University of Macau / Faculty of Health Sciences

Resumo: O carcinoma de células transicionais (CCT) da bexiga possui como principal característica o alto índice de recorrência (70% dos carcinomas superficiais). Desta forma, é necessário acompanhar rigorosa e periodicamente os pacientes acometidos por tal neoplasia, bem como empregar técnicas sensíveis para a detecção precoce da doença, tanto em pacientes submetidos à ressecção do tumor de bexiga, quanto em pacientes considerados como grupo de risco para o desenvolvimento do CCT. O presente estudo tem por objetivo utilizar o teste do micronúcleo como ferramenta para a avaliação de danos cromossômicos em células uroteliais obtidas por lavado vesical de pacientes com história de CCT. A freqüência de células uroteliais micronucleadas foi avaliada em 77 pacientes (não tabagistas, tabagistas atuais e ex-tabagistas) sem ou com história de CCT, mas com diagnóstico atual negativo para neoplasia. Foi detectado aumento significativo (P=0,003) de células micronucleadas somente nos pacientes não fumantes e com história de CCT, quando comparados aos indivíduos do grupo controle (não fumantes e sem história de CCT). Não foram detectados efeitos do tabagismo na freqüência de células micronucleadas, e nem associação desse hábito com o grau do tumor. Concluindo, indivíduos não tabagistas com história de neoplasia urotelial apresentaram freqüência aumentada de micronúcleos em células esfoliadas da bexiga, mesmo após a ressecção do tumor. Portanto, o epitélio citologicamente normal da bexiga de indivíduos com história de CCT, pode apresentar células geneticamente instáveis, que poderiam conferir um risco aumentado para desenvolvimento neoplásico. / Abstract: The main feature of transitional cell carcinoma (TCC) is the high recurrences rates of superficial carcinomas. Therefore, patients must be monitored regularly by periodic cystoscopies. The employment of sensible techniques are important for detecting bladder cancer and early disease in patients undergoing tumor resection and individuals with high risk for tumor development. To evaluate whether cytogenetic disorders can be evolved in the tumor development and recurrences, the frequency of micronucleated cells (MNC) was established in non-neoplastic exfoliated bladder cells from patients with history of TCC. Seventy-seven patients with and without history of bladder cancer, either smokers or non-smokers, with current diagnosis negative for neoplasia were included. The results showed a significant increase (P < 0.01) of MNC in patients with history of TCC and non-smokers when compared to counterpart group (without history of TCC and non-smokers). However, the same association was not observed in patients with TCC and smokers and in patients without history of TTC and smokers. Furthermore, was not observed correlation between smoking habits and tumor grade. These results suggesting that non smokers with history of urothelial tumor had an increase of MNC even after tumor resection. Thus, the macroscopically normal looking urothelium of patients of history of TCC, still could be harbored genetically instable cells that can be related to high risk for neoplastic development. / Orientador: Maria Luiza Cotrim Sartor De Oliveira / Coorientador: Daizy Favero Salvatori / Banca: Mário Sérgio Montovani / Banca: Rosa Marlene Viero / Mestre

Prostate cancer (PCa) is the second most common cause of cancer-related deaths in men in the Western world. Advanced PCa is initially managed by anti-androgen therapy however, resistance frequently develops resulting in progressive metastatic disease. The current standard of care for hormone-insensitive PCa includes the cytotoxic drugs docetaxel and mitoxantrone although resistance rapidly develops to all available therapies. We demonstrated that the replication-selective oncolytic adenoviral mutant AdΔΔ enhanced drug-induced cell killing in several preclinical cancer models. AdΔΔ is deleted in the viral E1ACR2 and E1B19K, to prevent pRb-binding and enhance drug-mediated apoptotic cell killing, respectively. In drug-insensitive PCa tumour-xenografts, in vivo administration of AdΔΔ greatly enhanced drug-mediated tumour regression. The aim of my thesis project was to investigate the role of apoptosis and pro-survival pathways, including drug-induced autophagy, in AdΔΔ-mediated drug-sensitisation. I have demonstrated that autophagy was activated in a dose-dependent manner in response to mitoxantrone in the human PCa cell lines PC3, PC3M and 22Rv1. Low doses of mitoxantrone (< EC50-values) caused initiation of autophagy, determined as increased conversion of LC3I to LC3II and increased number of acidic vesicles, indicating autophagosome formation. At higher doses degradation of p62 was also observed, suggesting autophagosome fusion with the lysosome. AdΔΔ attenuated the drug-induced activation of autophagy by restoring basal LC3II/I ratios, and increasing apoptosis, determined as increased PARP-cleavage and mitochondrial depolarization. The autophagyinducer rapamycin prevented AdΔΔ-mediated sensitization in PC3 cells increasing mitoxantrone EC50-values 3-fold and attenuating apoptosis induction. In contrast, the autophagy-inhibitor chloroquine further sensitized PC3 and 22Rv1 cells to the combinationtreatment, decreasing mitoxantrone EC50-values by 40% and increasing apoptotic cell death. Atg7 is a key-factor in the autophagy pathway and siRNA-mediated knockdown prevented increases in LC3II/I ratios. In siAtg7-transfected PC3 cells mitochondrial depolarization was further promoted in combination-treated cells, similar to the results with chloroquine. The cellular Bcl-2-protein has important roles as a mediator of both anti-apoptotic and antiautophagic functions. In cells transfected with siBcl-2 the LC3II/I ratios increased and AdΔΔ- mediated sensitization to mitoxantrone was prevented, indicating initiation of autophagy. In addition, mitoxantrone-induced degradation of Bcl-2 was attenuated by AdΔΔ infection, suggesting stabilization of the protein. The mechanisms for the AdΔΔ-mediated increases in cell killing were also demonstrated in 3-dimensional co-culture models of PC3 or 22Rv1 in combination with prostate stromal cells and extracellular matrix proteins using confocal microscopy. These data demonstrate that AdΔΔ attenuates drug-induced cell survival/rescue and promotes elimination of cancer cells through apoptosis and viral lysis, and that Bcl-2 was essential for the sensitisation.

Edar is a member of the death receptor subfamily of TNF receptors that plays an important role in the development of ectodermal appendages. Mutations in this signalling pathway cause the developmental disorder hypohidrotic ectodermal dysplasia (HED). HED has a similar phenotype in a number of mammals, and includes improper development of ectodermal appendages such as hair, teeth and glands. Edar signalling has not previously been linked with tumourigenesis, however, increased NFĸB signalling has been associated with a number of cancers, including breast cancer, and is known to be downstream of the Edar pathway. In addition, Edar signalling is important in mammary gland development, and enhanced Edar signalling in mice has been associated with precocious elongation and branching of mammary gland ducts. Recent research has shown that overexpression of Edar in Edartg951/951 mice leads to the development of mammary gland tumours, particularly in multiparous mice. These tumours were ER negative and displayed squamous metaplasia. EDAR expression was also found to be upregulated in a subset of human metaplastic breast cancers, suggesting that activation of Edar signalling might play a role in the aetiology of these tumours. In this investigation, immunohistochemical staining and qRT-PCR was used to show that the Edartg951/951 mammary gland tumours had high levels of Wnt and Edar signalling. Using DNA sequencing and Western blot assays, we showed that the tumours in Edartg951/951 mice all contained truncations in β-catenin, specifically deletions of exon 3 of the β-catenin gene. This mutation was found to be somatic, and specific to the mammary gland. The Edartg951/951 mice were found to have an increased number of dying cells in the lumen of the mammary gland during involution day 2, and were susceptible to mastitis during early involution. We have also shown using immunohistochemistry that the Edartg951/951 mammary glands with mastitis stained positively for phospho γ-H2A.X, with high levels of cytoplasmic β-catenin in the affected cells. The majority of Edartg951/951 mammary glands with mastitis showed an increase in mAID expression using qRT-PCR analysis, and a Western blot assay showed that the majority contain a truncated form of β catenin. These findings suggest that the mastitis may have generated a mutagenic environment, able to promote tumourigenesis via expression of mAID. Finally, a soft agar assay showed that Edar expression was transforming in EpH4 cells.

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No description available.

Colorectal cancer is a major public health problem and a leading cuase of cancer deaths in the western world. At present, three monoclonal antibodies (mAbs) namely anti-epidermal growth factor receptor (EGFR) cetuximab and panitumumab and anti-vascular endothelial growth factor mAb bevacizumab have been approved for the treatment of patients with metastatic colorectal cancer. However, many patients simply do not respond, or the cancer aquires resistance following a short course of therapy. The aim of this study was to develop a new panel of mouse monoclonal antibodies directed against other cell surface antigens which are over-exoressed on human colorectal tumour cells using hybridoma technology and to investigate their potential as diagnostic and therapetic agents. A panel of human colorectal cancer cell lines, established from colorectal cancer patients at different stages of the disease, were employed as the source of immunogen for immunisation of a group of mice. Seven novel mouse hybridomas were developed, secreting seven novel mAbs named KU2.77, KU2.90, KU3.39, KU3.47, KU3.61, KU4.76 and KU4.94. Following purification, the binding efficacy of each purified mAb to a large panel of colorectal tumor cells, human foreskin fibroblast (DE532) and human immortalized keratinocyte (HACAT) cells was determined using ELISA and flow cytometry. All seven mAbs are directed against cell surface antigens which are over-expressed on human colorectal cancer cells. The differential binding of these mAbs to the various human colorectal tumour cells and to the other cells suggests that these antibodies may be directed against at least three distinct over-expressed cell surface antigens. Interestingly, of the seven mAbs examined, KU3.47 and KU4.94 did not bind to DE532 and HACAT cells suggesting that the antigen recognised by these two antibidies may be tumour specific antigens. at 300nM, with the exception of mAb KU3.39 which induced slight growth inhibition of CCL-228 and Colo2 cells, the growth of other colorectal cancer cells were not inhibited by the any of the antibodies while only two mAbs (KU2.77 and KU2.90) inhibited the migration of CCL-228 cells. Out of the seven novel mAbs, KU3.47, KU4.94 and KU3.61 stained their respective antigens in formalin-fixed, paraffin-embedded, colorectal tumour cell pellets, suggesting that these antibodies are directed against a sequential determinant on the antigen and could therefore have potential for use as tools for investigating the expression pattern, prognostic significance and predictive value of such antigens in cancer patients. The preliminary results of immunoprecipitation followed by mass spectroscropy revealed that three newly developed mAbsKU2.77, KU3.39 and KU4.94 may recognise or cross react with integrin alpha-3, Large neutral amino acid transporter small subunit1 (LAT-1) and apo-lipoprotein A-IV respectively. Since all seven novel mAbs are directed against over-expressed cell surface antigens, they could form excellent tools for use in basic research for investigating the role of such antigens in the progression of human cancers. Further investigations are warranted to identify the target antigens recognised by each of the novel antibodies and to determine their full potential as diagnostic agents and therapeutic agents when conjugated to various toxins, drugs or radioisotopes.

Malgré les progrès dans les traitements contre le cancer, cette maladie demeure difficile à contrôler au stade métastatique. La migration des cellules tumorales est une étape majeure du processus métastatique qui implique la réorganisation du cytosquelette. Dans ce contexte, notre équipe a caractérisé une voie de signalisation par laquelle le récepteur oncogénique ErbB2 régule la migration cellulaire via le contrôle de la dynamique des microtubules (MT). La protéine EB1, qui s’associe aux extrémités + des MT, est un acteur majeur du réseau de protéines favorisant la capture des microtubules. Dans l’objectif de mieux définir les mécanismes qui régulent la capture des MT, nous avons caractérisé l’interactome d’EB1. J’ai ainsi identifié iASPP, un inhibiteur du suppresseur de tumeur p53, comme un nouveau partenaire d’EB1. Mes résultats montrent qu’iASPP participe à la capture des MT au cortex cellulaire lors de la migration. De manière intéressante, la déplétion de iASPP a aussi des conséquences sur la mitose, induisant un défaut du positionnement du fuseau, dû à un ancrage asymétrique des microtubules astraux au cortex. Ces fonctions de iASPP dépendent de son interaction spécifique avec EB1 via un motif SxIP, mais sont indépendantes de son association à p53. iASPP interagit aussi avec des protéines associées au cortex cellulaire dont la Myosine 1c, qui participe également au bon positionnement du fuseau mitotique. Nous proposons que le complexe EB1-iASPP-Myo1c contribue à la capture des MT lors de la division et de la migration des cellules tumorales et pourrait constituer à terme une cible thérapeutique. / Despite significant progress in the treatments of cancer, this disease is still difficult to manage at the metastatic grade. Tumor cell migration is a major step in the metastatic process which involves cytoskeleton reorganization. In this context, our group has characterized a signal transduction pathway driven by the oncogenic receptor ErbB2 which controls microtubule dynamics and cell migration. EB1, a protein associating with the +end of MT, is a key actor of proteins network promoting MT capture. In order to characterize mechanisms that regulate MT dynamics, we have defined EB1 interactome. I identified iASPP, an inhibitor of the p53 tumor suppressor as a partner of EB1. My results show that iASPP contributes to MT capture at the cell cortex during cell migration. Interestingly, iASPP depletion has consequences on mitosis, inducing a defect in mitotic spindle positioning most likely due to asymmetric astral MT anchoring at the cell cortex. iASPP functions are dependent on its specific interaction with EB1 via a « SxIP » motif, but are independent of its interaction with p53. iASPP interacts also with proteins associated to the cell cortex of which Myosin 1C participates, as well, to the correct positioning of the mitotic spindle. We propose that EB1-iASPP-Myo1c complex contribute to MT capture during division and migration of tumoral cells and could represent a therapeutic target.

Master of Dental Surgery / This work was digitised and made available on open access by the University of Sydney, Faculty of Dentistry and Sydney eScholarship . It may only be used for the purposes of research and study. Where possible, the Faculty will try to notify the author of this work. If you have any inquiries or issues regarding this work being made available please contact the Sydney eScholarship Repository Coordinator - ses@library.usyd.edu.au

I have been intensively involved in the research on the application of currently available knowledge for the improvement of cancer care. This research covers the types of treatment that are appropriate for different clinical conditions (benchmarking and guidelines), planning services to improve access for patients, monitoring service delivery through patterns of care studies and development of the knowledge and skills of the cancer workforce. I devoted considerable effort to better educating the cancer workforce by measuring cancer teaching and developing model curricula and innovative teaching programs. I have made substantial contributions to knowledge about best practice by developing clinical practice guidelines and have developed tools and plans for cancer service delivery and have had a major influence on the training of the undergraduate and specialist medical workforce about cancer.