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.

Os carcinomas de cabeça e pescoço (CECPs) compreendem um grupo de tumores que atingem vários sítios do trato aerodigestivo superior, incluindo cavidade oral, orofaringe, hipofaringe e laringe. Esses carcinomas são clinicamente heterogeneous e resultam de modificações cumulativas em genes que regulam proliferação, migração celular e apoptose. São estimados aproximadamente 500.000 novos casos de CECP anualmente no mundo. No Brasil, cerca de 14.000 novos casos são esperados em 2010, somente para cavidade oral. As taxas de morbidade e mortalidade e as limitações das estratégias terapêuticas enfatizam a necessidade de um melhor entendimento dos padrões moleculares envolvidos na iniciação e na progressão desses tumores, e de abordagens preventivas e terapêuticas efetivas. Infelizmente, apesar da intensa pesquisa nessa área, poucos marcadores moleculares são conhecidos que exibam sensibilidade e especificidade para diagnóstico e prognóstico de CECP. Em um estudo prévio, nós avaliamos dados de três bibliotecas SAGE de carcinoma de laringe com a finalidade de identificar eventos associados ao desenvolvimento e à agressividade de CECP. Utilizando abordagens estatísticas e de Bioinformática, nós identificamos 60 genes com expressão elevada ou reduzida em tumores metastáticos versus não-metastáticos e em ambos os grupos versus tecidos normais. O objetivo do presente estudo foi avaliar a expressão de quatro genes desta lista, os das queratinas 6A (KRT6A) e 19 (KRT19), da mesotelina (MSLN) e da calicreína 8 (KLK8), em um conjunto de 63 carcinomas primários de cabeça e pescoço e suas margens cirúrgicas e em quarto linhagens celulares (Hep-2, FaDu, SCC9 e UM-SSC-38) por RT-PCR em tempo real. Como amostra de referência para as linhagens, foram utilizados queratinócitos orais humanos normais, cultivados sobre uma camada de sustentação de fibroblastos irradiados. Todos os genes exibiram níveis de transcritos reduzidos ou ausentes nas linhagens celulares, exceto MSLN, que mostrou um padrão irregular de expressão. Em tumores primários, os genes KRT19 e MSLN apresentaram expressão diminuída em laringe, o mesmo sendo observado para o gene KLK8 em tumores de língua metastático. Além disso, foi detectada expressão elevada de MSLN e KLK8 em tumores não metastáticos de soalho de boca e expressão reduzida de KRT19 em tumores de soalho de boca e língua metastáticos. Os resultados levantam questões sobre o papel desses genes em processos biológicos associados com a tumorigênese de cabeça e pescoço e sobre sua participação no fenótipo neoplásico. / Head and neck squamous cell carcinomas (HNSCCs) encompass a group of tumors that affect a variety of sites in the upper aero-digestive tract, including oral cavity, oropharynx, hypopharynx and larynx. These carcinomas are clinically heterogeneous and result from cumulative changes in genes that regulate cell proliferation, migration and death. It is estimated that approximately 500,000 new cases of HNSCC are diagnosed worldwide each year. In Brazil, about 14,000 new cases are expected in the year 2010, only in oral cavity. The morbidity and mortality rates and the limitations of therapeutic strategies emphasize the need for a better understanding of the molecular pathways involved in the initiation and progression of these tumors and for effective preventive and therapeutic approaches. Unfortunately, despite intense research, few molecular markers are known to exhibit sensitivity and specificity for the diagnosis or prognosis of HNSCC. In a previous study, we evaluated data from three SAGE libraries of larynx carcinoma in order to identify events associated with the development and aggressiveness of HNSCCs. Using statistical and bioinformatic tools, we identified sixty top-up and 60 top-downregulated genes in metastatic versus non-metastatic tumors and in both these tumors versus normal tissues. The objective of the present study was to evaluate the expression of four genes from this list, keratin 6A (KRT6A), keratin 19 (KRT19), mesothelin (MSLN) and kallikrein 8 (KLK8), in a set of 63 primary carcinomas of head and neck and their surgical margins and in four cell lines (Hep-2, FaDu, SCC9 and UM-SSC-38) by real time RT-PCR. As a reference sample for cell lines, we used normal human oral keratinocytes grown on irradiated fibroblast feeder layer. All genes exhibited no or decreased levels of transcripts in the cell lines, except MSLN, which displayed an irregular pattern of expression. In primary tumors, KRT19 and MSLN genes were downregulated in larynx, and KLK8 in metastatic tongue tumors. In addition, MSLN and KLK8 were upregulated in non-metastatic floor of the mouth tumors and KRT19 was down regulated in metastatic floor of the mouth and tongue tumors. The results open questions about the role of these genes on biological processes related to head and neck tumorigenesis and on neoplastic phenotype.

Merkel cell carcinoma (MCC) is a highly aggressive skin cancer with rising global incidence. Merkel cell polyomavirus (MCV) was discovered in 2008 in 80% of MCC samples and since then a causal link between MCV and the majority of MCC cases has been established. microRNAs (miRNA, miR) are a family of small non-coding RNAs which play a key role in post-transcriptional regulation of gene expression and are considered significant players in disease and development in many species. Whilst the focus of MCV research has thus far been on the oncogenic MCV early proteins, large tumour (LT) and small tumour (sT) antigens, there is a knowledge gap regarding MCV miRNA and its functional significance in MCV pathogenesis. Given the emerging importance of viral miRNAs in virus-host interaction and pathogenesis, the aim of this doctoral research project was to investigate alterations in host cell transcripts induced by MCV miRNA and determine any functional significance these might have on virus-host cell interaction. RNA sequencing (RNA-Seq) in the presence and absence of MCV miRNA uncovered a multitude of downregulated cellular transcripts. Gene ontology analysis revealed that MCV miRNA targets transcripts associated with multiple cellular processes, however, regulation of immune response was overrepresented in our datasets. Validation of RNA-Seq data using MCV miRNA mimics and a synthetic, fully replicative MCV genome (MCVSyn) confirmed RNA-Seq data at mRNA and protein expression level for several targets, including the cytokine stimulating gene, SP100, and the neutrophil stimulator chemokine, CXCL8. Moreover, dual luciferase assays revealed that SP100 and MAPK10 (a member of mitogen-activated protein kinases (MAPK) family which is involved in regulation of CXCL8 expression) are directly and specifically targeted and downregulated by MCV miRNA. The MCV miRNA-dependent dysregulation of CXCL8 secretion is associated with impaired neutrophil migration, suggesting that the virus miRNA may be implicated in evasion of the host immune response.

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

A localização da podoplanina e da ezrina nas células malignas sugere uma ligação dessas proteínas nos processos de migração e invasão tumoral, ativadas mediante a fosforilação de Rho-A. O objetivo desse estudo foi avaliar a distribuição e a correlação da podoplanina, da ezrina e da Rho-A em 91 carcinomas espinocelulares de lábio inferior, e verificar a associação dessas proteínas com as variáveis clínicas e patológicas, com a evolução e com o prognóstico dos pacientes. Os pacientes foram analisados quanto ao gênero, idade, tabagismo, etilismo, classificação pelo sistema TNM, tratamento, ocorrência de recidivas locorregionais, segundo tumor primário, além da presença de embolização vascular, infiltração perineural, muscular, glandular e comprometimento linfonodal histopatológico. Analisou-se também as expressões imuno-histoquímicas de podoplanina, ezrina e Rho-A no front de invasão tumoral e o índice de malignidade tumoral. A associação entre a podoplanina, a ezrina e a Rho-A com as variáveis clínico-patológicas e a correlação entre as proteínas foram analisadas pelos testes do qui-quadrado e de Spearman, respectivamente. A análise da sobrevivência global em 5 e 10 anos foi feita pelo estimador produto-limite Kaplan-Meier e a comparação da curva de sobrevivência pelo teste log-rank. Os resultados demonstraram uma forte expressão de podoplanina, de ezrina e de Rho-A no front de invasão dos carcinomas espinocelulares de lábio inferior. Houve uma associação significativa entre a expressão citoplasmática de podoplanina com o etilismo (p=0,024), com a recidiva locorregional (p=0,028) e com comprometimento linfonodal histopatológico (p=0,010), porém não foi detectada nenhuma associação significativa entre a ezrina e a Rho-A com as variáveis clínicas e microscópicas analisadas. Uma correlação positiva e estatisticamente significativa entre a expressão de podoplanina membranosa (p=0,000 e r =0,384) e citoplasmática (p=0,000 e r=0,344) com a expressão de ezrina, e da podoplanina membranosa com a expressão de Rho-A (p=0,006 e r=0,282) foi detectada. Nenhuma das três proteínas se mostrou fator de prognóstico significativo para os pacientes com câncer de lábio. Concluímos que a forte expressão membranosa de podoplanina nos carcinomas espinocelulares de lábio inferior pode ajudar a identificar pacientes com menor risco de recidiva locorregional. Além disso, a correlação entre as expressões da podoplanina, da ezrina e da Rho-A pelas células neoplásicas sugere uma participação conjunta destas proteínas nos processos de movimentação celular e invasão tumoral do câncer de lábio. / Immunolocalization of podoplanin and ezrin suggests a connection between these proteins in migration and tumoral invasion process, activated through Rho-A phosphorylation. The aim of this study was to evaluate the distribution and the correlation of podoplanin, ezrin and Rho-A in 91 squamous cells carcinomas of the lower lip and to verify its association with clinical and pathological features, evolution and prognostic of the patients. Patients were analyzed concerning gender, age, tobacco, alcohol, TNM classification, local and regional recurrences, second primary tumor, perineural, muscle and glandular infiltration, and histopahological lymph node metastasis. The association of podoplanin, ezrin and Rho-A expressions at tumoral invasion front and histological risk assessment of tumors were verified by chi-square test. The association between podoplanin, ezrin and Rho-A expressions with clinical and pathological variables, and the correlation of these variables were analyzed by chi-square and Spearman test, respectively. Overall survival in 5 and 10 years was calculated by Kaplan Meier method and overall curves were compared by log rank test. The results showed strong expression of podoplanin, ezrin and Rho-A at tumoral invasion front of squamous cell carcinomas of the lower lip. A significant association of strong cytoplasmic podoplanin expression and alcoholism (p=0,024), local recurrences (p=0,028) and lymph node metastasis (p=0,010) was found, although ezrin and Rho-A expressions were not associated with clinical and microscopic features analyzed. A statistically significant correlation between membranous (p=0,000 e r =0,384) and cytoplasmic (p=0,000 e r=0,344) podoplanin expressions and ezrin, and membranous podoplanin and Rho-A (p=0,006 e r=0,282) was observed. None of the proteins analyzed can be considered as prognostic factor for lip cancer. We can conclude that strong membranous podoplanin expression in squamous cell carcinoma of the lower lip can help to identify patients with lower risk of local and regional recurrences. Furthermore, the correlation of podopolanin, ezrin and Rho-A expressions suggest a cooperative participation in cell movement and tumoral invasion.

A radiação ultravioleta ao atingir os seres humanos em grande quantidade e durante uma longa exposição pode provocar danos específicos ao DNA, sendo causa de várias lesões como o carcinoma epidermoide de lábio e a queilite actínica, que é considerada uma lesão precedente ao aparecimento da primeira. Entre os danos causados pela radiação UV está a alteração genética do TP53, provocando anomalias na proteína por ele codificada. A produção da proteína p53 somente é recrutada em situações de estresse como: radiação ionizante, hipoxia ou ativação de oncogenes, nas quais sua função é regular o ciclo celular e ativar vias de apoptose. Contudo, sabe-se que no processo de carcinogênese não somente as alterações não reparadas do DNA são responsáveis pelo aparecimento de uma lesão. Outro fator de extrema importância nesse processo são os mecanismos de apoptose, entre os quais estão as vias do Bcl2 e do Fas. A queilite actínica normalmente é classificada segundo seus graus de displasia, o que para alguns sugeriria os passos percorridos por essa lesão até o carcinoma epidermoide de lábio já que esta lesão possui um potencial de malignização. Os resultados deste trabalho demonstraram que não há diferença estatística na expressão gênica e imuno-histoquímica de p53 entre os diversos graus de displasia. Demonstraram ainda que as vias de apoptose de Bcl2 e Fas estavam ocorrendo normalmente. Sugere-se então que não há comprovação de que ocorra uma progressão para a malignização passando por todos os graus de displasia da queilite actínica tendo, todos a mesma possibilidade de se transformarem em um carcinoma epidermoide de lábio. / The long exposition of the human tissues to the ultraviolet radiation causes DNA damages and consequently several lesions might appear. Among them, lip squamous cell carcinoma and actinic cheilitis which is considered to be a condition that precedes squamous cell carcinomas. The TP53 mutation is a well-known effect of UV radiation, leading to the synthesis of an anomalous protein. The p53 production is only recruit in stress conditions like: ionizing radiation, hypoxia or oncogene activation, where it plays a role in cell cycle regulation and apoptosis activation. Apoptosis is also a fundamental mechanism associated to carcinogenesis. Bcl2 e Fas pathways are central in the regulation of this process. It is usual to classify the epithelial dysplasia degree in actinic cheilitis as a prognosticator. This type of classification suggests a one way path towards malignization. Our results have shown that there is no statistical difference in TP53 status and p53 expression among the different degrees of actinic cheilitis epithelial dysplasia and squamous cell carcinoma. Based on the findings, there is no prove that the malignant transformation occurs as the epithelial dysplasia progresses. Therefore, should be considered that any degree has the same probability to become a lip squamous cell carcinoma

O carcinoma basocelular (CBC) é uma neoplasia cutânea maligna de baixo potencial metastatizante, originada das células da camada basal da epiderme. Sua importância clínico-epidemiológica pode ser constatada pelo fato de constituir a neoplasia maligna mais comum na espécie humana, cujo principal fator etiológico é a exposição à radiação ultravioleta. Apesar da baixa incidência de metástases, a neoplasia pode adotar um comportamento localmente agressivo, com comprometimento de estruturas profundas e de forte apelo estético, como ocorre na região periocular. Uma das complicações advindas de sua infiltração neste sítio anatômico consiste na invasão de tecidos orbitários cujo tratamento é a exenteração, conduta mutiladora que consiste na retirada do globo ocular e das partes moles da órbita acometida. O objetivo deste estudo foi avaliar os casos de CBC com invasão orbitária que foram submetidos à exenteração no Hospital de Clínicas de Ribeirão Preto, no período de 1992 a 2012, para a possível identificação de fatores clínicos e morfológicos que possam predizer uma evolução desfavorável da neoplasia. Foi realizada uma coleta de dados clínicos, epidemiológicos e histopatológicos dos casos submetidos à exenteração a partir dos prontuários médicos dos pacientes. As lâminas referentes aos exames anátomo-patológicos foram revistas e foi realizado estudo imuno-histoquimico para os marcadores p53, bcl-2, actina de músculo liso e metaloproteinase-1. O grupo controle foi composto por pacientes com diagnóstico da neoplasia em topografia periocular, sem sinais de invasão orbitária. Para os casos com invasão orbitária o número de casos positivos marcados para p53 (0,21) e para actina de músculo liso (0,21) foi significantemente menor que o número de casos positivos para bcl-2 (0,63) e MMP-1 (0,58) (p= 0,0331). Entretanto, o número de casos positivos para bcl-2 (0,63) foi significantemente maior que o número de casos marcados por MMP-1 (0,58) (p=0,0126). Para os tumores sem invasão orbitária, o número de casos positivos para p53 (0,31) e actina (0,31) foi significantemente menor que o número de casos positivos para bcl-2 (0,63) eMMP-1 (0,50) (p=0,0273). Os resultados indicam que a invasão orbitaria por carcinoma basocelular palpebral ocorre com maior frequência no sexo masculino, em pacientes com longa história clínica de múltiplas lesões e submetidos a múltiplos procedimentos. Além disso, os marcadores estudados aparentemente não podem predizer um comportamento mais agressivo do tumor. / Basal cell carcinoma (BCC) is a malignant skin cancer of low metastasizing potential originated from the basal cells of the epidermis. Its clinical and epidemiological importance is evidenced by the fact that it is the most common malignancy in humans and it has as the main etiological factor the exposure to ultraviolet radiation. Despite the low incidence of metastases, the cancer can adopt a locally aggressive behavior with involvement of deep structures and it can have a strong aesthetic appeal, as in the periocular region. One of the complications arising from its infiltration in this anatomical site consists of orbital tissue invasion whose treatment is exenteration, a mutilating procedure consisting of the removal of the eyeball and the soft tissue of the affected orbit. The aim of this study was to evaluate the cases of BCC with orbital invasion that underwent exenteration at the Clinics Hospital of Ribeirão Preto Medical School, University of Sao Paulo, from 1992 through 2012, for possible identification of clinical and morphological factors that can predict an unfavorable evolution of the tumor. The clinical data were obtained from the patients\' charts and we have reviewed all the slides from exenteration specimens and performed immunohistochemical studies with p53, bcl-2, smooth muscle actin and metalloproteinase-1(MMP-1). The control group consisted of age-matched patients with eyelid basal cell carcinomas without orbital invasion. For cases with orbital invasion the number of positive cases labeled for p53 (0.21) and actin (0.21) was significantly lower than the number of positive cases for bcl-2 (0.63) and MMP -1 (0.58) (p = 0.0331). However, the number of positive cases for bcl-2 (0.63) was significantly greater than the proportion of positive cases for MMP-1 (0.58) (p = 0.0126). For cases without orbital invasion the number of positive cases for p53 (0.31) and actin (0.31) was significantly lower than the number of positive cases for bcl-2 (0.63) and MMP-1 (0.50) (p = 0.0273), even though the number of positive cases marked for MMP-1 (0.50) was not significantly different from number of positive cases for bcl-2 (0.63) (p = 0.059). The results indicate that orbital invasion of basal cell carcinoma of the eyelid was more frequent in male sex and that the patients have usually a long history of multiple lesions and were submitted to several procedures. In addition, our results suggest these markers can not predict an aggressive behavior for basal cell carcinomas of the periocular region.

A radiação ultravioleta ao atingir os seres humanos em grande quantidade e durante uma longa exposição pode provocar danos específicos ao DNA, sendo causa de várias lesões como o carcinoma epidermoide de lábio e a queilite actínica, que é considerada uma lesão precedente ao aparecimento da primeira. Entre os danos causados pela radiação UV está a alteração genética do TP53, provocando anomalias na proteína por ele codificada. A produção da proteína p53 somente é recrutada em situações de estresse como: radiação ionizante, hipoxia ou ativação de oncogenes, nas quais sua função é regular o ciclo celular e ativar vias de apoptose. Contudo, sabe-se que no processo de carcinogênese não somente as alterações não reparadas do DNA são responsáveis pelo aparecimento de uma lesão. Outro fator de extrema importância nesse processo são os mecanismos de apoptose, entre os quais estão as vias do Bcl2 e do Fas. A queilite actínica normalmente é classificada segundo seus graus de displasia, o que para alguns sugeriria os passos percorridos por essa lesão até o carcinoma epidermoide de lábio já que esta lesão possui um potencial de malignização. Os resultados deste trabalho demonstraram que não há diferença estatística na expressão gênica e imuno-histoquímica de p53 entre os diversos graus de displasia. Demonstraram ainda que as vias de apoptose de Bcl2 e Fas estavam ocorrendo normalmente. Sugere-se então que não há comprovação de que ocorra uma progressão para a malignização passando por todos os graus de displasia da queilite actínica tendo, todos a mesma possibilidade de se transformarem em um carcinoma epidermoide de lábio. / The long exposition of the human tissues to the ultraviolet radiation causes DNA damages and consequently several lesions might appear. Among them, lip squamous cell carcinoma and actinic cheilitis which is considered to be a condition that precedes squamous cell carcinomas. The TP53 mutation is a well-known effect of UV radiation, leading to the synthesis of an anomalous protein. The p53 production is only recruit in stress conditions like: ionizing radiation, hypoxia or oncogene activation, where it plays a role in cell cycle regulation and apoptosis activation. Apoptosis is also a fundamental mechanism associated to carcinogenesis. Bcl2 e Fas pathways are central in the regulation of this process. It is usual to classify the epithelial dysplasia degree in actinic cheilitis as a prognosticator. This type of classification suggests a one way path towards malignization. Our results have shown that there is no statistical difference in TP53 status and p53 expression among the different degrees of actinic cheilitis epithelial dysplasia and squamous cell carcinoma. Based on the findings, there is no prove that the malignant transformation occurs as the epithelial dysplasia progresses. Therefore, should be considered that any degree has the same probability to become a lip squamous cell carcinoma

A instabilidade genética é um importante evento associado ao carcinoma epidermóide de boca, sendo alterações na região cromossômica 11q constantemente relatadas. Neste estudo, genes localizados na região cromossômica 11q, especificamente os genes CTTN, PPFIA1, SHANK2, TAOS1 e MMP-7, foram investigados quanto a diferenças de expressão de transcritos entre carcinomas epidermóides de boca e suas margens correspondentes. A expressão desses genes foi relacionada com aspectos clínicos e histológicos, com critérios de agressividade estabelecidos, e com a sobrevida dos pacientes. Foram analisadas pela técnica de qRT-PCR 29 amostras congeladas de tumores e 25 margens. Todos os genes apresentaram maiores valores de expressão nos tumores em comparação com as margens, embora apenas o gene MMP-7 tenha exibido valores estatisticamente significantes. A expressão do gene MMP-7 mostrou fraca associação com tumores menos agressivos, e os outros genes apresentaram maiores valores de expressão em tumores mais agressivos, sem significância estatística. Não houve diferença estatística entre a freqüência das variáveis clínicas e histopatológicas com a expressão dos genes estudados, porém o PPFIA1 demonstrou maiores níveis de expressão em tumores de assoalho. Em relação à sobrevida, a expressão elevada de PPFIA1 pode implicar em um maior risco de óbito. Assim, é possível a participação do gene MMP-7 no desenvolvimento da neoplasia, e a relação do PPFIA1 com o risco de óbito, porém a expressão de transcritos dos genes CTTN, SHANK2, TAOS1 e MMP-7 não pode ser relacionada com agressividade tumoral e prognóstico. / Genetic instability is an important event associated with oral squamous cell carcinoma, and alterations in the chromosome region 11q are constantly reported. In this study, genes located on chromosome region 11q, specifically genes CTTN, PPFIA1, SHANK2, TAOS1 and MMP-7, were investigated for differences in the expression of transcripts in oral squamous cell carcinoma and their corresponding margins. The expression of these genes was correlated with clinical and histological aspects, aggressiveness criteria established, and with patient survival. Twenty-nine frozen samples of tumors and 25 samples of margin tissue were analyzed using qRT-PCR. All genes showed a higher expression in tumors, compared with the margins, although only the MMP-7 gene demonstrated statistically significant values. The expression of the MMP-7 gene showed weak association with less aggressive tumors, and the other genes showed higher expression in more aggressive tumors, without statistical significance. There was no statistical difference between the frequency of clinical and histopathological variables and the expression of genes studied, however the PPFIA1 gene demonstrated higher levels of expression in tumors of the floor of mouth. With regard to survival, the high expression of PPFIA1 may imply a greater risk of death. Thus, it is possible that the MMP-7 gene participates in the development of malignancy, and PPFIA1 expression may also be associated with risk of death, however, the expression of transcripts of the CTTN, SHANK2, TAOS1 and MMP-7 genes may not be related to tumor aggressiveness and prognosis.