Lornella Seeneevassen

Ciblage de la voie de signalisation Hippo dans les cellules souches cancéreuses gastriques: Le cancer gastrique (GC), 4ème cause de mortalité par cancer dans le monde, est un cancer de mauvais pronostic. Les tumeurs gastriques sont des entités hétérogènes constituées d’une petite sous population de cellules souches cancéreuses (CSCs), à l’origine de l’initiation et la croissance tumorale, ainsi que de la résistance aux traitements. Le GC est souvent diagnostiqué tardivement, à un stade métastatique, trop avancé pour les résections chirurgicales. Une partie des CSCs est aussi capable d’initier des métastases de par leur propriétés plastiques leur permettant de changer de phénotype cellulaire très rapidement. Le ciblage et la caractérisation de ces CSCs sont donc cruciaux au traitement du GC.En conséquence, la première partie de ma thèse a consisté au ciblage des CSCs en agissant sur les voies de signalisations cellulaires importantes au maintien de leur propriétés tumorigéniques. Pour cela, les deux stratégies choisies étaient l’activation des kinases de la voie et l’inhibition des effecteurs oncogènes YAP/TAZ-TEAD.Premièrement, le Leukaemia Inhibitory Factor (LIF) a été utilisé pour activer les kinases de la voie Hippo MST1/2 et LATS1/2. L’analyse de la formation de tumeursphères, de l’invasion 2D et 3D, et de l’expression des marqueurs associés aux CSCs et à la transition épithélio-mésenchymateuse (EMT) démontre que le LIF présente des propriétés anti-tumorigéniques, anti-chimiorésistantes, anti-invasives et anti-EMT sur les CSCs provenant de lignées cellulaires de GC et de cellules de tumeurs-dérivées de patients (PDX). De plus, le LIF améliore les effets anti-CSCs des chimiothérapies telles que la doxorubicine et le 5-fluorouracile et réduit l’activité des facteurs de transcription associés à l’EMT, ZEB1 et SNAIL. L’utilisation d’un inhibiteur de kinases de la voie Hippo MST1/2, XMU-MP-1, a démontré la participation de la voie Hippo aux effets anti-CSC du LIF. De plus, il a été démontré que les cellules préalablement traitées au LIF ont une capacité moindre à former des métastases dans des modèles in vivo d’injection intracardiaque.Par ailleurs, la Verteporfine (VP) a été utilisée en tant que stratégie d’inhibition des effecteurs oncogéniques de la voie Hippo de par sa capacité à altérer les interactions entre YAP/TAZ et le facteur de transcription TEAD. La VP diminue la prolifération des cellules GC ainsi que la population de CSCs et leurs marqueurs. Elle inhibe aussi la croissance tumorale dans des modèles de xénogreffes sous-cutanées et de manière intéressante les cellules résiduelles ne sont plus capables de former des tumeursphères ex vivo confirmant la diminution du pool de CSCs au sein des tumeurs.La deuxième partie de mon projet a consisté en la caractérisation des CSCs et plus particulièrement celles qui circulent, les cellules tumorales circulantes (CTCs) ; ceci dans le but de trouver de nouvelles stratégies de diagnostic, pronostic et de thérapie. Les CTCs, récupérées à partir de sang de patients atteints de GC, sont retrouvées seules ou en groupes dans le sang, par exemple regroupées avec des cellules immunitaires. En conséquence, une partie de ce projet, en collaboration avec l’équipe de N. Larmonier (Immunoconcept), a été d’explorer le rôle de l’interaction cellules myéloïdes suppressives (MDSCs)-CSCs et son importance dans les propriétés tumorigéniques et invasives des CSCs. Des expériences d’invasions et de tumeursphères ont démontré un premier effet pro-tumorigénique et pro-invasif des MDSCs sur les CSC gastriques.Dans l’ensemble, mon travail de thèse a exploré le ciblage de la voie Hippo comme stratégie thérapeutique anti-CSC dans le GC. Le LIF et la VP, deux méthodes visant différentes parties de la voie, représentent des approches intéressantes dans le traitement des CSCs tumorigéniques, chimio-résistantes et invasives. Enfin, le travail sur la caractérisation des CTCs et de leurs partenaires donnent des pistes pour le développement de thérapies contre les GC métastatiques.

CD44 has been described in many malignancies as a marker of cancer stem cells (CSCs). Several techniques can be used to detect these cells. Here we detail CD44 detection by flow cytometry, a precise technique allowing to determine the percentage of positive cells and the mean fluorescent intensity reflecting the CD44 expression by cells in the samples. The protocol explained here can be used to detect CD44 from cell suspensions prepared from tissues or in vitro cell cultures. Key words Cancer stem cell CD44 Immunofluorescence Flow cytometry Antibody Cancer

Uveal melanoma (UM) can remain in clinical dormancy for decades only to later produce lethal metastases. Using Gαq/11 ^mut /BAP1 ^wt UM xenograft models and human metastatic samples, we identified NR2F1 as a key inducer of UM disseminated cancer cell (DCC) dormancy. Dormant UM DCCs upregulate NR2F1, neural crest genes and, along with suppression of proliferation programs, NR2F1 silences YAP1/TEAD1 transcription by altering histone H3 activation marks. YAP1 can reciprocally repress NR2F1, but inhibiting Gαq/11 signaling or activating NR2F1 can arrest UM growth. NR2F1 knockout led to dormant DCC awakening and liver metastatic growth. NR2F1 and YAP1 inverse expression was confirmed in human livers carrying UM solitary, small DCC clusters as well as large metastases. Intriguingly, RNA-seq and Cut&Run analysis revealed that NR2F1 short-circuits oncogene signaling by repressing multiple G-protein signaling components. Our work provides previously unrecognized mechanistic insight into UM DCC dormancy and potential pathways for interception. Statement of significance: NR2F1 epigenetically suppresses genes associated with G-protein signaling, cell cycle, and YAP1/TEAD1 pathways, inducing dormancy in uveal melanoma (UM) disseminated cancer cells. This study unveils novel markers for UM dormancy and reactivation, positioning NR2F1 as a promising target for intercepting residual and UM metastatic disease.

Gastric cancer’s (GC) bad prognosis is usually associated with metastatic spread. Invasive cancer stem cells (CSC) are considered to be the seed of GC metastasis and not all CSCs are able to initiate metastasis. Targeting these aggressive metastasis-initiating CSC (MIC) is thus vital. Leukaemia inhibitory factor (LIF) is hereby used to target Hippo pathway oncogenic members, found to be induced in GC and associated with CSC features. LIF-treated GC cell lines, patient-derived xenograft (PDX) cells and/or CSC tumourspheres underwent transcriptomics, laser microdissection-associated proteomics, 2D and 3D invasion assays and in vivo xenograft in mice blood circulation. LIFR expression was analysed on tissue microarrays from GC patients and in silico from public databases. LIF-treated cells, especially CSC, presented decreased epithelial to mesenchymal transition (EMT) phenotype and invasion capacity in vitro, and lower metastasis initiation ability in vivo. These effects involved both the Hippo and Jak/Stat pathways. Finally, GC’s high LIFR expression was associated with better clinical outcomes in patients. LIF treatment could thus represent a targeted anti-CSC strategy to fight against metastatic GC, and LIFR detection in primary tumours could constitute a potential new prognosis marker in this disease.

The bacterial genotoxin, cytolethal distending toxin (CDT), causes DNA damage in host cells, a risk factor for carcinogenesis. Previous studies have shown that CDT induces phenotypes reminiscent of epithelial to mesenchymal transition (EMT), a process involved in cancer initiation and progression. We investigated different steps of EMT in response to Helicobacter hepaticus CDT and its active CdtB subunit using in vivo and in vitro models. Most of the steps of the EMT process were induced by CDT/CdtB and observed throughout the study in murine and epithelial cell culture models. CdtB induced cell-cell junctions’ disassembly, causing individualization of cells and acquisition of a spindle-like morphology. The key transcriptional regulators of EMT (SNAIL1 and ZEB1) and some EMT markers were upregulated at both RNA and protein levels in response to CDT/CdtB. CdtB increased the expression and proteolytic activity of matrix metalloproteinases, as well as cell migration. A range of these results were confirmed in Helicobacter hepaticus infected and xenograft murine models. In addition, colibactin, a genotoxic metabolite produced by Escherichia coli, induced EMT-like effects in cell culture. Overall, these data show that infection with genotoxin-producing bacteria elicits EMT process activation, supporting their role in tumorigenesis.

Background Gastric cancer (GC), the fourth leading cause of cancer-related death worldwide, with most deaths caused by advanced and metastatic disease, has limited curative options. Here, we revealed the importance of proprotein convertases (PCs) in the malignant and metastatic potential of GC cells through the regulation of the YAP/TAZ/TEAD pathway and epithelial-to-mesenchymal transition (EMT) in cancer stem cells (CSC). Methods The general PCs inhibitor, decanoyl-RVKR-chloromethyl-ketone (CMK), was used to repress PCs activity in CSCs of various GC cell lines. Their tumorigenic properties, drug resistance, YAP/TAZ/TEAD pathway activity, and invasive properties were then investigated in vitro, and their metastatic properties were explored in a mouse xenograft model. The prognostic value of PCs in GC patients was also explored in molecular databases of GC. Results Inhibition of PCs activity in CSCs in all GC cell lines reduced tumorsphere formation and growth, drug efflux, EMT phenotype, and invasive properties that are associated with repressed YAP/TAZ/TEAD pathway activity in vitro. In vivo, PCs’ inhibition in GC cells reduced their metastatic spread. Molecular analysis of tumors from GC patients has highlighted the prognostic value of PCs. Conclusions PCs are overexpressed in GC and associated with poor prognosis. PCs are involved in the malignant and metastatic potential of CSCs via the regulation of EMT, the YAP/TAZ/TEAD oncogenic pathway, and their stemness and invasive properties. Their repression represents a new strategy to target CSCs and impair metastatic spreading in GC.

Background: Cancer stem cells (CSCs) are at the origin of tumour initiation and progression in gastric adenocarcinoma (GC). However, markers of metastasis-initiating cells remain unidentified in GC. In this study, we characterized CD44 variants expressed in GC and evaluated the tumorigenic and metastatic properties of CD44v3+ cells and their clinical significance in GC patients. Methods: Using GC cell lines and patient-derived xenografts, we evaluated CD44+ and CD44v3+ GC cells molecular signature and their tumorigenic, chemoresistance, invasive and metastatic properties, and expression in patients-derived tissues. Results: CD44v3+ cells, which represented a subpopulation of CD44+ cells, were detected in advanced preneoplastic lesions and presented CSCs chemoresistance and tumorigenic properties in vitro and in vivo. Molecular and functional analyses revealed two subpopulations of gastric CSCs: CD44v3+ CSCs with an epithelial-mesenchymal transition (EMT)-like signature, and CD44+/v3– CSCs with an epithelial-like signature; both were tumorigenic but CD44v3+ cells showed higher invasive and metastatic properties in vivo. CD44v3+ cells detected in the primary tumours of GC patients were associated with a worse prognosis. Conclusion: CD44v3 is a marker of a subpopulation of CSCs with metastatic properties in GC. The identification of metastasis-initiating cells in GC represents a major advance for further development of anti-metastatic therapeutic strategies.

Background: Gastric cancer, the fifth most common cancer worldwide, is mainly linked to Helicobacter pylori infection. H. pylori induces chronic inflammation of the gastric mucosa associated with high oxidative stress. Our study aimed at assessing the implication of Nrf2, a major regulator of cellular redox homeostasis, in H. pylori-induced gastric carcinogenesis. Methods: Using three different gastric epithelial cell lines, a non-cancerous (HFE-145) and two different subtypes of gastric cancer (AGS and MKN74), we analyzed the modulation of Nrf2 expression over time. After invalidation of Nrf2 by CRISPR-cas9, we assessed its role in H. pylori-induced epithelial-to-mesenchymal transition (EMT). Finally, we evaluated the expression of Nrf2 and ZEB1, a central EMT transcription factor, in human gastric tissues. Results: We first demonstrated that the Nrf2 signaling pathway is differentially regulated depending on the infection stage. Rapidly and transiently activated, Nrf2 was downregulated 24 h post-infection in a VacA-dependent manner. We then demonstrated that Nrf2 invalidation leads to increased EMT, which is even exacerbated after H. pylori infection. Finally, Nrf2 expression tended to decrease in human patients’ gastric mucosa infected with H. pylori. Conclusions: Our work supports the hypothesis that Nrf2 downregulation upon H. pylori infection participates in EMT, one of the most important events in gastric carcinogenesis.

The Hippo pathway is one of the most important ones in mammals. Its key functions in cell proliferation, tissue growth, repair, and homeostasis make it the most crucial one to be controlled. Many means have been deployed for its regulation, since this pathway is not only composed of core regulatory components, but it also communicates with and regulates various other pathways, making this signalisation even more complex. Its role in cancer has been studied more and more over the past few years, and it presents YAP/TAZ as the major oncogenic actors. In this review, we relate how vital this pathway is for different organs, and how regulatory mechanisms have been bypassed to lead to cancerous states. Most studies present an upregulation status of YAP/TAZ, and urge the need to target them. A focus is made here on gastric carcinogenesis, its main dysregulations, and the major strategies adopted and tested to counteract Hippo pathway disbalance in this disease. Hippo pathway targeting can be achieved by various means, which are described in this review. Many studies have tested different potential molecules, which are detailed hereby. Though not all tested in gastric cancer, they could represent a real interest.