Rozprawy doktorskie na temat „Cancer tumer”

Le cancer de l'ovaire (CO) est la cinquième cause de décès chez les femmes qui se caractérise par son diagnostic tardif (stades FIGO III et IV) et par l’importance de ses métastases abdominales souvent observées au moment du diagnostic. Le traitement repose sur une chirurgie cytoréductive complète associée à une chimiothérapie. Malheureusement, parmi les patientes ayant une rémission clinique complète après la fin du traitement initial, 60% des personnes atteintes d'un cancer épithélial de l'ovaire (CEO) à un stade avancé rechuteront dans les cinq ans.L'importance de la néo-angiogenèse dans le développement des tumeurs a poussé les chercheurs à étudier d'autres stratégies. Les thérapies anti-angiogéniques ciblant le système vasculaire tumoral sont désormais utilisées en association avec la thérapie cytotoxique standard dans le traitement des CEO. Malheureusement, les progrès réalisés grâce à cette approche offrent encore un succès limité, qui peut s'expliquer en partie par l'interaction hétérotypique entre les cellules endothéliales et la tumeur. Plusieurs études suggèrent un dialogue complexe entre les cellules cancéreuses de l'ovaire (OCC) et les cellules endothéliales (EC) pouvant entraîner une sensibilité différente à la chimiothérapie et aux traitements anti-angiogéniques conduisant à la progression tumorale.L’objectif de la présente étude est d’étudier le rôle des interactions entre EC et OCC dans la prolifération et la chimiorésistance des CEO. Pour modéliser l'endothélium de la tumeur, nous avons utilisé notre modèle de cellules endothéliales AKT activées (E4+EC). Nous avons démontré en utilisant un modèle de coculture 2D que l’endothélium activé induit une prolifération et une chimiorésistance accrues dans les CEO par l’activation de la signalisation de Notch. Nous avons montré que l’expression et l’activation des récepteurs Notch étaient augmentées dans les cultures en coculture et dans les OCC résistantes à la chimiothérapie.L’accumulation d’ascite dans l’abdomen des patientes atteintes de CO semble jouer un rôle clé dans le mécanisme de propagation des OCC. Les OCC isolées flottent généralement dans l'ascite et forment des sphéroïdes multicellulaires. Dans ce contexte, nous avons développé un nouveau modèle 3D de sphéroïde pour étudier les interactions tumeur-endothélium dans un modèle plus proche des conditions in vivo. Nous avons démontré que les E4+EC et OCC formaient des angiosphères organisées avec un noyau de cellules endothéliales entourées par des OCC qui prolifèrent rapidement. Nous avons établi que l'activation de l'AKT dans les EC était nécessaire pour la formation d'angiosphères organisées. Fait intéressant, dans les ascites de patientes CEO, nous avons pu trouver des structures très similaires à nos angiosphères. De plus, dans une cohorte rétrospective de 59 patientes, nous avons montré que les EC étaient AKT activé chez des patientes atteintes de CEO, ce qui confirme l'importance de l'activation d’AKT dans la CEO. De plus, nous avons démontré l'importance du FGF2, de la Pentraxine 3 (PTX3), du PD-ECGF et du TIMP-1 dans l'organisation de l'angiosphères. Enfin, nous avons confirmé le rôle de Notch3/Jag1 dans le cross-talk des OCC-EC dans la prolifération et l'invasion des OCC au péritoine.En conclusion, notre étude illustre l’importance des EC AKT activé dans les CEO. Au vu des résultats mitigés des traitements anti-angiogéniques, se concentrer sur la normalisation vasculaire dans l'angiogenèse pathologique pourrait être plus efficace. Bien que l'AKT soit difficilement ciblable, la caractérisation génétique des tumeurs pourrait potentiellement identifier un sous-ensemble de tumeurs avec une signalisation NOTCH aberrante qui constituerait une cible idéale pour des inhibiteurs spécifiques. Alors que nous nous dirigeons vers la médecine personnalisée et de précision, il pourrait y avoir une place pour l'inhibition de NOTCH dans les CO en combinaison avec d'autres stratégies thérapeutiques
Ovarian cancer (OC) is the most lethal gynecologic malignancy in developed countries and the fifth cause of death among women. OC is a heterogeneous disease, which is characterized by its late diagnosis (FIGO III and IV stages) and the importance of abdominal metastases often observed at the time of diagnosis. The mainstay of treatment involves complete cytoreductive surgery associated with platinum and taxane-based chemotherapy. Unfortunately, among patients achieving complete clinical remission after completion of initial treatment, 60% with advanced epithelial ovarian cancer (EOC) will relapse within five years.The importance of neo-angiogenesis in tumor formation, growth and dissemination has driven researchers to investigate into alternative strategies. Anti-angiogenic therapies targeting tumor vasculature are now used in combination with standard cytotoxic therapy in the treatment of EOC. Unfortunately, the progress achieved by this approach still offers limited success which can partly be explained by the heterotypic interaction between the tumor and endothelial cells. Evidence suggests a complex cross-talk between ovarian cancer cells (OCCs) and endothelial cells (ECs) that can result in the emergence of a heterogeneous tumoral and endothelial population with different sensitivity to chemotherapy and anti-angiogenic therapies leading to an increase of OCC proliferation and dissemination.The objective of the present study is to investigate the role of ECs and OCCs interactions in the proliferation and chemoresistance of EOC. To model tumor endothelium, we used our model of Akt-activated endothelial cells (E4+ECs). We demonstrated using a 2D co-culture model that activated endothelium induces increased proliferation and chemoresistance in EOC through the activation of Notch signaling. We showed that Notch receptor expression and activation are increased in co-culture and in OCCs resistant to chemotherapy.The accumulation of ascites in the abdomen of an OC patient seems to play a key role in the mechanism of OCC spreading. Detached cancer cells usually float in ascites and form multicellular spheroids. In this context, we developed a new model of organized multicellular 3D spheroid to study tumor-endothelium interactions in a model closer to in vivo conditions. We demonstrated that when cocultured in 3D condition, E4+ECs and OCCs formed organized tumor angiospheres with a core of endothelial cells surrounded by highly proliferating OCCs. We established that AKT activation in ECs was mandatory for the formation of organized angiospheres. Interestingly, in EOC patient ascites, we were able to find structures that were very similar to our angiospheres. In addition, in a retrospective cohort of 59 patients, we showed that ECs were AKT activated in EOC patients which support the importance of AKT activation in EC in EOC. Besides, we demonstrated the importance of FGF2, Pentraxin 3 (PTX3), PD-ECGF and TIMP-1 in angiosphere organization. Finally, we confirmed the role of Notch3/Jagged1 in OCCs-ECs crosstalk for OCC proliferation but also during peritoneum invasion.Altogether, our study illustrates the importance of AKT activated ECs in EOC. In a context of poor results of anti-angiogenic therapies in clinical settings, focusing on vascular normalization in pathological angiogenesis could be more efficient. While AKT is hardly targetable, the genetic characterization of tumors could potentially identify a subset of tumors with aberrant NOTCH signaling that would constitute an ideal target for specific inhibitors. As we move toward personalized and precision medicine, there might be a place for notch inhibition in advanced ovarian cancer in combination with other therapeutic strategies

BRCA1 est l’un des deux gènes majeur de prédisposition au cancer du sein. Les multiples partenaires protéiques de BRCA1 lui confèrent de nombreuses fonctions par lesquelles elle peut assurer la surveillance de l’intégrité cellulaire. L’équipe dans laquelle j’ai mené ma thèse a identifié un nouveau partenaire protéique de BRCA1, la protéine de liaison au poly(A) des ARN messagers PABP1, et a ainsi mis en lumière l’implication de BRCA1 dans la régulation de la traduction [1]. Durant ma thèse, j’ai étudié cette nouvelle fonction de BRCA1 et essayé de comprendre si, et comment, elle pourrait contribuer à son rôle de suppresseur de tumeur. J’ai tout d’abord montré que BRCA1 est associée avec la fraction ribosomique des cellules, et plus particulièrement à la fraction subpolysomique, ce qui pourrait indiquer sa participation à l’initiation de la traduction. De plus, nos résultats indiquent que BRCA1 pourrait participer à un complexe non canonique d’initiation de la traduction. Dans la mesure où BRCA1 n’est pas un facteur canonique d’initiation de la traduction, il est peu probable qu’elle intervienne dans la traduction de tous les ARNm. Notre hypothèse est qu’elle pourrait réguler la traduction d’ARNm spécifiques codant pour des protéines impliquées dans ses différentes fonctions de surveillance de l’intégrité cellulaire. Afin d’identifier les cibles traductionnelles de BRCA1, j’ai réalisé une analyse transcriptomique comparative du contenu des polysomes de cellules épithéliales mammaires MCF7 exprimant de façon transitoire un ARN interférent dirigé contre BRCA1 ou contrôle. Parmi les ARNm cibles de l’activité traductionnelle de BRCA1, beaucoup codent effectivement pour des protéines impliquées dans les fonctions auxquelles elle participe. Ce travail confirme que la participation de BRCA1 à la régulation de la traduction pourrait être une autre voie par laquelle BRCA1 exerce son rôle de suppresseur de tumeur. De plus, l’analyse des cibles traductionnelle de BRCA1 pourrait conduire à l’identification de nouvelles fonctions de cette protéine ainsi qu’à la découverte de marqueurs tumoraux ou de cibles thérapeutiques pour les patients porteurs d’une mutation de BRCA1
BRCA1 is one of the two major genes of breast cancer susceptibility. The numerous binding partners of BRCA1 allow it to participate to several cellular pathways which globally contribute to its cell surveillance capacity. The team in which I performed my PhD identified a new bindind partner of BRCA1, the Poly(A)-Binding Protein 1 and, consequently, a new function of this tumor suppressor, namely, the translation regulation [1]. During my PhD, I studied this new function and try to elucidate if, and how, this function could participates to the BRCA1’s tumor suppressive activity. I first showed that BRCA1 is associated with the ribosomal fraction of the cell, and more precisely, with the subpolysomal fraction, which could indicate that BRCA1 participates to the initiation step of translation. Moreover, our results suggest that BRCA1 could participate to a non canonical initiation complex. Given that BRCA1 is not a canonical translation initiation factor, it is unlikely that BRCA1 regulates the translation of all mRNAs. Our hypothesis is that BRCA1 could regulate the translation of specific mRNAs involved in its various cell surveillance functions. In an attempt to identify the BRCA1 translational targets, we performed a microarray analysis of polysomes-bound mRNAs in MCF7 cells transiently expressing siRNA directed against BRCA1 or control siRNA. We found that, among the translational targets of BRCA1, many indeed encode proteins involved in the same main functions as BRCA1’s. Altogether, our results suggest that the involvement of BRCA1 in translation regulation could be another way by which BRCA1 exerts its tumor suppressor role. Moreover, the analysis of the BRCA1’s translational targets could lead to the identification of new functions for BRCA1 as well as to the discovery of new tumoral markers and therapeutic targets for the BRCA1 mutated patients

Les cancers du sein de stade précoce constituent un enjeu de santé publique du fait de leur fréquence élevée (70% des nouveaux cas diagnostiqués). Parmi eux, les cancers luminaux (exprimant les récepteurs hormonaux sans surexpression de l’HER2) de stade T1N0, de moins de 2cm sans envahissement ganglionnaire, ont un pronostic spontanément favorable et font ainsi l’objet de peu d’études dédiées. Néanmoins certaines de ces patientes (5 à 10% à 10 ans) vont récidiver avec des métastases à distance et mourir. L’identification de facteurs pronostiques pour ces tumeurs et une meilleure compréhension des mécanismes de récidive pourraient permettre de discriminer les tumeurs réellement indolentes pour lesquelles le traitement pourrait être diminué et au contraire, les tumeurs les plus à risque de récidive pour lesquelles un traitement devrait être renforcé. L’objectif de ce travail de thèse était d’identifier les mécanismes de la récidive métastatique dans les cancers du sein luminaux de stade T1b-cN0M0 au diagnostic en analysant plus spécifiquement le microenvironnement tumoral. Pour cela, nous avons constitué une cohorte de patientes ayant eu un cancer du sein T1b-cN0, de type luminal (exprimant les récepteurs hormonaux sans surexpression de l’HER2) de type cas (patientes avec récidive métastatique à distance) / témoin (patiente sans récidive à distance), à partir de la cohorte de patientes suivies à l’Institut Curie. Les témoins étaient choisis appariés aux cas sur les principaux facteurs pronostiques connus : âge, grade tumoral et prolifération tumorale estimée par le Ki67. Nous avons ensuite évalué les facteurs de récidive dépendant des cellules tumorales. En analyse univariée, on montre qu’une moins bonne différenciation (baisse de l’expression de la E-cadhérine) est associée à la récidive. Par ailleurs, le score de récidive prédit par la signature transcriptomique ProsignaTM est plus élevé chez les cas que chez les contrôles sans que la discrimination entre les deux ne soit parfaite pour cette population de cancers du sein. L’originalité de notre étude reposait sur l’étude détaillée du microenvironnement tumoral. Sur le plan immunologique, l’analyse systématique quantitative et qualitative, en immunohistochimie, de l’infiltrat immun montre une absence d'association avec les lymphocytes B, macrophages et cellules dendritiques. En revanche, on observe une baisse des lymphocytes T CD4+ tumoraux chez les cas, sans autres association concernant les différents sous-types de lymphocytes T. Dans la continuité des travaux déjà réalisé au laboratoire du Dr Fatima Mechta-Grigoriou, l’analyse des différents sous-types de fibroblastes associés au cancer (CAF) dans notre cohorte révélait que les cas étaient plus spécifiquement enrichis en CAF activés de type CAF-S1 alors que la quantité globale de stroma ne soit pas associée à la récidive. Ici, l’action des CAF-S1 est médiée par la cadhérine-11 qui augmente les capacités de migration et invasion des cellules tumorales in vitro par stratégie siRNA. En analyse multivariée, les caractéristiques du microenvironnement et en particulier l’enrichissement en CAF-S1 restent significativement associées à la récidive.En conclusion, nous avons identifié que la survenue de métastases dans les cancers du sein luminaux de stade précoce repose sur des mécanismes associés au micro-environnement tumoral tels que la présence de CAF-S1 et leur expression de la cadhérine-11, indépendamment des cellules tumorales elles-mêmes. L’enrichissement en CAF-S1 dans le stroma tumoral était également un facteur de mauvais pronostic
Purpose: Early luminal breast cancers (BC) represent 70% of newly diagnosed BC. Their prognosis is generally favorable but some patients (around 5 to 10% at 10 years) will relapse with distant metastases and most of them will die. Because this dismal prognosis concerns a small number of patients, T1N0 BC have been rarely studied. The aim of this work was to identify the mechanisms of metastatic recurrence in luminal breast cancers T1b-cN0M0 at diagnosis by deciphering characteristics of both epithelial cancer cells and their surrounding tumor microenvironment (TME). Experimental Design: We constituted a cohort of luminal T1b-cN0 BC patients with metastatic recurrence (defined as "cases”) and corresponding "controls" (i. e. patients without metastatic relapse) matched (1:1) to cases on the main known prognostic factors: age, tumor grade and tumor proliferation (assessed by Ki67). Results: We found that properties in both epithelial compartment and TME are indicative of relapse in early luminal breast cancers. In univariate analysis, the loss of differentiation (assessed by the reduced expression of CDH1/E-cadherin) in cancer cells is associated with recurrence, as also predicted by high ROR score using ProsignaTM test. In TME, quantitative and qualitative immunohistochemical analyzes reveals that “cases” are characterized by a significant decrease in CD4+ T lymphocytes and an accumulation of a particular subset of Cancer Associated Fibroblasts (CAF-S1) compared to “controls”, without any other association of T lymphocyte subtypes, B lymphocytes, macrophages or dendritic cells. In multivariate analysis, TME features, in particular CAF-S1 enrichment, remain significantly associated with recurrence, which demonstrates their biological and clinical relevance. Finally, by performing functional analyses, we demonstrated that CAF-S1 pro-metastatic properties are -at least in part- mediated by CDH11/Osteoblast cadherin, consistent with the fact that the bones are a major site of metastases in these patients. Conclusions: Distant recurrence in early luminal BC is strongly associated with TME features, such as the presence of CAF-S1 and their expression of CDH11. This is independent of tumor cells and represents a new prognostic factor of distant relapse in early luminal BC patients. This could justify targeted therapies against CAF-S1 or CDH11 in these cases

La thérapie photodynamique, basée sur l'affinité pour les tissus tumoraux d'un agent photosensibilisant est un traitement anticancéreux prometteur mais encore en phase de recherche. Parmi les paramètres inconnus, l'action de l'oxygène est un élément essentiel a la définition d'une dosimétrie efficace de la lumière. En effet, sa détermination permet une optimisation de l'action phototoxique ainsi que la détermination d'un seuil de réussite thérapeutique. A ces fins, le développement d'un système adapte aux mesures par voie endoscopique, seul accès au site tumoral, permettant l'évaluation de l'état d'oxygénation des tissus au cours du traitement en temps réel et in situ est nécessaire. Reposant sur le principe de différenciation optique, éprouve par les oxymétries dits de pouls, la saisie des données s'effectue par un capteur original issu de la technologie fibres optique. La métrologie associée comporte une phase de modélisation de la réponse de la masse tumorale a une excitation lumineuse. Cette démarche s'apparente parfaitement au traitement de signal physiologique, puisque les tissus tumoraux, soumis a un échelon de lumière, sont identifies par un modèle de type arx. Celui-ci a été valide tant par des simulations numériques que par des expérimentations sur fantômes optiques. Il ouvre d'ailleurs de nouveaux horizons a l'oxymétrie pulsée en général. La gestion des données est assurée par le biais d'un logiciel débouchant sur une surveillance en temps réel du site opératoire. L'optimisation du système comporte une gestion dynamique de l'espace mémoire par un échantillonnage sélectif des données expérimentales. L'élaboration de fantômes a base de colorants, destines a la validation du principe est décrite. Enfin un protocole de mesures in vivo confirme les bons résultats obtenus in vitro

Immunotherapy of cancer has been shown to be promising in prolonging patient survival. However, complete elimination of cancer and life-long relapse-free survival remain to be major challenge for anti-cancer therapeutics. We have previously reported that ex vivo reprogramming of tumor-sensitized immune cells by bryostatin 1/ionomycin (B/I) and the gamma-chain (γ-c) cytokines IL-2, IL-7, and IL-15 resulted in the generation of memory T cells as well as CD25+ NKT cells and CD25+ NK cells. Adoptive cellular therapy (ACT) utilizing these reprogrammed immune cells protected FVBN202 mice from tumor challenge, and overcame the suppressive functions of myeloid-derived suppressor cells (MDSCs). We then demonstrated that the presence of CD25+ NKT cells was required for anti-tumor efficacy of T cells as well as their resistance to MDSCs. Similar results were obtained by reprogramming of peripheral blood mononuclear cells (PBMC) from patients with early stage breast cancer, demonstrating that an increased frequency of CD25+ NKT cells in reprogrammed immune cells was associated with modulation of MDSCs to CD11b-HLA-DR+ immune stimulatory cells. Here, we tested the efficacy of immunotherapy in a therapeutic setting against established primary breast cancer (Chapter One), experimental metastatic breast cancer (Chapter Three) as well as against minimal residual disease (MRD) in patients with multiple myeloma (Chapter Two). We evaluated the ability of reprogrammed immune cells, including CD25+ NKT cells, to convert MDSCs to myeloid immune stimulatory cells, in vivo; this resulted in the identification and characterization of a novel antigen presenting cell (APC). These novel immune stimulatory cells differed from conventional APCs, including dendritic cells (DCs) and macrophages. We have also demonstrated that enhancing immunogenicity of mammary tumors by treatment with Decitabine (Dec) along with overcoming MDSCs by utilizing reprogrammed T cells and NKT cells in ACT prolongs survival of animals, but fails to eliminate the tumor. However, targeting cancer during a setting of MDR, when tumor cells are dormant, results in objective responses as evidenced in our multiple myeloma studies. This suggests that targeting breast cancer with immunotherapy following conventional therapies, in a setting of residual disease when tumor cells are dormant, may be effective in eliminating such residual cells or maintaining dormancy and extending time-to-relapse for breast cancer patients.

Le cancer du sein est le cancer le plus fréquent chez la femme et représente la principale cause de mortalité liée au cancer. Le décés est habituellement causé par le développement de résistance aux traitements et la propagation métastatique de la maladie. Malgré la pertinence clinique, la complexité moléculaire de la maladie et sa dynamique restent à ce jour peu connues.Depuis longtemps, l’hétérogénéité du cancer du sein a été observée au niveau histologique et du profil évolutif clinique, et ces différences ont servi de base pour la classification de la maladie. Avec le développement des technologies à haut débit, telles que les puces à damier (microarrays) et le séquençage à haut débit, cette classification a été affinée et une complexité génétique jusqu'alors inconnue a été révélée.Des études utilisant ces techniques ont montré que des différences moléculaires existent non seulement entre les différentes patientes atteintes d’un cancer du sein (hétérogénéité inter-tumorale), mais aussi chez la même patiente (hétérogénéité intra-tumorale). En outre, l'hétérogénéité intra-tumorale peut exister non seulement entre les différentes parties d'une tumeur (hétérogénéité intra-tumorale spatiale) mais elle peut aussi résulter de l’évolution moléculaire d'une tumeur au cours du temps (hétérogénéité intra-tumorale temporelle). Cette complexité pourrait avoir un impact important sur la façon dont les patientes atteintes d’un cancer du sein sont prises en charge et traitées.La recherche que j’ai menée dans le Breast Cancer Translational Research Laboratory sous la direction du Professeur Christos Sotiriou avait deux objectifs principaux. Le premier était de déterminer l'ampleur et les implications cliniques de l'hétérogénéité intra-tumorale dans deux scénarios cliniques courants, à savoir: les cancers du sein multifocaux (MFBCs) et les cancers du sein métastatiques ER positif / HER2 négatif. Le deuxième était d'étudier l'impact de l'édition de l'ARN dans la détermination de l'hétérogénéité inter-tumorale, phénomène encore peu caractérisé. Notre recherche a notamment montré que:1) Les lésions de tous les MFBCs que l’on a étudiés partagent une origine commune. Malgré cela, et malgré des caractéristiques pathologiques similaires, chez un tiers des patientes, les lésions multifocales d’une même patiente ne partageaient aucune substitution et aucune insertion/déletion. De plus, l’hétérogénéité inter-lésion a été observée pour des mutations oncogéniques dans des gènes tels que PIK3CA, TP53, GATA3 et PTEN;2) En se concentrant sur un nombre défini de gènes associés au cancer, une concordance substantielle des mutations et du nombre de copies des gènes a été observée entre les lésions primaires et métastatiques appariées de cancers du sein ER positif / HER2 négatif. Des différences entre les lésions appariées ont cependant été trouvées pour les niveaux d’expression de certains gènes. Dans les lésions primaires, seuls les niveaux d’expression de quelques gènes et un niveau élevé d'amplification de FGFR1 ont été associés à la survie;3) L'édition de l’ARN est une source généralisée de variation du transcriptome dans le cancer du sein. Dans ce cancer, et potentiellement dans tous les cancers, l'édition de l’ARN est principalement contrôlée par deux facteurs, à savoir l'amplification de 1q et l'inflammation, qui sont toutes deux très répandues parmi les cancers humains. La magnitude de l'édition de l’ARN, en combinaison avec la conservation des sites d'édition détectés dans les tissus et les patientes, suggère qu'il pourrait y avoir des implications cliniques et thérapeutiques pour un large éventail de patientes atteintes d’un cancer.Nos résultats suggèrent qu'une caractérisation moléculaire approfondie des cancers du sein multifocaux et métastatiques est importante pour apprécier leur complexité, et que dans la recherche sur le cancer du sein, plus d’importance devrait être accordée à l'édition de l'ARN, un phénomène encore peu étudié qui pourrait influencer notre connaissance sur le développement et l'évolution de la maladie.
Breast cancer still represents the most frequently diagnosed cancer and the leading cause of cancer-related mortality in women. Death is usually caused by the development of resistance to treatments and the resulting metastatic spread of the disease. Despite the clinical relevance, little is known about the molecular complexity of the disease and its dynamics.Breast tumor heterogeneity has been observed at the level of the histology and the natural history of the disease for a long time, and these differences have served as the basis for disease classification. With the advent of high-throughput technologies, such as gene expression microarrays and massively parallel sequencing, this classification has been refined and a previously unknown genetic complexity has been revealed. Studies implementing these technologies have shown that molecular dif¬ferences exist not only between different breast cancer patients (inter-tumor heterogeneity), but also within the same patient (intra-tumor heterogeneity). Furthermore, intra-tumor heterogeneity could occur either between different regions of a tumor (spatial intra-tumor heterogeneity), or as the result of the molecular evolu¬tion of a tumor over time (temporal intra-tumor heterogeneity). This complexity might have a profound impact on the way breast cancer patients are managed and treated. The research work that I carried out in the Breast Cancer Translational Research Laboratory under the direction of Prof Christos Sotiriou had two main aims. The first was to determine the extent and the clinical implications of intra-tumor heterogeneity in two common clinical scenarios, namely: multifocal breast cancers (MFBCs) and metastatic ER positive/HER2 negative breast cancers. The second was to investigate the potential impact of yet poorly characterized phenomenon, such as RNA editing, in determining inter-tumor heterogeneity. For this purpose, I have conducted three main projects, which resulted in three manuscripts.We showed that:1) The lesions of all the investigated MFBCs shared a common origin. Despite this, and despite having similar pathological features, in up to a third of the patients the lesions of the same MFBC didn’t share any substitution/indels, and inter-lesion heterogeneity was observed for oncogenic mutation(s) in genes such as PIK3CA, TP53, GATA3, and PTEN; 2) When focusing on a defined number of cancer-associated genes, a substantial concordance for mutations and copy number aberrations could be found between primary and matched metastatic lesions of ER positive/HER2 negative breast cancers. Differences between matched pairs could however be found for the level of expressions of few genes. In primary lesions, only the expression levels of few genes and high FGFR1 amplification levels were associated with OS;3) A-to-I RNA editing is a pervasive source of transcriptome variation in breast cancer. In breast and potentially all cancers, A-to-I editing is mainly controlled by two factors, namely 1q amplification and inflammation, both of which are highly prevalent among human cancers. The wide-spread editing observed, in combination with the conservation of editing sites detected across tissues and patients, suggests that there might be clinical and therapeutic implications for a wide range of cancer patients.Our results suggest both that a thorough molecular characterization of multifocal and metastatic breast cancers is important to appreciate their genomic complexity, and that in breast cancer research more relevance should be given to RNA editing, a yet poorly investigated phenomenon that has the potential to impact the development and the evolution of the disease.
Doctorat en Sciences médicales (Médecine)
info:eu-repo/semantics/nonPublished

Background: An increasing number of studies have established that circulating tumor cells (CTCs) are a heterogeneous population in which cells have different degrees of metastatic potential mainly due to epithelial-mesenchymal transition (EMT). This study aimed to assess the feasibility of circulating esophageal cancer cells identification, characterization and to evaluate their prognostic value in esophageal cancer Methods: In order to closely mimic CTC heterogeneity, MC10A and HMEL cell lines differently forced in EMT are used. Single cell analysis was conducted by DEPArray. We assigned a specific phenotypic tag to each potential CTC population: Epithelial-tag, Mesenchymal/stem-tag. We evaluated the basal cell phenotype and after EMT induction. Subsequently, the Grab all assay was performed on peripheral blood samples from patients with esophageal cancer. This feasibility study enrolled 11 patients (4 M1, 7 M0). Analyses were conducted on 3 peripheral blood samples (15/20 ml) per patients. Blood samples from non-metastatic patients were taken before and after primary neo-adjuvant therapy and after secondary treatment (surgery). Samples from metastatic patients were taken before and after first line therapy and after second line therapy. CTCs were identified and sorted singly by DEPArray system. Results: The assay was able to detect the phenotypic changes in cell lines mimicking CTC heterogeneity. Before therapy, CTCs were found in 3/4 metastatic patients. Of the 7 non-metastatic patients, 3 were positive for CTCs before therapy. Examining CTC status at different clinical time points, it was possible to suggest a correlation between the presence of CTCs and disease progression. Conclusions: Data showed that the assay is feasible, capable to analyze the phenotypic tags by DEPArray using a multiple staining without aspecific signals. Experiments carried out from both metastatic/non metastatic cancer patients showed the ability of the Grab-allassay to identify subpopulations of CTCs with different epithelial/stem/mesenchymal or hybrid phenotypes potentially related to disease progression.

Background: An increasing number of studies have established that circulating tumor cells (CTCs) are a heterogeneous population in which cells have different degrees of metastatic potential mainly due to epithelial-mesenchymal transition (EMT). This study aimed to assess the feasibility of circulating esophageal cancer cells identification, characterization and to evaluate their prognostic value in esophageal cancer Methods: In order to closely mimic CTC heterogeneity, MC10A and HMEL cell lines differently forced in EMT are used. Single cell analysis was conducted by DEPArray. We assigned a specific phenotypic tag to each potential CTC population: Epithelial-tag, Mesenchymal/stem-tag. We evaluated the basal cell phenotype and after EMT induction. Subsequently, the Grab all assay was performed on peripheral blood samples from patients with esophageal cancer. This feasibility study enrolled 11 patients (4 M1, 7 M0). Analyses were conducted on 3 peripheral blood samples (15/20 ml) per patients. Blood samples from non-metastatic patients were taken before and after primary neo-adjuvant therapy and after secondary treatment (surgery). Samples from metastatic patients were taken before and after first line therapy and after second line therapy. CTCs were identified and sorted singly by DEPArray system. Results: The assay was able to detect the phenotypic changes in cell lines mimicking CTC heterogeneity. Before therapy, CTCs were found in 3/4 metastatic patients. Of the 7 non-metastatic patients, 3 were positive for CTCs before therapy. Examining CTC status at different clinical time points, it was possible to suggest a correlation between the presence of CTCs and disease progression. Conclusions: Data showed that the assay is feasible, capable to analyze the phenotypic tags by DEPArray using a multiple staining without aspecific signals. Experiments carried out from both metastatic/non metastatic cancer patients showed the ability of the Grab-allassay to identify subpopulations of CTCs with different epithelial/stem/mesenchymal or hybrid phenotypes potentially related to disease progression.

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas
A aspirina, cuja substância ativa é o ácido acetilsalicílico, representa um dos medicamentos mais utilizados em todo o mundo. Desde a Idade Média até aos dias de hoje, têm vindo a ser descritas inúmeras propriedades terapêuticas nomeadamente: analgésica, antipirética, anti-inflamatória, anti-plaquetária, entre outras. No presente, para além do seu uso no alívio da dor ligeira a moderada, febre e inflamação, a aspirina assume um importante papel na diminuição do risco de acidentes cardiovasculares. Entretanto, vários ensaios têm vindo a sugerir novos efeitos benéficos da aspirina. Descobertas recentes demonstraram que a aspirina, administrada por vários anos, pode reduzir, a longo prazo, o risco de alguns tipos de cancro, particularmente o cancro colorretal. Várias evidências apoiam a hipótese de que esse efeito é conseguido com doses baixas de aspirina, as mesmas utilizadas na prevenção de eventos cardiovasculares, posicionando a ação antiplaquetária da aspirina no centro de sua eficácia anti-tumoral. Contudo, ainda não há dados suficientes sobre o risco/benefício e, como tal, ainda não foram feitas recomendações definitivas relativamente ao seu uso, sendo portanto, necessários novos estudos para que se possa definir qual a dose mínima efetiva, qual a duração do tratamento, qual a idade para o iniciar e qual a população alvo em que o benefício é superior ao risco. Nesta revisão, irão ser abordados os conhecimentos atuais sobre o papel da aspirina na prevenção do cancro, especialmente o cancro colorretal. Em particular, serão explorados alguns dos possíveis mecanismos responsáveis por este efeito: uns dependentes das prostaglandinas e da ciclooxigenase (COX), nomeadamente da COX-2, e outros provavelmente independentes, destacando novos caminhos para pesquisas futuras. Aspirin, which active substance is the acetylsalicylic acid, is one of the most widely used medications in the world. From the middle Ages to the present day, several therapeutic properties have been described for this molecule, including: analgesic, antipyretic, anti-inflammatory and anti-platelet action, among others. At present, in addition to its use in the relief of mild to moderate pain, fever and inflammation, aspirin plays an important role in reducing the risk of cardiovascular events. Interestingly, other beneficial effects of aspirin have been recently put forward. For instance, new findings have demonstrated that aspirin intake, administered over several years, may reduce the long term risk of certain types of cancer, particularly colorectal cancer. Indeed, it has been argued that such effect may be achieved using small amounts of aspirin (in proportions similar to ones used in the prevention of cardiovascular events), placing the antiplatelet action of aspirin at the center of its anti-tumor efficacy. However, much uncertainty remains concerning the risk/benefit of aspirin usage in cancer prevention and, as such, no definitive recommendations have been made to date. Consequently, further studies are crucially needed so that it can be defined the minimum effective dose, duration of treatment, what age to start and what the target population in which the benefit is greater than the risk. In this review, it will be considered the current knowledge regarding the role of aspirin in cancer prevention, with a special focus on colorectal cancer. In particular, it will be explored some of the possible mechanisms responsible for this effect while highlighting new paths for future research.

La présence de métastases est un facteur de mauvais pronostic dans les cancers solides et une meilleure compréhension de la dissémination tumorale est nécessaire afin d'améliorer la prise en charge de ces formes avancées. Les cellules tumorales circulantes (CTC) représentent un intérêt majeur dans la pathologie tumorale, d'une part sur le plan clinique en tant que marqueur prédictif et pronostique et d'autre part sur le plan de la compréhension des mécanismes impliqués dans la formation des métastases. Les CTC sont rares et hétérogènes et restent mal caractérisées, et ce, particulièrement dans le cancer colorectal. Une partie de ces cellules aurait un phénotype de cellules initiatrices de tumeur (CIT) leur permettant de former des métastases, de résister aux traitements et par conséquent d'être responsables des rechutes. Une meilleure connaissance des CTC possédant un phénotype de CIT représente donc un enjeu majeur. L'objectif de ce travail a été d'identifier et de caractériser les CTC avec un potentiel de cellules initiatrices de tumeur dans le cancer colorectal en se basant sur les propriétés fonctionnelles des CIT. Nous avons ainsi, pour la première fois, pu établir deux modèles permettant de répondre à cet objectif. D'une part des lignées de CTC avec un fort potentiel de CIT obtenues à partir d'échantillons sanguins de patients atteints de cancer colorectal, et d'autre part, nous avons mis en place un modèle murin de dissémination tumorale par xénogreffe orthotopique permettant d'isoler les CTC
Liver or lung metastases represent a poor prognosis in colorectal cancer patients and better understanding tumor spreading became essential to improve patient care. Circulating tumor cells (CTC) is considered as a promising tool, both as prognostic marker and as tool to study mechanisms involved in metastasis development. CTCs are rare and heterogeneous and remain poorly characterized especially in colorectal cancer. It is accepted that at least some of the CTC have a tumor initiating cell (TIC) phenotype that could be responsible for metastasis, chemoresistance and consequently lead to relapse. A deep characterization of CTC became thus an urgent unmet need. The aim of this work was to identify and characterize CTC with TIC properties in colorectal cancer, on the basis of their functional properties. To reach this aim, we established for the first time and characterized CTC lines from blood sample of colorectal cancer patient, and we also developed an orthotopic xenograft mouse model in which tumoral cells are circulating in the blood

Mestrado em Bioquímica - Bioquímica Clínica
According to the World Health Organization, around 8.2 million people die each year with cancer. Most patients do not perform routine diagnoses and the symptoms, in most situations, occur when the patient is already at an advanced stage of the disease, consequently resulting in a high cancer mortality. Currently, prostate cancer is the second leading cause of death among males worldwide. In Portugal, this is the most diagnosed type of cancer and the third that causes more deaths. Taking into account that there is no cure for advanced stages of prostate cancer, the main strategy comprises an early diagnosis to increase the successful rate of the treatment. The prostate specific antigen (PSA) is an important biomarker of prostate cancer that can be detected in biological fluids, including blood, urine and semen. However, the commercial kits available are addressed for blood samples and the commonly used analytical methods for their detection and quantification requires specialized staff, specific equipment and extensive sample processing, resulting in an expensive process. Thus, the aim of this MSc thesis consisted on the development of a simple, efficient and less expensive method for the extraction and concentration of PSA from urine samples using aqueous biphasic systems (ABS) composed of ionic liquids. Initially, the phase diagrams of a set of aqueous biphasic systems composed of an organic salt and ionic liquids were determined. Then, their ability to extract PSA was ascertained. The obtained results reveal that in the tested systems the prostate specific antigen is completely extracted to the ionic-liquid-rich phase in a single step. Subsequently, the applicability of the investigated ABS for the concentration of PSA was addressed, either from aqueous solutions or urine samples. The low concentration of this biomarker in urine (clinically significant below 150 ng/mL) usually hinders its detection by conventional analytical techniques. The obtained results showed that it is possible to extract and concentrate PSA, up to 250 times in a single-step, so that it can be identified and quantified using less expensive techniques.
De acordo com dados disponibilizados pela Organização Mundial de Saúde, cerca de 8,2 milhões de pessoas morrem anualmente com cancro. A elevada taxa de mortalidade associada ao cancro resulta da maioria dos pacientes não efetuar exames de rotina e porque a manifestação dos sintomas, na maioria dos casos, acontece quando o paciente já se encontra numa fase avançada da doença. Atualmente, o cancro da próstata representa a segunda maior causa de morte entre indivíduos do sexo masculino em todo o mundo. Tendo em conta que não existe cura para casos avançados de cancro da próstata, a estratégia passa por um diagnóstico precoce que permita aumentar a taxa de sucesso dos tratamentos. O antigénio prostático específico (PSA) é um biomarcador importante do cancro da próstata que pode ser detetado em fluidos biológicos, nomeadamente sangue, urina e sémen. No entanto, os kits comerciais disponíveis utilizam amostras de sangue e os métodos analíticos normalmente utilizados na sua deteção e quantificação requerem pessoal especializado, equipamento específico e um processamento extensivo das amostras, resultando em processos com um elevado custo associado. Assim, o objetivo deste mestrado passou por desenvolver um método simples, eficiente e menos dispendioso para a extração e concentração de PSA a partir de amostras de urina utilizando sistemas aquosos bifásicos (SAB) constituídos por líquidos iónicos. Numa fase inicial, determinaram-se os diagramas de fases de um conjunto de sistemas aquosos bifásicos constituídos por um sal orgânico e por líquidos iónicos. Em seguida, avaliou-se a capacidade dos mesmos para a extração do PSA. Os resultados obtidos demonstram que, nos sistemas em estudo, o antigénio prostático específico é totalmente extraído para a fase rica em líquido iónico num único passo. Por fim, averiguou-se a aplicabilidade dos SAB estudados para a concentração do PSA a partir de soluções aquosas e de urina. A baixa concentração deste biomarcador na urina (clinicamente significativo abaixo de 150 ng/mL) dificulta a sua deteção através de técnicas analíticas convencionais. Os resultados obtidos demonstraram que é possível extrair e concentrar PSA até 250 vezes, numa única etapa, sendo este detetável através de técnicas menos dispendiosas.

Cancer-associated cachexia is a condition defined by a sustained net-negative energy imbalance. Although the different types of adipose tissue – white, beige, and brown – have been implicated in contributing to cancer-associated cachexia, the mechanisms of these maladaptive changes and their impact on whole-body energy expenditure have not been fully elucidated. Using breast cancer as our model, we demonstrate white adipose tissue browning in murine and human breast cancer; furthermore, we demonstrate that this effect is extremely localized and takes place early in tumor progression. We utilized in vitro cell culture techniques and demonstrate that cancer secreted factors and cross-talk with white adipocytes decrease expression of classic white adipose tissue-related genes. We also demonstrate in murine and human culture models that cancer secreted factors reduce white adipocyte lipid droplet size, and cross-talk between cancer cells and adipocytes results in an increase in lipolysis-related gene expression. Interestingly, our results strongly suggest that in mice, neither cancer secreted factors nor cross talk with adipocytes can induce white adipose tissue browning, indicate that this process likely occurs independently of direct cancer interactions with local white adipocytes. We demonstrate that interleukin 6, a cytokine with previous implications in white adipose tissue browning, induces interleukin 6-mediated signaling; however, that signaling alone is not enough to directly induce white adipose tissue browning. We present preliminary data suggesting that immune cell population shifts within the white adipose tissue of mice with breast cancer tumors may be source of white adipose tissue browning. We show that the Virginia Commonwealth University Health System has an identifiable population of patients with cancer with what we hypothesize as maladaptive thermogenic adipose tissue activity, and discuss ongoing experiments aimed at understanding the implications of these changes on whole body energy expenditure in human patients. Lastly, in a case of autoimmune diabetes mellitus in the setting of an extra-adrenal paraganglioma, we demonstrate that the interaction between cancer and whole-body metabolism is multifaceted. Together, these experiments demonstrate that adipose tissue plasticity occurs in breast cancer (and other cancers), and that different drivers for individual changes exist within the tumor microenvironment. We predict that further exploration of the exact mechanisms and translational implications will provide useful information to lead to new therapeutic treatments for patients with cancer-associated cachexia.

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas
Introdução – O cancro da próstata tem tido uma enorme prevalência a nível mundial, justificando um elevado número de estudos não só na procura de novos tratamentos mais eficazes como também dos agentes que causam esta patologia, visto ser essencial saber a sua origem para a introdução de estratégias de prevenção. As causas que levam ao seu aparecimento são ainda desconhecidas, embora se saiba já a influência de alguns fatores, tais como a dieta, a idade, os antecedentes familiares, entre outros. Algumas linhas de investigação têm proposto uma hipótese que envolve alguns vírus na etiologia do cancro da próstata, tal como acontece noutros tipos de cancro, tendo como exemplo no cancro do colo do útero com o vírus do papiloma humano. Objetivos – Esta dissertação visa efetuar uma revisão com atualização do conhecimento científico sobre os mecanismos víricos que possam ser considerados como uma das causas do cancro da próstata. Material e métodos – Realizou-se uma pesquisa bibliográfica que incidiu na pesquisa eletrónica de numerosos artigos de jornais e revistas científicas e clínicas e principalmente na base de dados Medline, com interface de pesquisa Pubmed. Efetuou-se uma análise quantitativa e qualitativa dos agentes virais relacionados com o cancro da próstata descritos na literatura. Resultados e discussão – Na literatura, o vírus mais associado ao aparecimento do cancro da próstata é o vírus XMRV. Vários mecanismos de ação foram propostos como forma de infetar os tecidos humanos, principalmente as células da próstata. A presença de mutações no gene RNAseL, tal como uma preferência local por determinados locais de integração levando a uma influência andrógena foram mecanismos defendidos por vários autores. Uma possível transmissão sexual e uma ligação do vírus indireta à tumorigénese apresentam uma menor incidência de citação, mas que não deve ser ignorada. Foram identificados fármacos que poderiam ser utilizados numa terapia contra este vírus, tal como fatores de restrição existentes no humano que impedem a replicação viral por parte de XMRV. Conclusão – Há um elevado número de artigos que defendem uma relação causal entre o vírus XMRV e o cancro da próstata. Contudo, muitos outros autores sugerem que este vírus em humanos seja encontrado apenas devido a contaminação laboratorial. Introduction – Prostate cancer´s worldwide prevalence has justified a high number of studies not only seeking new, more effective treatment but also concerning the pathogenesis since it is of utmost importance reconnaissance of its underlying causes in order to apply preventing measures. Those causes are yet unknown even though some factors influence such as diet, age or family history. Some research have set forward a hypothesis where some viruses take part in prostate´s cancer etiology, similar to other kinds of cancer like the uterine cancer with the HPV. Objectives – The dissertation here presented aims to review the scientific updates on viral mechanisms which are considered as a potencial cause for prostate cancer. Methods and materials – A bibliographical research was conducted with particular focus on online scientific and clinical journals and magazines, using primarily Medline database through Pubmed research interface. A quantitative and qualitative analyses of the prostate cancer related viral agents described in literature was made. Results and Discussion – Literature shows the XMRV as being the virus most associated with prostate cancer. Several mechanisms were purposed for the infection events of human tissue, particularly prostate cells. The presence of mutation on the RNAseL gene and a local preference by determined integration spots leading to an androgenic influence were mechanisms defended by several authors. A possible sexual transmission and an indirect link of the virus to the tumorgenesis are fewer times presented but its significance should not be ignored. Drugs to be used in therapy against XMRV were identified as well as restriction factor present in humans wich stop its replication. Conclusion- There is a high number of articles defending a causal relation between XMRV and prostate cancer. Nevertheless, many authors suggest this virus is only found in humans due to laboratorial contamination.

Tumors cells grow in nutrient and oxygen-deprived microenvironments and adapt to the suboptimal growth conditions by altering their metabolic pathways. The adaptation process commonly creates a tumor phenotype of high glycolytic potential and aggressive growth characteristics which facilitate metastasis and confer resistance to radiation and chemotherapy. Understanding the mechanisms that allow tumors to adapt and survive in their microenvironment is crucial to cancer prevention and control. It was hypothesized that the tumor microenvironment would induce signaling and enzymatic changes, which if manipulated could improve treatment outcome. The results presented here demonstrate that exposure of tumor cells to chronic low pH or hypoxic conditions induced signaling cascades and altered enzyme profiles which resulted in a pro-survival phenotype. Three key adaptation events were observed and included 1) the up regulation of the metabolic stress and glycolytic proteins AMP-activated protein kinase (AMPK) and 6-Phosphofructo-2-Kinase/Fructose-2,6-Biphosphatase 3 (PFKFB3), respectfully. 2) The upregulation of p53 and 3) changes in the ratios of the bioreductive enzymes were also found to be important in the adaptation. The tumor suppressor p53 played a central role in adaptation because it induced the transcription of anti-glycolytic proteins to control glycolysis and minimize tumor cell acidosis. The ratio of bioreductive enzymes was also altered by changes to the microenvironment. Hypoxia had the greatest effect on protein levels and caused a decrease in the ratio of NAD(P)H:quinone oxidoreductase 1 (NQO1): cytochrome p450 reductase. The increase in cytochrome p450 reductase, a one electron bioreduction enzyme, has been shown to increase toxicity of bioreductive drugs in hypoxic tumors. Micronutrients also had an effect on p53 homeostasis because increasing NQO1 activity by riboflavin supplementation induced a p53-stabilizing effect by enhancing binding of p53 to NQO1, protecting the tumor suppressor from degradation. Taken together, these results indicate the changes that occur in tumor adaptation to the microenvironment require signaling and enzymatic changes that work in concert to regulate metabolism and apoptosis. Many of these changes present therapeutic targets that could be exploited to enhance therapy or prevent adaptation and subsequent tumor growth.

The tumor microenvironment encompasses all of the factors and accessory cells which interact with a tumor and, more often than not, are co-opted by tumor-secreted factors to help the cancer grow. In this thesis, we examined elements of the tumor microenvironment, specifically the cancer-promoting M2 macrophages (MΦs) and tumor glucose supply and metabolism. In the vast majority of advanced cancer patients and tumor-bearing animals, their tumors contain MΦs that are profoundly skewed to a cancer-promoting M2 phenotype, which often correlates with a poor prognosis. As well, these same tumor tissues are more dependent on high glucose levels for energy and survival than most normal tissues. Although MΦ phenotype and tumor cell glucose metabolism are quite disparate fields of study, we employed similar strategies to uncover what regulates them in order to explore mechanisms to reduce tumor growth. In our M2 MΦ studies, we assessed the phenotypic plasticity of mature IL-4-induced M2 MΦs and found their phenotype (i.e., cell-surface markers, cytokine secretion, and T cell stimulatory properties) could be fully reversed with IL-4 withdrawal and subsequent IFN-γ priming, demonstrating that M2 MΦs, indeed, can be reprogrammed even if they are phenotypically polarized. As well, we uncovered a circuit of M2-MΦ generation which may be relevant in vivo in the M2-skewed SHIP-/- mouse model. This circuit involves the sensitization of MΦs and MΦ progenitors, via IgG and TGF-β-containing mouse plasma, to low levels of constitutive IL-4 uniquely secreted by SHIP-/- basophils. In a similar vein, and based on the Warburg effect, which describes the propensity of cancer cells to consume glucose via glycolysis rather than oxidative phosphorylation (OXPHOS), we designed low carbohydrate (CHO) diets to limit the glucose supply to tumors. We found that mice fed our low CHO diets had lowered blood glucose, insulin, and lactate, and this correlated with a slower growth rate of implanted tumors, and a lower cancer incidence in a spontaneous mouse mammary carcinoma model. Furthermore, our diets worked additively with known anti-cancer agents (i.e.,Temsirolimus, Celebrex) to slow tumor growth.

The following thesis is based on my study of two candidate prostate cancer tumor markers, namely telomerase and CD9. Accordingly, the thesis will be divided in two main chapters describing the results obtained for each protein. Although these proteins are de-regulated in prostate cancer cells, little is known about their regulation and function in prostate cells. For example, telomerase is generally inactive in normal cells and reactivated in cancer cells and CD9 is a transmembrane protein that is lost as prostate cancer progresses. In chapter 2, I will show that introduction of normal human chromosomes into mouse melanoma cells can shut down its telomerase activity by inhibiting hTERT transcription and that the gene encoding this regulator is likely located on human chromosome 17p11.1. Additionally, following further characterization of CD9 partners in chapter 3, we will learn more about the likely mechanism by which CD9 induces mitotic catastrophe when over-expressed in prostate cancer cells.

"Anaplastic thyroid cancer (ATC) is the most aggressive form of thyroid cancer, with very high mortality rate. Tumor-associated macrophages (TAMs) can represent up to 70% of ATC’s tumor mass, making them an interesting target for novel therapies. In this thesis, the aim was to further understand the crosstalk between ATC and TAMs. To achieve that, cell surface proteomics of two ATC cell lines (C3948 and T235), in mono- or co-culture with THP-1-derived macrophage-like cells, was performed. The protein Spry-4, an inhibitor of the MAPK-ERK pathway, was found to be downregulated in C3948 cells upon co-culture. Spry-4 protein levels were further evaluated by Western blot; only T235 in co-culture showed a trend for downregulation, although without statistical significance.(...)"
N/A

Tumor biologist have long appreciated that both cell to cell and cell to extracellular matrix (ECM) interactions are involved in the invasive and metastatic events that are characteristic of malignancy. Cancer cell attachment to and invasion of an ECM has been associated with metastatic potential of cell lines of the Dunning rat prostate model. It was postulated that differences observed in the metastatic potential of four Dunning cell lines may correlate with cell-matrix interactions. Four cell lines, highly metastatic ML, MLL, AT-3 and non-metastatic AT-1 were studied. The adhesive, invasive and chemoinvasive capability of each cell line was compared. Cell adhesion was examined by plating the cells on plastic dishes coated with various components of the ECM (fibronectin, laminin and collagen) as well as EHS Natrix (a natural ECM) . Invasion was determined by examining cells ability to traverse a matrigel barrier. Correlations were found between the cells' adhesive and invasive abilities in response to the ECM. These observations suggest that ECM components are highly involved in prostate cancer cell activities and loss may contribute to tumor progression and metastasis.

Pancreatic and prostate cancer are both prevalent cancers in the United States with pancreatic being one of the most aggressive of all cancers and prostate cancer being one of the most common, ranking as the number one cancer in men. Treatment of both cancers can be quite challenging as the anatomy of the pancreas and prostate, as well as the development and diagnosis of the disease can greatly limit treatment options. Therefore, it is necessary to develop new cancer treatments to help manage and prevent these cancers. Irreversible electroporation is a new non-thermal focal ablation therapy utilizing short, pulsed electric fields to damage cell membranes leading to cell death. The therapy is minimally invasive, involving the insertion of needle electrodes into the region of interest and lasts less than two minutes. Heat sink effects that thermal therapies experience near large blood vessels do not affect irreversible electroporation. This allows the treatment to be used on tumors near vasculature as well as critical structures without harming these vital regions. While irreversible electroporation is a promising new cancer therapy, further developments are necessary to improve treatment planning models. This work aims to further understand the electric field thresholds necessary to kill different types of cancer cells with a focus on pancreatic and prostate cancer. The work is done using an in vitro tumor (hydrogel) model as this model is better than traditional cell suspension studies, with added benefits over the immediate use of tissue and animal models.
Master of Science

INTRODUCTION: Breast cancer is the most common cancer in women worldwide and metastatic dissemination is the principal factor related to death by this disease. Breast cancer stem cells (bCSC), defined in this work as the ALDH1high/LIN-/ESA+ population, are thought to be responsible for metastasis and chemoresistance. The objective of this work is to find gene master regulators, in particular transcription factors (TFs), which are controlling the bCSC phenotype. METHODS: We used in this work two groups of datasets with transcriptome data, the discovery dataset group contains one dataset obtained by ourselves containing three paired samples comparing the bCSC and the bulk of the tumor (My Data - bCSC/Bulk dataset), a dataset with eight paired samples comparing the bCSC and cancer cells (Wicha - bCSC/CC dataset) and a dataset with 115 samples of breast cancer tissue (clinical response dataset). The second group, validation datasets, contains the BRCA-TCGA dataset with information of 621 samples, 4142 breast cancer samples of the Kmplot tool, 17 primary samples of BasL subtype and their information of grafting in patient derived xenografts and analyzes of cell lines (MF10A and HMLE). For the analyzes we used the paired t-test in the Limma R package, the ARACNE algorithm for the inference of regulons in the clinical response dataset, MRA-FET to define the master regulators of the bCSC phenotype, and GSEA to identify the biological meaning of the findings in the different datasets. RESULTS: We identified 12 TFs as master regulators of the bCSC phenotype, with nine of them forming two highly interconnected networks, one positively related with the bCSC phenotype formed by SNAI2, TWIST, PRRX1, BNC2 and TBX5 with its regulons, defined here as the mesenchymal transcription network and one negative correlated to the phenotype formed by SCML4, ZNF831, SP140 and IKZF3, defined as the immune response transcription network, totally unknown in the context of breast cancer in the literature. Although still with weak evidence, ZEB1 seems to control the two networks and can be responsible for the expression of ALDH1 and of the three remaining TFs: ID4, HOXA5 and TEAD1. As their names portray, our data showed in the different datasets, and independently of the molecular subtype and of the platform used, that the mesenchymal transcription network seems to be responsible for the bCSC phenotype and the immune response transcription network to the adaptive immune response in the tumor and a better prognosis for the patients. We also defined 10 membrane proteins as new markers and/or therapeutic targets of the bCSC. CONCLUSION: We found and described two TF networks that seem to control the bCSC phenotype, one of them totally unknown until now and correlated to a good prognosis. Our findings have a clear potential for clinical use.
INTRODUÇÃO: O cancer de mama é no mundo o câncer mais comum em mulheres e a disseminação metastática é o principal fator relacionado com a morte pela doença. Acreditasse que as células tronco do câncer de mama - bCSC, na sigla em inglês e definida neste trabalho com a população ALDH1high/LIN-/ESA+ - é responsável pela metástase e pela quimioresistência. O objetivo deste trabalho é encontrar genes que são essenciais para o controle do fenótipo das bCSC, em particular fatores de transcrição. MATERIAIS E MÉTODOS: Nesse trabalho nós utlizamos dois grupos de datasets com dados do transcriptoma, o grupo de datasets de descoberta contém um dataset gerado por nós com 3 amostras pareadas comparando as bCSC com o tumor total (My Data - bCSC/Bulk dataset), um dataset com 8 amostras pareadas comparando as bCSC com as células cancerígenas (Wicha - bCSC/CC dataset) e um dataset com 115 amostras de tecido de câncer de mama (Clinical Response dataset). O segundo grupo, grupo de validação, contém o dataset BRCA-TCGA com 621 amostras, as 4142 amostras de câncer de mama da ferramenta Kmplot, as 17 amostras humanas primárias do subtipo BasL e sua informação sobre a geração, ou não, de tumores em camundongos imunosuprimidos e a análise de linhagens celulares (MF10A e HMLE). Para a análise dos dataset utilizamos o test-t pareado no pacote Limma da liguagem R, o algoritmo ARACNE para a inferência de regulons no dataset Clinical Response, a análise MRA-FET para definir os Reguladores Mestres para o fenótipo das bCSC e a análise GSEA para identificar o significado biológico de nosso achados nos diferentes datasets. RESULTADOS E DISCUSSÃO: Nós identificamos 12 TFs como reguladores mestres, com 9 deles formando duas redes altamente conectadas, uma positivamente relacionada ao fenótipo bCSC formada por SNAI2, TWIST, PRRX1, BNC2 e TBX5 com seus regulons, e definida aqui como a rede de transcrição mesenquimal, e uma rede correlacionada negativamente, formada por SCML4, ZNF831, SP140 e IKZF3, definida aqui como a rede de transcrição da resposta imune e totalmente desconhecida da literatura no contexto do câncer de mama. Embora ainda com fraca evidencia, ZEB1 para controlar as duas redes e ser responsável pela expressão de ALDH1 e dos 3 TFs restantes: ID4, HOXA5 e TEAD1. Como mostram seus nomes, e independente do dataset, do subtipo molecular ou da plataforma utilizada, a rede de transcrição mesenquimal, parece ser responsável pela manutenção do fenótipo de células tronco cancerígenas e a rede de transcrição da resposta imune pela resposta imune adaptativa ao tumor e a um bom prognóstico para as pacientes. CONCLUSÃO: Nós encontramos e descrevemos duas redes de fatores de transcrição que parecem controlar o fenótipo das bCSC, uma delas totalmente desconhecida até agora e relacionada a um bom prognóstico. Nosso achados possuem um claro potencial para uso clínico.

Cancers develop in complex microenvironments whose importance was emerging during last years. In fact, cancer microenvironment influences tumor progression and leads to the raising of chemotherapics resistance. Thus, a shift of the focus from cancer cells to cancer cells in their environment is crucial for studying the molecular and metabolic features of tumors in physiological contexts. Within the microenvironment, cancer associated fibroblasts (CAF) are attracting the attention of scientific community since, up to date, it is clear that they are the main component involved in the organization of tumor tissue and that they interact with cancer cells and affect their behavior. Importantly, stromal cell types within the microenvironment are genetically stable, contrary to the tumor counterpart that frequently shows genetic instability and seriously altered physiological mechanisms. Accordingly, they represent a valuable therapeutic target. For these reasons, in this work we investigated the interplay between prostate cancer cells and tumor microenvironment, focusing on CAFs' role and on alterations of cancer cells occurring due their presence. In the first part of the study we collected expression profile data from cultures and co-cultures as well as metabolic data in order to characterize prostate cancer cells (PC3), CAFs and normal fibroblasts and alterations occurring in these cells n co-culture conditions. We found deregulated genes, strictly related to activation of processes which ease conditions favoring tumor progression in PC3 cells and in CAFs. More, we found deregulated genes in CAFs which usually are described as deregulated in transformed cells from patients and studied in PC3s, highlighting the importance of considering the stromal component as playing a major role in driving tumor progression. Through analyses of metabolic data we shed light on metabolic needs of PC3s and CAFs and on the metabolic behavior adopted by CAFs to enhance PC3s' growth. In the second part of the study, the retrieved data were used to build a metabolic model which in turn was used to validate an algorithm we developed, Metpath, designed toto improvestudies of unknown pathways' alterations. The models we built, a single cell model for PC3 and S9 and one model representing interacting PC3 and CAF cells, showed to be able to recapitulate the main metabolic reactions occurring in the corresponding conditions and represent a valid starting ground on which refined models for further studies could be developed. Moreover, MetPathrevealed altered metabolic pathways in PC3 and in CAF, which deserve further studies to deepen the knowledge about the way they can confer advantages to tumor. Taken together, all the results obtained in this work demonstrate the importance of considering and studying the tumors not only as a set of cancer cells but as part ofa complex microenvironment which influences tumor's progression. Importantly, this workhighlights the importance of the modern approaches to cancer study. In fact, simulation studies on --omics integrated models, like the ones elaborated in this work, can really push forward research in the multidisciplinary field of cancer research.

Les mutations du gène suppresseur de tumeur MEN1 sont connues depuis de nombreuses années, pour être à l'origine du syndrome des Néoplasies endocriniennes multiples de type 1 (Syndrome des NEM1). Ce syndrome constitue une maladie héréditaire associée à une perte d'hétérozygotie progressive du gène MEN1, affectant principalement les organes endocrines. Plus récemment, l'implication du gène MEN1 a émergé dans la tumorigénèse d'autres organes, et plus particulièrement dans dans le cancer du sein et le cancer de la prostate, où son rôle reste encore très controversé. Pour mieux déterminer le rôle joué par menin dans les cellules prostatiques (oncogène ou gène suppresseur), mon projet de thèse avait donc pour but de caractériser un nouveau modèle murin d'invalidation du gène Men1 spécifiquement dans les cellules luminales de la glande prostatique, le modèle Men1F/F Nkx3.1CreERT2+/-. Les examens anatomopathologiques réalisés sur ce nouveau modèle murin ont montré que la perte d'expression du gène Men1 conduisait à une accélération de la tumorigénèse dans la glande prostatique par rapport aux souris contrôles. D'autre part les analyses moléculaires issues de l'étude de notre nouveau modèle murin, ont montré que l'expression du récepteur aux androgènes (RA) était diminué dans les cellules déficientes pour le gène Men1 . Des analyses menées in vitro ont montré que la protéine menin, codée par le gène MEN1, joue le rôle de régulateur transcriptionnel du RA. De la même manière, mes travaux ont mis en évidence, que la protéine menin semble réguler l'expression du récepteur aux estrogènes alpha (RE?), en liant la région promotrice de ce dernier dans des lignées cellulaires du cancer du sein. De plus, les analyses cliniques ont révélé que l'expression réduite de menin corrélée avec la survenue du sous type luminal B du cancer du sein, connue pour exprimer faiblement le RE??Ainsi mes travaux de thèse ont permis de conforter notre hypothèse sur le rôle oncosuppressif du gène MEN1 dans le glande prostatique. D'autre part, nous avons mis en évidence l'implication de la protéine menin dans la régulation de l'expression des récepteurs nucléaires, dans le cancer du sein et de la prostate
For a long time, mutations of the MEN1 gene have been known to be responsible of the Multiple Endocrine Neoplasia type 1 (MEN1 syndrome), a hereditary disease affecting mainly endocrine organs. Recent advances highlighted the involvement of the MEN1 gene in the development of the breast cancer and prostate cancer. Nevertheless, the role played by the MEN1 gene in prostate cancer still remains unclear, described as on oncogene by some studies, or as a tumor suppressor by others. To further adress this issue, we generated a novel and inductible mouse model, Men1F/F-Nkx3.1Cre-/+, in which the Men1 gene can be specifically disrupted in luminal prostatic cells upon tamoxifen injection. Anatomopathologic examination of our model showed that the Men1 gene disruption accelerate the tumorigenesis in the prostatic gland compared to the control mice. Moreover, molecular analyses showed that the expression of androgen receptor (AR) decreased in Men1-deficient cells. In vitro study perfomed in prostate cancer cell lines showed that menin protein encoded by the Men1 gene is involved in the transcriptionnal regulation of AR.Similarly, my work showed that menin protein also involved in the transcriptionnal regulation of the estrogen receptor alpha (ER?) expression, through its binding on the promoter of the ER??gene. Moreover, clinical study revealed that decrease in menin expression correlates with the occurrence of luminal B subtype of breast cancer, in which ER??expression is reduced. Thus this thesis work, allowed to better characterized the oncosuppressive role of the Men1 gene in the prostatic gland. This work, also highlighted for the first time the involvement of menin protein in the regulation of nuclear receptor expression, in prostate and breast cancer

Bakgrund: I Sverige drabbas 8000 kvinnor av bröstcancer varje år. Dessa kvinnor utsätts för både kroppsliga och emotionella biverkningar och förändringar. Syfte: Syftet med denna litteraturöversikt är att belysa hur kvinnor som drabbas av bröstcancer upplever att deras sexualitet påverkas av sjukdom och behandling. Metod: Metoden var en litteraturstudie. Resultat: Ett huvudtema framkom en förändrad identitet; och fem subteman: kroppsliga förändringar, känna sig okvinnlig, relationens betydelse, kommunikationens betydelse för att komma vidare och sjukvårdens betydelse. Slutsats: Resultatet visade att kvinnorna upplevde att de förlorat sin sexuella identitet och sin kvinnlighet. Förlusten gjorde att de inte kunde identifiera sig med den kvinna som de tidigare varit. De upplevde flera fysiska förändringar som ledde till minskad sexuell aktivitet. Men trots den minskade sexuella aktiviteten upplevde många av kvinnorna en ökad närhet till sin partner och stödet från en partner ansågs viktigt. Många kvinnor saknade dock stöd och information från sjukvården angående sexuella problem. De upplevde att vårdpersonalen tyckte det var genant eller oviktigt att tala om kvinnornas sexualitet. Då sjuksköterskans primära mål är att främja hälsa och välbefinnande bör sjuksköterskan beakta kvinnornas sexualitet eftersom sexualiteten är en viktig dimension för upplevelsen av hälsa.
Background: In Sweden 8000 women are afflicted by breast cancer every year. These women are subject to both physical and mental side effects and alternations. Aim: The objective of this literature review is to highlight how women in the case of breast cancer experience that their sexuality is affected by the disease and treatment. Method: The method is a review of the related literature. Results: One major theme emerges: a changed identity, and five minor themes: physiological changes, feeling unfeminine, the significance of relationship, the significance of communication in order to move on, and the significance of medical care. Conclusion: It turns out that these women experienced a loss of their sexual identity and their femininity. The deprival resulted in an inability to identify themselves with the women they once were. They also experienced physiological changes, e.g. dryness of the vaginal mucous membrane, pain from the removed breast, and less sexual arousal which impeded and reduced the sexual activity of the women. But despite reduced sexual activity, many of these women experienced acquiring a greater proximity to their partner, and that the support from a partner was regarded as important. Many of the women, though, lacked support and information from the medical institutions concerning sexual problems. They experienced that the medical staff thought that conversing upon the women’s sexuality was something embarrassing or of no importance. Since the primary objective of the nurse is to promote health and well-being, the nurse should take heed to and attend to the sexuality of the women, considering that sexuality is an important dimension in regard to experiencing health.

Background: Neuroblastoma (NB) is a pediatric tumor in infants and young children. The survival rate is only around 50 percent for high-risk NB despite advanced and intense multi-modal therapy. Current research aims to find new effective treatment additional to modern therapy to improve prognosis of high-risk NB in children. As such, SU11248 may be a valuable approach for improving treatment and survival as growth factors have crucial roles in tumor growth, angiogenesis, and metastasis. Aim: The aim of this investigation was to examine tissues from SU11248 treated and nontreated tumor-bearing animals on the abundance of tumor-associated macrophages (TAMs) in metastases found on vital organs. Our hypothesis is that if SU11248 could cause “deactivation” or “silencing” of the stroma of metastases particularly by acting on stromal immune cells such as TAMs. Methods: Paraffin-embedded metastases developed in an orthotopic xenograft model in beige SCID mice were stained with a monoclonal rat anti-mouse antibody as a marker of TAMs. Morphological analysis of tissue slides, and macrophage quantification was performed using a microscope. Statistical analysis was achieved using an unpaired two tailed t-test.  Results: Macrophages were stained nicely, but the number of macrophages in the metastases were not statistically different between the vehicle treated controls and SU11248 treated metastases. Conclusion: In patients with high-risk NB, SU11248 may be a useful therapeutic supplement. We believe that further research into mechanisms that target critical factors for angiogenesis and metastasis in NB, such as TAMs, is an important step toward improving patient outcomes in high-risk NB.

Chemoresistance remains a major clinical obstacle to effective management of ovarian cancer. Cancer stem cells (or tumor-initiating cells, TICs) have been discovered recently, and have played a pivotal role in changing the view of cancer development; however, the molecular mechanisms by which these cells escape conventional therapies remain elusive. In this study, TICs were isolated from ovarian cancer cells as tumor spheres with specific stem properties under TIC-selective conditions. Unlike non-TICs, TICs strongly express stem cell factor (SCF) and c-Kit. Blocking SCF-c-Kit by SCF neutralizing antibodies, c-Kit small interfering RNA (siRNA) or imatinib (Gleevec), a clinical drug that inhibits c-Kit signaling, significantly inhibited TIC proliferation. Although cisplatin and paclitaxel killed the non-TICs, they did not eliminate TICs. Importantly, the combination of cisplatin/paclitaxel with c-Kit siRNA or imatinib inhibited the growth of both non-TICs and TICs. Similar results were obtained when patient-derived TICs were used. The findings also indicate that tumor-predisposing microenvironment, such as hypoxia, may promote ovarian TICs through upregulating c-Kit expression. Furthermore, I have showed that c-Kit expression induced activation of Phosphatidylinositol 3-kinases (PI3K)/Akt, -catenin, and ATP-binding cassette G2, which could be reversed by treatment with the PI3K/Akt inhibitor or -catenin siRNA. I further studied potential gene expression in TICs using cDNA and microRNA (miRNA) microarrays. The result from these microarrays provided a general profile in gene expression of TICs compared with the bulk tumor cells. In particular, let-7a, b, and c were shown to be downregulated in TICs compared to bulk tumor cells, suggesting that their loss may contribute to ovarian cancer development. Together, this study reveals a previously undescribed therapeutic effect of SCF-c-Kit signaling blockade to prevent ovarian cancer progression by eliminating TICs and the altered genes or miRNAs may represent possible molecular targets.
published_or_final_version
Biological Sciences
Master
Master of Philosophy

Les carcinomes sont des cancers touchant plusieurs organes du corps humain, notamment les seins, le pancréas, les poumons, l'intestin… et sont issus de la transformation de cellules épithéliales en cellules tumorales. Au cours du développement d'une tumeur, les cellules cancéreuses, contrairement aux cellules normales, acquièrent la capacité de se déplacer dans le corps humain, jusqu'à coloniser des organes voisins. Ces colonies sont appelées métastases. Le processus métastatique est responsable de 90% des décès dans le cadre des carcinomes. Ce processus n'est pas dû à l'action isolée des cellules cancéreuses mais est aussi le résultat d'une coopération entre la tumeur et son voisinage – le microenvironnement tumoral – favorisant la survie et la migration des cellules cancéreuses. Les fibroblastes sont une population cellulaire du microenvironnement tumoral. Il a été démontré que les fibroblastes sont activés à proximité des cellules cancéreuses ; on les qualifie de fibroblastes associés au cancer ou CAFs. Dans des tissus de patients, les tumeurs les plus agressives corrèlent avec un enrichissement en fibroblastes et une matrice plus dense. Mon projet de thèse illustre un nouveau mécanisme de coopération entre CAFs et cellules cancéreuses. Cibler l’action des fibroblastes pourrait ralentir la progression tumorale, voire bloquer la formation de métastases
Cancer-associated fibroblasts (CAFs) are the most abundant cells of the tumor stroma. Their capacity to contract the matrix and induce invasion of cancer cells has been well-documented. However, it is not clear if CAFs remodel the matrix by other means (degradation, matrix deposition or stiffening). This project demonstrates that CAFs induce cancer cell invasion through assembly of FN into the matrix. CAFs assembled fibronectin (FN) mainly via integrin α5 but integrin αvβ3 was necessary for initial mechanosensing and fibrillar adhesion formation. In the absence of FN, contractility of the matrix by CAFs is preserved. When degradation is impaired, CAFs retain the capacity to induce invasion in a FN-dependent manner. In all cases, the levels of expression of integrin β3 and the amount of assembled FN was directly proportional to the invasion induced by fibroblast populations. Our results highlight FN assembly and integrin β3 as new hallmarks of CAFs. We also noticed that cancer cells migrate towards CAFs suggesting a possible chemotactic response. Using Dunn’s chemotaxis chamber, we found that cancer cells migrate along a gradient of CAF-conditioned media and a gradient of fibronectin. Finally, orthotopic injections of cancer cells and CAFs in the colon wall of mice revealed that CAFs stimulate metastasis of cancer cells to the liver. In conclusion, our data show that CAFs promote cancer cell invasion by depositing fibronectin that can guide cancer cells favoring metastasis formation

La membrane joue un rôle clé dans un large nombre de processus physiologiques. Lors d’une activation cellulaire, l’externalisation de PS sur la membrane plasmique est suivie par l’émission de microvésicules membranaires (MV). Les MV reflétant le profil antigénique de la cellule et de la stimulation dont elles sont originaires. Le microenvironnement tumoral est enrichi en MV libérées par les cellules qui constitue le tissu cancéreux. En nous focalisant sur l’implication des fibroblastes nous proposons un modèle in vitro dans lequel les cellules normales et cancéreuses communiquent entre elles via MV. Le but de cette étude est d’élucider un mécanisme, dans lequel les cellules du cancer de la prostate influencent le comportement de cellules normales du stroma, et ces dernières affectent l’agressivité des cellules de carcinome, Le tout via la libération mutuelle de MV pour promouvoir ou soutenir la création d’une niche favorable au développement de la tumeur
The plasma membrane plays a pivotal role in a large number of physiological processes. After cellular stimulation, externalization of PS in the exoplasmic leaflet of plasma membrane is followed by the shedding of membrane microvesicles (MV) in almost all cell types. MV composition reflect the antigenic profile of cells which they originate from, depending on the stimulus apply. Tumour microenvironment is highly enriched in MV shed from cells infiltrating the tumour tissue. Fibroblasts are associated with tumour cells at all stages of cancer progression. Focusing on fibroblasts implication in cancer, we propose an in vitro model in which cancer and normal cells communicate each other via MV. The aim of this study is to elucidate a mechanism, in which prostate cancer cells influence the behaviour of normal stromal cells that in turn affect the aggressiveness of carcinoma cells by mutual MV shedding, promoting or support the creation of a niche favourable for tumour development

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Lung cancer (LC) is the leading cause of cancer mortality worldwide and second most common type of cancer in the United States in both genders. Moreover, Head and neck cancer (HNC) is the sixth most common cancer worldwide and 4% of all malignancies in the United States. The role of FDG-PET-CT has recently increased in oncology for diagnosis, treatment monitoring and patient prognosis. FDG Metabolic parameters sued to assess patient care include maximum standard uptake value (SUVmax), metabolic tumor volume (MTV), and total glycolytic activity. This study attempts to prove FDG-metabolic parameters reliability in LC and HNC patients before and after treatment among readers with different levels of experience. Three readers, 2 experienced and 1 inexperienced, read before and after treatment scans of 74 FDG-PET-CT scans from 13 lung cancer patients and 24 head and neck cancer patients. Lesion location was provided beforehand and reliability was tested using intra-class correlation coefficients (ICC) and ANOVA analysis. For every case, ICC was >0.81 (almost perfect agreement) among all readers and ANOVA showed no statistical significance (p>0.05) on the any of the measurements among all readers as well. We concluded that FDG-PET-CT metabolic parameters (SUVmax, MTV, and TGA) are reliable measurements for treatment response in LC and HNC patients and are independent of reader experience as long as lesion location is provided. These parameters have been found to accurately correlate with tumor behavior and patient prognosis; therefore, reliability on its accurate measurement provides strength to FDG-PET-CT as an imaging modality of choice for oncology patients.

La spleen tyrosine kinase (Syk) est une protéine kinase cytoplasmique qui intervient dans la signalisation immunitaire. Notre équipe a montré pour la première fois que Syk est exprimée aussi dans les cellules épithéliales mammaires et que son expression est perdue au cours de l’acquisition d’un phénotype invasif/métastatique. Syk agit comme un suppresseur de tumeurs et de métastases dans des modèles de xénogreffes de cancer du sein. Ces observations ont été étayées par des études cliniques qui montrent que la perte d’expression de Syk correspond à un risque accru de développement de métastases (facteur de mauvais pronostic) dans le cancer du sein et d’autres carcinomes. Par une approche de phospho-protéomique quantitative (SILAC), nous avons pu identifier de nouveaux substrats potentiels de Syk dans les cellules de cancer du sein. De façon intéressante, ressortent de nombreuses protéines impliquées dans l'adhésion intercellulaire (E-cadhérine/caténines) et la polarisation épithéliale (eg ZO3, occludine, claudine-3). Ces protéines, qui se localisent aux jonctions d'adhésion et d'occlusion, sont connues comme composants plate-formes de signalisation et exercent souvent une fonction de suppresseur de tumeur.Dans ce travail de thèse je me suis focalisé principalement sur :(i) le rôle de l’activité kinase de Syk dans la régulation du complexe E-cadhérine/caténines et(ii) les conséquences de l’invalidation conditionnelle de Syk dans la glande mammaire murine (développement mammaire et tumorigenèse).Par une approche de phosphorylation in vitro, nous avons montré que la E-cadhérine et différentes caténines sont des substrats directs de Syk. Les résidus tyrosines phosphorylés dans ces protéines ont été identifiés par spectrométrie de masse et les anticorps phospho-spécifiques correspondants ont été générés. En immunofluorescence, Syk endogène colocalise avec la E-Cadhérine au niveau des jonctions adhérentes et la surexpression de Syk stimule la phosphorylation de la E cadhérine et différentes caténines au niveau des jonctions intercellulaires. Des expériences d’immunoprécipitation montrent que les protéines E-cadhérine et caténines phosphorylées restent associées dans un complexe au niveau des jonctions adhérentes. L’extinction de Syk par shRNA dans une lignée de cancer de sein inhibe partiellement la ré-agrégation intercellulaire (2D/3D) et augmente l’invasion et la migration cellulaires et la croissance en 3D dans le Matrigel. Inversement, la surexpression de Syk inhibe la migration et l’invasion et favorise l'adhérence intercellulaire. Syk semble par la phosphorylation du complexe E-cadhérine/caténines consolider leurs interactions renforçant ainsi les jonctions intercellulaires et l’intégrité de l’épithélium, ce qui pourrait révéler un mécanisme majeur responsable de son activité anti-invasive. Leurs mécanismes moléculaires ont été explorés.Ces modèles cellulaires in vitro ont ensuite été étendus vers un modèle intégré murin. L'invalidation homozygote du gène SYK étant létale, nous avons développé un modèle d’invalidation conditionnelle de Syk dans la glande mammaire (Syk-flox:WAP-Cre). Ce modèle nous a permis tout d’abord d’étudier le rôle de Syk dans le développement et la physiologie de la glande mammaire au cours de la lactation et de l’involution, les glandes Syk-négatives montrant des défauts de développement. Il permet à plus long terme également d’évaluer l'implication de Syk dans la formation et la progression du cancer du sein chez des souris cKO Syk, après croisement ou non avec des souris transgéniques exprimant l’oncogène MMTV-Neu/Her2.Déterminer si Syk est un bona fide suppresseur de tumeurs est crucial car un inhibiteur de Syk est en cours d’étude clinique pour le traitement de l’arthrite rhumatoïde. L’identification des voies de signalisation gouvernées par Syk pourrait ultérieurement déboucher sur le développement de nouvelles thérapies ciblant ces protéines et bloquant l'évolution cancéreuse
The spleen tyrosine kinase (Syk) is a cytoplasmic protein kinase involved in immune-response signaling. Our team showed for the first time that Syk is also expressed in mammary epithelial cells and that its expression is lost during acquisition of an invasive/metastatic phenotype. Syk acts as a tumor and metastasis suppressor in breast cancer xenograft models. Clinical studies corroborated that loss of Syk expression is correlated with a decreased survival and an increased risk of metastasis development (poor prognosis) in breast cancer and other carcinomas. Using a quantitative phospho-proteomic SILAC approach in breast cancer cells, our group identified new potential Syk substrates. Interestingly, many proteins are involved in intercellular adhesion (E-cadherin/catenin) and epithelial polarization (eg ZO3, occludin, claudin-3). These proteins are localized at the adherens and tight junctions and are known as signaling platforms and components often presenting a tumor suppressor function.In this thesis I mainly focused on:(i) the role of the Syk kinase activity in the regulation of the E-cadherin/catenin complex and(ii) the consequences of the conditional Syk knockout in the mouse mammary gland on breast development and tumorigenesis.Using in vitro kinase assays, we demonstrated that E-cadherin (E-Cdh) and different catenins are direct Syk substrates. The phosphorylated tyrosine residues were identified by mass spectrometry and corresponding phospho-specific antibodies were generated. By immunofluorescence, we observed that endogenous Syk and E-Cdh colocalize at adherens junctions (AJ) and that Syk overexpression stimulates Syk-dependent phosphorylation of E-cadherin and different catenins at AJ. Immunoprecipitation experiments indicate phosphorylated E-cadherin and catenin proteins are associated in a complex. Using functional tests, Syk knockdown by shRNA in breast cancer cells partially inhibited intercellular re-aggregation (2D/3D) and increased cell invasion, migration and 3D-growth in Matrigel. Conversely, Syk overexpression inhibited migration and invasion and promoted intercellular adhesion. Thus, Syk seems to strengthen the intercellular junctions and the integrity of the epithelium via the phosphorylation of the E-cadherin/catenin complex of which its molecular mechanisms were explored. This could be a major mechanism responsible for its anti-invasive activity.These in vitro observations were subsequently extended to an integrated mouse model. As the homozygous SYK gene knockout is lethal; we developed a conditional Syk deletion model in the murine mammary gland (Syk-flox:WAP-Cre).This model allowed us to study the role of Syk in the development and physiology of the mammary gland during lactation and involution, the Syk-negative glands showing developmental defects. On a long-term basis, it also allows to assess the involvement of Syk in the formation and progression of breast cancer in aging cKO Syk mice, bred or not with transgenic mice expressing the MMTV-Neu / Her2 oncogene.Whether Syk is a bona fide tumor suppressor is a crucial issue as Syk inhibitors are being evaluated in clinical studies for the treatment of rheumatoid arthritis. Identification of the signaling pathways governed by Syk could lead to the development of new therapies targeting these proteins and blocking tumor development and progression

Tese especialmente elaborada para obtenção do grau de Doutor em Ciências Veterinárias, especialidade de Ciências Biológicas e Biomédicas
Jagged1 (Jag1) is a Notch signaling ligand, which has been described as essential for developmental angiogenesis and to play an important role in several aspects of tumor biology. However the underlying mechanism related to Jagged1/Notch signaling still remain incompletely understood. Therefore this thesis analyzed Jagged1 driven Notch signaling enrolment in adult angiogenesis settings, and in tumor development. To address the role of endothelial Jag1 in physiological and tumoral angiogenesis, endothelial-specific Jag1 mutants driven angiogenic phenotypes were assessed in skin wound healing and in transplanted tumors and prostatic autochthonous tumor growth, respectively. An extensive transcription and expression analysis of Notch signaling members in the tumorigenic development of the mouse prostate was also performed to identify ectopically expressed Notch members. Lastly, the therapeutic potential of an Anti-Jagged1/2 antibody in mouse prostate cancer was evaluated. Altogether, results presented here demonstrate that Jagged1 is a pro-angiogenic ligand due to its ability to antagonize Dll4/Notch1 mediated signaling. It also has a pro-maturation function by both endothelial Notch4 and perivascular Notch3 mediated signaling. Both these functions contribute to accelerated wound healing and tumor growth, by inducing a more functional vasculature. Moreover, we have identified a new angiocrine function for endothelial Jagged1, mediated through Notch3/Hey1 activation in tumor cells. Finally, we have demonstrated that either by mediated endothelial-specific angiocrine function or by tumor cells mediated Jagged1 ectopic expression, this ligand regulated tumor cell proliferation, apoptosis, de-differentiation, epithelial-to-mesenchymal transition and cancer stem-like cells proliferation and survival. Ultimately, we have demonstrated that blocking Jagged-mediated Notch signaling inhibited development and progression of mouse prostate cancer and therefore constitutes a promising therapeutic approach in prostate cancer treatment.
RESUMO - Avaliação do papel do ligando Jagged1 na regulação da angiogénese do adulto e no desenvolvimento de tumores sólidos - A via Notch é uma via de sinalização intercelular altamente conservada que está envolvida na determinação do destino, e na regulação da proliferação e diferenciação celulares. Jagged1 é um ligando desta via, que tem sido descrito como essencial ao processo de angiogénese durante o desenvolvimento embrinário e que desempenha um papel crucial em diversos aspectos de biologia tumoral. No processo do desenvolvimento angiogénico da retina, Jagged1 foi descrito como tendo um efeito contrário ao de outro ligando da via, o Dll4, mas esta interacção continua por demonstrar noutros contextos angiogénicos. Além de ser expresso na vasculatura, Jagged1 também é detectado em células epiteliais de vários órgãos. Em tumores já foi descrito que a expressão epitelial de Jagged1 aumenta ao longo do desenvolvimento tumoral. Jagged1 é desta forma considerado um marcador de mau prognóstico e de elevado potencial metastático em diversos tipos de cancro, nomeadamente da mama e da próstata. No entanto, o mecanismo intrínseco de sinalização Jagged1/Notch nos contextos acima referidos ainda permanence pouco compreendido. Como tal, o trabalho apresentado nesta tese descreve o papel do ligando Jagged1 na regulação da angiogénese fisiológica e sua interacção com o ligando Dll4. Descreve ainda o seu papel na angiogénese tumoral do adulto, no desenvolvimento de tumores da próstata e finalmente o potencial terapêutico do bloqueio dos ligandos Jagged no tratamento do cancro da próstata. Para investigar o papel do ligando Jagged1 no processo angiogénico do adulto recorreu-se ao uso de mutantes endoteliais específicos de ganho e perda-de-função de Jag1 (eJag1OE e eJag1cKO, respectivamente) num modelo de cicatrização de feridas cutâneas. Ainda neste contexto, para investigar as interacções entre os dois ligandos Notch, Jagged1 and Dll4, os mesmos modelos genéticos foram combinados com inibição farmacológica de Dll4 ou Jagged1, respectivamente. Além disso, os mesmos mutantes endoteliais específicos foram ainda utilizados na investigação do papel do ligando Jagged1 na angiogénese tumoral. Para este fim recorreu-se a dois modelos diferentes de tumores no ratinho: o modelo de tumores transplantados subcutaneamente de células Lewis Lung Carcinoma (LLC) e o modelo autóctone de tumor da próstata murino- Transgenic Adenocarcinoma of the Mouse Prostate (TRAMP). Em relação aos processos angiogénicos foram avaliados nos diferentes mutantes, e combinações, diversos parâmetros tais como: densidade, maturidade, funcionalidade e permeabilidade vasculares. Simultaneamente, as populações endoteliais e perivasculares dos diferentes mutantes foram isoladas e efectuada uma análise específica de transcrição de genes envolvidos na regulação da angiogénese nestes tipos celulares. Na avaliação do contributo do ligando Jagged1 expresso no endotélio para o desenvolvimento de tumores da próstata foram ainda analisados os pesos da próstata nos términos das experiências, as lesões classificadas histológicamente bem como outros aspectos de biologia tumoral, tais como proliferação, apoptose e diferenciação celulares e transição epitélio-mesenquimal. Foi também efectuada uma análise da transcrição e expressão dos membros da via de sinalização Notch, incluindo ligandos (Dll1, 3, 4 e Jagged1 e 2), receptores (Notch1-4) e efectores (Hes1, 2, 5 e Hey1, 2, L), em próstatas de ratinhos saudáveis e de ratinhos TRAMP. Esta análise foi efectuada para descrever a dinâmica dos membros da via de sinalização Notch na tumorigénese da próstata de forma a poder identificar membros ectopicamente expressos na próstata tumoral quando comparados com a próstata normal. Foram ainda isoladas as diferentes subpopulações celulares da próstata, basal, luminal e do estroma e os seus perfis transcripcionais comparados entre próstatas normais e com lesões tumorais (TRAMP). Por fim, avaliou-se o potencial terapêutico de um anticorpo anti-Jagged1/2 no tratamento do cancro da próstata, administrando um anticorpo bloqueador anti-Jagged1/2 aos ratinhos TRAMP. Estes ensaios terapêuticos foram realizados em duas janelas temporais distintas: numa fase precoce do desenvolvimento de tumores da próstata, das 12 às 18 semanas de idade e numa fase mais tardia, das 18 às 24 semanas de idade, simulando uma detecção precoce e tardia da doença nos humanos. A eficácia terapêutica foi avaliada pelos pesos da próstata no término da experiência, classificação histopatológica das lesões, análise da vasculatura, proliferação, apoptose e diferenciação celulares, alterações nos compartimentos celulares da próstata, transição epitélio-mesenquimal, e alterações da população de células cancerosas estaminais (CSCs). Nestes trabalhos foram utilizadas técnicas de imunofluorescência, imunohistoquímica, “fluorescent associated cell sorting” (FACS), e “quantitative real time PCR” (qRT-PCR). Colectivamente, os resultados aqui apresentados demonstram que Jagged1 é um ligando pró-angiogénico em contextos de angiogénese do adulto, tanto fisiológica como tumoral. No modelo de cicatrização de feridas foi demonstrado que os mutantes de ganho-de-função (eJag1OE) exibiam um processo de cicatrização de feridas cutâneas e de desenvolvimento de tumores sólidos acelerado, relativamente aos respectivos controlos, enquanto os mutantes de perda-de-função exibiam o fenótipo oposto. Estas diferenças fenotípicas entre os mutantes deveram-se aos primeiros exibirem uma vasculatura mais densa, com maior número de células de suporte, e portanto mais funcional e menos permeável, enquanto que os segundos exactamente o oposto. O papel pró-angiogénico do ligando Jagged1 foi também demonstrado ser exercido através de complexas interações com diferentes receptores Notch. Utilizando o modelo de cicatrização de feridas foi demonstrado que Jagged1 presente no endotélio regula negativamente a transcrição e activação de Notch1, e portanto bloqueia a activação de Notch1 mediada por Dll4. Ao contrário do que acontece com Notch1, foi também demonstrado que Jagged1 regula positivamente a transcrição e activação de Notch4. Esta observação foi averiguada tanto no contexto angiogénico fisiológico como tumoral. Adicionalmente, a sinalização por Jagged1/Notch4 foi implicada na maturação vascular, uma vez que quer as feridas e tumores dos mutantes eJag1OE quer as feridas de ratinhos administrados com um anticorpo Notch4 agonista exibiram aumento da maturação vascular. Também foi demostrado que Jagged1 endotelial é capaz de activar Notch3/HeyL expresso nas células perivasculares e assim também contribuir para a regulação do recrutamento dessas mesmas células de suporte, tornando a vasculatura mais funcional e menos permeável, contribuindo para a cicatrização e desenvolvimento tumorais. Adicionalmente, foi demonstrado que o ligando endotelial Jagged1 contribui para a displasia tumoral não só pela sua função pró-angiogénica, limitando o aporte de nutrientes e oxigénio às células tumoriais, mas também por uma função angiócrina mediada pela activação de Notch3 e expressão de Hey1 nas células tumorais. Desta forma, este ligando regula ainda a proliferação, apoptose e diferenciação das células tumorais prostáticas, assim como o processo de transição epitélio-mesenquimal. Inclusivamente, através do estudo da expressão dos diferentes components Notch na próstata tumoral versus próstata normal, o eixo Jagged1/2/Notch3/Hey1 foi identificado como estando ectopicamente expresso no tecido prostático tumoral. Esta observação sugere então que para a regulação dos processos tumorigénicos referidos anteriormente, não só é importante o papel pró-angiogénico e angiócrino do ligando Jagged1 endotelial, mas também o seu papel directo decorrente da expressão aumentada ao nível das células tumorais. Por último, foi demonstrado que o bloqueio da sinalização Notch mediada pelos ligandos Jagged inibe o desenvolvimento e progressão de tumores da próstata do ratinho e que como tal, pode constituir uma nova potencial abordagem terapêutica no tratamento do cancro da próstata. O bloqueio de Jagged1/2 mimetizou o fenótipo vascular decorrente da perda-de-função endotelial específica de Jagged1, produzindo uma neo-vascularização menos densa, imatura e consequentemente menos funcional e mais permeável, inibindo fortemente o desenvolvimento de tumores da próstata murinos. De forma semelhante ao verificado nos mutantes de perda-de-função, o bloqueio de ambos os ligandos Jagged levou à inibição do crescimento celular próstático, restingindo a proliferação e promovendo a apoptose celular e inibindo a transição epitélio-mesenquimal. Adicionalmente, o tratamento com o anticorpo bloqueador também teve um efeito protector relativamente às alterações nos compartimentos celulares decorrentes da displasia prostática. O tratamento minimizou a perda da identidade celular luminal, inibiu a proliferação do compartimento basal e a des-diferenciação de um fenótipo luminal para um mais basal. O bloqueio de Jagged1/2 também apresentou um efeito benéfico ao nível da regulação do “pool” de células cancerosas estaminais da próstata, apresentado um efeito inibitório sobre a sua proliferação e sobrevivência. Adicionalmente, as amostras tratadas com anti-Jagged1/2 apresentaram uma significativa sub-expressão de Notch3 e Hey1 nesta subpopulação celular. Em conclusão, os resultados aqui apresentados contribuem para uma compreensão mais abrangente do papel do ligando Jaged1 no organismo adulto, fora do sistema nervoso central, inclusivamente em cenários tumorais, como o caso do cancro da próstata. De forma mais específica, a sinalização mediada por Jagged1, foi demonstrada ser uma rede complexa envolvendo múltiplos aspectos em diferentes tipos celulares e dependente do receptor envolvido. No endotélio Jagged1 actua como um ligando pró-angiogénico através de um efeito antagonístico a Dll4/Notch1 e mediado quer pela activação de Notch4/Hey1 no endotélio quer pela activação de Notch3/HeyL nas células perivasculares. Também foi importante verificar que ambas estas funções da sinalização por Jagged1/Notch são altamente conservadas em diferentes contextos no adulto, incluindo nos processos angiogénicos fisiológicos e patológicos. O ligando Jagged1 expresso por células endoteliais exerce ainda um papel angiócrino mediado pela activação de Notch3 e expressão de Hey1 na regulação do desenvolvimento tumoral da próstata. Esta função angiócrina juntamente com a expressão ectópica dos ligandos Jagged nos tecidos tumorais da próstata do ratinho constituem importantes fontes de regulação dos diversos aspectos da biologia tumoral. O bloqueio de Jagged1 pode desta forma vir a constituir um nova e promissora forma terapêutica no tratamento do cancro da próstata.
CIISA - Centro de Investigação Interdisciplinar em Sanidade Animal

L’impatto della dieta (in particolare quella associate a cicli di restrizione calorica) sui benefici della salute sono stati già dimostrati. Questi includono il miglioramento delle malattie cardiovascolari, insulino resistenza, diabete, disordini immuni, rallentamento dei processi di invecchiamento ed in particolare il ridotto rischio per il cancro. Recenti studi in modelli animali ed in modelli in vitro hanno scoperto un nesso tra i cicli di restrizione calorica ed il ridotto rischio di cancro ed un migliorata efficacia della chemioterapia che è stata già descritta per alcuni tipi di cancro. L’obiettivo di questo progetto di ricerca è stato quello di elucidare il ruolo dei cicli di restrizione calorica nelle vie di segnale intracellulari coinvolti nei meccanismi di chemioresistenza del cancro del pancreas che è tra i cancri più aggressivi ed è classificato come la quarta causa di morte cancro-correlata al fine di implementare una formulazione nutrizionale che mimasse la restrizione calorica in grado di reversare la chemioresistenza o di inibire la crescita tumorale. Avvantaggiandoci di modelli animali xenograft e di linee cellulari del cancro del pancreas, mediante l’utilizzo di approcci biochimici e biomolecolari abbiamo inizialmente cercato di comprendere a fondo il ruolo dei cicli di restrizione calorica nella progressione del cancro del pancreas in un modello murino predisposto e poi elucidato i meccanismi molecolari coinvolti nella chemioresistenza. A doggi è di fondamentale importanza identificare i targets potenziali che posso essere utilizzati come predittori di malattia utili per la prevenzione, la prognosi ed il trattamento. I risultati di questo progetto saranno di aiuto per gli scienziati impegnati nell’identificazione di nuovi target terapeutici.
The impact of nutrition (particularly associated with short term starvation (STS)) on major health benefits have been already demonstrated. These include amelioration of cardiovascular diseases, diabetes, insulin resistance, immune disorders, slowing of the aging process and in particular reduced risks of cancer. Recent studies in rodent and in in vitro models uncovered a potential link between STS and improved efficacy of chemotherapy which has already been demonstrated for some types of cancer. The broader objective of the research project developed during the PhD program was to elucidate the role of fasting (or short term starvation, STS) on the intracellular signaling events involved in the chemo-resistance of pancreatic cancer (PC) amidst the most aggressive types of cancer ranked as the fourth leading cause of cancer-related deaths worldwide, in order to implement a new diet formulation, mimicking calories restriction, in order to reverse chemoresistance or inhibit tumor growth. Taking advantage of in vivo xenograft mouse model for pancreatic cancer and in vitro PC cell lines, using biochemical and biomolecular approaches we first aimed to understand in depth the role of STS during the onset of pancreatic cancer in an ad hoc murine model and we then elucidate the molecular mechanisms involved in PC chemoresistance. It is important to systematically identify potential targets, which could serve as biomarkers for cancer prevention, prognosis and treatment. By elucidating the mechanisms involved in PC chemoresistance the results of this study will help scientists to identify new therapeutic targets.

A treatment strategy for cancer is the suppression of tumor growth by directing an immune response to the tumor vessels, which will destroy the tissue. In this thesis we describe the development of a vaccine that targets antigens expressed around angiogenic vasculature in most solid tumors. These antigens are alternative spliced extra domains of glycoproteins present in the extracellular matrix; e.g. the extra domain-B (ED-B) and extra domain-A (ED-A) of fibronectin and the C-domain of tenascin-C (TNCC). We show that it is possible to break self-tolerance and induce a strong antibody response against ED-B by vaccination. Furthermore, tumor growth was inhibited and the changes observed in the tumor tissue were consistent with an attack of the tumor vasculature by the immune system. For clinical development of therapeutic vaccines, targeting self-molecules like ED-B, a potent but non-toxic biodegradable adjuvant is required. The squalene-based Montanide ISA 720 (M720) in combination with CpG DNA fulfilled these requirements and induced an equally strong anti-self immune response as the preclinical golden standard Freund’s adjuvant. We have further characterized the immune response against ED-B generated with the adjuvant M720/GpG.  The ED-B vaccine also inhibited tumor growth in a therapeutic setting in a transgenic mouse model of pancreatic insulinoma in which tumorigenesis was already initiated. Furthermore, antibodies against ED-A and TNCC could be induced in mice and rabbits. We analyzed the expression of ED-A in breast tumors of transgenic MMTV-PyMT mice, a metastatic breast cancer model, with the aim to use this model to study the effect of an ED-A vaccine on metastasis. We also detected ED-B in canine mammary tumor tissue. Therefore vascular antigens might also represent potential therapeutic targets in dogs.  All together our preclinical data demonstrate that a vaccine targeting tumor blood vessels is a promising new approach for cancer treatment.

The Wilms’ tumor gene 1 (WT1) was first reported as a tumor suppressor gene in Wilms’ tumor. However, later studies have shown the oncogenic properties of WT1 in a variety of tumors. It was recently proposed that WT1 was a chameleon gene, due to its dual functions in tumorigenesis. We aimed to investigate the clinical significance of WT1 as biomarker in acute myeloid leukemia (AML) and clear cell renal cell carcinoma (ccRCC) and to elucidate the function of WT1 as an oncogene in squamous cell carcinoma of head and neck (SCCHN). In AML, it was suggested that WT1 expression was an applicable marker of minimal residual disease (MRD). In adult patients with AML, we found a good correlation between WT1 expression levels normalized to two control genes, β-actin and ABL. Outcome could be predicted by a reduction in WT1 expression in bone marrow (≥ 1-log) detected less than 1 month after diagnosis, when β-actin was used as control. Also, irrespective of the control gene used, outcome could be predicted by a reduction in WT1 expression in peripheral blood (≥ 2-log) detected between 1 and 6 months after treatment initiation. Previous studies in RCC demonstrated that WT1 acted as a tumor suppressor. Thus, we tested whether single nucleotide polymorphisms (SNPs) or mutations in WT1 might be associated with WT1 expression and clinical outcome in patients with ccRCC. We performed sequencing analysis on 10 exons of the WT1 gene in a total of 182 patient samples, and we identified six different SNPs in the WT1 gene. We found that at least one or two copies of the minor allele were present in 61% of ccRCC tumor samples. However, no correlation was observed between WT1 SNP genotypes and RNA expression levels. Moreover, none of the previously reported WT1 mutations were found in ccRCC. Nevertheless, we found that a favorable outcome was associated the homozygous minor allele for WT1 SNP. We then further investigated whether WT1 methylation was related to WT1 expression and its clinical significance. Methylation array and pyrosequencing analyses showed that the WT1 promoter region CpG site, cg22975913, was the most frequently hypermethylated CpG site. We found a trend that showed nearly significant correlation between WT1 mRNA levels and hypermethylation in the 5’-untranslated region. Hypermethylation in the WT1 CpG site, cg22975913, was found to be associated with patient age and a worse prognosis. One previous study reported that WT1 was overexpressed in SCCHN. That finding suggested that WT1 might play a role in oncogenesis. We found that both WT1 and p63 could promote cell proliferation. A positive correlation between WT1 and p63 expression was observed, and we identified p63 as a WT1 target gene. Furthermore, several known WT1 and p63 target genes were affected by knocking down WT1. Also, co-immunoprecipitation analyses demonstrated a protein interaction between WT1 and p53. In summary, WT1 gene expression can provide useful information for MRD detection during treatment of patients with AML. In RCC, our results suggested that the prognostic impact of WT1 SNPs was limited to the subgroup of patients that were homozygous for the minor allele, and that WT1 promoter hypermethylation could be used as a prognostic biomarker. In SCCHN, WT1 and p63 acted as oncogenes by affecting multiple genes involved in cancer cell growth.

Induction of an endogenous antibody response by therapeutic vaccination could provide an alternative to cost-intensive monoclonal antibody-based treatments for cancer. Since the target of a cancer vaccine will most likely be a self-antigen, self-tolerance of the immune system must be circumvented. Using fusion proteins consisting of the self-antigen to be targeted and a part derived from a foreign antigen, it is possible to break tolerance against the self-antigen. Furthermore, a potent adjuvant is required to support an immune response against a self-molecule. Currently no adjuvant suitable for this purpose is approved for use in humans. This thesis describes the development of a therapeutic vaccine targeting the vasculature of tumors. As tumor cells have developed strategies to escape immune surveillance, targeting of molecules associated with the tumor stroma is an interesting alternative. The alternatively spliced extra domain-A and B (ED-A and ED-B) of fibronectin and the glycan-binding protein galectin-1 are selectively expressed during events of tumor angiogenesis. We have designed recombinant proteins to target ED-B, ED-A and galectin-1, containing bacterial thioredoxin (TRX) as a non-self part, resulting in TRX-EDB, TRX-EDA and TRX-Gal-1. Vaccination against ED-B induced anti-ED-B antibodies and inhibited growth of subcutaneous fibrosarcoma. Immunization against ED-A decreased tumor burden and reduced the number of lung metastases in the MMTV-PyMT model for metastatic mammary carcinoma in a therapeutic setting. Analysis of the tumor tissue from ED-B and ED-A-immunized mice indicated an attack of the tumor vasculature by the immune system. Finally, we show that galectin-1 immunization reduced tumor burden and increased leukocyte numbers in the tumor tissue. Galectin-1 is pro-angiogenic and immunosuppressive, and therefore allows simultaneous targeting of fundamental characteristics of tumorigenesis. We furthermore show that the biodegradable squalene-based Montanide ISA 720 combined with CpG oligo 1826 (M720/CpG) is at least as potent as Freund’s adjuvant with respect to breaking self-tolerance, when comparing several immunological parameters. Freund’s is a potent but toxic adjuvant used in the majority of preclinical studies. The work presented in this thesis shows that therapeutic cancer vaccines targeting the tumor vasculature are a feasible and promising approach for cancer therapy.

L'activation de TLR9 par injection directe de nucléotides CpG non méthylés dans une tumeur peut induire une réponse immunitaire thérapeutique, mais les lymphocytes T régulateurs (Tregs) inhibent ensuite la réponse immunitaire antitumorale et limitent ainsi le pouvoir des stratégies d'immunothérapies contre le cancer.Chez des souris porteuses de tumeurs, nous avons constaté que les Tregs dans la tumeur expriment préférentiellement les marqueurs cellulaires de surface CTLA-4 et OX40. Nous montrons que la co-injection intratumorale d'anti-CTLA-4 et anti-OX40 avec du CpG en intra-tumoral aboutit à l’élimination des Tregs infiltrant la tumeur. Cette immunomodulation in situ, réalisée avec de faibles doses d'anticorps dans une tumeur unique, génère une réponse immunitaire antitumorale systémique capable d’éradiquer la maladie disséminée chez la souris. De plus, cette modalité de traitement est efficace contre des lésions de lymphome du SNC avec métastases leptoméningées, des sites qui sont généralement considérés comme des sanctuaires de cellules tumorales pour les traitements systémiques conventionnels.Ces résultats démontrent que les effecteurs immunitaires anti-tumoraux activés par immunomodulation locale peuventt éradiquer des cellules tumorales siègeant dans des sites éloignés. Nous proposons que, plutôt que d'utiliser des anticorps monoclonaux pour cibler les cellules cancéreuses par voie systémique, des anticorps monoclonaux pourraient être utilisés pour cibler les cellules immunitaires infiltrant la tumeur localement, provoquant ainsi une réponse immunitaire systémique
Activation of TLR9 by direct injection of unmethylated CpG nucleotides into a tumor can induce a therapeutic immune response; however, regulatory T-cells (Tregs) eventually inhibit the antitumor immune response and thereby limit the power of cancer immunotherapies. In tumor-bearing mice, we found that Tregs within the tumor preferentially express the cell surface markers CTLA-4 and OX40. We show that intratumoral coinjection of anti–CTLA-4 and anti-OX40 together with CpG depleted tumor-infiltrating Tregs. This in situ immunomodulation, which was performed with low doses of antibodies in a single tumor, generated a systemic antitumor immune response that eradicated disseminated disease in mice. Further, this treatment modality was effective against established CNS lymphoma with leptomeningeal metastases, sites that are usually considered to be tumor cell sanctuaries in the context of conventional systemic therapy. These results demonstrate that antitumor immune effectors elicited by local immunomodulation can eradicate tumor cells at distant sites. We propose that, rather than using mAbs to target cancer cells systemically, mAbs could be used to target the tumor infiltrative immune cells locally, thereby eliciting a systemic immune response

Le cancer bronchique représente le cancer le plus fréquent dans le monde. Le tabagisme en est la principale cause. Le développement des cellules tumorales provoque une réaction stromale tissulaire contenant des cellules inflammatoires dont la majorité sont des lymphocytes infiltrant la tumeur. Dans cette étude, quatre approches ont été utilisées afin d'étudier la modulation tumeur-dépendante du système immunitaire : l'immunophénotypage du répertoire Vβ du TCR par cytométrie de flux et l'électrophorèse en gel de gradient dénaturant (DGGE/TTGE) des segments VJγ du TCR, l'exploration phénotypique des tissus sains, tumoraux et ganglionnaires en immunofluorescence sur coupes à la congélation, et l'amplification par RT-PCR de l'ARN de diverses cytokines. Les résultats obtenus montrent qu'il existe une oligoclonalité des lymphocytes T dans les tissus sains, tumoraux et ganglionnaires. Un clone Vβ13. 1 et un clone gamma semblent spécifiques des carcinomes épidermoïdes alors que deux clones gamma semblent spécifiques des adénocarcinomes. De plus, l'expression de CD3/TCR est significativement diminuée dans les tumeurs. De même, les molécules β2 microglobuline, HLA-DR et -DQ sont fortement exprimées par les pneumocytes sains mais sont peu présentes sur les cellules tumorales. Diverses cytokines ayant des effets antagonistes sont produites dans les tumeurs en particulier le TGFβ et l'IL-10 favorisant la croissance tumorale et le TNFalpha et l'IFNγ potentialisant la réponse immune anti-tumorale. En conclusion, les clones détectés dans les tissus sains pourraient être spécifiques de lésions très précoces prétumorales liées au tabac. Il semble exister des clones spécifiques des tumeurs et des types tumoraux. Cependant, la surveillance immune a été mise en échec par un certain nombre de mécanismes et en particulier la diminution d'expression des molécules impliquées dans la synapse immunologique ainsi que la sécrétion de diverses cytokines ayant des effets antagonistes
Lung cancer is the most frequent type of cancer in the world. Smoking is clearly the major cause of this pathology. The proliferation of tumor cells induces an inflammatory stromal reaction comprising numerous tumor-infiltrating lymphocytes. In this study, four complementary approaches have been used to study the tumor-dependent modulation of the immune system : TCR Vβ repertoire usage in flow cytometry, TCRγ gene clonal rearrangements in denaturing gradient gel electrophoresis (DGGE/TTGE), tumor and healthy lung tissue infiltration as well as lymph nodes characteristics in immunohistology and cytokine production by RNA RT-PCR. The results obtained have demonstrated the oligoclonality of T-cells in the three types of tissues tested. A Vβ13. 1 clone and a gamma clone appeared to be specifics of epidermoid carcinoma. Similarly, two TCRγ clones appeared to be restricted to adenocarcinoma. Moreover, the CD3/TCR complex was clearly down regulated in tumors compared to healthy tissue or lymph nodes. Similarly, HLA-DR, HLA-DQ and β2 microglobuline, strongly expressed on healthy pneumocytes were nearly absent from tumor cells. Several cytokines with antagonistic effects were detected within tumoral tissue, especially TGFβ and IL-10, which favour tumor growth and TNFalpha and IFNγ which potentialize the anti-tumoral immune response. In conclusion, the clones identified in healthy lung tissue could be specific of early pre-tumoral lesions induced by tobacco smoke, and some of the clones appear to be tumor-specific. However, the immune system has been defeated by several mechanisms, including a decrease of the expression of partners of the immunological synapse and the production of antagonistic cytokines

Parity is associated with a short–term increase in breast cancer (BC) risk followed by a long–term decrease in risk. BC diagnosed 5–7 years after a completed pregnancy is associated with worse outcomes. BC is not a single disease. The dual effect of pregnancy could account for the BC characteristics at presentation (i.e. younger age and more advanced disease) and worse outcomes observed among Hispanics, relative to Non–Hispanic Whites. The purpose of this study was to investigate the association of reproductive characteristics by tumor subtype in a case series of women of Mexican–descent. Cases diagnosed ≤10 years following a birth had nearly 3 times the odds of a diagnosis with HER2+ tumors, relative to ER+/PR+ tumors. HER2+ tumors are associated with reduced survival compared to ER+/PR+ tumors. Diagnosis within a recent pregnancy may contribute to the aggressiveness of BC observed among women of Mexican descent ≤50 years of age.