As alterações genéticas mais frequentes em câncer de pulmão são mutações pontuais que ativam o oncogene KRAS. Embora estas mutações estejam causalmente relacionadas à oncogênese, até hoje diferentes abordagens para inibir as proteínas RAS diretamente não obtiveram sucesso. Portanto, para que melhores alvos terapêuticos para o câncer de pulmão se tornem disponíveis é necessário identificar os mecanismos moleculares ativados por KRAS que estão diretamente envolvidos com a aquisição de propriedades malignas importantes, como o desenvolvimento e a manutenção de um fenótipo tronco-tumoral pelas células iniciadoras de tumor (CITs). CITs, também conhecidas como células tronco-tumorais, são definidas como uma subpopulação de células tumorais capazes de se autorrenovar, iniciar a formação de tumores e sustentar o crescimento tumoral. O desenvolvimento de estratégias terapêuticas dirigidas a estas células é imprescindível para melhorar a eficácia da terapia antitumoral. Uma vez que KRAS está associada a manutenção de um fenótipo tronco-tumoral e ativa o fator de transcrição NF-kB através da quinase IKKβ para promover a tumorigênese pulmonar, nós hipotetizamos que a quinase IKKβ contribui para o fenótipo tronco-tumoral induzido por KRAS em câncer de pulmão. Nós utilizamos ensaios de formação de tumoresferas para enriquecer e avaliar a função de CITs das linhagens pulmonares positivas para KRAS A549 e H358. As células A549 e H358 formaram tumoresferas em cultura de baixa aderência e, quando comparadas às células derivadas da cultura aderente, as células oriundas da cultura de tumoresferas apresentaram maior crescimento clonogênico, maior expressão de genes associados ao fenótipo tronco por qPCR e maior atividade da quinase IKKβ. A inibição da atividade de IKKβ através de um inibidor farmacológico altamente específico (Composto A) diminuiu levemente a proliferação de células A549 e H358, sem resultar em morte celular significativa. Entretanto, a inibição da atividade ou da expressão de IKKβ por interferência de RNA reduziu a expressão de genes associados ao fenótipo tronco e diminuiu a formação de tumoresferas. A inibição da expressão de IKKβ em células A549 reduziu também a capacidade de autorrenovação de CITs. Estes resultados sugerem que IKKβ desempenha um papel importante na manutenção do fenótipo tronco-tumoral de CITs pulmonares induzidas por KRAS. Em seguida, nós demonstramos que a inibição da atividade de IKKβ afetou preferencialmente a proliferação celular e o crescimento clonogênico de células oriundas da cultura de tumoresfera, sugerindo que IKKβ desempenha um papel mais importante em CITs do que em células derivadas da cultura aderente. A análise por citometria de fluxo identificou que células derivadas da cultura de tumoresfera apresentam um enriquecimento para células CD24+ na linhagem A549 e células CD44+ na linhagem H358, sugerindo que estes possam ser marcadores promissores para purificação de CITs nestas linhagens. Adicionalmente, demonstramos, por ensaios de wound-healing de células A549 e H358, que a inibição da atividade de IKKβ reduziu a migração celular, uma outra uma propriedade aumentada em CITs. Além disso, mostramos que a atividade da quinase IKKβ em células A549 e H358 não depende das vias da MAPK ou PI3K/Akt. Interessantemente, a inibição combinada de IKK (um efetor downstream de KRAS) e de EGFR/ERRB2 (reguladores upstream de KRAS que ativam as vias MAPK e PI3K/Akt) reduziu de forma aditiva a formação de tumoresferas, proliferação e migração celular. Quando avaliados em conjunto, nossos resultados sugerem que a quinase IKKβ desempenha um papel importante na biologia de CITs pulmonares portadoras de KRAS oncogênica e que a inibição desta quinase sozinha ou em combinação com a inibição de outras vias pode representar uma estratégia terapêutica promissora a ser explorada para reduzir a recidiva e metástase no câncer de pulmão induzido por KRAS. / The most frequent genetic alterations in lung cancer are point mutations that activate the KRAS oncogene. Although these mutations are causally related to oncogenesis, different approaches to inhibit RAS proteins directly have not been successful to date. Therefore, for better therapeutic targets for lung cancer to become available, it is necessary to identify the molecular mechanisms activated by KRAS that are directly involved with important malignant features, such as the development and maintenance of a cancer stem-like phenotype by the tumour-initiating cells (TICs). TICs, also known as cancer stem cells, are defined as a subpopulation of tumour cells able to self-renew, promote tumour initiation, and sustain tumour growth. The development of therapeutic strategies to target these cells is imperative to improve the efficacy of antitumor therapy. Since KRAS is associated with the maintenance of a cancer stem-like phenotype and activates the transcription factor NF-kB through the IKKβ kinase to promote lung tumourigenesis, we hypothesised that IKKβ kinase contributes to the cancer stem-like phenotype induced by KRAS in lung cancer. We used tumoursphere formation assays to enrich and evaluate the function of TICs of KRAS-mutant cell lines A549 and H358. A549 and H358 cells formed tumourspheres in low adhesion culture and, when compared to cells grown in adherent culture, sphere-derived cells displayed increased clonogenic growth, higher expression of stemness genes by qPCR, and increased IKKβ kinase activity . Inhibition of IKKβ activity through a highly specific pharmacological inhibitor (Compound A) slightly decreased proliferation of A549 and H358 cells without inducing significant cell death. On the other hand, inhibition of IKKβ activity or expression by RNA interference reduced the expression of stemness genes and decreased tumoursphere formation. Inhibition of IKKβ expression in A549 cells also reduced TICs self-renewal . These results suggest that IKKβ plays an important role in maintaining the cancer stem-like phenotype of KRAS-driven lung TICs. Next, we demonstrated that IKKβ inhibition preferentially reduced cell proliferation and clonogenic growth of sphere-derived cells, suggesting that IKKβ plays a more important role in TICs than in adherent culture-derived cells. Flow cytometry analysis identified that sphere-derived cells display an enrichment for the surface marker CD24 in A549 cells and CD44 in H358 cells, indicating that these could be promising markers for the purification of TICs in these cell lines. Furthermore, we have shown by wound-healing assays of A549 and H358 cells that IKKβ inhibition reduced cell migration , another feature increased in TICs. In addition, we have shown that IKKβ activity in A549 and H358 cells does not depend on the MAPK or PI3K/Akt pathways. Interestingly, combined inhibition of IKKβ (a downstream effector of KRAS) and EGFR/ERBB2 (upstream regulators of KRAS that activate the MAPK and PI3K/Akt pathways) additively reduced tumoursphere formation, cell proliferation and migration. Taken together, our results suggest that IKKβ kinase plays an important role in the biology of KRAS-driven lung TICs, and that inhibition of this kinase alone or in combination with inhibition of other signalling pathways may represent a promising therapeutic strategy to be explored in order to reduce tumour recurrence and metastasis in KRAS-driven lung cancer.

Classic Hodgkin Lymphoma (cHL) has an annual incidence of 2.4 cases per 100 000 population in the UK, and is one of the most common malignancies diagnosed in young adults aged 15 to 34. The majority of younger patients have a good long-term outcome with between 80 and 90% disease-specific survival but cHL also affects older adults in whom the prognosis is significantly poorer. The role of tumour-associated macrophages (TAM) in cHL has gained much interest, with several studies reporting an association between high numbers of CD68-positive TAM and poor prognosis. There is also a question over the prognostic significance of Epstein-Barr Virus (EBV) infection which is implicated in up to 50% of cHL cases in developed countries. Published data suggests that EBV positivity in elderly patients may be associated with a poorer outcome, whereas in younger adults may be of prognostic benefit. Differences related to age are of interest particularly as an age-related decline in immunity has been linked with the development of certain subtypes of Non-Hodgkin Lymphoma in older patients. In a retrospective study, two separate cohorts of patients with cHL were examined with the aim of identifying: • Differences in the cellular composition of the tumour microenvironment in cHL which has arisen in young and elderly adult patients; • Differences in the cellular composition of the tumour microenvironment in cHL associated with or without EBV infection; • Factors within the tumour microenvironment which may influence prognosis and may be targeted for novel treatments. One group consisted of patients aged between 15 and 34 years at diagnosis and the other, of those aged 60 or over at presentation. Tissue obtained at the time of diagnosis was examined with regard to a number of factors related to the malignant cells and the surrounding microenvironment, including the number and phenotype of macrophages, the number of plasmacytoid dendritic cells and the number of malignant Hodgkin Reed-Sternberg (HRS) cells and non-malignant ‘background’ cells undergoing apoptosis. Comparisons were made between the two age groups, also taking into account the EBV-status of tumours, cHL subtype and gender. Results confirmed the current understanding that EBV-positive cHL is more common in older patients and has a strong, but not exclusive, association with the MCHL subtype. In addition, a strong link between young males and EBV-positive disease was shown. Macrophages were found to vary between the two age groups, in number and phenotype and there were clear differences associated with the presence or absence of EBV infection. While no definite link with outcome and macrophages was identified it was apparent that the implications of macrophages in the tumour microenvironment may differ between the two age groups. The number of apoptotic cells correlated closely with the number of macrophages and in the young the number of HRS cells was associated with prognosis. Investigation of the tumour microenvironment is complex and caution is needed in interpreting studies which do not differentiate between patients according to age, as tumour characteristics may have variable implications in different age groups. In this thesis a number of clinicopathological differences were identified between the two age groups. These point to the need for further larger studies to delineate how such age-related differences may or may not be associated with immune function and how this information could be translated into treatments to improve outcomes.

p16INK4a (p16) traps the cell at the restriction point of the cell cycle by binding to cyclin-dependent kinase 4/6 thus preventing the phosphorylation of the retinoblastoma protein (pRB). As p16 accumulates the cell stops dividing and becomes senescent. This study investigates the modulation of p16 function by the putative membrane protein ABCB5 and a group of five putative oncogenic microRNAs (oncomiRs). ABCB5 is a poorly characterised member of the B-subfamily of human ATP Binding Cassette transporters. ABCB5 is reportedly transcribed into four transcripts, one of which could potentially encode a full-length transporter (ABCB5fl) whilst a second could encode a half-transporter (ABCB5β). The other two transcripts (ABCB5α and ABCB5γ) could only encode short polypeptides. Exogenous expression of ABCB5fl and ABCB5β was achieved in HEK293T cells, but the recombinant protein expressed poorly and localised to the endoplasmic reticulum. Point mutations introduced into the ATP catalytic domain failed to improve expression levels suggesting that protein function was not deleterious to the cell. Exogenous expression in HEK293T cells also allowed commercial antibodies purportedly raised against ABCB5 isoforms to be tested. Several were found not to recognise ABCB5 necessitating re-interpretation of published data. However, one antibody recognised both ABCB5fl and ABCB5β, and was subsequently used to evaluate protein expression levels in other cell types.siRNA knockdown of ABCB5 in human mammary epithelial cells (HMECs) caused a concomitant reduction in p16 expression and an increase in cellular proliferation. Differential siRNAs and RT-qPCR analyses demonstrated ABCB5β to be the relevant transcript with respect to the reduction in p16 expression; however, no native ABCB5β protein was detected in HMECs. Together these data lead to the hypothesis that the ABCB5β transcript may act as a long noncoding RNA to regulate p16. Exogenous expression of each of five distinct putative oncomiRs in HMECs was found to increase cellular proliferation and, surprisingly, increase p16 expression. These results mirror a phenotype commonly observed in p16-positive basal-like breast cancer (BLBC), an aggressive form of breast cancer with poor prognosis and few treatment options. Bioinformatic analysis of the predicted target genes for these oncomiRs identified multiple transcriptional regulators of pRB. These predictions, together with the work performed in a cellular model of p16-positive BLBC, suggest that the oncomiRs may cause unrestricted cell proliferation by indirectly reducing transcription of the pRB gene, RB1. In the absence of pRB, p16 expression is induced via a previously reported oncogeneinduced senescence-like positive feedback loop. These data, and previously published observations, suggest that a similar mechanism may explain the basis of p16-positive BLBC.

Pancreatic Ductal Adenocarcinoma (PDAC) affects approximately 8000 people every year in the UK and is the fifth leading cause of cancer related death. At a molecular level PDAC is characterized by a significant immune infiltrate. Tumour-associated macrophages (TAMs) infiltrate the tumour and contribute to a worse prognosis by promoting growth, metastasis and resistance to chemotherapy. TAMs are derived from circulating ‘classical’ CD14++ CD16- monocytes in the peripheral blood. Current work in murine models suggests targeting monocyte recruitment in PDAC can reduce TAM infiltration and disease burden therefore improving survival. This project aims to identify markers specific to monocytes from PDAC patients and to investigate their biological relevance and potential for therapeutic intervention. Gene expression and metabolomics analysis was carried out on classical CD14++ CD16- monocytes from locally advanced PDAC patients and age matched healthy donors. Transcriptomic profiling revealed a significantly altered gene expression profile in classical monocytes from patients and genes with the highest fold change difference were chosen for validation using qPCR. Validated gene targets were investigated further in vitro and large-scale gene expression analysis from pancreatic tumours assessed. The results from my work demonstrate that the gene expression profile of classical monocytes from PDAC patients is significantly different compared to healthy volunteers. Identification and validation of up-regulated genes and their biological relevance may represent a relevant novel novel biomarker or therapeutic strategyies to target monocytes and myeloid recruitment in cancer.

The MYC onocogene is frequently overexpressed in human cancer due to its capacity to promote cell growth and cell proliferation. MYC overexpression activates the p53 tumour suppressor pathway, which resists the pro-tumourigeneic program elicited by MYC. How MYC overexpression engages p53 is yet to be elucidated, and in this study I carried out a large metabolic siRNA screen to determine whether p53 responds to a specific MYC-driven metabolic pathway. Two clear lipid metabolic pathways emerged from the siRNA screen: PPARγ/arachidonate metabolism and de novo sphingolipid synthesis. Knockdown or inhibition of PPARγ increased p53 levels, and PPARγ ligands decreased following MYC overexpression. Knockdown of ceramide synthesis depleted p53 levels, and MYC overexpression increased de novo ceramide synthesis. This demonstrated that MYC-driven ceramide synthesis positively regulates p53, and highlights the role of cell metabolism in the tumour suppressor response to MYC deregulation.

Conventional treatments for prostate cancer have significant limitations making it difficult to control the disease. Cyclopropabenzindoles (CBI) are more biologically potent, stable and synthetically accessible analogues of cyclopropapyrroloindole (CPI) anti-tumour antibiotics, such as duocarmycin-SA and CC1065. A polymeric prodrug carrying a CBI drug attached to the polymeric backbone through a PSA cleavable linker peptide has two modes of selectivity: activation by PSA and the EPR effect. To synthesise a 5-amino-seco-CBI analogue, 2,4-dinitronaphthalen- 1-ol gave di-Boc-1-iodonaphthalene-2,4-diamine in five steps (triflation, SNAr displacement with iodide, reduction (loss of iodine), protection and restoration of the iodine. For the amino-seco-CBI, it was important to discriminate between N2 and N4. Acidic removal of the Boc-group(s) resulted in deiodination. NMR investigations showed an unexpected Wheland-like cationic intermediate. N3 of naphthalene-1,3-diamine was selectively trifluoroacetylated and N1 was masked with Boc. Electrophilic iodination gave an orthogonally protected 1-iodonaphthalene-2,4-diamine. Allylation at the trifluoroacetamide was followed by free radical cyclisation with TEMPO trap. Removal of the trifluoroacetyl group allowed coupling to 5-(2-(dimethylamino)ethoxy)-1H-indole-2-carboxylic acid. Reductive removal of 2,2,6,6-tetramethylpiperidine, substitution of the exposed hydroxy group with chloride and removal of the Boc-group gave the amino-seco-CBI drug, 5-amino-1-chloromethyl-3-(5-(2-dimethylaminoethoxy)indole-2-carbonyl)-2,3-dihydro-1H-benz[e]indole. A DNA-melting assay confirmed that it binds very strongly to dsDNA causing a 13 deg. C increase in melting temperature. The drug was a highly potent cytotoxin in vitro, with IC50 = 18 nM against LNCaP prostate cancer cells. The polymeric prodrug system involved the synthesis of the pentapeptide SSKLQ. The amide side chain of glutamine can be masked as the nitrile and this can be quantitatively hydrated to the γ-carboxamide of L-Gln with hydroperoxide. The pentapeptide was coupled to 4-methoxynaphthalen-1-amine and to poly(ethylene glycol) as a model polymeric prodrug system. Efficient release of the model drug from the polymeric prodrug by PSA will allow this polymeric prodrug system to be adopted for the synthesised amino-seco-CBI drug.

Intratumoural heterogeneity complicates the molecular interpretation of biopsies, as multiple distinct tumour genomes are sampled and analysed at once. Ignoring the presence of these populations can lead to erroneous conclusions, and so a correct analysis must account for the clonal structure of the sample. Several methods to reconstruct tumour clonality from sequencing data have been proposed, spanning methods that either do not consider phylogenetic constraints or posit a perfect phylogeny. Models of the first type are typically latent feature models that can describe the observed data flexibly, but whose results may not be reconcilable with a phylogeny. The second type, instead, generally comprises non-parametric mixture models, with strict assumptions on the tumour’s evolutionary process. The focus of this dissertation is on the development of a phylogenetic latent feature model that can bridge the advantages of these two approaches, allowing deviations from a perfect phylogeny. The work is recounted by three statistical models of increasing complexity. First, I present a non-parametric model based on the Indian Buffet Process prior, and highlight the need for phylogenetic constraints. Second, I develop a finite, phylogenetic extension of the previous model, and show that it can outperform competing methods. Third, I generalise the phylogenetic model to arbitrary copy-number states. Markov chain Monte Carlo algorithms are presented to perform inference. The models are tested on datasets that include synthetic data, controlled biological data, and clinical data. In particular, the copy-number generalisation is applied to longitudinal circulating tumour DNA samples. Liquid biopsies that leverage circulating tumour DNA require sensitive techniques in order to detect mutations at low allele fractions. One method that allows sensitive mutation calling is the amplicon sequencing strategy TAm-Seq. I present bioinformatic tools to improve both the development of TAm-Seq amplicon panels and the analysis of its sequencing data. Finally, an enhancement of this method is presented and shown to detect mutations de novo and in a multiplexed manner at allele fractions less than 0.1%.

There is a need for informative biomarkers in localised urological cancers. At present, no method can accurately distinguish between indolent and aggressive prostate cancers, and men often require repeated biopsies. Patients with muscle invasive bladder cancer undergo neo-adjuvant chemotherapy (NAC) to improve survival. However many do not respond to NAC, delaying definitive treatment. Cell-free mutant DNA (mutDNA) analysis represents an opportunity for non-invasive monitoring of cancer through tumour genome analysis. MutDNA derived from plasma can monitor tumour burden. There is emerging evidence that mutDNA can identify mutations from multiple clones and is abundant in adjacent body fluids. This work explores the utility of plasma and urinary mutDNA in localised prostate and bladder cancers. This thesis describes the optimisation of urinary mutDNA analysis by assessing urinary DNA processing and extraction methods using healthy volunteer and bladder cancer patient urine samples. Primer panels were designed and validated to target frequently mutated regions in prostate and bladder cancers, as well as for analysis of patient-specific mutations. Sequencing-based methods and dPCR were employed to analyse clinical samples including plasma and urine, to detect and quantify mutDNA. Molecular and clinical data were integrated to explore potential areas of application of mutDNA analysis. For bladder cancer, mutDNA was analysed from liquid-biopsy samples including plasma, cell pellets from urine and urine supernatant from multiple time-points of 17 MIBC patients undergoing NAC. I showed that mutDNA was more frequently detected and was present at higher AFs in urine compared to plasma samples. Of potential clinical relevance, I showed that the presence of mutDNA after starting NAC was associated with disease recurrence. This original contribution to knowledge could offer patients an opportunity to expedite surgical resection in a timely manner, if corroborated in large-scale trials. For prostate cancer, a TP53 specific panel was applied to men with metastatic disease, to demonstrate that clones containing TP53 mutations, which are dominant in at the metastatic stage were present in historical prostatectomy samples taken when then patient was believed to have localised disease only. Furthermore, I showed that these TP53 mutations could be detected at the localised stage of disease. To investigate the ability of mutDNA detection private clonal mutations I developed a method for higher sensitivity analysis (MRD-Seq). This was applied to a clinical cohort of 2 men with multi-focal localised prostate cancer to demonstrate the though the overall levels of mutDNA is low, private clonal mutations may be detectable. Taken together, these original contributions to knowledge could allow for less invasive surveillance of men with low risk prostate cancer and warrants further investigation. In this thesis, I used a range of molecular methods were applied to small cohorts of clinical samples from patients with urological malignancies, in an exploratory analysis. The molecular data was analysed in conjunction with clinical information to draw hypotheses on the biology and natural history of these cancer, and to suggest possible utility of mutDNA analysis in their clinical management. Some of the findings suggest areas of potential utility, which merit further validation or investigation in larger cohorts or clinical studies.

Orientador: Sandra Aparecida Drigo Linde / Resumo: Introdução: Apesar dos avanços no tratamento, cerca da metade dos pacientes com câncer de reto (CR) desenvolverá metástase à distância. No entanto, as vias biológicas envolvidas na progressão do câncer não são totalmente conhecidas. Neste estudo, investigamos os perfis moleculares e imunológicos em adenocarcinomas de reto relacionados à progressão metastática visando identificar biomarcadores moleculares e/ou alvos terapêuticos. Pacientes e Métodos: O transcritoma de 15 tecidos de CR metastático (M) e não-metastático (NM) pré-tratamento e de duas amostras de tecido de reto normais foi avaliado utilizando a plataforma Clariom D. Os genes foram considerados diferencialmente expressos quando a alteração de expressão era maior que 2 vezes e o valor de p <0,05 e detectados com o pacote limma. As funções moleculares e vias biológicas foram determinadas com a ferramenta Enricher. Os achados foram validados utilizando dados do TCGA e o perfil imunológico determinado com o algorótimo xCell. Resultados: A comparação entre os grupos M e NM revelou 52 genes diferencialmente expressos, sendo 27 regulados positivamente e 25 regulados negativamente. O gene ANLN foi detectado com o maior valor de fold change nos tumores metastáticos. Além disso, expressão aumentada de ANLN foi associada com menor sobrevida em pacientes com CR. A via do fator de crescimento endotelial vascular (VEGF) foi detectada como alterada nos tumores M. Validação dos resultados com dados do TCGA confirmou o gene ANLN co... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Introduction: Despite advances in treatment, about half of patients with rectal cancer (RC) will develop distant metastasis. However, the biological pathways underpinning the cancer progression are not fully understood. In this study, we sought to identify molecular and immunological profiles in rectal adenocarcinomas related to metastatic progression aiming to identify molecular biomarkers and/or therapeutic targets. Patients and Methods: Transcriptome analysis of 15 pre-treatment metastatic (M) and non-metastatic (NM) rectal cancer tissues and two normal rectal tissue samples was evaluated using Clariom D platform. Genes were considered differentially expressed when presenting 2-fold change and p<0.05 and were obtained with limma package . Molecular function and biological pathways with the Enricher package. Our findings were validated from the TCGA database and the immunological profile was determined using the xCell algorithm. Results: The comparison of M with NM groups revealed 52 differentially expressed genes, being 27 up-regulated and 25 down-regulated. ANLN gene was detected as the top gene upregulated in M tumours. Additionally, ANLN overexpression was associated with shorter survival in RC patients. Vascular endothelial growth factor (VEGF) pathway was detected as altered in M tumours. Cross-study validation with TCGA dataset confirmed ANLN gene as associated with M tumours. Furthermore, KIF14, XRCC2 and GPX3 genes, which have important carcinogenesis functions, we... (Complete abstract click electronic access below) / Mestre

A matriz extracelular (MEC) pode ser definida como um complexo de proteínas e glicoproteínas que envolve as células nos mais diversos tecidos e tem papel importante na diferenciação e atividade celular, bem como no processo de mineralização e nos processos neoplásicos. Os componentes não colágenos da MEC têm sido abundantemente estudados, visando conhecer os minuciosos detalhes da biologia dos tecidos e assim entender os mecanismos envolvidos em suas patologias. Neste contexto, o presente trabalho tem como objetivo estudar a expressão e distribuição dos seguintes componentes não colágenos da MEC dos tecidos dentais: biglican, decorin, fibromodulin, osteonectina (ONC), osteopontina (OPN), sialoproteína óssea (BSP) e osteocalcina (OCC) no ameloblastoma e no tumor odontogênico cístico calcificante (cisto de Gorlin). Para ta nto foi utilizada a técnica da imunoistoquímica, com o método da estreptavidina-biotina-peroxidase, e anticorpos contra as proteínas anteriormente citadas. Os resultados mostraram que o biglican, o decorin e a BSP foram expressos somente nas células epiteliais metaplásicas, nas células fantasmas e células fantasmas em processo de calcificação, no estroma dos ameloblastomas e no ectomesênquima neoplásico do tumor odontogênico cístico calcificante. Já o fibromodulin e a OC foram predominantemente negativos no componente epitelial e no mesenquimal, com exceção para as células fantasmas, células fantasmas em processo de calcificação e áreas de hialinização próximas ao epitélio. A ONC foi positiva na maioria das células epiteliais, com exceção das células estrelárias dos ameloblastomas folicular e acantomatoso, e também no componente mesenquimal de ambas neoplasias. Já a OPN apresentou positividade somente nos focos de calcificação presentes no tumor odontogênico cístico calcificante. As proteínas estudadas apresentaram distribuição semelhante em neoplasias caracterizadas por padrões de crescimento diferentes, levando a crer que, apesar de participarem ativamente do mecanismo de crescimento neoplásico intra-ósseo, isoladamente não exercem papel decisivo na determinação do tipo de padrão de crescimento. Outro fato digno de relevância é a baixa expressão dessas proteínas nas células epiteliais neoplásicas quando comparada com a expressão no estroma e ectomesênquima, levando-nos a crer que as células epiteliais atuem principalmente como estimuladores da expressão dessas proteínas, que, por sua vez, podem atuar de forma agonista ou antagonista ao crescimento neoplásico. / The extracellular matrix (ECM) can be defined as a complex of proteins and glycoproteins that involves the cells in all tissues. It has a key role in cell differentiation and activity, as well as in mineralization and neoplastic processes. The non-collagenous components of the ECM have been abundantly studied to know the details of the biology of tissues and thus to understand the mechanisms involved in its pathologies. The aim of the present work is to study the expression and distribution of the following noncollagenous components of the ECM of dental tissues: biglycan, decorin, fibromodulin, osteonectin (ONC), osteopontin (OPN), bone sialoprotein (BSP) and osteocalcin (OCC) in ameloblastoma and the calcifying cystic odontogenic tumour. The streptavidin-biotinperoxidase method of immunohistochemistry was used with antibodies against the antigens previously cited. The results show that biglican, decorin and BSP had been expressed only in metaplastic epithelial cells, in ghost cells and ghost cells in calcification process, stroma of ameloblastomas and neoplastic ectomesenchyma of the calcifying cystic odontogenic tumour. The fibromodulin and the OCC showed predominantly negative expression in the epithelial and mesenchymal components, with exception for the ghost cells, ghost cells in calcification process and hyalinization areas next to the epithelium. The ONC was positive in the majority of the epithelial cells, with exception of the central cells of follicular and acanthomatous ameloblastomas, and also in the mesenchymal component of both tumours. OPN presented positivity only in the calcification focus of the calcifying cystic odontogenic tumour. The proteins studied presented similar distribution in tumours characterized by different patterns of growth, leading to believe that although they participate actively of the mechanism of intraosseous growth, separately they do not exert a key role in the determination of the type of growth pattern. Another relevance fact is the low expression of these proteins in the neoplastic epithelial cells when compared to the expression in stroma and ectomesenchyma, which make us believe that the epithelial cells act mainly as stimulators of the expression of these proteins, which in turn can act as agonist or antagonist to the tumour growth.