The expression of CCR7 was evaluated in different cancer cell lines by using flow cytometry, western blot, Immunofluorescence and immunohistochemistry. We showed for the selected cell lines that the expression is maintained in cells grown as spheroids, and xenoplanted in mice. Furthermore, we showed the expression of CCR7 correlates with stage of the disease in patient derived head and neck cancer tissue. We also showed that expression of CCR7 in cancer cell lines correlates with migratory aptitude towards CCL21 in a scratch assay, Boyden chamber assay and spheroid invasion assay. We then showed that the expression of CCR7 is elevated under serum starvation and under hypoxia in cancer cell lines grown as monolayers and as spheroids; and that there is a correlation between hypoxia and CCR7 expression in spheroids, xenografted cells and clinical cancer tissue. However, we found that in cell line OSC-19, the increase in the expression of CCR7 did not correlate to increased migration. Our investigations following this observation showed that whilst hypoxia increases the expression of CCR7, it concurrently causes a decrease in reactive oxygen species (ROS) which strongly abrogates migratory aptitude in OSC-19, resulting in an overall loss of migration in OSC-19 cells. In addition, we characterised OSC-19 as a suitable model to evaluate small molecule CCR7 antagonists using a number of different assays. In particular, we showed that ICT13069 antagonised response of this cell line across a number of drivers of malignancy such as migration, invasion in 2D and 3D models.

Le développement du parasite Plasmodium falciparum, responsable du paludisme, nécessite la synthèse de phospholipides et plus particulièrement de phosphatidylcholine (PC) et phosphaditylethanolamine (PE) qui représentent environ 85% de la totalité des phospholidipes du parasite. Leur synthèse s'effectue principalement par les voies métaboliques de novo, voies de Kennedy, en trois étapes enzymatiques. Les enzymes CTP: phosphoethanolamine cytidylyltransferase (ECT) et CTP: phosphocholine cytidylyltransferase (CCT) catalysent les étapes limitantes des deux voies de biosynthèse de la PE et de la PC, respectivement. Ces deux enzymes sont essentielles à la survie du parasite murin, P. berghei et représentent ainsi des cibles thérapeutiques potentielles. La PfCCT est constituée de deux domaines cytidylyltranférases (CT) répétés alors que l'enzyme homologue chez l'homme est composée d'un seul domaine. En revanche, pour la ECT, la présence de deux domaines CT est retrouvée chez toutes les espèces mais les analyses de séquences et de structures ont montré que des résidus importants du site catalytique liant le substrat n'étaient pas conservés dans le domaine CT C-terminal de la PfECT. Ce travail a eu pour but de déterminer les propriétés enzymatiques et les caractéristiques cellulaires de la PfECT et de la PfCCT. Les paramètres cinétiques de ces enzymes ont été quantifiés in vitro à l'aide protéines recombinantes ainsi que sur les enzymes endogènes à l'aide d'extraits parasitaires. Grâce à l'utilisation de protéines recombinantes ponctuellement mutées, nous avons montré que seul le domaine CT N-terminal de la PfECT est catalytiquement actif. Chez P. falciparum, la PfECT et la PfCCT sont exprimées tout au long du cycle intra-érythrocytaire du parasite. La PfECT est présente dans la fraction soluble du parasite alors que la PfCCT apparait aussi bien dans la fraction soluble qu'insoluble. Des expériences d'immunofluorescence ont montré que la PfECT est cytosolique. L'ensemble des résultats présentés apportent un éclairage important sur les fonctions et les propriétés de ces deux cibles potentielles et constituent les premières étapes indispensables à l'élaboration d'une approche thérapeutique. / Phospholipids are essential for the growth and development of Plasmodium falciparum malaria parasite. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) are its major structural phospholipids. This study focused on CTP: phosphoethanolamine cytidylyltransferase (ECT) and CTP: phosphocholine cytidylyltransferase (CCT) that catalyzes the rate-limiting steps of the de novo Kennedy pathways for PE and PC biosynthesis respectively. Both ECT and CCT are essential in the rodent malaria parasite P. berghei and constitute potential chemotherapeutic targets to fight against malaria. PfCCT consists of two very similar cytidylyltransferase (CT) domains whereas the human enzyme consists of only one CT domain. The presence of two CT domains in ECT seems to be widespread in all the organisms. Sequence and structural analysis showed that the C-terminal CT domain of ECT lacks key residues in the substrate binding motif. This study aimed at unravelling the enzymatic properties and cellular characteristics of PfECT and PfCCT enzymes. In addition, these studies addressed the key question if C-terminal CT domain of PfECT is catalytically active. Kinetic parameters of the enzymes were evaluated in vitro on native proteins as well as on recombinant proteins, the latter being produced in bacterial system. Cellular characterisation studies using polyclonal antisera showed that PfECT and PfCCT are expressed throughout the intra-erythrocytic life cycle of the parasite. PfECT is found mainly in soluble form in the parasite while PfCCT is present in soluble as well as insoluble forms in the parasite. Furthermore, immunofluorescence studies for PfECT revealed that it is mainly cytosolic. To assess the contribution of each CT domain to overall PfECT enzyme activity, recombinant PfECT mutants were generated by site-directed mutagenesis. Kinetic studies on these mutants indicated that the N-terminal CT domain was the only active domain of PfECT. Collectively, these results bring new insights into the kinetic and cellular properties of the enzymes and will pave the way in developing a future pharmacological approach.

Les leishmanioses sont des maladies tropicales négligées provoquées par un protozoaire parasite du genre Leishmania, et transmises par un insecte vecteur, le phlébotome. Les leishmanioses menacent 310 millions de personnes dans 98 pays à travers le monde. Les traitements antileishmaniens actuels sont limités et présentent des problèmes majeurs de toxicité et d'émergence de chimiorésistance. Dans ce contexte, il est nécessaire de développer de nouveaux agents antileishmaniens spécifiquement dirigés contre une cible thérapeutique chez le parasite. La GDP-Mannose Pyrophosphorylase (GDP-MP) est une cible thérapeutique essentielle à la survie du parasite à la fois in vitro et in vivo. Plusieurs différences ont été identifiées dans le site actif de GDP-MPs leishmaniennes par rapport à l'enzyme humaine, montrant ainsi l'intérêt de cette cible thérapeutique dans le développement de nouveaux traitements contre la leishmaniose. La GDP-MP catalyse la synthèse du GDP-mannose, la forme activée du mannose, brique moléculaire importante dans les processus de glycosylation et la synthèse de glycoconjugués essentiels à la reconnaissance hôte-parasite. Ce travail de thèse a consisté à produire et purifier les GDP-MPs de 3 espèces de parasites (Leishmania infantum, Leishmania donovani et Leishmania mexicana) ainsi que l'homologue humaine dans le but de comparer leurs propriétés enzymatiques. A partir d'inhibiteurs potentiels conçus et synthétisés sur la base de modèles moléculaires de GDP-MPs leishmaniennes et humaine, 100 composés ont été évalués sur les enzymes purifiées et sur les parasites in vitro. Cette analyse nous a permis de sélectionner des composés spécifiquement dirigés contre la cible du parasite et présentant une activité antileishmanienne. Nous avons également initié une étude de l'expression et de la localisation de la GDP-MP après traitement par les composés les plus intéressants. Ces composés pourront par la suite être utilisés comme outils pharmacologiques pour le développement de nouveaux agents antileishmaniens spécifiques. / Leishmaniases are Neglected Tropical Disease (NTD)caused by a protozoan parasite of the genus Leishmania and transmitted by an insect vector, the phlebotomine sandfly. Leishmaniases threaten 310 millions people in 98 countries around the world. Current antileishmanial treatments are limited and present major issues of toxicity and drug resistance emergence. In this context, it is necessary to develop new specific antileishmanial drugs specifically directed against a therapeutic target in the parasite.The GDP-Mannose Pyrophosphorylase (GDP-MP) is a therapeutic target which has been described to be essential for parasite survival both in vitro and in vivo. Several differences have been identified in the active site of leishmanial GDP-MPs compared to the human counterpart, showing the prominence of this therapeutic target in the development of new treatments against leishmaniasis. The GDP-MP catalyzes the synthesis of GDP-Mannose,the activated form of mannose, an important molecular constituent of the glycosylation processes involved in the biosynthesis of glycoconjugates which are essential for host-parasite recognition. My thesis work consisted in the production and purification of GDP-MPs from 3 Leishmania species (Leishmania donovani,Leishmania infantum and Leishmania mexicana)and from humanin order to compare their enzymatic properties. From potential inhibitors designed and synthesized on the basis of leishmanial and human GDP-MP molecular models, 100 compounds were evaluated on purified enzymes and on parasites in vitro. These analyses allowed us to select some compounds which are specifically directed against the parasite target and presenting antileishmanial activities. We have also initiated a study of expression and localization of GDP-MP after treatment with the most potent compounds. These compounds will be used as pharmacological tools for the development of new specific antileishmanial drugs.

The expression of CCR7 was evaluated in different cancer cell lines by using flow cytometry, western blot, Immunofluorescence and immunohistochemistry. We showed for the selected cell lines that the expression is maintained in cells grown as spheroids, and xenoplanted in mice. Furthermore, we showed the expression of CCR7 correlates with stage of the disease in patient derived head and neck cancer tissue. We also showed that expression of CCR7 in cancer cell lines correlates with migratory aptitude towards CCL21 in a scratch assay, Boyden chamber assay and spheroid invasion assay. We then showed that the expression of CCR7 is elevated under serum starvation and under hypoxia in cancer cell lines grown as monolayers and as spheroids; and that there is a correlation between hypoxia and CCR7 expression in spheroids, xenografted cells and clinical cancer tissue. However, we found that in cell line OSC-19, the increase in the expression of CCR7 did not correlate to increased migration. Our investigations following this observation showed that whilst hypoxia increases the expression of CCR7, it concurrently causes a decrease in reactive oxygen species (ROS) which strongly abrogates migratory aptitude in OSC-19, resulting in an overall loss of migration in OSC-19 cells. In addition, we characterised OSC-19 as a suitable model to evaluate small molecule CCR7 antagonists using a number of different assays. In particular, we showed that ICT13069 antagonised response of this cell line across a number of drivers of malignancy such as migration, invasion in 2D and 3D models. / Zarqa University / The full text was made available at the end of the embargo, 3rd December 2019

Submitted by admin tede (tede@pucgoias.edu.br) on 2018-06-20T17:27:19Z No. of bitstreams: 1 THAIS APARECIDA GOMES ALMEIDA.pdf: 1404797 bytes, checksum: 66238507a88b256c3d88b7850c4a0842 (MD5) / Made available in DSpace on 2018-06-20T17:27:19Z (GMT). No. of bitstreams: 1 THAIS APARECIDA GOMES ALMEIDA.pdf: 1404797 bytes, checksum: 66238507a88b256c3d88b7850c4a0842 (MD5) Previous issue date: 2018-03-12 / Thhyroid cancer is the most common malignant neoplasm of the endocrine system and accounts for the majority of deaths reported among these tumors. Papillary thyroid carcinoma (PTC) is the most common histological type, accounting for 85% of all thyroid cancers. The expression of two important genes, CITED-1 and P300, involved in the regulation of several transcriptional factors, has been shown in several tumors; however, to date, no studies have been found in the literature that elucidated the role of P300 and CITED-1 in PTC. The present study aimed to evaluate the expression of the messenger RNA (mRNA) of P300 and CITED-1 in PTC using reverse transcription and real-time quantitative PCR. P300 expression was similar in both tissues, normal (median = 0.052, IIQ = 0.094) and tumor (median = 0.041, IIQ = 0.048) (p = 0.481). In relation to the expression of CITED-1, expression was higher in tumor tissues (median = 0.780, IIQ = 0.881) compared to normal tissues (median = 0.017, IIQ = 0.086) (p <0.0001). Significant association was observed between CITED-1 expression and tumor size, however no association was evidenced between P300 expression and histopathological characteristics of the tumors. P300 is described as a molecule associated with the regulation of CITED-1 and several other genes. CITED-1 is a transcription-specific cofactor, which may explain its overexpression in the tumors evaluated. CITED-1 hyperexpression in PTC suggests that this gene is a candidate for integrating the PTC biomarkers profile, and the fact that CITED-1 is associated with tumor size in later stage tumors may turn it an important candidate as a therapeutic target for these tumors. / O câncer da glândula tireoide é a neoplasia maligna mais comum do sistema endócrino e representa a maioria dos óbitos relatados entre estes tumores. O carcinoma papilífero de tireoide (CPT) é o tipo histológico mais comum, sendo responsável por 85% de todos os cânceres de tireoide. Dois importantes genes, CITED-1 e P300, envolvidos na regulação de diversos fatores transcricionais, foram evidenciados em diversos tumores, no entanto, até o momento, não foram encontrados na literatura estudos que elucidaram o papel de P300 e CITED-1 nos CPT. O presente estudo teve como objetivo avaliar a expressão do RNA-mensageiro (mRNA) de P300 e CITED-1 em CPT utilizando transcrição reversa e PCR quantitativa em tempo real. A expressão de P300 foi semelhante em ambos os tecidos, normal (mediana = 0,052, IIQ = 0,094) e tumoral (mediana = 0,041, IIQ = 0,048) (p = 0,481). Em relação à expressão de CITED-1, a expressão foi maior nos tecidos tumorais (mediana = 0,780, IIQ = 0,881) em relação aos tecidos normais (mediana = 0,017, IIQ = 0,086) (p < 0,0001). Associação significativa foi observada entre a expressão de CITED-1 e o tamanho dos tumores, contudo nenhuma associação foi evidenciada entre expressão de P300 e as características histopatológicas dos tumores. P300 é descrita como uma molécula associada à regulação de CITED-1 e de vários outros genes. CITED-1 é um co-fator específico da transcrição, o que pode explicar sua hiperexpressão nos tumores avaliados. A hiperexpressão de CITED-1 nos CPT sugere que o gene é um candidato a compor o perfil de marcadores dos CPT, e o fato de CITED-1 estar associado ao tamanho tumoral em tumores em estágios mais avançados, pode torná-lo um candidato importante como alvo terapêutico para esses tumores.

PKD1 est une sérine-thréonine kinase codée par le gène PRKD1. Elle appartient à la famille des protéines kinase D (PKD). La famille des PKD comprend également deux autres membres, PKD2 et PKD3 qui partagent une grande similarité structurale avec PKD1. Certaines études suggèrent que la PKD1 est impliquée dans de nombreuses voies oncogéniques telles que MAPK, NF-κB, HDAC ... Les données préliminaires de notre équipe avait montré que PKD1 est fortement exprimée dans les lignées cellulaires de mélanome ayant un phénotype mésenchymateux. Nous avons également montré que PKD1 participe à l’indépendance vis-à-vis des oestrogènes dans le cancer du sein et que la forte expression des transcrits PRKD1 était associée à une moins bonne survie dans les tumeurs traitées au Tamoxifène. L'objectif de ma thèse est d'analyser le rôle de la PKD1 dans le maintien du phénotype mésenchymateux dans le mélanome et de déterminer si PKD1 pourrait être un facteur pronostique et une cible thérapeutique pertinente pour certaines sous-populations de cancer du sein. Nous avons analysé les marqueurs moléculaires impliqués dans le phénotype mésenchymateux et épithélial (cadhérine, β-caténine ...) ainsi que les caractéristiques fonctionnelles des cellules de mélanome ayant un phénotype mésenchymateux après inhibition pharmacologique de PKD1. Les résultats ont montré que l'inhibition de PKD1 induit une réversion du phénotype mésenchymateux vers un phénotype pseudo-épithélial moins agressif. L'expression de PRKD1, PRKD2 et PRKD3 a été analysée par RT-qPCR dans 527 tumeurs de sein. Nous avons montré que les niveaux plus élevés d'ARNm de PRKD1 étaient associés à un mauvais pronostic dans la population globale de cancer du sein et plus particulièrement dans les tumeurs ERα- et les tumeurs triples négatives. Nous avons criblé des inhibiteurs pharmacologiques de PKD1 en collaboration avec la société pharmaceutique AB Science, et nous avons évalué l'activité anti-tumorale de plusieurs d’entre eux in vitro dans la lignée cellulaire de cancer du sein triple négative MDA-MB-436 et in vivo dans un modèle de PDX (xénogreffe dérivée du patient) de cancer du sein triple négatif. L’inhibition pharmacologique de PKD1 ou à l’aide de siRNA a réduit la capacité des cellules MDA-MB-436 à former des colonies. L'inhibition pharmacologique de PKD1 a également réduit la croissance tumorale in vivo dans le modèle de PDX. Finalement, nous avons analysé l'expression de PRKD1 dans différentes cohortes multi-cancers. Nos résultats ont montré qu'une forte expression de PRKD1 est retrouvée dans le mélanome et le glioblastome. En conclusion, les résultats ont montré que PKD1 pourrait être une cible thérapeutique pertinente pour le mélanome, le cancer du sein et le glioblastome. / PKD1 is a serine-threonine kinase encoded by the PRKD1 gene. It belongs to the protein kinase D (PKD) family. The PKD family also includes PKD2 and PKD3 which share a high structural similarity with PKD1. Some studies suggest that PKD1 is involved in many oncogenic pathways such as MAPK, NF-κB, HDAC... Recent data from our group showed that PKD1 is more expressed in melanoma cell lines with mesenchymal phenotype compared to melanoma cell lines with epithelial phenotype. We also showed that PKD1 participates in the development and/or maintenance of the estrogen-independent phenotype in breast cancer, and that high PKD1 mRNA levels were associated with a worse outcome in tamoxifen-treated tumors. The objective of my thesis is to analyze the role of PKD1 in the maintenance of mesenchymal phenotype in melanoma and to determine whether PKD1 could be a prognostic factor and/or a therapeutical target for the treatment of breast cancer subpopulations. We analyzed molecular markers involved in mesenchymal and epithelial phenotype (cadherin, β-catenin …) and the functional characteristics of mesenchymal melanoma cells after PKD1 pharmacological inhibition. Results suggest that PKD1 inhibition induced a mesenchymal to epithelial like transition in these melanoma mesenchymal cell lines. In addition, the expression of PRKD1, PRKD2 and PRKD3 was analyzed by RT-qPCR in 527 breast cancers. We showed that higher PRKD1 mRNA levels were associated with a lower metastasis-free survival in the overall breast cancer population. PRKD1 prognostic value was even stronger in ERα- and in triple negative tumors. Then, we tested PKD1 inhibitors in collaboration with the AB Science pharmaceutical company. We assessed the anti-tumoral activity of PKD1 inhibitors in vitro in TNBC (triple negative breast cancer) cell lines and in vivo in a TNBC PDX (patient derivated xenograft) model. PKD1 pharmacological inhibition or depletion by siRNA reduced colony forming units in MDA-MB-436 TNBC cells. PKD1 pharmacological inhibition also reduced tumor growth in vivo in TNBC PDX model. Following those results, we analyzed PRKD1 expression in different multi-cancer cohorts. Our results showed that higher PRKD1 expression is found in melanoma and glioblastoma. In conclusion, results showed that PKD1 could participate in the maintenance of mesenchymal phenotype in melanoma. And that PKD1 could be an interesting therapeutic target in melanoma, breast cancer and glioblastoma.

Much interest has recently accumulated of the role of adult stem cells in both normal tissue homeostasis and carcinogenesis. Whereas normal and cancerous mammary epithelial stem cells have been identified and isolated from bulk primary tissue, little remains known about their regulation in vivo. Here we describe the molecular profile of mammary epithelial stem cells cultured in vitro and that of their tumourigenic counterparts, breast cancer stem cells. Our studies of gene transcription reveal potential mechanisms that may cooperate in the regulation of normal and cancer stem cells in vitro, and may also reflect their in vivo behaviour. These data bear consequences for the design of novel breast cancer therapeutics, as cancer stem cells are thought to resist conventional treatments and persist thereafter, causing disease relapse and seeding metastases. To address this issue we have devised a functional genomics approach to screen for novel biomarkers and therapeutic targets of breast cancer modeled in vitro; this culture system is centered on bona fide stem cells and may therefore offer improved relevance to human disease when compared with breast cancer cell lines. / Thesis / Master of Science (MS)

La radiothérapie par microfaisceaux (MRT) synchrotron est une méthode de radiothérapie alternative pour les tumeurs cérébrales, qui présente l'avantage unique de pouvoir déposer de très hautes doses d'irradiation (plusieurs 100aines de Gy) au niveau de la masse tumorale. En effet, le fractionnement spatial des rayons X en microfaisceaux parallèles de quelques dizaines de micromètres s'est montré efficace dans le traitement des tumeurs cérébrales du rongeur tout en préservant le tissu cérébral péritumoral. Pour autant, son mode d'action sur le plan biologique n'est qu'en partie connu. Si l'effet différentiel de cette irradiation sur les vaisseaux sains et tumoraux a pu être démontré ces dernières années, il ne peut expliquer à lui seul l'efficacité de la MRT. Dans ce travail, nous avons établi une description de la réponse transcriptomique précoce des tissus sains et tumoraux (gliosarcome 9L) à la MRT et les fonctions biologiques et voies de signalisation associées. Ces résultats constituent une base de données interrogeable à partir d'hypothèses précises. Cette base a ainsi permis d'identifier des transcrits impliqués dans la réponse de la tumeur à la MRT et dont l'inhibition n'interfèrerait pas avec la réparation des tissus sains : nous avons proposé 3 cibles potentielles qui permettraient d'augmenter l'index thérapeutique de la MRT. (i) L'inhibition radio-induite d'un groupe de 13 gènes (Plk1, Cdc20, Ccnb1, Pttg1, Bub1, Dlgap5, Cenpf, Kif20a, Traf4af1, Depdc1b, Mxd3, Cenpe et Cenpf), participerait au contrôle tumoral précoce après MRT par la perturbation de la division cellulaire et pourrait être amplifié pour prolonger l'inhibition de la croissance tumorale. (ii) La mise à profit de l'activation du promoteur de Clecsf6 au sein des tumeurs irradiées permettrait la surexpression locale, via les monocytes modifiés et infiltrés, de protéine d'intérêt thérapeutique. (iii) Areg (codant pour l'Amphiréguline) est surexprimé au sein du tissu tumoral après MRT et son implication connue dans la chimio/radiorésistance nous conduit à considérer que son inhibition pourrait être une stratégie de renforcement des effets de la MRT. Par ailleurs, nous avons montré que la MRT engendrait de meilleurs résultats sur le contrôle tumoral et la survie animale qu'une irradiation synchrotron en champ plein (avec une dose équivalente à la vallée MRT). Cependant, aucune différence transcriptomique ne pouvant soutenir cet effet n'a pu être mis en évidence. / Synchrotron Microbeam Radiation Therapy (MRT) is a novel form of radiosurgery of brain tumors which allows high dose deposition (few hundreds of Gy) in pathologic tissues. The spatial fractionation of the incident beam into arrays of near-parallel microbeams has shown efficiency on brain tumors implanted in rodents while sparing normal tissues. The preferential effects observed on tumor vessels could not entirely explain the efficiency of MRT and other biological mechanisms might be involved in tumor control. In this work, we described the early whole transcriptomic responses of normal and tumoral (9L gliosarcoma) tissues to MRT and the associated biofunctions and pathways. This provides a questionable data base which can be used by the whole MRT community. This base allows to identify transcripts involved in tumor response to MRT and which inhibition would have no consequence in healthy tissue repair. We identified 3 relevant targets which might increase the therapeutic index of MRT. (i) The radio-induced inhibition of a cluster of 13 genes (Plk1, Cdc20, Ccnb1, Pttg1, Bub1, Dlgap5, Cenpf, Kif20a, Traf4af1, Depdc1b, Mxd3, Cenpe and Cenpf) may be involved in tumor control after MRT through the deregulation of cell division and could be amplified to continue the tumor growth inhibition. (ii) We might benefit from the activation of the Clecsf6 promoter in irradiated tumors by delivering, via modified and injected monocytes, some therapeutic proteins. (iii) Finally, Areg (encoding for Amphiregulin) is overexpressed in tumors after MRT and its involvement described in chimio/radioresistance enable to consider that its inhibition might help in tumor control after irradiation. We also showed that MRT induces a greater tumor control and survival rates compared with similar broad beam irradiations but no differences in transcriptomic responses have been highlighted.

O câncer cervical é uma neoplasia muito prevalente na população feminina e está associado à infecção pelo papilomavírus humano (HPV). As oncoproteínas E6 e E7 de HPV de alto risco são as principais responsáveis pelas alterações celulares que levam ao desenvolvimento deste tipo tumoral. A dipeptidil peptidase IV (DPPIV/CD26) é uma enzima que exerce importantes funções relacionadas à progressão tumoral. Diversos estudos demonstram alterações na expressão e atividade desta proteína em diferentes tipos de câncer. Tendo em vista a relação entre a DPPIV/CD26 e o câncer, e que ainda não existem estudos relacionando esta proteína ao câncer cervical, neste estudo inicialmente investigamos a expressão e atividade da DPPIV/CD26 em linhagens celulares de carcinoma cervical humano (SiHa, HeLa e C33A) e em queratinócitos imortalizados (HaCaT). Nossos resultados demonstram uma baixa expressão da DPPIV/CD26 nas linhagens celulares estudadas, sendo praticamente indetectável na linhagem HeLa. Foi verificada a atividade enzimática dipeptidilpeptidásica tanto ligada à membrana quanto solúvel em todas as linhagens. Na presença do inibidor de DPPIV/CD26 (fosfato de sitagliptina) observamos que a linhagem SiHa apresentou um aumento na migração celular, e assim sugerimos que ao menos em parte a migração nesta linhagem é regulada pela atividade enzimática da DPPIV/CD26. A fim de investigar a relação da expressão da DPPIV/CD26 com as oncoproteínas E6 e E7 do HPV, avaliamos sua expressão em queratinócitos normais e transduzidos com estas oncoproteínas. Verificamos que queratinócitos expressando E6 de HPV de alto risco apresentam uma redução na expressão da DPPIV/CD26, e esta regulação parece ser dependente da degradação da p53. Considerando que as linhagens celulares estudadas apresentam baixa expressão e atividade da DPPIV/CD26, para melhor compreender a importância da expressão desta proteína, nós induzimos a superexpressão da DPPIV/CD26 em linhagem de câncer cervical (HeLa) para posterior avaliação dos efeitos em diferentes mecanismos tumorais. Os resultados demonstram uma redução no crescimento de células expressando DPPIV/CD26, sendo este efeito independente da atividade enzimática. Além disso, foi demonstrado que a indução da expressão de DPPIV/CD26 não afeta os mecanismos de migração e adesão celular na linhagem HeLa. Sendo assim, acreditamos que o esclarecimento do papel da DPPIV/CD26 no contexto do câncer cervical possibilita que novas abordagens diagnósticas e terapêuticas sejam implementadas no futuro. / Cervical cancer is a very prevalent neoplasm in female population and is associated with human papillomavirus (HPV) infection. The high risk HPV E6 and E7 oncoproteins are responsible for cellular alterations that lead to the development of this tumor type. The dipeptidyl peptidase IV (DPPIV/CD26) is an enzyme that exerts important functions related to tumor progression. Several studies have shown changes in the expression and activity of this protein in different types of cancer. Considering the relationship between DPPIV/CD26 and cancer, and that there are still no studies relating this protein to cervical cancer, in the present study we first investigated the DPPIV/CD26 expression and activity in human cervical carcinoma cell lines (SiHa, HeLa and C33A) and in immortalized keratinocytes (HaCaT). Our results demonstrate a low DPPIV/CD26 expression in the studied cell lines, being almost undetectable in HeLa cell line. The dipeptidylpeptidasic enzymatic activity was verified both membrane bound and in the soluble form in all cell lines. In the presence of the DPPIV/CD26 inhibitor (sitagliptin phosphate) we observed that SiHa cell line showed an increase in cell migration, thus we suggest that at least in part cell migration in this cell line is regulated by DPPIV/CD26 enzymatic activity. In order to investigate the relationship between DPPIV/CD26 expression and HPV E6 and E7 oncoproteins, we evaluated the expression of this protein in normal keratinocytes or transduced with these oncoproteins. We have found that keratinocytes expressing high-risk HPV E6 present a reduction in DPPIV/CD26 expression, and this regulation appears to be dependent on p53 degradation. Considering that the cell lines studied have low DPPIV/CD26 expression and activity, in order to better understand the importance of the expression of this protein, we induced the DPPIV/CD26 overexpression in a cervical cancer cell line (HeLa) for further evaluation of the effects on different tumor mechanisms. The results demonstrate a reduction in cell growth of DPPIV/CD26 expressing cells, being this effect independent of the enzymatic activity. In addition, it has been demonstrated that the induction of DPPIV/CD26 expression does not affect the cell migration and adhesion mechanisms in the HeLa cell line. Thus, we believe that the elucidation of the DPPIV/CD26 role in the context of cervical cancer enables new diagnostic and therapeutic approaches to be implemented in the future.

Donada la resistència a la quimioteràpia que presenta el melanoma i el carcinoma d’endometri avançat, és important la investigació destinada a buscar dianes terapèutiques. En aquest treball hem investigat els efectes de d’anàlegs de la somatostatina (SA), d’inhibidors del proteasoma (PI) com Bortezomib, de l’inhibidor de receptors tirosina cinasa Sunitinib i la combinació de Sunitinib i Bortezomib en línies cel•lulars de melanoma. Aquesta combinació també l’hem testat en el carcinoma d’endometri. S’observa que les cel•lules de melanoma expressen varis receptors de la somatostatina. Els PI són eficients a l’hora d’induir una parada en el cicle cel•lular i apoptosi. Les línies cel•lulars que presenten PDGFRα i VEGFR2 són sensibles a Sunitinib, i aquest interacciona amb Bortezomib de forma sinèrgica. En el carcinoma d’endometri, Sunitinib indueix una parada en el cicle cel•lular, apoptosi, inhibeix la via NFκB i interacciona sinèrgicament amb Bortezomib. Els SAs i PI podrien ser útils en combinació amb altres agents i l’ús de Bortezomib i Sunitinib ha mostrat ser efectiu en determinats casos de melanoma i en el carcinoma d’endometri. / Dada la resistencia a la quimioterapia que presenta el melanoma y el carcinoma de endometrio avanzado, es importante aquella investigación destinada a buscar dianas terapéuticas. En este trabajo hemos investigado los efectos de análogos de la somatostatina (SAs), de inhibidores del proteasoma (PI) como Bortezomib, del inhibidor de receptores tirosina cinasa Sunitinib y la combinación de Sunitinib y Bortezomib en líneas celulares de melanoma. Esta combinación también la hemos testado en el carcinoma de endometrio. Se observa que las células de melanoma expresan varios receptores de la somatostatina. Los PI son eficientes a la hora de inducir una parada en el ciclo celular y apoptosis. Las líneas celulares que presentan PDGFR α i VEGFR2 son sensibles a Sunitinib, y éste interacciona con Bortezomib de forma sinérgica. En el carcinoma de endometrio, Sunitinib induce una parada en el ciclo celular, apoptosis, inhibe la via NFκB e interacciona sinérgicamente con Bortezomib. Los SAs y PI podrían ser útiles en combinación con otros agentes y el uso de Bortezomib y Sunitinib ha demostrado ser efectivo en determinados casos de melanoma y en el carcinoma de endometrio. / As advanced melanoma and endometrial carcinoma is highly resistant to chemotherapy it is important the research focused to the search of therapeutic targets. In the present work, we have investigated the effects of somatostatin analogues (SAs), proteasome inhibitors (PI) such as Bortezomib, the tyrosine kinase receptor inhibitor Sunitinib and the combined therapy of Sunitinib and Bortezomib. This combination has been also tested in endometrial carcinoma. It is observed that melanoma cell lines express various somatostatin receptors. In addition, proteasome inhibitors induce a cell cycle arrest and apoptosis. Those cells presenting activated PDGFRα or VEGFR2 are more sensitive to Sunitinib and this interacts synergistically with Bortezomib. In endometrial carcinoma, Sunitinib induces a cell cycle arrest, apoptosis, inhibits NFκB pathway and interacts synergistically with Bortezomib. The SAs and PI could be useful combined with other agents and the use of Bortezomib and Sunitinib has shown to be effective in certain cases of melanoma and in endometrial carcinoma.