Yam Hin-Fai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1990. / Bibliography: leaves 138-157. / ABSTRACT --- p.2 / ACKNOWLEDGEMENT --- p.5 / CONTENT --- p.6 / Chapter I. --- INTRODUCTION --- p.8 / Chapter II. --- LITERATURE REVIEWS / Chapter 1. --- Esophagus and Esophageal Carcinoma --- p.11 / Chapter 2. --- Characterization of Cell Line --- p.23 / Chapter 3. --- Membrane Surface --- p.26 / Chapter 4. --- Differentiation and Cancer --- p.36 / Chapter 5. --- Calcium Ion --- p.42 / Chapter III. --- MATERIALS AND METHODS / Chapter 1. --- Characterizations of EC/CUHK2 Cell Line --- p.48 / Chapter 2. --- SQM1 Localization on EC/CUHK2 Cells --- p.57 / Chapter 3. --- SQM1 Localization on Other Cells and Cell Lines --- p.62 / Chapter 4. --- Characterizations of EC/CUHK2 Cells in Different Extracellular Calcium Ion Concentrations --- p.65 / Chapter 5. --- SQM1 Localization on EC/CUHK2 Cells in Different Extracellular Calcium Ion Concentrations --- p.71 / Chapter 6. --- SQM1 Localization on EC/CUHK2 Cells with Changes of Extracellular Calcium Ion Concentrations --- p.73 / Chapter IV. --- RESULTS / Chapter 1. --- Characterizations of EC/CUHK2 Cell Line --- p.74 / Chapter 2. --- SQM1 Localization on EC/CUHK2 Cells --- p.81 / Chapter 3. --- SQM1 Localization on Other Cells and Cell Lines --- p.83 / Chapter 4. --- Characterization of EC/CUHK2 Cells in Different Extracellular Calcium Ion Concentrations --- p.87 / Chapter 5. --- SQM1 Localization on EC/CUHK2 Cells in Different Extracellular Calcium Ion Concentrations --- p.96 / Chapter 6. --- SQM1 Localization on EC/CUHK2 Cells with Changes of Extracellular Calcium Ion Concentrations --- p.105 / Chapter V. --- DISCUSSIONS / Chapter 1. --- Characterizations of Carcinoma Cell Line --- p.107 / Chapter 2. --- SQM1 Distribution on Esophageal Cancer Cells --- p.118 / Chapter 3. --- SQM1 Distribution on Other Cells --- p.122 / Chapter 4. --- Calcium-Induced Differentiation of Esophageal Carcinoma Cells --- p.125 / Chapter 5. --- SQM1 Distribution on Calcium-Induced Esophageal Carcinoma Cells 6 --- p.132 / Chapter VI. --- CONCLUSION --- p.136 / Chapter VII. --- REFERENCES --- p.138 / Chapter VIII. --- ILLUSTRATIONS --- p.158

Organ‐transplant recipients (OTRs) on a new class of immunosuppressants, rapamycin and its analogues, have reduced cutaneous Squamous Cell Carcinomas (cSCCs). Rapamycin, an mTORC1 inhibitor, is also a known autophagy inducer in experimental models. Autophagy, which literally means self‐eating, is a cell survival mechanism but can also lead to cell death. Therefore, the main hypothesis behind this work is that rapamycin prevents epidermal tumourigenesis by either affecting epidermal mTOR regulation of autophagy and/or selectively affecting epidermal AKT isoform activity. Epidermal keratinocytes move from the proliferating basal layer upwards to the granular layers where they terminally differentiate, forming a layer of flattened, anucleate cells or squames of the cornified layer which provides an essential environmental barrier. However, epidermal terminal differentiation, a specialised form of cell death involving organelle degradation, is poorly understood. The work presented in this thesis shows that analysis of the autophagy marker expression profile during foetal epidermal development, indicates autophagy is constitutively active in the terminally differentiating granular layer of epidermis. Therefore, I hypothesize that autophagy is a mechanism of organelle degradation during terminal differentiation of granular layer keratinocytes. In monolayer keratinocytes, activation of terminal differentiation is accompanied by autophagic degradation of nuclear material, nucleophagy. This suggests that constitutive autophagy is a pro‐death mechanism required for terminal differentiation. In cultured keratinocytes and in epidermal cultures, rapamycinmediated mTORC1 inhibition strongly increases AKT1 activity as well as up‐regulates constitutive granular layer autophagy promoting terminal differentiation. Therefore, autophagy is an important fundamental process in keratinocytes which may be the mechanism by which terminally differentiating keratinocytes of the epidermal granular layer degrade their organelles required for barrier formation. This may have implications for the treatment of patients with barrier defects like psoriasis. In immunosuppressed OTRs, rapamycin may promote epidermal autophagy and AKT1 activity adding to its anti‐tumourigenic properties.

Mutations in RHBDF2, the gene encoding inactive rhomboid protein iRHOM2, result in the dominantly inherited condition Tylosis with oesophageal cancer (TOC). TOC causes plamoplantar keratoderma, oral precursor lesions and up to a 95 % life-time risk of oesophageal squamous cell carcinoma (SCC). The role of iRHOM2 in the epidermis is not well characterised, although we previously showed dysregulated epidermal growth factor receptor (EGFR) signalling and accelerated migration in TOC keratinocytes, and a role for iRHOM2 was shown in trafficking the metalloproteinase ADAM17. Substrates of ADAM17 include EGFR ligands and adhesion molecules. iRHOM2 localisation and function were investigated in frozen sections and keratinocyte cell lines from control and TOC epidermis. Although iRHOM2 was predicted to be an ER-membrane protein, it showed cell-surface expression in control epidermis, with variable localisation in TOC. Increased processing and activation of ADAM17 was seen in TOC keratinocytes compared with control cells, suggesting that increased ADAM17-mediated processing of EGFR ligands may cause the changes in EGFR signalling. Downstream of iRHOM2-ADAM17, Eph/Ephrin and NOTCH signalling also appeared affected. Additionally, desmosomes in TOC epidermis lacked the electron-dense midline of the mature desmosomes seen in normal skin; this was accompanied by increased processing of desmoglein 2, a substrate of ADAM17. Expression and localisation of iRHOM2 was also investigated in TOC and sporadic SCC. iRHOM2 expression varied between SCC cell lines, and appeared to correlate with ADAM17 and NOTCH1 expression in oesophageal SCC and head and neck SCC cells. In summary, iRHOM2 mutations in TOC appear to be gain-of-function in nature, resulting in increased ADAM17 processing and enhanced EGFR signalling. Questions remaining include the reason why iRHOM2 is found at the plasma membrane. Future study of the iRHOM2-ADAM17 pathway may provide additional insight into the mechanism of epidermal wound healing and the pathogenesis of oesophageal SCC.

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.

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.