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<p>There is increasing evidence that prostate tumours are organized as a hierarchy with rare cancer stem cells (CSCs) implicated in initiating and maintaining the tumour. However, prospective prostate cancer stem cells (PCSCs) have not been thoroughly characterized from primary tissue specimens. Using the DU145 cell line, PCSCs have been propagated as non-adherent spheres <em>in vitro</em>. Approximately 1.25% of monolayer DU145 cells formed primary spheres while 26% of sphere cells formed subsequent spheres; a measure of PCSC self-renewal capacity. Spheres are enriched for cells expressing prostate basal and luminal cytokeratins and CSC markers (CD44, CD24, integrin alpha2beta1). PCSCs initiate xenograft tumours with enhanced capacity compared to monolayer cells. While epidermal growth factor (EGF) promoted PCSC propagation, basic fibroblast growth factor (bFGF) inhibited these events. Activation of EGF receptor (EGFR) signalling, following EGF treatment or expression of constitutively-active EGFR (EGFRvIII), increased sphere formation. Conversely, attenuation of EGFR signalling inhibited PCSC self-renewal. Consistent with the MEK-ERK pathway being a major target of EGFR signalling, the MEK-ERK pathway contributes to EGFR-facilitated PCSC propagation. Inhibition of ERK activation following MEK inhibitor treatment, expression of dominant-negative MEK1(K97M), or knockdown of ERK1 or ERK2 reduced PCSC propagation. Therefore, EGFR signalling promotes PCSC self-renewal by activating the MEK-ERK pathway.</p> <p>SOX2 is an essential transcription factor for stem cells, however, its role in PCSCs remains unclear. SOX2 protein is upregulated in PCSCs propagated as spheres, and its expression is regulated by EGFR signalling. EGFR activation, following EGF treatment or expression of constitutively-active EGFRvIII, increased SOX2 expression and PCSC self-renewal, while being attenuated by EGFR inhibitor treatment. Ectopic SOX2 expression enhanced EGF-induced PCSC self-renewal, while SOX2 knockdown renders PCSCs non-responsive to EGF-induced self-renewal and reduced their anchorage-independent growth. Furthermore, SOX2 expression is associated with the ability of PCSCs to form aggressive xenograft tumours. Collectively, SOX2 regulates EGFR-mediated PCSC self-renewal.</p> / Doctor of Philosophy (PhD)

Les immunothérapies de type immune checkpoint blockade (ICB) ont révolutionné les approches thérapeutiques en oncologie médicale et ont largement contribué au fait que l’immunothérapie est maintenant considérée comme le quatrième pilier des traitements anticancer, aux côtés d’approches traditionnelles telles que la chirurgie, la radiothérapie et la chimiothérapie. Malgré les résultats encourageants des études cliniques évaluant ce type d’immunothérapie, la majorité des patients décèderont des suites de leur maladie. Conséquemment, le domaine de recherche visant à comprendre les mécanismes de résistance aux immunothérapies est en expansion constante. Plusieurs stratégies visant à améliorer les issues cliniques ont été proposées, parmi lesquelles figurent: 1) la recherche de nouvelles cibles thérapeutiques dans le microenvironnement immun tumoral et; 2) les études de combinaisons thérapeutiques où une immunothérapie est jumelée à d’autres types de modalités thérapeutiques potentiellement synergiques. Chacune des études présentées dans cette thèse de recherche s’apparente à l’une ou l’autre de ces stratégies. Dans le cadre de notre première étude, nous démontrons que la protéine WISP1 représente une cible prometteuse à l’intérieur du microenvironnement de plusieurs types de tumeurs solides étant donné son association avec différentes variables pronostiques et proinflammatoires, ainsi qu’avec un programme épigénétique complexe, la transition épithélialemésenchymateuse (Epithelial-Mesenchymal Transition; EMT). De plus, nous démontrons que les niveaux d’expression de WISP1 sont significativement plus élevés au sein des tumeurs démontrant une résistance primaire aux immunothérapies de type ICB, particulièrement lorsque qu’une signature reliée à l’EMT peut être retrouvée de façon concomitante. Pour notre deuxième étude, nous avons utilisé des modèles murins in vivo de cancer pulmonaire non à petites cellules KRAS-mutés afin de tester différentes combinaisons thérapeutiques jumelant une thérapie dite ciblée (i.e., un inhibiteur de MEK) a différentes immunothérapies de type ICB. Nos résultats démontrent que l’ajout d’une immunothérapie anti-CTLA-4 à l’inhibiteur de MEK AZD6244 (selumetinib) et une immunothérapie anti-PD-L1 augmente significativement la survie, et que ces bénéfices sont associés à une diminution de marqueurs reliés à l’EMT. Il existe donc un lien commun entre ces deux études qui repose sur l’importance de l’EMT comme facteur favorisant la résistance thérapeutique aux immunothérapies. De plus, nous démontrons pour la première fois que les bénéfices associés à la triple combinaison thérapeutique susmentionnée peuvent être corrélés à une diminution d’expression de marqueurs liés à l’EMT. Par conséquent, nos résultats sont discutés en tant que base potentielle pour de futures études visant à réduire la résistance thérapeutique reliée à l’EMT. Nous discutons également de la valeur translationnelle de nos résultats à travers le développement d’une étude clinique. / Immune checkpoint blockade (ICB) has revolutionized therapeutic approaches in the field of medical oncology and has largely contributed to the fact that immunotherapy is now being regarded as the fourth pillar of cancer treatment alongside surgery, radiotherapy and chemotherapy. Despite encouraging results from clinical trials using ICB, most patients ultimately relapse or succumb to their disease. Therefore, the field of immunotherapeutic resistance research is rapidly expanding. Many strategies to improve ICB responses have been undertaken, including: 1) the search for novel, actionable targets in the immune tumor microenvironment (TME) and; 2) therapeutic combination studies where an ICB backbone is combined with different, synergistic treatment modalities. Each of the studies presented in this research thesis embraces one of these strategies. In our first study, we show that WISP1 represents a promising TME target in multiple solid tumor types by demonstrating its association with prognostic and pro-inflammatory variables, as well as to a complex epigenetic program termed Epithelial-Mesenchymal Transition (EMT). Furthermore, we show that increased WISP1 expression is associated to primary resistance to ICB, particularly when EMT-related signatures are found concomitantly. In our second study, we used in vivo mouse models of KRAS-mutant Non-Small Cell Lung Cancer (NSCLC) to test different therapeutic combinations of targeted therapies (i.e., MEK inhibitor) and ICB. We found that the addition of anti-CTLA-4 ICB to MEK inhibitor AZD6244 (selumetinib) and anti-PD-L1 ICB increases survival, and that these benefits are associated with the downregulation of EMT-related markers. Therefore, there exists a common link between these studies, which relies on the significance of EMT as a detrimental factor within the TME and its association with ICB resistance. Moreover, we show for the first time that the benefits of ICB combination therapy can be associated to the downregulation of EMT markers in vivo. Consequently, we discuss how our results may constitute the basis for future work aiming at reducing EMT-mediated therapeutic resistance, as well as the translational relevance of our pre-clinical results through the development of a clinical trial.

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Albert Font Haro ABSTRACT Modulation of plasmacytoid dendritic cell function: role of immunoreceptors TIM-3 and BDCA-2 Plasmacytoid dendritic cells (pDCs) are key players in the antiviral response as well as in linking innate and adaptive immune response. They express endosomal toll-like receptors 7 and 9, which can detect ssRNA and unmethylated CpG DNA, respectively. Due to the constitutive expression of the transcription factor IRF7, pDCs are able to rapidly produce massive quantities of type I (α, β, ω) and type III (1, 2, 3, 4) interferons (IFN-I and IFN-III) as well as pro- inflammatory cytokines such as IL-1, IL-6 and TNF-α. After maturation, they also function as antigen-presenting cells. Despite intense research, the mechanisms of IFN and pro-inflammatory cytokines production and regulation are still poorly understood. Using the pDC cell line GEN2.2 and also primary human pDCs, we shed light on the role of kinases MEK and SYK in IFN-I production and regulation. We found that SYK is not only involved in the regulatory receptor (RR)-mediated BCR-like pathway that represents the negative regulation of IFN-I and IFN-III secretion but also in the positive TLR7/9-mediated signal transduction pathway that leads to IFN-I production, representing the immunogenic function. We also found that MEK plays a...

Kolorektale Karzinome (CRC) zeigen eine heterogene Ätiologie. Die Progression prämaligner Vorläufer zu CRC unterscheidet (U) sich in Morphologie, molekularen Veränderungen und Interaktion mit der Tumorumgebung. CRC weisen oft onkogene Mutationen in KRAS und BRAF auf. Diese steigern die MAPK Signalwegaktivität (Mpa). Obwohl sie im selben Signalweg wirken, sind KRAS und BRAF auf die CRC-Entitäten U verteilt. Dabei ist KRAS häufiger im sogenannten konventionellen und BRAF im serratierten Weg zu CRC mutiert. In dieser Studie nutzte ich murine intestinale Organoide (iO), die induzierbare (Ind) KRAS oder BRAF Onkogene exprimieren. Große U zwischen KRAS und BRAF zeigten sich sowohl in Signaltransduktion (ST) als auch im Phänotyp. Phosphoprotein-, ERK-Reporter-, scRNA-Seq und EM-Analysen ergaben eine starke Mpa durch BRAF, die zu hoher Expression von MAPK-Zielgenen und Verlust der epithelialen Integrität führte. iO nach KRAS-Ind blieben intakt, korrelierend mit moderater, zelltypspezifischer (ZS) Mpa in sekretorischen und undifferenzierten Zellen. Die meisten Enterozyten waren Mpa-negativ. ERK-Reporter zeigten: Das ZS Muster der Mpa ist nicht nur gegenüber KRAS, sondern auch dem Entzug von Wachstumsfaktoren stabil. Dies spricht für eine intrinsische, robuste Regulierung der Mpa. BRAF-Ind Mpa setzte die ZS Regulierung der MAPK außer Kraft und schädigte das Gewebe, im Einklang mit einer oberen Grenze tolerabler Mpa. Die ZS Mpa wurde in CRC-Zelllinien bestätigt, deren Mpa durch KRAS aber nicht BRAF U ausfiel. Ferner, nutzte ich iO mit bCatenin+KRAS-Ind, um den konventionellen Weg zu CRC zu modellieren. Die Kombination führte zu synergistischen Effekten, die sich in EGFR-unabhängigem Wachstum und der Aufhebung der ZS Mpa-Blockade äußerten, die durch eine Verschiebung der Differenzierung zu mehr Progenitorzellen bewirkt wurde. Zusammenfassend konnte ich U in der Mpa durch KRAS oder BRAF im Darmepithel feststellen, was dazu beiträgt, deren Rollen in der CRC-Genese zu bestimmen. / Colorectal cancer (CRC) is a disease with heterogeneous etiology. Premalignant lesions follow distinct routes of progression to carcinoma reflected by differences in morphology, molecular alterations and the tumor environment. Mutant KRAS and BRAF are frequent, leading to MAPK pathway activation (Mpa), which is relevant for CRC therapy. Despite acting in the same pathway, mutant KRAS and BRAF segregate to different entities, as KRAS is more frequent in the conventional- and BRAF being specific for the serrated route to CRC. I used murine intestinal organoids (iO) expressing inducible oncogenic KRAS or BRAF to study the impact of oncogenes in primary cells. I found marked differences in signal transduction and phenotype. Phospho-protein, ERK-reporter, scRNA-seq and EM data showed strong Mpa upon BRAF induction followed by ERK-target gene expression leading to tissue disruption. In contrast, KRAS left the tissue intact resulting in less and cell type-dependent Mpa limited to secretory cells, a subset of late-stage enterocytes and undifferentiated crypt cells. Most enterocytes were irresponsive to KRAS. The pattern of Mpa was robust towards KRAS or growth factor depletion arguing in favor of intrinsic, resilient MAPK regulation. In iO, BRAF-induced Mpa could break this cell type-specific regulation, indicating an upper limit of tolerable Mpa. I validated these findings in CRC cell lines that differed in Mpa in response to oncogenic KRAS but not BRAF. Finally, I used iO expressing an inducible form of stabilized bCatenin in combination with KRAS to mimic events frequently found in the conventional pathway to CRC. Expression of KRAS and bCatenin synergized in driving EGFR independent growth and breaking the villus-specific block of Mpa by altering differentiation towards progenitor cell types. In summary, this study emphasizes differences between Mpa induced by oncogenic KRAS or BRAF which helps clarifying their nature in different etiological routes to CRC genesis.

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