Chapter 3.4 --- Assessment of DNA ploidy --- p.23 / Chapter 4. --- Cell proliferation and its assessment by immunohistochemical methods --- p.34 / Chapter 4.1 --- Proliferation activity and tumor growth --- p.34 / Chapter 4.2 --- Basic principles of immunohistochemistry (IHC) --- p.34 / Chapter 4.3 --- "Ki 67, a cell proliferation marker" --- p.39 / Chapter 4.4 --- "p27kipl, a cell cycle arrest marker" --- p.41 / Chapter Chapter 3 --- Aims of the study --- p.44 / Chapter Chapter 4 --- Materials and Methods --- p.46 / Chapter 1. --- Tissue samples --- p.47 / Chapter 1.1 --- Sample retrieval --- p.47 / Chapter 1.2 --- Tissue processing --- p.47 / Chapter 1.3 --- Preparation of tissue sections --- p.47 / Chapter 2. --- Methods for quantitative DNA analysis --- p.49 / Chapter 2.1 --- Instrumentation --- p.49 / Chapter 2.2 --- Procedures for quantitative DNA analysis --- p.50 / Chapter 3. --- Immunohistochemical (IHC) studies of proliferation activity of RCC --- p.59 / Chapter 3.1 --- Antibodies used --- p.59 / Chapter 3.2 --- Other reagents --- p.60 / Chapter 3.3 --- Unmasking of antigens --- p.62 / Chapter 3.4 --- ABC method for monoclonal antibodies with a avidin/biotin blocking --- p.62 / Chapter 3.5 --- Interpretation and scoring of immunostaining --- p.64 / Chapter 4. --- Clinical data retrieval --- p.64 / Chapter 5. --- Statistical analysis --- p.65 / Chapter Chapter 5 --- Results --- p.66 / Chapter 1. --- Clinical information --- p.67 / Chapter 2. --- Pathological features --- p.67 / Chapter 2.1 --- Histological subtypes --- p.67 / Chapter 2.2 --- Nuclear grading --- p.68 / Chapter 2.3 --- Clinical stage --- p.68 / Chapter 3. --- DNA ploidy analysis --- p.76 / Chapter 3.1 --- By flow cytometry --- p.76 / Chapter 3.2 --- By static image cytometry using cytospin preparations --- p.82 / Chapter 3.3 --- By static image cytometry using tissue sections --- p.87 / Chapter 4. --- Immunohistochemistry --- p.92 / Chapter 4.1 --- Ki 67 (MIB-1) --- p.92 / Chapter 4.2 --- p27kipl --- p.96 / Chapter 5. --- Statistical analysis --- p.101 / Chapter 5.1 --- DNA ploidy analysis --- p.101 / Chapter 5.2 --- Ki 67 (MIB-1) --- p.108 / Chapter 5.3 --- p27kipl --- p.110 / Chapter 5.4 --- Nuclear grade and nuclear area --- p.112 / Chapter 5.5 --- Stage --- p.115 / Chapter 5.6 --- Survival analysis --- p.117 / Chapter Chapter 6 --- Discussion --- p.118 / Chapter 1. --- DNA ploidy analysis --- p.119 / Chapter 1.1 --- Flow cytometry --- p.119 / Chapter 1.2 --- Image analysis using cytospin preparations --- p.123 / Chapter 1.3 --- Image analysis using tissue sections --- p.126 / Chapter 1.4 --- Intratumoral heterogeneity --- p.130 / Chapter 1.5 --- Comparison of the results from three methods --- p.131 / Chapter 1.6 --- The potential significance of the DNA ploidy status --- p.137 / Chapter 2. --- Proliferation activity of RCC --- p.139 / Chapter 2.1 --- Ki 67 --- p.139 / Chapter 2.2 --- p27kipl --- p.140 / Chapter 3. --- Nuclear grade --- p.142 / Chapter 4. --- Stage --- p.143 / Chapter Chapter 7 --- Conclusion --- p.144 / Chapter Chapter 8 --- Further studies --- p.147 / References --- p.149

Renal cell carcinoma (RCC) is the most deadly of urological malignancies. While metastatic disease affects one third of patients at diagnosis, a further third of patients who undergo extirpative surgery with curative intent subsequently develop metastatic disease. Inconsistency in the clinical course ensures predicting subsequent metastasis is notoriously difficult, despite the routine use of prognostic clinico-pathological parameters in risk stratification. With greater understanding of pathways involved in disease pathogenesis, a number of biomarkers have been proposed to be of prognostic significance; however there are currently no molecular prognostic markers in clinical use. Genetic intra-tumoural heterogeneity (genetic ITH) has been described in clear cell RCC (ccRCC) and may limit the clinical translation of biomarkers. There has been no assessment of ITH at other molecular levels. The aim of this work was to define and compare proteomic, transcriptomic and DNA methylation ITH in ccRCC, and identify potential prognostic biomarkers. Using reverse phase protein arrays to study protein expression in multiple spatially separate regions of primary and metastatic ccRCC, proteomic ITH was demonstrated for the first time. Interestingly there was no significant difference in proteomic ITH in metastatic ccRCC tumour deposits compared to primary tumours. However, on analysis of differential protein expression between primary and metastatic ccRCC tissue using a tissue microarray and automated analysis of immunofluorescence, there was significantly greater expression of Ki67, p53, VEGFR1, SLUG and SNAIL in the metastases compared to the primary tumours. Subsequent profiling of gene expression and DNA methylation in multiple areas of the same primary tumours confirmed transcriptomic and methylomic ITH. On comparison of this multimolecular ITH, significantly greater proteomic ITH was seen compared to gene expression and DNA promoter methylation heterogeneity. Recent evidence suggests DNA methylation may be prognostically important in RCC and given the lower methylomic ITH in ccRCC, the identification of prognostic DNA methylation changes in ccRCC were pursued using the Infinium HumanMethylation450K Beadchip. Following development of an analysis pipeline, identification and validation of prognostic differentially methylated regions (DMR) was performed on an experimental cohort and published dataset respectively. Five DMRs, which were associated with disease recurrence in ccRCC, were identified. NEFM gene promoter methylation was the only DMR associated with cancer specific survival, independent of TNM stage and nuclear grade on multivariate analysis, which was confirmed on a third independent published dataset. This thesis therefore demonstrates multi-molecular ITH in ccRCC for the first time. Despite this, NEFM promoter methylation may be a useful independent prognostic marker of cancer specific survival.

Telomeres are protective structures at the end of our chromosomes, composed of multiple repeats of the DNA sequence TTAGGG. They are essential for maintaining chromosomal stability by preventing damage and degradation of the chromosome ends. Telomeres are normally shortened with each cell division until a critical length is reached, at which stage cell cycle arrest is induced. Telomere shortening can be prevented in the presence of the telomere-­‐elongating enzyme telomerase. Telomerase is expressed during embryogenesis and in certain normal cell types, but most somatic cells exhibit undetectable levels of telomerase activity. In contrast, most cancer cells express telomerase enabling them to proliferate indefinitely. There is a search for reliable molecular markers that can be used to help predict cancer risk and outcome. The interest of investigating telomere length as a potential biomarker in malignancy has grown rapidly, and both tumors and normal tissues have been in focus for telomere length measurements. In this thesis, telomere length was investigated in breast cancer patients and in patients with renal cell carcinoma (RCC). The breast cancer patients were found to have significantly longer mean telomere length in peripheral blood cells (i.e. immune cells) compared to a tumor-­‐free control group. Moreover, patients with the longest blood telomere length had a significantly worse outcome compared to patients with shorter blood telomeres. In a patient group with clear cell RCC, telomere length was investigated in peripheral blood cells, in tumors and in corresponding kidney cortex. Again, patients with the longest blood telomere length had a significantly worse prognosis compared to those with shorter blood telomeres. In contrast, telomere length in tumor and kidney cortex tissues did not predict outcome per se. Immunological components may play a role in telomere length dynamics as well as in cancer development. We aimed to investigate a possible association between telomere length and certain immunological parameters, including various cytokines and peripheral levels of a blood cell type with suppressor function [regulatory T cells (Tregs)]. In our patients with clear cell RCC, three cytokines correlated significantly with tumor telomere length, but not with telomere length in peripheral blood cells. In a separate patient group with various RCC tumors, blood telomere length correlated positively with the amount of Tregs. It might be speculated that a subset of patients with long blood telomeres has a less efficient immune response due to high Treg levels, contributing to a worse prognosis. Another aim of this thesis was to explore telomere length changes over time. Evaluation of blood samples collected at a 6-­‐month interval from 50 individuals, showed that half of the participants experienced a decline in mean telomere length during the time period. This group had longer telomere length at baseline compared to those who demonstrated increased/stable telomere length. In a separate group of five blood donors, a remarkable drop in telomere length was detected in one donor over a 6-­‐month period, whereas the other donors exhibited only small fluctuations in telomere length. In conclusion, the results of this thesis indicate that blood telomere length has potential to act as an independent prognostic marker in malignancy. Adding to the complexity is the fact that changes in blood telomere length might occur within relatively short time spans, indicating that telomere length is a dynamic character.

The epidermal growth factor receptor (EGFR) family consists of four (EGFR, ErbB2, Erbb3, and ErbB4) receptor tyrosine kinases (RTK) whose signalling is important for physiological and malignant cellular functions such as proliferation, survival, migration, and differentiation. EGFR and ErbB2 in particular are established oncogenes in many solid tumours and are targets for anti-cancer treatment. LRIG1 (leucine-rich repeats and immunoglobulin-like domains-1) is a protein that negatively regulates the EGFR-family, and other RTKs and is a proposed tumour suppressor. This thesis examines the expression of the EGFR-family members and LRIG1 in renal cell carcinoma (RCC) and in prostate cancer (PC). In RCC, up-regulation of EGFR was shown for all RCC types analysed: clear cell (ccRCC), papillary (pRCC), and chromophobe (chRCC). ErbB2 was down-regulated in ccRCC. ErbB3 expression was low in non-neoplastic kidney and not significantly altered in RCC. ErbB4 was strongly down-regulated in the vast majority of RCCs of all types. LRIG1 was down-regulated in ccRCC. No prognostic value was found for any of these factors in RCC. In prostate cancer cells, LRIG1 was shown to be up-regulated by androgen stimulation and suppressed the growth of prostate cancer cells. In prostate cancer, the expression and prognostic value of LRIG1 was investigated in two patient series, one with untreated patients and one with patients who had undergone prostatectomy. In the untreated patient series, LRIG1 correlated with malignancy grade (Gleason score) and poor outcome for patients (both cancer specific and overall survival), being an independent prognostic factor. In contrast, in the series of patients who had undergone prostatectomy, LRIG1 expression correlated with a good outcome (overall survival). Thus in RCC, there were alterations in gene-expression of the EGFR-family members and LRIG1 between kidney cortex and RCC and between the RCC types. Despite few associations with clinical factors, these alterations are likely to be of biological importance. In prostate cancer LRIG1 was up-regulated by androgen stimulation and inhibited cell proliferation. LRIG1 expression had prognostic value in prostate cancer, maybe as a secondary marker of androgen receptor activation or because of growth inhibition of prostate cancer cells. Contradicting findings in untreated patients and patients treated with prostatectomy poses the question of whether the prognostic value of LRIG1 and other markers vary depending on the specific biological and clinical circumstances in the materials studied.

Telomerase is a ribonucleoprotein complex that catalyses telomeric repeat addition at the ends of chromosomes. The catalytic subunit, hTERT, acts as a key determinant for telomerase activity control; the induction of hTERT expression is required for telomerase activity. hTERT participates in cellular immortalization and is elevated in certain malignant tissues. Several tumours exhibit telomerase activity, which contributes to the infinite proliferation capacity that promotes tumour progression. Renal cell carcinoma (RCC) represents 2% of all adult malignancies and has a high mortality rate. The WHO classifies RCC into several sub-types based on cytogenetic aberrations and morphological features; the most prevalent sub-types are clear cell (ccRCC), papillary (pRCC), and chromophobe RCC (chRCC). The aims of this thesis were to study the expression patterns of various signalling molecules, to elucidate the functional links among them, and to define the roles of these signalling molecules in the regulation of hTERT gene expression and telomerase activity in RCC. The first paper included in this thesis revealed mRNA overexpression of DJ-1 (a PTEN inhibitor), cMyc, and hTERT in clinical ccRCC samples compared to tumour-free kidney cortex tissues. Significant, positive correlations were detected for DJ-1, cMyc, and hTERT mRNA levels in ccRCC, but not in pRCC. In vitro knockdown of DJ-1 by siRNA in ccRCC cells induced downregulation of p-Akt, cMyc, hTERT, and telomerase activity. Forced overexpression of DJ-1 in an ovarian carcinoma cell line was followed by increased hTERT promoter activity, which appeared to be dependent on cMYC binding to the promoter. Collectively, the in vitro studies verified a functional link among DJ-1, cMyc, and hTERT as implied in the clinical ccRCC samples. The second paper included in this thesis demonstrated overexpression of NBS1 mRNA levels in ccRCC compared to the kidney cortex. NBS1 mRNA levels exhibited significant, positive correlations with DJ-1, cMyc, and S phase, but not with hTERT. In vitro experiments suggested that DJ-1 could regulate NBS1 gene expression. The role of the hTERT transcriptional repressor WT1 in RCC was evaluated in the third paper included in this thesis. ccRCC samples displayed low WT1 mRNA levels compared to kidney cortex samples. Interestingly, WT1 expression was negatively associated with hTERT and cMyc both of which were elevated in ccRCC. Forced overexpression of WT1 isoforms in a ccRCC cell line increased the expression of several negative transcriptional regulators of hTERT and diminished the expression of hTERT positive regulators. In consequence, hTERT mRNA levels and telomerase activity were reduced. Chromatin immunoprecipitation verified direct binding of WT1 to the cMyc, Smad3, and hTERT promoters. Taken together, these data suggested that in ccRCC, WT1 affects hTERT at the transcriptional level via a combined effect on both positive and negative regulators. In conclusion, DJ-1 can regulate hTERT and telomerase activity through the PI3K pathway encompassing PTEN, NBS1, p-Akt, and cMyc in ccRCC, but not in pRCC. WT1 negatively regulates hTERT and telomerase activity directly and indirectly through multiple pathways in ccRCC.