Desmoglein 3 (Dsg3) belongs to the desmoglein subfamily and functions as an adhesion molecule in desmosomes. Two pools of Dsg3 have been identified, detergent soluble and insoluble proteins. Recent studies show that DSG3 is upregulated in squamous cell carcinoma (SCC). However, its biological function in cancer remains poorly understood. The aim of this study was to investigate the extra-junctional functions of Dsg3, in particular its roles in signalling that regulates cell morphology and locomotion in cancer cells. This study adopted a unique cancer cell model with Dsg3 gain-of-function and has discovered two novel regulatory signal pathways that may play a crucial role in the control of cell invasion and metastasis in Dsg3 associated cancers. Firstly, Dsg3 regulates the phosphorylation of Ezrin at Thr567 in a PKCdependent manner that is crucial for its activation and regulation of actin based membrane projections and accelerated cell locomotion in SCC. Secondly, Dsg3 modulates the transcriptional activity of cJun:AP1 that is responsible for regulating a cohort of genes to confer an invasive phenotype. It is likely that these two pathways are closely linked in that the Dsg3-mediated activation of cJun:AP1 elicits PKCdependent Ezrin activation that in turn enable it to form a complex with Dsg3 at the plasma membrane to promote membrane projection and cell locomotion. Several lines of evidence support these conclusions: Dsg3 forms a complex with Ezrin at the plasma membrane and induces phosphorylation of Ezrin resulting in augmented membrane protrusions and cell migration. Dsg3 silencing inhibits junction formation concomitant with collapse of membrane protrusion. Furthermore, Dsg3 regulates the activity of cJun:AP1. Collectively, these findings provide new insight regarding Dsg3 in cancer, suggesting it acts as a key regulator of cell invasion and metastasis in SCC. Therefore, targeting Dsg3 could be a potential new strategy in the control of cancer progression and metastasis.

Cheung Tsz Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 187-203). / Abstracts in English and Chinese. / Acknowledgements --- p.is / Abbreviations --- p.II / Abstracts --- p.V / List of Figures --- p.IX / List of Tables --- p.XIII / Contents / Chapter Chapter 1. --- General Introduction / Chapter 1.1 --- Cancer --- p.1 / Chapter 1.2 --- Growth Factor --- p.2 / Chapter 1.3 --- Growth Factor and Growth Factor Receptor --- p.4 / Chapter Chapter 2. --- Alteration of EGF Responses and EGFR Signaling in EGF-conditioned A431 cells / Chapter 2.1 --- Background Information / Chapter 2.1.1 --- Epidermal Growth Factor (EGF) --- p.6 / Chapter 2.1.2 --- Epidermal Growth Factor Receptor (EGFR) --- p.10 / Chapter 2.1.2.1 --- The Structure of EGFR --- p.10 / Chapter 2.1.2.2 --- The EGFR Family --- p.11 / Chapter 2.1.2.3 --- EGFR Activation --- p.13 / Chapter 2.1.3 --- The Intracellular Signal Transduction Pathways in EGFR Signaling --- p.18 / Chapter 2.1.3.1 --- The Ras/Raf/MAPK Pathway (MAPK pathway) --- p.19 / Chapter 2.1.3.2 --- The Jak/Stat Pathway --- p.23 / Chapter 2.1.3.3 --- The PI3K/Akt Pathway --- p.28 / Chapter 2.1.4 --- EGFR and Cancer --- p.31 / Chapter 2.1.5 --- EGFR-targeted Cancer Therapy --- p.35 / Chapter 2.1.5.1 --- Monoclonal Antibody (MAb) --- p.36 / Chapter 2.1.5.2 --- Immunotoxin Conjugates --- p.37 / Chapter 2.1.5.3 --- Bispecific Antibody --- p.37 / Chapter 2.1.5.4 --- Small-molecule EGFR Tyrosine Kinase Inhibitor (EGFR-TKI) --- p.38 / Chapter 2.1.5.5 --- Antisense Oligonucleotide --- p.39 / Chapter 2.2 --- Objectives --- p.41 / Chapter 2.3 --- Materials and Methods / Chapter 2.3.1 --- Materials --- p.42 / Chapter 2.3.2 --- Methods / Chapter 2.3.2.1 --- Cell Lines --- p.44 / Chapter 2.3.2.1.1 --- Establishment of Epidermal Growth Factor (EGF)-conditioned A431 Cells (EGF-conditioned Cells) ´ؤ AC Cells --- p.44 / Chapter 2.3.2.2 --- Growth Curve between A431 Parent Cells and EGF-conditioned Cells --- p.45 / Chapter 2.3.2.3 --- Epidermal Growth Factor (EGF) Sensitivity Assay --- p.45 / Chapter 2.3.2.4 --- Western Blot Analysis --- p.47 / Chapter 2.3.2.4.1 --- Protein Samples Preparation --- p.47 / Chapter 2.3.2.4.2 --- Protein Assay (by BCA Protein Assay Reagent) --- p.48 / Chapter 2.3.2.4.3 --- Protein Electrophoresis --- p.49 / Chapter 2.3.2.4.4 --- Electroblot (Protein Transfer) --- p.50 / Chapter 2.3.2.4.5 --- Antibody Probing (Immunoblotting) --- p.51 / Chapter 2.4 --- Results / Chapter 2.4.1 --- Growth Curve --- p.53 / Chapter 2.4.2 --- EGF Responses of A431 Parent Cells and EGF-conditioned Cells by MTT Assay --- p.55 / Chapter 2.4.3 --- The EGFR Expression Levels in A431 Parent Cells and EGF-conditioned Cells by Western Blot Analysis --- p.57 / Chapter 2.4.4 --- EGF-induced Protein Tyrosine Phosphorylation Pattern in A431 Parent Cells and EGF-conditioned Cells by Western Blot Analysis --- p.59 / Chapter 2.4.5 --- The Expression Profiles of EGFR Signaling Molecules in A431 Parent Cells and EGF-conditioned Cells by Western Blot Analysis --- p.61 / Chapter 2.4.5.1 --- The Ras/Raf/MAPK Pathway --- p.62 / Chapter 2.4.5.2 --- The Jak/Stat Pathway --- p.63 / Chapter 2.4.5.3 --- The PI3K/Akt Pathway --- p.64 / Chapter 2.4.6 --- The Cellular Responses to the Modifiers that Targeting the EGFR Signaling --- p.68 / Chapter 2.4.6.1 --- The Sensitivity of A431 Parent Cells and EGF-conditioned Cells to Various Signaling Modifiers --- p.69 / Chapter 2.4.6.2 --- The Influence of EGFR Signaling Modifiers on EGF --- p.76 / Chapter 2.5 --- Discussion --- p.85 / Chapter Chapter 3. --- The Inter-relationship between the Differential Anti-cancer Drugs Sensitivity and Alteration of EGFR Signaling in EGF-conditioned A431 Cells / Chapter 3.1 --- Background Information / Chapter 3.1.1 --- Drug Resistance and its Mechanisms in Tumor Cells --- p.90 / Chapter 3.1.2 --- Anti-cancer Drugs ´ؤ Introduction / Chapter 3.1.2.1 --- Camptothecin (CPT) --- p.93 / Chapter 3.1.2.2 --- Methotrexate (MTX) --- p.95 / Chapter 3.1.2.3 --- 5-fluorouracil (5-Fu) --- p.98 / Chapter 3.1.2.4 --- Vincristine (VCR) and Taxol --- p.104 / Chapter 3.1.2.5 --- Cisplatin (cis-DDP) --- p.108 / Chapter 3.2 --- Objectives --- p.110 / Chapter 3.3. --- Materials and Methods / Chapter 3.3.1 --- Materials --- p.112 / Chapter 3.3.2 --- Methods / Chapter 3.3.2.1 --- Cell Lines --- p.115 / Chapter 3.3.2.2 --- Determination of Drug Sensitivity by MTT Assay --- p.115 / Chapter 3.3.2.2.1 --- Determination the Influence of EGFR Signaling Modifiers on the Differential Anticancer Drugs Sensitivity by MTT Assay --- p.115 / Chapter 3.3.2.3 --- Semi-quantitative RT-PCR --- p.116 / Chapter 3.3.2.3.1 --- Preparation of RNA Samples --- p.116 / Chapter 3.3.2.3.2 --- RT-PCR --- p.117 / Chapter 3.3.2.4 --- DNA Fragmentation Assay --- p.118 / Chapter 3.3.2.5 --- Western Blot Analysis --- p.120 / Chapter 3.3.2.6 --- Northern Blot Analysis --- p.120 / Chapter 3.4 --- Results / Chapter 3.4.1 --- The Responses to Various Anti-cancer Drugs / Agents in A431 Parent Cells and EGF-conditioned Cells --- p.122 / Chapter 3.4.2 --- The Expressions of Classical Cellular Drug Resistant Factors in EGF-conditioning-associated Differential Anti-cancer Drugs Sensitivity --- p.126 / Chapter 3.4.2.1 --- Camptothecin Sensitivity --- p.126 / Chapter 3.4.2.2 --- Methotrexate Sensitivity --- p.130 / Chapter 3.4.2.3 --- 5-fluorouracil Sensitivity --- p.135 / Chapter 3.4.2.4 --- Vincristine and Taxol Sensitivity --- p.141 / Chapter 3.4.3 --- EGFR Signaling Modifiers and Differential Anti-cancer Drugs Sensitivity by MTT Assay --- p.143 / Chapter 3.4.3.1 --- Methotrexate --- p.143 / Chapter 3.4.3.2 --- Vincristine --- p.147 / Chapter 3.4.3.3 --- Taxol --- p.149 / Chapter 3.5 --- Discussion --- p.153 / Chapter Chapter 4. --- Identification of Differentially Expressed Genes in A431 Parent Cells and EGF-conditioned Cells by Differential Display (DD) / Chapter 4.1 --- Introduction 一 Differential Display (DD) --- p.156 / Chapter 4.2 --- Objectives --- p.160 / Chapter 4.3 --- Materials and Methods / Chapter 4.3.1 --- Materials --- p.161 / Chapter 4.3.2 --- Methods / Chapter 4.3.2.1 --- Cell Lines --- p.163 / Chapter 4.3.2.2 --- RT-PCR-based mRNA Differential Display --- p.163 / Chapter 4.3.2.2.1 --- Preparation of RNA Samples --- p.163 / Chapter 4.3.2.2.2 --- Identification of Differentially Expressed Genes by RT-PCR --- p.164 / Chapter 4.3.2.2.3 --- Reamplification of cDNA Probes --- p.164 / Chapter 4.3.2.2.4 --- Subcloning of the Differentially Expressed cDNAs --- p.165 / Chapter 4.3.2.2.4.1 --- Preparation of the Ultra-competent E.coli Cells for Transformation --- p.165 / Chapter 4.3.2.2.4.2 --- Preparation of Cloning Vector --- p.166 / Chapter 4.3.2.2.4.3 --- Transformation --- p.166 / Chapter 4.3.2.2.5 --- Verification of cDNA Differentially Expression by Colony-PCR and Northern Blot Analysis --- p.167 / Chapter 4.3.2.2.5.1 --- Colony-PCR --- p.167 / Chapter 4.3.2.2.5.2 --- Preparation of Cloned Plasmid cDNA and Bacterial Glycerol Stocks --- p.167 / Chapter 4.3.2.2.5.3 --- Preparation of cDNA Probes for Northern Blot Analysis --- p.168 / Chapter 4.3.2.2.5.4 --- Northern Blot Analysis --- p.168 / Chapter 4.3.2.2.6 --- Sequencing of the Desired Cloned cDNA Inserts --- p.170 / Chapter 4.3 --- Results --- p.171 / Chapter 4.4 --- Discussion --- p.180 / Chapter Chapter 5. --- General Conclusion and Future Perspectives / Chapter 5.1 --- General Conclusion --- p.182 / Chapter 5.2 --- Future Perspectives --- p.185 / References --- p.187

Kindlin-1 (Kin1) is an epithelial focal adhesion protein that plays a key role in integrin-mediated anchorage of cells to the extracellular matrix. Congenital loss of Kin1 leads to Kindler Syndrome (KS), whose symptoms include progressive epidermal atrophy, reduced keratinocyte proliferation, skin blistering and increased incidence of aggressive Squamous Cell Carcinoma (SCC). Objectives of this study were to examine the role of Kin1 in skin homeostasis and in the development of aggressive SCC in KS, as the molecular aetiologies for these pathologies are yet to be clearly understood. We first examined whether the recently discovered role of Kin1 in mitosis contributes to reduced keratinocyte proliferation observed in KS epidermis. We discovered that short-term loss of Kin1 in adult mouse epidermis reduced keratinocyte proliferation. We also found that Kin1 loss increased mitotic spindle misorientation that, according to the model of cell division in skin homeostasis, decreases cell proliferative potential, and, thus, may account for the reduced proliferation in our model. As spindle misorientation can stem from microtubule instability, we believe that the reduction in acetylated α-tubulin (ac-tub), a known marker of stable microtubules, that we also observed in mouse epidermis following Kin1 loss could account for the defective spindle orientation phenotype. The role of Kin1 in spindle orientation was also evident in vitro. Moreover, data from our lab revealed showed reduction in spindle ac-tub following Kin1 depletion, mirroring our in vivo observation. Additionally to orientation defects, in vitro depletion of Kin1 led to enhanced chromosome missegregation, which is likely to result from reduced microtubule stability due to low levels of ac-tub. We showed that role of Kin1 in spindle orientation and chromosome segregation is dependent on HDAC6, a known inhibitor of ac-tub. Overall, our results uncover an in vitro and in vivo role of Kin1 in mitotic spindle fidelity that could be crucial to skin homeostasis, and, when disturbed, may lead to reduced keratinocyte proliferation. Interestingly, our in vitro studies also revealed that in mitosis Kin1 and Kindlin-2 (Kin2) had overlapping, but also distinct roles, which is in line with various reports that show different biological functions for the two protein isoforms. Our next and final aim was to explore the roles of Kin1 in the development and progression of SCC, which would help us comprehend the reason behind the cancer's aggressive nature in KS. By employing in vitro and in vivo SCC growth assays and tumour immunohistochemical staining we found that absence of Kin1 in SCC cells and tumours enhanced proliferation and growth, and enhanced tumour vascularisation. RNA sequencing of tumour material revealed that lack of Kin1 increased expression of matrix metalloproteinases and chemokines, which have been implicated in tumour progression via promotion of angiogenesis and invasion in a plethora of studies, and of various angiogenesis markers. Together this provides an insight into the mechanisms via which Kin1 controls tumour microenvironment and, ultimately, SCC tumour growth and development. Overall, we report an in vitro and in vivo role for Kin1 in mitotic spindle stability, which affects a variety of mitotic processes and may be linked to reduced keratinocyte proliferation observed in epidermis of KS patients, thus contributing to skin homeostasis. Moreover, we describe a role for Kin1 in regulation of SCC tumour growth and progression, which may ultimately offer an explanation for the aggressive and life-threatening nature of SCC developed in KS.

FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgico / Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Angiogenesis is the development of new blood vessels from pre-existing capillaries, being an essential step in tumor growth for supplying nutrition and oxygen to cells in proliferation. A cell that may be involved in this process is the mast cell (MC), since besides the defense function, acts in the blood vessels regulation. The MC participation in the induction of angiogenesis has been suggested in various malignant tumors. The purposes of this study was to evaluate angiogenesis and mast cell density in oral epithelial dysplasia and squamous cell carcinoma (SCC). This is an observational, retrospective and quantitative study using the sample selection from the archives of the Department of Legal Medicine and Pathology and Laboratory of Oral Pathology, both from the Federal University of CearÃ. For MC evaluation , the sample was consisted of 73 paraffin blocks, distributed between SCC (n=30), epithelial dysplasia (n=23) and hyperplasias fibroepithelial (HFE) (n = 20), as control, and for angiogenesis the sample was 65 blocks, consisted of 24 SCC, 19 epithelial dysplasias and 22 HFE. Immunohistochemistry was performed using the MC-tryptase, CD31 and CD34 antibodies. For quantification, digital images were captured and then counting was performed using Image J software. The antibody staining percentage was determined using SAMM software. With regard to mast cells, there was a lower density in malignant lesions in relation to HFE and dysplasia (p = 0.0092). Evaluating angiogenesis, CD31 expression showed differences between epithelial dysplasia and SCC and between SCC and HFE, with a greater percentage of vessels in SCC (p <0.0001). However, CD34 expression did not differ between groups. The CD31 antibody was shown to be a better angiogenesis marker in oral mucosa than CD34. Increased vascularity in oral squamous cell carcinoma suggests that angiogenesis is necessary for tumor growth, increasing when the malignant transformation starts. However, no correlation was found between mast cells and angiogenesis. / AngiogÃnese à o surgimento de um novo vaso sanguÃneo a partir de capilares prÃ-existentes, sendo um passo essencial no crescimento tumoral por fornecer nutriÃÃo e oxigÃnio Ãs cÃlulas em proliferaÃÃo. Uma cÃlula que pode estar envolvida nesse processo à o mastÃcito, pois, alÃm da funÃÃo de defesa, atua na regulaÃÃo de vasos sanguÃneos. Sua participaÃÃo na induÃÃo da angiogÃnese tem sido sugerida em vÃrios tumores malignos. Os objetivos deste trabalho foram avaliar a angiogÃnese e a densidade de mastÃcitos em displasias epiteliais e no carcinoma espinocelular (CEC) de boca. Trata-se de um estudo observacional, retrospectivo e quantitativo, realizado atravÃs da seleÃÃo de amostra proveniente dos arquivos do Departamento de Patologia e Medicina Legal e do laboratÃrio de Patologia Bucal do curso de Odontologia, ambos da Universidade Federal do CearÃ. Para a avaliaÃÃo dos mastÃcitos, a amostra foi constituÃda por 73 blocos parafinados, distribuÃdos entre CEC (n=30), displasias epiteliais (n=23) e hiperplasias fibroepiteliais (HFE) (n=20), como controle, e para a angiogÃnese a amostra foi de 65 blocos, sendo 24 de CEC, 19 de displasias epiteliais e 22 de HFE. Foi realizada imunohistoquÃmica utilizando-se os anticorpos anti-triptase, para mastÃcitos e anti-CD31 e anti-CD34, para vasos sanguÃneos. Para quantificaÃÃo, foram capturadas imagens digitais e, em seguida, utilizados softwares para auxiliar na contagem dos mastÃcitos (Image J) e para determinaÃÃo do percentual de marcaÃÃo do anticorpo (SAMM). Com relaÃÃo aos mastÃcitos, houve menor densidade destes nas lesÃes malignas em relaÃÃo Ãs HFE e displasias (p=0,0092). Avaliando angiogÃnese, a expressÃo de CD31 mostrou diferenÃa entre os grupos CEC e displasia epitelial e entre CEC e HFE, havendo um maior percentual de vasos nos CEC (p<0,0001). Contudo, o CD34, nÃo mostrou diferenÃa entre os grupos. O anticorpo CD31 mostrou-se melhor marcador de angiogÃnese em mucosa oral do que CD34. O aumento da vascularizaÃÃo em CEC oral sugere que a angiogÃnese à necessÃria ao crescimento tumoral, aumentando à medida que inicia o processo de malignizaÃÃo. NÃo foi encontrada correlaÃÃo entre mastÃcitos e angiogÃnese.

O carcinoma oral de células escamosas (OSCC Oral Squamous Cell Carcinoma) é o câncer de cabeça e pescoço mais comum. Somente no Brasil, foram estimados 14.160 novos diagnósticos para o ano de 2009. O HPV está associado com o aumento no risco do câncer oral, mas seu papel na carcinogênese ainda é controverso. A BubR1, uma proteína importante para o checkpoint de fuso mitótico (SAC Spindle Assembly Checkpoint), tem sido associada com algumas proteínas codificadas por espécies virais e com o câncer. O objetivo do presente estudo foi avaliar a expressão de BubR1 em lesões orais benignas e amostras de OSCC com e sem metástase associadas com infecção pelo HPV. Nós realizamos imunoistoquímica para BubR1 em 16 biópsias de lesão oral benigna e em 70 biópsias de OSCC divididas em três grupos (tumores in situ, tumores invasivos sem metástase e tumores invasivos com metástase), com os respectivos linfonodos das amostras com metástase. A técnica de Nested PCR foi realizada com finalidade de detectar DNA do HPV. Nas lesões malignas, foi observada uma significante superexpressão de BubR1 associada com menor sobrevida (p = 0.0479). Houve também correlação significante (r = 1.000) de BubR1 entre as lesões com metástase e seus respectivos linfonodos. Noventa por cento dos OSCC e 100% das lesões benignas foram HPV positivos. HPV 16 e HPV 18 foram detectados em, respectivamente, 13% e 24% das amostras com OSCC HPV-positivas. O HPV teve maior prevalência (76%) nas amostras com alta expressão de BubR1 e a ausência de DNA viral não influenciou no padrão de expressão de BubR1. Esses resultados sugerem uma provável associação do HPV com a superexpressão de BubR1 em OSCC, o que não se aplica para lesões orais benignas. / Oral squamous cell carcinoma (OSCC) is the most common head and neck cancer. Only in Brazil, the estimate is that 14,160 new diagnoses will be made in 2009. HPV is associated with increasing risk of oral cancer, but its role in carcinogenesis is still controversial. BubR1, an important protein in the mitotic Spindle Assembly Checkpoint (SAC), has been associated with some virus-encoded proteins and cancer. The aim of the present study was to evaluate the expression of BubR1 in non-malignant oral lesions and OSCC with and without metastasis associated with HPV infection. We performed immunohistochemistry for BubR1 in 16 non-malignant oral lesion biopsies and in 70 OSCC biopsies divided into three groups (in situ tumors, invasive tumors without metastasis and invasive tumors with metastasis) with their respective lymph nodes from samples with metastasis. Nested PCR was performed in order to detect HPV DNA. Significantly higher BubR1 expression associated with shorter survival (p = 0.0479) was observed in malignant lesions. There was also a significant correlation (r = 1.000) with BubR1 expression in lesions with metastasis and their lymph nodes. Ninety percent of OSCC and 100% of benign lesions were HPV positive. HPV 16 and HPV 18 were present in 13% and 24% of HPV-positive OSCC samples, respectively. HPV was more prevalent (76%) in samples with high BubR1 expression and the absence of viral DNA had no influence on BubR1 expression. These findings suggest that HPV could be associated with overexpression of BubR1 in OSCC, but not in benign oral lesions.

O ameloblastoma é definido como uma neoplasia benigna relativamente rara com origem relacionada à reativação das estruturas odontogênicas. Sua classificação histológica é baseada em características microscópicas e na distribuição arquitetural das células neoplásicas. A importância da metaplasia escamosa e queratinização tem sido questionada nos ameloblastomas. Este estudo analisou aspectos clínicos e histopatológicos de 85 ameloblastomas com ênfase em aspectos de metaplasia escamosa e queratinização e sua relação com o epitélio odontogênico do germe dental normal. Dados clínicos/demográficos dos 85 casos de ameloblastomas foram obtidos nos prontuários médicos. A análise microscópica de todos os casos teve como ênfase os aspectos de metaplasia escamosa e queratinização, assim como cortes histológicos representativos dos estágios da odontogênese humana também foram comparados com os aspectos histológicos dos ameloblastomas. Dos 85 casos de ameloblastomas, 46 (54,12%) foram diagnosticados no gênero masculino com faixa etária média de 37 anos. A maioria dos doentes eram leucodermas, 56 (65,88%), e a mandíbula foi afetada em 68 (80%) dos casos. Cinqüenta e oito (68,23%) ameloblastomas apresentaram típico aspecto multilocular e foram classificados como sólidos/multicísticos, 26 (30,58%) possuíam imagem unilocular bem definida sendo classificados como unicísticos. Dos casos analisados, a maioria apresentou áreas de metaplasia escamosa e queratinização, similares a áreas de queratinização da lâmina dental durante a odontogênese. Recidivas ocorreram em um total de 16 casos não havendo relação com os aspectos de queratinização do tumor. Os aspectos de queratinização encontrados nos ameloblastomas foram similares aos da lâmina dental, e estes aspectos morfológicos não apresentaram impacto no comportamento do tumor. / Ameloblastoma is defined as a benign odontogenic neoplasm with origin reputed to reactivation of odontogenic structures. Histological classification is based on its microscopic features and architectural distribution of neoplastic cells. The importance of squamous metaplasia and keratinisation has been disputed in ameloblastomas. The present study evaluated the clinical and histopathological aspects of 85 ameloblastomas, with special attention to the presence of squamous metaplasia and keratinisation. Keratinisation features of the tumours were compared with aspects normal tooth germ epithelium. Clinical-demographical information of 85 cases of ameloblastomas were gleaned form the medical records. Microscopic analysis of all cases was carried out with emphasis on the keratinisation aspect of each tumour. Histological sections representative of human odontogenesis stages were compared with the aspects of ameloblastomas. Most cases of ameloblastomas (54.12%) were diagnosed in male patients with mean age of 37 years. Most patients were Caucasian (n=56, 65.88%) and mandible was affected in 68 (80%) cases. Fifty-eight (68.23%) ameloblastomas presented the typical multiloculated aspect and were classified as solid/multicystic; 26 (30.58%) had a well-defined unilocular image, and were classified as unicystic. Most cases analyzed presented areas of squamous squamous metaplasia and keratinisation. These areas were similar to areas of keratinisation of the dental lamina during the odontogenesis. Recurrence was detected in 16 cases ant it was not related to the keratinisation aspects of the tumour. Keratinisation aspects in ameloblastomas are similar to the dental lamina and occur. This morphological aspect seems to have no with impact in tumour behaviour.

Philosophiae Doctor - PhD / Purpose: High risk human papillomavirus (HR-HPV) is well established as a causative agent of squamous cell carcinoma (SCC) of the orophaynx. HR-HPV has also been reported in periocular cancers and precancers, but controversy exists about its overall incidence and clinicopathologic profile. The purpose of this study is to evaluate the role of HR-HPV infection in periocular cancers and precancers, using multiple methods of detection. Design: Retrospective observational case series with laboratory investigations. Methods: Sequential surgical samples of 87 carcinomas (invasive SCC, SCC in situ and sebaceous carcinoma) from three different periocular sites (conjunctiva, lacrimal sac and the eyelid) diagnosed over a 15-year period (2000-2015) were selected for evaluation. Unstained paraffin sections of 87 cases of periocular carcinomas were analyzed with immunohistochemistry (IHC) for p16 as a screening test. p16 positive conjunctival- and lacrimal sac SCC were further evaluated for HR-HPV using DNA in situ hybridization (DNA ISH), and a subset was also analyzed by DNA Polymerase Chain Reaction (DNA PCR). p16 positive periocular sebaceous carcinomas (SC) were analyzed with PCR, and a subset of 18cases was further studied with a novel method of mRNA ISH, an advanced technique with an enhanced sensitivity and specificity. Relevant patient clinical information was obtained from review of the electronic medical records.

Head and neck cancers account for 3% of all newly diagnosed cancers, of which 90% are squamous cell carcinomas (SCC). Improvements in surgery, radiotherapy and chemotherapy have done little to improve the mortality of this disease over the past 20 years while current clinicopathological predictors of disease outcome are sub-optimal. Identifying molecular targets of prognostic and therapeutic significance in head and neck squamous cell carcinomas (HNSCC) may help direct novel therapies to patients whom it is most likely to benefit. Accrued knowledge of the biology of HNSCC has highlighted specific aberrations in pRb and p53 pathways which warrant further study. An immunohistochemical analysis (IHC) in a cohort of 145 patients with SCC of the anterior tongue was performed. Protein expression of the pRb and p53 pathways and related molecules that directly or indirectly influence cell cycle progression at the G1/S phase checkpoint was assessed. We determined that over-expression of E2F-1 occurred in &gt35% of these cancers and associated with improved overall survival on univariate analysis. The strongest multivariate model included: regional lymph node status, tumour grade, p16INK4A, cyclin D1 and p14ARF. This is the first study to determine that p14ARF is an independent marker of both improved diseasefree survival and overall survival in a cohort of SCC of the anterior tongue. Unrecognized molecular heterogeneity is thought to account for the unpredictable clinical response to ZD1839, an EGFR tyrosine kinase inhibitor. We explored the anti-proliferative effects following ZD1839 treatment alone or in combination with radiotherapy in cyclin D1 and E2F-1 over-expressing SCC9 HNSCC cells. SCC9 cells over-expressing cyclin D1 or E2F-1 were highly resistant to ZD1839 treatment, while E2F-1 clones were also radioresistant. Combined therapy in SCC9 controls had a greater anti-proliferative effect than each individual treatment. These data showed that cyclin D1 and E2F-1 may have utility as markers of ZD1839 resistance. The data in this thesis contribute to our knowledge of the clinical behaviour and molecular pathology of HNSCC. Specifically the molecular data identifies novel markers of outcome in SCC of the anterior tongue such as p14ARF, and therapeutic response to ZD1839 such as cyclin D1 and E2F-1. This study addresses in part, the current issues and limitations of management in HNSCC and has the potential to contribute to strategies that may be developed to improve the outcome for patients who develop HNSCC in the future.

Introduction Avec plus de 7000 cas diagnostiqués par an en Belgique, le cancer bronchique est l’un des cancers les plus fréquents chez l'homme. Son pronostic est très réservé, la survie à 5 ans tous stades confondus étant inférieure à 15 %. Ce faible taux de guérison s’explique en grande partie par le fait que le diagnostic se réalise généralement à un stade avancé de la maladie. Une méthode de détection précoce, l’endoscopie en autofluorescence, exploite des variations de la fluorescence bronchique sous l'effet d'un laser et permet ainsi de dévoiler des lésions précancéreuses invisibles en lumière blanche. Réalisée à l’Institut Jules Bordet depuis 1996, la photodétection nous a permis de constituer une banque de biopsies d’épithélium bronchique à tous les stades de la carcinogenèse bronchique, conservées initialement en blocs paraffinés et, depuis 2003, par congélation. Hypothèse Nous avons émis l’hypothèse que la compréhension de la genèse du carcinome épidermoïde bronchique par la caractérisation de l’évolution des anomalies moléculaires dans les lésions précancéreuses et cancéreuses de l'arbre bronchique nous permettrait d’identifier de nouvelles cibles de détection et éventuellement de chimioprévention pour le cancer bronchique. L'objectif de nos travaux a donc été de caractériser les modifications moléculaires successives et/ou cumulatives sous-jacentes à la transition entre les différents stades histologiques de la carcinogenèse épidermoïde bronchique (épithélium bronchique normal du fumeur, hyperplasie, métaplasie, dysplasies légère, modérée et sévère, CIS et les carcinomes invasifs). Différentes techniques complémentaires ont été successivement utilisées à cet effet : 1) étude de l’expression protéique par immunohistochimie (IHC) et immunofluorescence (IF), 2) étude de l’expression des ARNs messagers par microdamiers, 3) étude de l’expression des microARNs par RT-PCR quantitative. Travaux réalisés Afin de sélectionner les protéines à étudier aux différents stades de la carcinogenèse épidermoïde bronchique, nous avons réalisé une étude méthodologique de la littérature chez les patients atteints de cancer bronchique non à petites cellules (CBNPC). En effet, d’une part, la petite taille des biopsies bronchiques que nous projetions d’étudier et, d’autre part, le faible taux de découvertes de lésions de haut grade lors de la réalisation de bronchoscopies en fluorescence limitent le nombre d’analyses réalisables. Les revues systématiques de la littérature, intégrant une analyse méthodologique des études publiées et une méta-analyse de leur impact pronostique en terme de survie, ont permis de choisir une série de marqueurs sur base de leur intérêt potentiel grâce à la puissance apportée par l’agrégation de grands nombres de cas. La mutation de KRAS identifiée par PCR constitue un facteur péjoratif pour la survie des patients atteints de CBNPC et plus spécifiquement des adénocarcinomes (ADC). Etant donné que notre projet est consacré à la genèse du carcinome épidermoïde bronchique (CEB), nous n’avons pas étudié ce facteur dans nos biopsies. Par contre, les revues systématiques ont montré un impact en terme de survie dans les CBNPC, y compris dans les CEB, pour l’angiogenèse, des récepteurs de facteurs de croissance épithéliale (EGFR et c-erbB-2) et des marqueurs de la prolifération cellulaire (Ki-67). Nous avons donc étudié l’expression de ces protéines par IHC dans les lésions précurseurs de CEB et dans les CEB. L’expression de Ki-67 est augmentée aux stades de dysplasie sévère et de CIS par rapport à ceux de dysplasies légère et modérée. La mesure de l’expression de Ki-67 dans ces biopsies aide donc à les classifier en lésions de bas et de haut grade. L’étude de la corrélation de l’expression de plusieurs protéines (EGFR, c-erbB-2 et Ki-67) dans les mêmes lésions bronchiques a montré que la majorité des biopsies de haut grade (dysplasies sévères et CIS) expriment anormalement EGFR et/ou Ki-67. Par contre, l’homologue d’EGFR, c-erbB-2, n’apparaît que plus tardivement, dans les carcinomes invasifs. Nous avons également caractérisé l’expression de protéines impliquées dans les voies de l'apoptose dans les lésions précurseurs de CEB. L’expression du facteur suppresseur de tumeurs p53 s’est avérée être un marqueur pronostique péjoratif important pour la survie des patients atteints de CNBPC et augmente dès les premières étapes de la tumorigenèse bronchique et même déjà dans l’épithélium morphologiquement normal du fumeur. L'altération de l'expression de p53 est donc un événement très précoce dans la genèse du CEB et la positivité de p53 augmente ensuite avec la sévérité des lésions. Dans le but de caractériser les voies de contrôle de p53 au cours de la carcinogenèse épidermoïde bronchique, nous avons analysé l'expression de 3 autres protéines, MDM2, p14arf et la nucléophosmine (NPM), dans une série de 200 biopsies. L’expression de MDM2, NPM et p14arf est respectivement altérée aux stades de dysplasie légère, modérée et sévère. Au stade de dysplasie sévère, l’expression de p14arf, inhibiteur de MDM2, peut soit disparaître, soit se concentrer dans les nucléoles. Tant la perte de p14arf que sa concentration dans les nucléoles sont associées à une augmentation de l’expression de MDM2. Nous avons également observé que la délocalisation de NPM depuis le nucléoplasme vers le nucléole est hautement corrélée à celle de p14arf. En IF, NPM et p14arf colocalisent dans le nucléoplasme dans les échantillons de bas grade ou dans les nucléoles dans les lésions de haut grade. Par contre, la protéine MDM2 n’est détectée dans les nucléoles quelles que soient les localisations de p14arf ou de NPM et quel que soit le stade. Ces données sont en faveur de l’hypothèse selon laquelle la localisation de p14arf dans les nucléoles empêcherait la formation des complexes p14arf–MDM2 et pourrait ainsi faciliter la carcinogenèse pulmonaire. Nos résultats suggèrent également que la présence de NPM dans le nucléoplasme pourrait jouer un rôle protecteur contre le dommage cellulaire et la transformation maligne tandis que sa délocalisation vers les nucléoles et ce, peut-être par la délocalisation de p14arf, favoriserait la carcinogenèse bronchique. Enfin, suite à la réalisation d’une méta-analyse montrant le rôle de la cyclooxygénase 2 (COX-2) en tant que facteur pronostique dans les CBNPC de stade précoce, l’expression de la protéine COX-2 a été analysée par IHC dans 106 biopsies. Cette étude montre que cette protéine s’accumule exclusivement à partir du stade de dysplasie sévère, permettant ainsi de séparer les lésions bronchiques de bas et de haut grade. Avec une haute valeur prédictive positive (100 %) et une bonne valeur prédictive négative (82,35 %), COX-2 apparaît donc comme un marqueur précoce potentiel à tester pour le dépistage du cancer bronchique. En parallèle à ces travaux, nous avons collecté des biopsies fraîchement congelées aux différents stades de la carcinogenèse épidermoïde pour une analyse du transcriptome. La première étape a consisté à définir le profil d’expression génique par la technique des microdamiers. Après son extraction et sa rétrotranscription, l’ARN a été marqué et hybridé sur des lames commercialisées par Agilent Technologies. Au total, 122 échantillons, dont minimum 12 de chaque catégorie, ont été hybridés avec un contrôle commun (issu d’un mélange de biopsies bronchiques normales de non-fumeurs). Une analyse en composante principale a montré que la hiérarchie de la classification histologique était bien représentée par les profils d’expression génique des biospies aux différents stades. Quelle que soit la liste de gènes de départ sélectionnée pour réaliser le regroupement hiérarchisé, nous avons observé, de manière constante et robuste, une différence majeure de profil d’expression génique entre, d’une part, les tissus bronchiques les plus « normaux » (normal normofluorescent, histologiquement normal mais hypofluorescent et hyperplasie) et, d’autre part, toutes les autres catégories histologiques dites « anormales ou modifiées » à partir de la métaplasie jusqu’au carcinome invasif. Parmi les épithéliums bronchiques « modifiés », deux grands groupes se distinguent : d’un côté, les métaplasies et dysplasies légères, qui sont très souvent bénignes et réversibles pour la plupart, et de l’autre côté, les dysplasies sévères, les CIS et les carcinomes invasifs, lésions à risque beaucoup plus élevé d’évoluer vers un cancer ou déjà malignes. Les échantillons au stade de dysplasie modérée se classent tantôt dans le deuxième groupe avec les dysplasies légères tantôt dans le troisième avec les dysplasies sévères. Il semble donc que le stade histologique de la dysplasie modérée soit un groupe hétérogène n’ayant pas de réalité biologique et qu’il serait plus adéquat de les reclasser dans un des 2 autres groupes sur base des arguments moléculaires. Nous avons obtenu les listes de gènes dont l’expression varie de manière statistiquement significative entre les différentes étapes de la carcinogenèse épidermoïde bronchique. Par ailleurs, nous avons pu mettre en évidence un profil d’expression génique permettant de discriminer les lésions bronchiques de mauvais pronostic (dysplasies sévères ou plus) de celles de meilleur pronostic (tissu normal et anomalies morphologiques de bas grade). Les gènes qui constituent ce profil permettant d’identifier les lésions de mauvais pronostic sont des candidats pour la détection précoce du cancer bronchique. De plus, nos données de génomique confirment la surexpression de certains ARNs messagers correspondant à des protéines dont la surexpression a été rapportée dans les études d’IHC comme COX-2, MDM2, Ki-67, les cytokératines, les métallopeptidases, les cyclines. Par ailleurs, certaines protéines dont le rôle dans les cancers pulmonaires invasifs a déjà été décrit mais pas aux stades pré-invasifs, parmi lesquelles la protéine PTH-like, des protéines liées à TNF ou d’autres liées à IGF, E2F ou à Wnt, semblent impliquées aux stades précoces de la carcinogenèse épidermoïde bronchique. Enfin, de nouveaux acteurs possibles de ce processus ont été mis en évidence. Nous avons également étudié l’évolution de l’expression des microARNs au cours de la carcinogenèse bronchique par des RT-PCR quantitatives (LDA) sur 60 des biopsies dont nous avons étudié le transcriptome (6 par catégorie) et en utilisant la classification en 3 groupes issue des analyses des microdamiers d’expression génique. Cette étude montre que plusieurs miARNs sont différentiellement exprimés durant la carcinogenèse bronchique. De plus, deux étapes successives et distinctes ont été mises en évidence pour l’évolution de l’expression des miARNs au cours de la carcinogenèse bronchique. Aux stades les plus précoces, correspondant à la progression de l’épithélium normal du non-fumeur vers l’épithélium histologiquement normal du fumeur et l’hyperplasie jusqu’au groupe des anomalies morphologiques relativement bénignes (métaplasie, dysplasies légère et modérée), on observe une réduction significative de l’expression de la grande majorité des miARNs. Aux stades plus tardifs de la carcinogenèse (dysplasie sévère, CIS et CEB), même si 74 % des miARNs altérés restent encore régulés négativement par rapport à leur niveau d’expression dans l’épithélium normal du non-fumeur, la proportion de miARNs dont l’expression est augmentée (43 %) s’accroît par rapport à leur niveau d’expression dans les stades qui les précèdent (métaplasie, dysplasies légère et modérée). En outre, lorsque l’on compare ce dernier groupe à celui des lésions plus sévères (dysplasie sévère et CIS), qui progressent fréquemment vers des carcinomes invasifs, 80 % des miARNs augmentent leur niveau d’expression. Par ailleurs, l’expression de certains microARNs évolue de manière linéaire. En particulier, l’expression de miR 34c, cible transcriptionnelle de p53, et celle de miR 15a, inhibiteur de Bcl2, diminuent progressivement entre les différents stades à partir du tissu bronchique normal du non-fumeur jusqu’au CEB. Enfin, les profils d’expression des miARNs permettent de prédire avec précision la classification histologique non seulement entre les lésions de bas grade (métaplasie, dysplasies légère et modérée) et de haut grade (dysplasie sévère et CIS) mais également entre les CIS et les carcinomes invasifs. Les miARNs qui constituent ces signatures sont donc des candidats potentiels pour la détection précoce du cancer bronchique. Conclusions Nos travaux montrent que les anomalies moléculaires apparaissent aux stades les plus précoces de la transformation maligne de l’épithélium bronchique. L’expression protéique des marqueurs étudiés -p53, MDM2, p14arf, NPM, Ki-67, COX-2, c-erbB-2, et EGFR- permet d’affiner la classification des lésions bronchiques précancéreuses et de mieux comprendre les voies de la carcinogenèse précoce. Les profils d'expression des gènes et des microARNs apportent une approche originale des étapes successives du processus de carcinogenèse épidermoïde bronchique et ont mis en évidence des signatures permettant de discriminer les lésions qui sont à très haut risque de progression vers un cancer invasif ou qui sont déjà néoplasiques de celles de meilleur pronostic. Les marqueurs qui constituent ces profils sont des candidats potentiels pour la détection précoce du cancer bronchique.

Sox2 is the most frequently amplified oncogene in lung squamous cell carcinoma (SCC). Lung SCC arises in the proximal to central airways and is thought to originate from the p63-positive basal progenitor cells. Since Sox2 amplification occurs early in SCC pathogenesis, we investigated the oncogenic role of Sox2 using normal primary human lung basal progenitor cells. Although Sox2 is highly expressed in normal basal progenitors in a quiescent tracheal epithelium in vivo, we found that Sox2 expression decreases substantially during in vitro proliferation. When Sox2 expression is elevated in the proliferating basal cells in vitro to a level clinically observed in lung SCCs, Sox2 causes hyperplasia and promotes both squamous and Mucin16-positive glandular lineages at the expense of ciliated cell differentiation. Furthermore, our data suggest that the squamous and glandular-differentiating activity of Sox2 is differentially modulated by Receptor tyrosine kinase (RTK) and/or PI3-kinase signaling to promote squamous metaplasia of basal progenitor cells during SCC development.