Des mutations acquises du gène TET2 ont été décrites dans les hémopathies malignes humaines. La fréquence de ces anomalies dans les hémopathies myéloïdes est de 10 à 20%, atteignant 50% dans les échantillons de leucémies myélo-monocytaires chroniques (LMMC). Les mutations observées sont inactivatrices, ce qui suggère que TET2 est un gène de type suppresseur de tumeur et que les mutations retrouvées conduisent à une perte de fonction de la protéine. Ce gène code pour une enzyme capable de modifier les cytosines méthylées. Il participerait ainsi au contrôle de la méthylation de l'ADN et donc à la régulation épigénétique de l’expression génique. Afin de mieux comprendre son rôle au cours de l’hématopoïèse, deux modèles murins d'inactivation du gène Tet2 ont été développés. Des expériences de greffe de cellules médullaires dans des souris syngéniques montrent que les cellules déficientes pour ce gène présentent un avantage compétitif par rapport aux cellules sauvages. L’analyse des souris invalidées pour ce gène montre une amplification des populations hématopoïétiques immatures, ainsi que des anomalies de la différenciation des lignages myéloïdes et également des lignages lymphoïdes. Une fraction des souris invalidées pour Tet2 âgées de plus de six mois développe des hémopathies malignes ressemblant à la LMMC humaine. Des anomalies équivalentes sont retrouvées dans les souris hémizygotes pour Tet2 et dans des souris portant un allèle hypomorphe du gène. L’ensemble de ces résultats montre qu’une dérégulation de l'activité de Tet2 conduit à des anomalies précoces de l'hématopoïèse, mais n'entraine pas directement la transformation des cellules progénitrices immatures. La latence du développement de ces tumeurs suggère la nécessité d'une coopération avec d'autres évènements oncogéniques, comme des anomalies d’autres acteurs épigénétiques / Acquired loss-of-function mutations of TET2 gene are frequently observed in patients with myeloid malignancies, including acute myeloblastic leukemia, myeloproliferative neoplasm, myelodysplastic syndrome, and chronic myelomonocytic leukemia (CMML). The Ten-Eleven-Translocation (TET) family proteins are 2-oxoglutarate/Fe(II)-dependent dioxygenases that catalyze the conversion of 5-methyl-cytosine into 5-hydroxymethyl-cytosine, which is proposed to constitute a first step toward cytosine demethylation. To study the function of Tet2 in murine hematopoiesis, we developed two mouse models in which the catalytic domain of the protein is disrupted. In both models, Tet2 deficiency leads to the progressive expansion of the immature hematopoietic compartment that includes stem cell and multipotent progenitors. In addition, both Tet2-deficient animals display abnormalities of erythroid, megakaryocytic, myelo-monocytic and lymphoid lineages, recapitulated in competitive transplantation assays. With age, Tet2-deficient mice develop bona fide myeloid tumors. All these properties were shown to be cell-autonomous by bone marrow cells transplantation and in vitro assays. Together these data suggest that TET2 activity is essential for normal homeostasis of the hematopoietic system. Its inactivation results in the development of hematologic disorders resembling human CMML myeloid disorders. TET2 deficiency endows the cells with a competitive advantage over wild type cells, induces hematopoietic differentiation abnormalities but is not responsible for full cellular transformation. The latency observed for CMML development in mouse models of Tet2 deficiency suggests a requirement for cooperating mutations, such other epigenetic regulator alterations.

As a master regulator of the epithelial state, p63 is a family member of the well-known tumor suppressor p53. It has previously been connected to a cancer-associated process, epithelial-to-mesenchymal transition (EMT), and here we find that it can be regulated by oncogenes involved in breast tumorigenesis. Specifically, activated forms of PIK3CA and H-RAS are able to strongly repress expression of ∆Np63α, which is the major p63 isoform in epithelial cells. In mammary epithelial lines, this oncogene downregulation occurs at the transcriptional level, and complete repression occurs over the course of several days. As p63 is repressed, the cells undergo EMT and acquire the ability to invade individually through a 3D collagen matrix. Strikingly, even when p63 is suppressed but no oncogene action is present, these cells undergo a mesenchymal shift, suggesting the importance of this gene in maintaining the epithelial state. Furthermore, it is particularly interesting that p63 protein and RNA levels are often low in breast tumors. By connecting H-RAS and PIK3CA signaling to p63, it is hypothesized that such oncogene suppression could account for tumor progression in cases where p63 levels are low. Here, it is proposed that p63 acts in a tumor-suppressive manner, although it can be overcome by oncogenes leading to changes in differentiation state and migratory capability, therefore drastically affecting breast carcinogenesis.

The p53 tumor suppressor protein has been well-characterized for its role in inducing growth arrest, senescence, and apoptosis upon various types of stresses. Recently, however, roles of p53 have expanded beyond the canonical functions, and now include cellular processes such as metabolism, oxidative balance, and ferroptosis. Through RNA-seq screening, we first identified phosphoglycerate dehydrogenase (PHGDH), a rate-limiting enzyme in the serine biosynthesis pathway, as a novel metabolic target of p53. p53 suppresses PHGDH expression and inhibits de novo serine biosynthesis. Notably, upon serine starvation, p53-mediated cell death is significantly enhanced in response to Nutlin-3 treatment. Moreover, PHGDH has been demonstrated to be frequently amplified in human melanomas. We found that PHGDH overexpression significantly suppresses the apoptotic response, whereas RNAi-mediated knock-down of endogenous PHGDH promotes apoptosis under the same treatment. Together, our findings demonstrate an important role of p53 in regulating serine biosynthesis through suppressing PHGDH expression, and reveal serine deprivation as a novel approach to sensitize p53-mediated apoptotic responses in human melanoma cells. In addition, we also identified spermidine/spermine N1-acetyltransferase 1 (SAT1) as a novel metabolic target of p53. SAT1 is a rate-limiting enzyme in polyamine catabolism critically involved in the conversion of spermidine and spermine back to putrescine. Surprisingly, we found that activation of SAT1 expression induces lipid peroxidation and sensitizes cells to undergo ferroptosis upon reactive oxygen species (ROS)-induced stress, which also leads to suppression of tumor growth in xenograft tumor models. Notably, SAT1 expression is down-regulated in human tumors, and CRISPR-cas9-mediated knockout of SAT1 partially abrogates p53-mediated ferroptosis. Moreover, SAT1 induction is correlated with the expression levels of arachidonate 15-lipoxygenase (ALOX15), and SAT1-induced ferroptosis is significantly abrogated in the presence of PD146176, a specific inhibitor of ALOX15. Together, these data indicate a novel regulatory role of p53 in polyamine metabolism and provide insight into the regulation of p53-mediated ferroptotic responses. Our studies on PHGDH and SAT1 led us to the question of whether these unconventional functions of p53 contribute to its role as a tumor suppressor. In fact, previous view regarding the mechanism of p53-mediated tumor suppression, which was long thought to be growth arrest, apoptosis, and senescence, has recently been challenged by several knockout and knock-in mouse studies. Previously, we established mice (p533KR/3KR) in which p53 acetylation at lysine residues K117, K161, and K162 were abolished by replacing lysine with arginine. p533KR/3KR mice completely lost p53-mediated cell cycle arrest, apoptosis, and senescence functions in response to stresses. However, unlike p53-null mice which rapidly develop spontaneous thymic lymphomas, all of the p533KR/3KR mice remain tumor-free, indicating that other aspects of p53 functions are sufficient to prevent tumor formation. Notably, p533KR retains the ability to regulate metabolic targets including TIGAR and SAT1, as well as ferroptosis regulator SLC7A11. In this study, we have identified two novel acetylation sites- K98 and K136, in the mouse p53 DNA-binding domain. Whereas loss of K98 or K136 acetylation (p53K98R, p53K136R) alone has modest effect on p53 transcriptional activity, simultaneous mutations at all of these acetylation sites (p534KR98: K98R+3KR, p534KR136: K136R+3KR, p535KR: K98R+K136R+3KR) completely abolish the ability of p53 to regulate TIGAR, SAT1, and SLC7A11. In addition, p534KR98, p534KR136, and p535KR are defective in Erastin-induced ferroptosis. Notably, p534KR98/4KR98, p534KR136/4KR136, and p535KR/5KR knock-in mice lost intact tumor suppression and developed spontaneous tumors. This suggests that p53-mediated ferroptosis may function as a critical barrier to prevent tumor formation independently from growth arrest, apoptosis, and senescence. Interestingly, both p534KR98/4KR98 and p534KR136/4KR136 mice displayed significantly delayed tumorigenesis comparing with p53-null and p535KR/5KR mice. We found that unlike p535KR, p534KR98 retains the capacity to inhibit mammalian target of rapamycin (mTOR) signaling pathway through activating the expression of two mTOR negative regulators, Sestrin2 and DDIT4. Altogether, our findings underscore the extensive scope of p53 functions in metabolic regulation, oxidative stress response, and ferroptosis, and provide novel insights into the tumor suppression mechanism of p53.

An important hallmark of cancer is the inactivation of tumor suppressor genes. The most common genetic alteration in cancer is the mutation of the TP53 gene occurring in about half of all cancers, but very little progress has been made on how to therapeutically target the signaling defects in these cancers. Additionally, the PTEN tumor suppressor is mutated in a wide variety of cancer types, and its expression is often lost in the absence of mutation. PTEN is a haploinsufficient tumor suppressor that exhibits dose-dependent effects in cells. In the context where PTEN is lost or downregulated, PI3K signaling and downstream signaling through AKT is overactive, leading to an increase in cell growth and proliferation, among other effects. Acting as both a protein and lipid phosphatase, loss of PTEN also affects the PI3K-independent signaling of PTEN, and results in an increase of migration and invasion phenotypes. Importantly, PTEN transcript level is the key determinant for PTEN protein expression, and downregulation of PTEN is part of a poor-prognosis gene expression signature in breast cancer. Downregulation of tumor suppressor gene expression represents a reversible change that is often sufficient to drive tumorigenesis. However, our understanding of the broad molecular mechanisms by which the expression of these tumor suppressors is lost remains limited, but is required to develop effective therapeutic strategies to target malignancies driven by tumor suppressor loss. In Chapter 2, we characterize the problem of transcriptional downregulation of PTEN in breast cancer. We investigate the expression of PTEN in various normal and tumor cells at both the transcript and protein level. We identify various model systems that we believe are suitable to model normal PTEN expression and the PTEN downregulation that mimics what is observed in tumors. We employ a sophisticated approach that couples RNA-sequencing with Nanostring nCounter analysis in order to obtain a detailed and thorough transcriptional profile of the PTEN and pseudogene PTENP1 genomic loci, as well as expression of the poor-prognosis gene signature associated with PTEN downregulation. In this study, we obtained an understanding of the changes in the PTEN transcriptional profile that occur in the progression from normal to cancer, and we believe this approach could be applied to other key tumor suppressor genes. In Chapter 3, we discovered that basally expressed p53 maintains expression of thirteen well-validated tumor suppressors. p53 is expressed at low levels under normal, low-stress conditions, and is expressed at much higher levels under enhanced stress, leading to the activation of stress-response genes. We begin the study by highlighting an association between TP53 mutation and downregulation of PTEN expression. Upon performing chromatin immunoprecipitation coupled with next generation sequencing for p53 under normal, low-stress conditions, we found that p53 binds in the vicinity of thirteen tumor suppressor genes, including PTEN. Basally expressed p53 binds to classic consensus binding sites in enhancers and promoters of target tumor suppressors to maintain their expression at baseline. CRISPR/Cas9-mediated knockout of the endogenous basal p53 binding site upstream of PTEN led to a decrease in PTEN expression and an increase in tumorigenic phenotypes. Given that mutation of TP53 leads to tumorigenesis in mice, but loss of p53 stress-response targets or loss of the ability of p53 to activate these stress-response targets does not lead to spontaneous tumorigenesis, it is likely that these tumor suppressor targets of basal p53 contribute to p53-mediated tumor suppression. In Chapter 4, we identified yet another mechanism by which transcriptional repression of PTEN occurs in triple-negative breast cancer (TNBC) through polycomb repressive complex 2 (PRC2)-mediated repression of the PTEN promoter and upstream regulatory region. Previous research has shown that mutated NOTCH1 represses PTEN through the HES-1 transcription factor in acute myeloid leukemia (AML), and that NOTCH translocations are frequent in TNBC and are sufficient for transformation in vitro. We discovered that NOTCH1 and NOTCH2 mutations and translocations correlate with PTEN downregulation by immunohistochemistry in a cohort of TNBC cases. The TNBC cell line exhibiting PRC2-mediated repression of PTEN also harbors a SEC22B-NOTCH2 translocation that creates a gene product resembling the NOTCH2 intracellular domain. The NOTCH target HES-1 co-localizes on the PTEN promoter with EZH2 (the lysine methyltransferase involved in PRC2-mediated transcriptional repression), and knockdown of NOTCH2 in this cell line led to decreased expression of EZH2, and restoration of PTEN expression at the transcript and protein level. We also demonstrated that EZH2 inhibitors, HDAC inhibitors, and DNA hypomethylating agents robustly restore PTEN transcript levels. Taken together, these results elucidate another mechanism by which PTEN is transcriptionally repressed in the highly aggressive and poor-prognosis TNBC subtype of breast cancer that may be applicable to other cancer types. The results also suggest that this repression is reversible by pharmacological approaches, highlighting a promising therapeutic avenue. Taken together, the studies presented in this thesis begin to unravel the complex mechanisms of transcriptional repression of tumor suppressor genes in cancer. As is the case with PTEN and p53, multiple regulatory mechanisms can influence expression in combination or in a context-dependent manner. The loss of expression of tumor suppressor genes is one of the key hallmarks of cancer, yet very few of the therapeutic approaches used in the clinic today aim to restore tumor suppressor expression. Our results demonstrate proof of concept that restoration of tumor suppressor expression is a plausible and promising therapeutic approach for many different types of cancer, but requires a detailed understanding of the underlying molecular mechanisms of transcriptional regulation.

Hui Wai Ying. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 158-189). / Abstracts in English and Chinese. / Abstract / Acknowledgements / List of Tables / List of Figures / Table of Contents / Table of Contents / Chapter Chapter I: --- Introduction --- p.1 / Chapter I. --- Aim of Study --- p.1 / Chapter II. --- Literature Review --- p.3 / Chapter 1. --- Background --- p.3 / Chapter A. --- Epidemiology --- p.3 / Chapter B. --- Histopathology --- p.3 / Chapter C. --- Etiology --- p.4 / Chapter i. --- Environmental Factors --- p.5 / Chapter ii. --- Epstein-Barr Virus (EBV) Infection --- p.6 / Chapter iii. --- Genetic Factors --- p.9 / Chapter 2. --- Molecular Genetics of NPC --- p.11 / Chapter A. --- Genome-Wide Studies --- p.11 / Chapter i. --- Comparative Genomic Hybridization (CGH) --- p.11 / Chapter ii. --- Loss of Heterozygosity (LOH) Studies --- p.12 / Chapter iii. --- Homozygous Deletion Study --- p.12 / Chapter B. --- NPC-related Oncogenes and Tumor Suppressor Genes --- p.13 / Chapter i. --- Oncogenes --- p.13 / Chapter ii. --- Tumor Suppressor Genes --- p.14 / Chapter 3. --- Chromosome 14q and NPC --- p.19 / Chapter A. --- Tumor Suppressor Loci and Cancer-Related Genes on Chromosome14q --- p.20 / Chapter i. --- Tumor Suppressor Loci on Chromosome 14q --- p.20 / Chapter ii. --- Cancer-Related Genes on Chromosome 14q --- p.26 / Chapter 4. --- Chromosome 16q and NPC --- p.28 / Chapter A. --- Tumor Suppressor Loci and Candidate Tumor Suppressor genes on Chromosome16q --- p.28 / Chapter i. --- Tumor Suppressor Loci on Chromosome 16q --- p.28 / Chapter ii. --- Metastasis Suppressor Loci on Chromosome 16q --- p.34 / Chapter iii. --- Candidate Tumor Suppressor Genes on Chromosome 16q --- p.34 / Chapter Chapter II: --- Materials and Methods --- p.40 / Chapter I. --- Cell Lines and Xenografts --- p.40 / Chapter 1. --- Cell Lines --- p.40 / Chapter 2. --- Xenografts --- p.41 / Chapter 3. --- DNA Extraction --- p.42 / Chapter II. --- Patients and Biopsy Specimens --- p.44 / Chapter 1. --- Manual Microdissection --- p.44 / Chapter 2. --- Laser Captured Microdissection (LCM) --- p.46 / Chapter 3. --- DNA Extraction --- p.46 / Chapter III. --- Comprehensive Screening for Homozygous Deletion Regions on Chromosomes 14q32.12-32.33 and 16q23.1-24.3 in Human Cancers --- p.48 / Chapter 1. --- DNA of Human Cancer Cell Lines --- p.48 / Chapter 2. --- Sequence-Tagged Sites (STS) Markers --- p.48 / Chapter 3. --- Polymerase Chain Reaction (PCR) --- p.49 / Chapter IV --- . Investigation of Inactivation of Potential Tumor Suppressor Genes on Chromosome 14q32.12-32.33 and 16q23.1-24.3 --- p.58 / Chapter 1. --- Detection of Homozygous Deletion --- p.58 / Chapter 2. --- Expression Analysis --- p.58 / Chapter A. --- RNA Extraction --- p.58 / Chapter B. --- Reverse-Transcription (RT) PCR --- p.61 / Chapter i. --- DNase I Digestion --- p.62 / Chapter ii. --- First-strand cDNA Synthesis and RNase Digestion --- p.62 / Chapter iii. --- Reverse-Transcription (RT)-PCR --- p.63 / Chapter C. --- Real-Time RT PCR --- p.63 / Chapter 3. --- Methylation Analysis --- p.68 / Chapter A. --- Sodium Bisulfite Modification --- p.68 / Chapter B. --- Methylation-Specific PCR (MSP) --- p.69 / Chapter C. --- Bisulfite Sequencing --- p.70 / Chapter D. --- Combined Bisulfite Restriction Analysis (COBRA) --- p.75 / Chapter E. --- 5 -aza-2' -deoxycytidine Treatment --- p.76 / Chapter ChapterIII： --- Results --- p.78 / Chapter I. --- Comprehensive Screening for Homozygous Deletion Regions in Human Cancers --- p.78 / Chapter 1. --- Chromosome 14q32.12-3233 --- p.78 / Chapter 2. --- Chromosome 16q23.1-243 --- p.79 / Chapter II. --- Investigation of Inactivation of Potential Tumor Suppressor Genes in NPC --- p.86 / Chapter 1. --- Chromosome 14q --- p.86 / Chapter A. --- "WW Domain-Containing Protein, 45-kD （WW45)" --- p.86 / Chapter B. --- Apoptosis Stimulating Protein of p53（ASPP1) --- p.88 / Chapter 2. --- Chromosome 16q --- p.92 / Chapter A. --- WW Domain-Containing Oxidoreductase （WWOX) --- p.92 / Chapter i. --- Homozygous Deletion Screening of WWOX --- p.92 / Chapter ii. --- Expression of Aberrant Splicing Transcripts of WWOX in NPC --- p.94 / Chapter iii. --- Sequencing of WWOX Aberrant Transcripts --- p.95 / Chapter iv. --- Quantitative Analysis of WWOX Transcripts in NPC --- p.95 / Chapter v. --- Methylation Analysis --- p.99 / Chapter B. --- H-Cadherin (CDH13) --- p.102 / Chapter i. --- Analysis of H-cadherin Deletion on Cancer Cell Lines and Xenografts --- p.102 / Chapter ii. --- Expression Analysis of H-Cadherin by RT-PCR and Real-Time RT-PCR --- p.102 / Chapter iii. --- Analysis of Promoter Hypermethylation by Methylation-Specific PCR (MSP) and Bisulfite Sequencing in NPC Cell Lines and Xenografts --- p.104 / Chapter iv. --- Demethylation Study of H-Cadherin in C666-1 Cell Line --- p.105 / Chapter v. --- Methylation Analysis of H-Cadherin in Primary Tumors --- p.105 / Chapter vi. --- Methylation Analysis of H-Cadherin in Human Cancer Cell Lines --- p.106 / Chapter C. --- Myeloid Translocation Gene on Chromosome 16 (MTG16) --- p.113 / Chapter i. --- Deletion Analysis of MTG16 in Cancer Cell Lines and Xenografts --- p.113 / Chapter ii. --- Differential Expression of MTG16a and MTG16b Transcripts in NPC Cell Lines and Xenografts --- p.113 / Chapter iii. --- Methylation Analysis of MTG16b in NPC Cell Lines and Xenografts --- p.118 / Chapter iv. --- Sequencing of MTGl 6b RT-PCR Products --- p.119 / Chapter v. --- Demethylation Study of MTG16b in HK-1 Cell Line --- p.119 / Chapter vi. --- Promoter Methylation Analysis of MTG16b by MSP in Primary NPC and Cancer Cell Lines --- p.120 / Chapter Chapter IV: --- Discussion --- p.124 / Chapter I. --- Comprehensive Homozygous Deletion Screening of Chromosomes 14q32.12-32.33 and 16q23.1-24.3 in Human Cancer Cell Lines and Xenografts --- p.124 / Chapter II. --- Investigation of Candidate Tumor Suppressor Genes on Chromosome 14q in NPC --- p.128 / Chapter III. --- Alterations of Candidate Tumor Suppressor Genes on Chromosome 16q in NPC --- p.133 / Chapter 1. --- Expression of Aberrant Transcripts of WWOX in NPC --- p.133 / Chapter 2. --- Methylation-Associated Silencing of H-Cadherin and MTG16b in NPC --- p.140 / Chapter Chapter V: --- Conclusion --- p.154 / Chapter Chapter VI: --- References --- p.158

Ng Yeung Lam. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 180-209). / Abstracts in English and Chinese. / acknowledgements --- p.i / abstract --- p.ii / abstract in chinese --- p.vi / table of contents --- p.ix / list of tables --- p.xiii / list of figures --- p.xi v / list of abbreviations --- p.xvi / Chapter 1 --- chapter1 introduction and literature review --- p.1 / Chapter 1.1 --- Introduction of brain tumors --- p.1 / Chapter 1.2 --- Oligodendroglial tumors (OTs) --- p.3 / Chapter 1.2.1 --- Oligodendroglioma (OD) and anaplastic oligodendroglioma (AOD) --- p.3 / Chapter 1.2.1.1 --- WHO's definition and grading --- p.3 / Chapter 1.2.1.2 --- "Incidence, age, sex distribution, tumor location and survival rate" --- p.3 / Chapter 1.2.1.3 --- Clinical presentation --- p.4 / Chapter 1.2.1.4 --- Macroscopy and histopathology --- p.4 / Chapter 1.2.1.5 --- Immunohistochemistry --- p.5 / Chapter 1.2.1.6 --- Treatment --- p.6 / Chapter 1.2.2 --- Oligoastrocytoma (OA) and anaplastic oligoastrocytoma (AOA) --- p.11 / Chapter 1.2.2.1 --- WHO's definition and grading --- p.11 / Chapter 1.2.2.2 --- "Incidence, age, sex distribution, tumor location and survival rate" --- p.12 / Chapter 1.2.2.3 --- Clinical features --- p.12 / Chapter 1.2.2.4 --- Macroscopy and histopathology --- p.12 / Chapter 1.3 --- Overview of Genetic and Epigenetic Aberrations of OTs --- p.14 / Chapter 1.3.1 --- Chromosomal and genetic aberrations in OTs --- p.14 / Chapter 1.3.2 --- Candidate regions and genes on 1 p --- p.15 / Chapter 1.3.3 --- Candidate regions and genes on 19q --- p.20 / Chapter 1.3.4 --- Other aberrations in WHO grade II OTs --- p.24 / Chapter 1.3.5 --- Progression-associated aberrations in ODs --- p.25 / Chapter 1.3.6 --- Chromosomal and genetic aberrations in OAs --- p.29 / Chapter 1.4 --- Correlation of genetic alterations with response to therapy and survival --- p.31 / Chapter 1.4.1 --- Response to PCV chemotherapy correlates with lp and combined lp/19q status in patients with AODs --- p.31 / Chapter 1.4.2 --- Survival of patients with AODs correlates with lp/19q status --- p.32 / Chapter 1.4.3 --- WHO grade II ODs behavior and lp/19q status --- p.32 / Chapter 1.4.4 --- Response to other therapies (temozolomide and radiotherapy) and lp/19q status in patients with ODs --- p.33 / Chapter 1.4.5 --- lp and 19q loss in OAs and diffuse astrocytomas --- p.34 / Chapter 1.5 --- Microarray-based expression profiling of OTs --- p.35 / Chapter 1.6 --- Description of p73 protein --- p.37 / Chapter 1.6.1 --- Introduction of p73 --- p.37 / Chapter 1.6.2 --- p73: gene structure and splicing variants --- p.37 / Chapter 1.6.3 --- Signaling in p73 --- p.40 / Chapter 1.6.4 --- Regulation ofp73 protein stability and transcriptional activity --- p.43 / Chapter 1.6.4.1 --- Regulation by DNA damage --- p.43 / Chapter 1.6.4.2 --- Regulation by oncogenes --- p.44 / Chapter 1.6.4.3 --- Interaction with viral proteins --- p.44 / Chapter 1.6.5 --- Role of p73 in the nervous system --- p.45 / Chapter 1.6.6 --- p73 in cancer --- p.45 / Chapter 1.6.6.1 --- p73 knockout mice --- p.45 / Chapter 1.6.6.2 --- Alteration of p73 expression in human cancers --- p.46 / Chapter 1.6.7 --- p73 and chemosensitivity --- p.50 / Chapter CHAPTER2 --- AIMS OF STUDY --- p.51 / Chapter CHAPTER3 --- MATERIALS AND METHODS --- p.53 / Chapter 3.1 --- Tumor and blood samples --- p.53 / Chapter 3.2 --- Cell culture --- p.53 / Chapter 3.3 --- DNA extraction from frozen tissues and blood samples --- p.54 / Chapter 3.4 --- Detection of allelic loss of chromosome lp --- p.58 / Chapter 3.4.1 --- LOH analysis --- p.58 / Chapter 3.4.2 --- Fluorescence in situ Hybridization (FISH) analysis on Paraffin and Frozen Sections --- p.60 / Chapter 3.6 --- DNA sequencing analysis --- p.62 / Chapter 3.7 --- Analysis of Methylation --- p.63 / Chapter 3.7.1 --- Bisulfite sequencing --- p.63 / Chapter 3.7.2 --- Methylation-specific polymerase chain reaction (MSP) --- p.66 / Chapter 3.8 --- Northern Blot analysis --- p.68 / Chapter 3.9 --- RNA isolation and cDNA preparation --- p.70 / Chapter 3.10 --- Laser microdissection and RNA extraction from microdissected tumor cells --- p.71 / Chapter 3.10.1 --- Conventional RT-PCR --- p.71 / Chapter 3.11 --- Primer design for TP73 and its isoforms --- p.74 / Chapter 3.12 --- Real-time RT-PCR --- p.77 / Chapter 3.12.1 --- Real-time RT-PCR for TP73 and its isoforms --- p.78 / Chapter 3.12.2 --- Real-time RT-PCR for KIAA0495 --- p.79 / Chapter 3.13 --- Statistical analyses --- p.81 / Chapter CHAPTER4 --- RESULTS --- p.82 / Chapter 4.1 --- Genes annotated in the minimally deleted regions --- p.82 / Chapter 4.2 --- Expression analyses of TP73 and its isoforms in ODs by quantitative real-time RT-PCR --- p.85 / Chapter 4.3 --- Methylation analysis of TP73 in ODs by methylation sensitive PCR (MSP) --- p.97 / Chapter 4.4 --- A rapid screen of candidate genes for aberrant expression in microdissected tumors --- p.100 / Chapter 4.5 --- Quantitative real-time RT-PCR of KIAA0495 gene --- p.103 / Chapter 4.6 --- Mutation analysis of KIAA0495 gene --- p.110 / Chapter 4.7 --- Methylation analysis of KIAA0495 in ODs by bisulfite sequencing…… --- p.112 / Chapter 4.8 --- Detection of allelic loss of lp by LOH analysis and interphase FISH --- p.121 / Chapter 4.9 --- Two-hit inactivation of KIAA0495 gene in ODs --- p.126 / Chapter 4.10 --- Tissue distribution of KIAA0495 gene --- p.130 / Chapter 4.11 --- Bioinformatics of KIAA0495 --- p.133 / Chapter CHAPTER5 --- DISCUSSION --- p.146 / Chapter 5.1 --- Expression analysis of TP73 and its isoforms in ODs by isoform-specific RT-PCR --- p.148 / Chapter 5.2 --- Methylation status ofTP73 in ODs --- p.153 / Chapter 5.3 --- A rapid screening of candidate genes for aberrant expressionin microdissected tumors --- p.156 / Chapter 5.4 --- Expression pattern of KIAA0495 mRNA in a large cohort of ODs --- p.157 / Chapter 5.5 --- No somatic mutation in coding region of KIAA0495 --- p.158 / Chapter 5.6 --- Methylation status of putative promoter region of KIAA0495 in ODs --- p.159 / Chapter 5.7 --- Status of chromosome lp in ODs --- p.161 / Chapter 5.8 --- Two-hit inactivation of KIAA0495 gene in ODs by promoter hypermethylation and allelic loss of lp --- p.162 / Chapter 5.9 --- Evaluation of expression of KIAA0495 gene as a marker for the response to chemotherapy and prognostic marker in patients with OTs --- p.164 / Chapter 5.10 --- Tissue distribution of KIAA0495 --- p.166 / Chapter 5.11 --- "KIAA0495 cDNA sequence, protein sequence and potential functional features" --- p.167 / Chapter 5.12 --- Candidate tumor suppressor genes on lp in other type of tumors with loss of lp --- p.171 / Chapter CHAPTER6 --- CONCLUSIONS --- p.174 / Chapter CHAPTER7 --- FUTURE STUDIES --- p.177 / Chapter CHAPTER8 --- REFERENCES --- p.180

Les récepteurs de la famille HER jouent un rôle majeur dans le développement du cancer. Alors qu’EGFR, HER2 et HER3 sont très bien étudiés et ciblés par des anticorps thérapeutiques, le dernier récepteur de cette famille, HER4, n’est que peu étudié et son implication dans la cancérogénèse est controversée. Il n’existe à ce jour pas d’anticorps thérapeutique anti-HER4 sur le marché ou en phase clinique. Ce récepteur est présent à la surface en quatre isoformes (JMa/CYT1 ; JMa/CYT2 ; JMb/CYT1 ; JMb/CYT2). Les isoformes JMa sont activées par clivage du récepteur, contrairement aux deux isoformes JMb. Le clivage de ces isoformes conduit à la libération de la partie intracellulaire du récepteur, appelée 4ICD. Ce fragment peut être dirigée au noyau ou dans d’autres compartiments cellulaires, impliquant HER4 dans des signalisations oncogéniques ou suppresseurs de tumeur. La littérature décrivant une activité pro-apoptotique de 4ICD et de la NRG1, le principal ligand de HER4, nous avons étudié la signalisation de ces isoformes afin de déterminer leurs rôles au niveau tumoral. Nos résultats indiquent que la NRG1 induit une signalisation suppresseur de tumeur via JMa/CYT1 et une signalisation oncogénique via JMa/CYT2. Sur la base de ces résultats, nous avons développé un criblage original d’anticorps anti-HER4 par phage display, sur des cellules exprimant l’isoforme JMa/CYT1 et stimulées par la NRG1. Nous avons caractérisés quatre anticorps anti-HER4, dont l’activité et les signalisations de certains sont modulées par la NRG1. Deux de ces anticorps, caractérisés comme étant des agonistes du récepteur HER4, induisent la mort des cellules tumorales par des mécanismes que nous sommes en train d’élucider. De manière similaire a la NRG1, un des anticorps induit la relocalisation de 4ICD-CYT1 a la mitochondrie pour induire la mort cellulaire. Ces résultats prometteurs ouvrent la voie à un ciblage thérapeutique du récepteur HER4 a l’aide d’anticorps agonistes pour le traitement des cancers / HER family is composed by four members which play a major role in cancer development. EGFR, HER2 and HER3 are well described and targeted with therapeutic monoclonal antibodies. HER4, the last one, is poorly described with a contentious role in cancerogenesis. Nowadays, there is no therapeutic antibody targeting HER4 in clinic. Four isoforms of the receptor are addressed to the plasma membrane and are called JMa/CYT1; JMa/CYT2; JMb/CYT1 and JMb/CYT2. JMa isoforms are activated by cleavage, but not JMb isoforms. Following their activation, JMa isoform cleavage releases the intracellular part of the receptor called 4ICD. This part can be directed to the nucleus or others subcellular compartments, involving HER4 in oncogenic or tumor suppressor signalling. Because a pro-apoptotic activity of 4ICD and its main ligand NRG1 have been described, we studied JMa isoforms signaling to determine their roles in cancer. We demonstrated that NRG1 induce a tumor suppressor signalling from JMa/CYT1 and an oncogenic signalling from JMa/CYT2. Based on these results, we developed an innovative screening for anti-HER4 antibodies by whole cell panning with phage display. To this end, we used NRG1- stimulated cells expressing JMa/CYT1 isoforms. We characterized four anti-HER4 antibodies and functions of some of them are affected and modulated by NRG1. Two antibodies were characterized as agonistic anti-HER4 antibodies and induce cell death of cancer cells by different mechanisms. Like NRG1, one of them induce mitochondrial localization of 4ICD-CYT1 to induce cell death. These promising results pave the way to a therapeutic targeting of HER4 receptor with agonistic antibodies to treat cancer

Os cistos e tumores odontogênicos sempre tiveram grande importância dentro da Odontologia, seja pela grande prevalência clínica seja pelo grande acometimento do indivíduo afetado pela lesão. A nova classificação da Organização Mundial de Saúde trouxe a mudança de categoria do queratocisto, que recebe agora a nômina de tumor odontogênico queratocístico, e que figura não mais na categoria de cisto odontogênico de desenvolvimento, mas sim de tumor odontogênico. Certa precipitação nessa mudança levou alguns autores a sugerirem a necessidade de estudos que esclareçam as características clínicas e histopatológicas dessa lesão para que se tenha uma classificação realmente precisa. O grande paradigma dessa lesão é: ao mesmo tempo em que apresenta características histológicas de um cisto, possui um comportamento clínico agressivo mais comumente observado nos tumores. O que realmente difere esta lesão das outras lesões que se inseriam no mesmo grupo é o padrão de crescimento diferenciado do tumor odontogênico queratocístico em relação às outras lesões císticas. Sendo assim, poderia se suspeitar que essa lesão possua um potencial proliferativo maior do que as outras que anteriormente pertenciam ao mesmo grupo, denotando uma regulação diferenciada do mecanismo proliferação-apoptose. Este estudo teve como objetivo comparar o tumor odontogênico queratocístico com uma lesão cística - o cisto dentígero - e com uma lesão tumoral - o ameloblastoma ? por meio de marcadores imunoistoquímicos para supressão tumoral e anti-apoptose. Os resultados demostraram que a maior diferença entre essas lesões está principalmente na atividade apoptótica, já que somente o resultado de bcl2 foi estatisticamente significante entre essas lesões. / Odontogenic cysts and tumors have always had a great importance in Dentistry, for its high clinical prevail and for its noticeable invasive behavior. The new classification released by WHO rearranged keratocyst, that is named now odontogenic keratocystic tumor, classifying it no longer as a development cyst, but now as odontogenic tumor. Certain haste in this change brought some authors to suggest the necessity of more studies to clarify the feautures of such lesions to determine a more accurate classification. The greatest paradigm of this lesion is that it shows cyst-like histological characteristics and simultaneuosly it has an aggressive clinical behavior, which is more commonly observed in tumors. The main difference between this lesion and the others cystic lesions is the growth pattern, which suggests that the odontogenic keratocystic tumor has higher proliferative potential than other cystic lesions. The aim of this research was to compare odontogenic keratocystic tumor with a cystic lesion ? dentigerous cyst ? and with a tumoral lesion ? ameloblastoma ? using tumor suppressor and anti-apoptosis immunohistochemical expression. Results show that the more important difference among the analysed lesions is apoptosis activity, since only bcl2 staining was significantly different among them.

A proteína ribossomal L10 (RP L10) é uma forte candidata a ser incluída na classe de proteínas supressoras de tumor. Também denominada QM, a proteína em questão é conhecida por participar da ligação das subunidades ribossomais 60S e 40S e da tradução de mRNAs. Possui massa molecular entre 24 a 26 kDa e ponto isoelétrico (pI) 10,5. A seqüência da proteína QM é bastante conservada em mamíferos, plantas, invertebrados, insetos e leveduras indicando que esta possui funções críticas na célula. Com função supressora de tumor, a proteína RP L10 foi estudada em linhagens de tumor de Wilm (WT-1) e em células tumorais de estômago, nas quais se observou uma diminuição na quantidade de seu mRNA. Mais recentemente a RP L10 foi encontrada em baixas quantidades nos estágios iniciais de adenoma de próstata e com uma mutação em câncer de ovário, indicando uma participação no desenvolvimento destas doenças. Como proteína, já foi descrito que esta interage com as proteínas c-Jun e c-Yes, inibindo a ação ativadora de fatores de crescimento e divisão celular. Este trabalho tem um papel importante no estabelecimento da expressão desta proteína solúvel, para estudos posteriores que tenham como objetivo avaliar a ação de regiões específicas que atuam na ligação das subunidades ribossomais 60S e 40S e tradução, bem como nas regiões que se ligam a proto-oncogenes. O cDNA para proteína QM foi amplificado por PCR e clonado no vetor de expressão periplásmica p3SN8. A proteína QM foi expressa em E.coli BL21 (DE3) no citoplasma e periplasma bacteriano e na melhor condição, a expressão de QM de bactérias transformadas pelo plasmídeo recombinante p1813_QM em 25°C ou 30°C, a proteína foi obtida solúvel e com quantidad es muito pequenas de contaminantes. Os ensaios de estrutura secundária demonstraram que a proteína QM tem predominância de a-hélice, mas quando do seu desenovelmento, essa condição muda e a proteína passa a ter característica de folhas β. / The ribosomal protein L10 (RP L10) is a strong candidate to be included in the class of tumor suppressor proteins. This protein, also denominated as QM, is known to participate in the binding of ribosomal subunits 60S and 40S and the translation of mRNAs. It has a molecular weight that varies between 24 and 26 kDa and an isoelectric point of (pI) 10.5. The sequence of the protein QM is highly conserved in mammals, plants, invertebrates, insects and yeast which indicates its critical functions in a cell. As a tumor suppressor, RP L10 has been studied in strains of Wilm\'s tumor (WT-1) and tumor cells in the stomach, where was observed a decrease in the amount of its mRNA. More recently, the RP L10 was found in low amounts in the early stages of prostate adenoma and showed some mutation in ovarian cancer, what indicates its role as a suppressor protein in the development of these diseases. It has also been described that this protein interacts with c-Jun and c-Yes inhibiting growth factors and consequently, cell division. This work has an important role on the establishment of soluble expression of QM to give base information for further studies on expression that aim to evaluate the specific regions where it acts binding the 60S and 40S ribossomal subunits and translation, as well as its binding to proto-oncogenes. The cDNA for QM protein was amplified by PCR and cloned into periplasmic expression vector p3SN8. The QM protein was expressed in E. coli BL21 (DE3) in the region of cytoplasm and periplasm, the best condition was obtained from the expression of the recombinant plasmid QM p1813_QM at 25°C or 30°C, the soluble protein was obtained with small amounts of contaminants. The assays of secondary structure showed that the QM protein is predominantly alpha-helix, but when it loses the folding, this condition changes and the protein is replaced by β- sheet feature.

Zhong, Lan. / Thesis Ph.D. Chinese University of Hong Kong 2014. / Includes bibliographical references (leaves 122-154). / Abstracts also in Chinese. / Title from PDF title page (viewed on 24, October, 2016). / Zhong, Lan.