Colony-stimulating factor 1 receptor (CSF1R, Fms) is an integral transmembrane glycoprotein with tyrosine specific protein kinase activity that it is found on the mononuclear phagocytes to promote their survival, proliferation and differentiation. Colony-stimulating factor 1 (CSF-1), also known as M-CSF, is a protein ligand that acts on the CSF1R. There is a variable association of Fms with the stem cell marker CD34 on acute myeloid leukemia (AML) cells and this suggests different structures of the AML hierarchy in different patients. Mouse stromal cells (MS-5) were transduced with a plasmid containing human CSF-1 because mouse CSF-1 is inactive on human CSF1R. Results show that AML cells cultured with CSF-1-expressing stroma had a much better growth and survival than the control stroma, suggesting that CSF-1 might be a stimulating factor for the growth of leukemic stem cells.

We sought to determine whether activation of the Wnt signaling pathway altered the function of hNSCs in vitro. We took three approaches to activate Wnt signaling: Wnt3a, constitutively stabilized β-catenin (ΔN90), and the GSK3 inhibitor BIO. While Wnt3a and ΔN90 had no effect on proliferation in both stem cell (+EGF/FGF) and differentiating (-EGF/FGF) conditions, BIO reduced proliferation in both. All methods of Wnt signaling activation promoted neuronal lineage commitment during hNSC differentiation. Furthermore, BIO was able to induce mild neuronal differentiation in stem cell conditions, suggesting that GSK3-inhibition interferes with several pathways to regulate hNSC fate decisions. We also probed BTSC function using BIO-mediated GSK3 inhibition. We found that in stem cell conditions, BIO was able to induce neuronal differentiation, decrease proliferation, and induce cell cycle arrest. Together this data suggests that GSK3-inhibition, possibly through activation of Wnt signaling, may offer a novel mechanism for the differentiation treatment of glioblastomas.

We sought to determine whether activation of the Wnt signaling pathway altered the function of hNSCs in vitro. We took three approaches to activate Wnt signaling: Wnt3a, constitutively stabilized β-catenin (ΔN90), and the GSK3 inhibitor BIO. While Wnt3a and ΔN90 had no effect on proliferation in both stem cell (+EGF/FGF) and differentiating (-EGF/FGF) conditions, BIO reduced proliferation in both. All methods of Wnt signaling activation promoted neuronal lineage commitment during hNSC differentiation. Furthermore, BIO was able to induce mild neuronal differentiation in stem cell conditions, suggesting that GSK3-inhibition interferes with several pathways to regulate hNSC fate decisions. We also probed BTSC function using BIO-mediated GSK3 inhibition. We found that in stem cell conditions, BIO was able to induce neuronal differentiation, decrease proliferation, and induce cell cycle arrest. Together this data suggests that GSK3-inhibition, possibly through activation of Wnt signaling, may offer a novel mechanism for the differentiation treatment of glioblastomas.

Glioblastoma multiforme (GBM) is one of the most common and aggressive primary brain tumours, with a median patient survival time of 6-12 months in adults. It has been recently suggested that a typically small sub-population of brain tumour cells, in possession of certain defining properties of stem cells, is responsible for initiating and maintaining the tumour. More recent experiments have studied the interactions between this subpopulation of brain cancer cells and tumour microenvironmental factors such as hypoxia and high acidity. In this thesis a computational approach (based on Gillespie’s algorithm and cellular automata) is proposed to investigate the tumour heterogeneities that develop when exposed to various microenvironmental conditions of the cancerous tissue. The results suggest that microenvironmental conditions highly affect the characterization of cancer cells, including the self-renewal, differentiation and dedifferentiation properties of cancer cells.

Les cellules souches cancéreuses (CSC) sont les cellules responsables du pouvoir tumoral et/ou métastasique, et résistent à la plus part des molécules anticancéreuses. L’expression de facteurs de transcription impliqués dans l’auto-renouvellement des cellules souches embryonnaires tels que Oct4 ou Nanog, indique toujours un mauvais pronostic quelle que soit l’origine de la tumeur. Ne pouvant pas isoler ces CSC à signature embryonnaire à l’aide des marqueurs de surface « traditionnels », le laboratoire a créé un modèle murin qui permet de sélectionner les cellules exprimant Oct4 à partir de tumeurs se développant spontanément dans différents tissus. A partir de ce modèle, nous avons cherché une classe de molécule pouvant cibler ces cellules. Nous montrons que les inhibiteurs de la protéase du VIH et principalement le Lopinavir, ciblent spécifiquement les CSC murines exprimant une signature embryonnaire. Ces cellules expriment aussi CXCR4, un récepteur au facteur chimiotactique CXCL12, impliqué dans la migration des cellules tumorales. Bien que préliminaires, nos résultats indiquent que CXCR4 joue un rôle tout comme Oct4 dans le maintien de l’auto-renouvellement des CSC exprimant une signature embryonnaire. De plus, nous proposons un mécanisme pour expliquer l’inter-dépendance entre ces deux facteurs dans le maintien des propriétés souches de ces CSC. Des travaux sur la transposition de ce modèle murin à un modèle humain sont actuellement en cours. / Cancer Stem Cells (CSC) bear the tumorigenic and/or metastatic potential and are resistant to most of the chemotherapeutic drugs. CSC expressing embryonic transcription factors such as Oct4 or Nanog are always associated to tumours with poor prognosis. As it is not possible to isolate them based on the expression of common cell surface markers, our lab has developed a mouse model selecting Oct4 expressing cells from tumours of diverse origins. Based on this model, we looked for a class of molecules that were able to target these cells. Here we show that HIV protease inhibitors, especially Lopinavir, specifically target CSC expressing an embryonic signature. These cells also express CXCR4, which is a receptor for the CXCL12 chemotactic factor implicated in cell migration including tumour cells. Although preliminary, our results indicate an unexpected role of CXCR4 in maintaining self-renewal of CSCs expressing an embryonic signature. We propose a model to explain the inter-dependence between Oct4 and CXCR4 to maintain stem cell properties in this population of CSC. We are now trying to transpose our mouse model to a human model.

Structured abstract Hypothesis Cancer stem cells (CSCs) are subpopulations of cells which could contribute to tumor growth, metastasis formation and chemoresistance. CSCs can be detected by surface markers assessed by immunohistochemistry methods. A typical surface marker for CSCs is CD44 (standard form). We assumed, that CD44(s) could serve as a prognostic factor and marker of chemoresistance in patients with epithelial ovarian cancer. The aim of study 1. To recruit group of patients with histologically verified epithelial ovarian carcinoma. 2. To evaluate prognostic significance of known prognostic factors in our series of patients. 3. To assess the expression of CD44 in specimens of primary tumors and specimens of implantation metastasis using immnunohistochemistry and analyze their correlation. 4. To evaluate the expression of CD44 in relation to known prognostic factors. To analyze the significance of CD44 expression evaluation for overall survival, disease-free interval and chemoresistance. To find CD44 positivity cut-off by using statistical methods Materials and Methods A retrospective study was performed on 87 patients with histologically verified EOC. All patients were tested for primary tumor specimens, 48 of them were tested with regard to both specimens of primary tumor and implantation...

Le cancer gastrique (CG) est une maladie multifactorielle, fréquemment associée à l’infection chronique par des souches CagA+ d’Helicobacter pylori. La transition épithélio-mésenchymateuse (EMT) est un processus réversible dans lequel une cellule épithéliale polarisée acquiert un phénotype mésenchymateux. L’EMT est à l’émergence de cellules souches cancéreuses (CSC) qui expriment CD44 et présentent une activité ALDH élevée. L’infection des cellules épithéliales gastriques humaines (CEGs) par CagA+ H. pylori induit des cellules CD44+ avec des propriétés des CSCs via une EMT. La voie Hippo est composée par les kinases MST et LATS, et leurs cibles, les YAP1 et TAZ. Suite à la phosphorylation, YAP1 et TAZ sont inhibés. YAP1 et TAZ activés lient les facteurs TEAD pour promouvoir la croissance cellulaire et l’inhibition de l’apoptose.Notre premier objectif était de rechercher si H. pylori change l’état d’activation de la voie Hippo et l'effet sur l’EMT et les CSC in vitro et in vivo. Le deuxième but est la caractérisation du rôle de YAP1/TEAD dans les propriétés de CSCs gastriques in vitro et les conséquences de son inhibition dans la croissance tumorale in vivo.Pour étudier la régulation de la voie Hippo pendant l’infection par H. pylori, LATS2, YAP1 et CD44 ont été évalués dans la muqueuse gastrique de sujets non-infectés et infectés par H. pylori, qui ont été augmentés avec l’infection et leur surexpression a été associée avec la gastrite et la métaplasie intestinale. Dans les CEGs l’expression de gènes de la voie Hippo a été altérée par l’infection. La régulation de la voie Hippo par H. pylori a une cinétique diphasique et dépendante de CagA. Dans l’infection précoce, H. pylori déclenche l’activité transcriptionelle de YAP1. Cette période d’inactivité de la voie Hippo est suivi de son activation progressive, soutenue par l’accumulation de LATS2 et la phosphorylation inhibitrice de YAP1. La répression de LATS2 avec siRNAs a accéléré l’acquisition du phénotype mésenchymateux après l’infection, l’augmentation de marqueurs de l’EMT (Zeb1 et Snail1), et la diminution des miR-200 épithéliaux. Les CSC induites par H. pylori ont été potentialisées par l’inhibition de LATS2, ce qui suggère que LATS2 limite l’EMT et le phénotype de CSC acquis pendant l’infection. L’inhibition de LATS2 ou YAP1 diminue l’expression de ces deux protéines, révélant ainsi une boucle de régulation positive. Dans des coupes de tissu de CG, l’expression de LATS2 et YAP1 est hétérogène et positivement corrélée, fait qui a été confirmé dans 38 CEGs de la CCLE. L’expression LATS2 est fortement corrélée à celle de CTGF et CYR61, ce qui suggère que LATS2 peut aussi être un gène cible de YAP1/TEAD.La verteporfine (VP) est capable d’interrompre l’interaction YAP1/TEAD, et donc d’inhiber son activité transcriptionelle. In vitro, utilisant CEGs et des cellules de tumeurs de patients amplifiées chez la souris (patient-derived xenograft PDX), le traitement à la VP a diminué la croissance cellulaire, l’expression de gènes cible de YAP1/TAZ/TEAD, l’activité du rapporteur TEAD-luciférase et la capacité de formation de sphères. L’activité de la VP a été testée in vivo par injection péri-tumorale dans un modèle de greffe sous-cutanés des CEGs MKN45 et MKN74 et le PDX GC10 chez la souris NSG. La croissance tumorale a été diminuée. Le poids des tumeurs, l’analyse par IHC (CD44, ALDH, Ki67) et la capacité de formation de sphères des CSCs résiduelles ont été diminuées. Ces résultats montrent une activité inhibitrice de la VP sur les CSCs gastriques in vitro et in vivo.Ce travail montre pour la première fois que l’axe LATS2/YAP1/TEAD est précocement activé pendant l’infection chronique avec H. pylori et que celui-ci contrôle l’EMT et les propriétés de CSC. Le ciblage de la voie Hippo a été montré comme étant efficace dans la prévention de la croissance tumorale, mettant en évidence le potentiel de son inhibition dans le traitement du cancer gastrique. / Gastric cancer (GC) is a multifactorial disease, most frequently associated to chronic infection with CagA-positive Helicobacter pylori strains. Epithelial-to-mesenchymal transition (EMT) is reversible process in which polarized epithelial cells acquire a mesenchymal phenotype. EMT is at the origin of cancer stem cells (CSC). In GC, CSCs express CD44 and high aldehyde-dehydrogenase (ALDH) activity. Infection with H. pylori of human gastric cancer cell lines (hGECs) in vitro induces the emergence of a population of CD44+ cells with CSC-properties through an EMT process in a CagA-dependent manner. The Hippo pathway is composed by the kinases MST and LATS, and their phosphorylation targets,YAP1 and TAZ. Upon phosphorylation by LATS, YAP1 and TAZ are inhibited. Active YAP1 and TAZ bind to TEAD transcription factors to promote the expression of genes that regulate cell growth and apoptosis.The first aim of this work was to investigate whether H. pylori affects the activation state of the Hippo pathway, and its effect on the EMT process and the CSCs. Second, we intended to characterize the role of YAP1/TEAD in gastric CSC properties in vitro and the consequences of its pharmacological inhibition on tumor growth in vivo.To study the Hippo pathway regulation during infection, LATS2, YAP1 and CD44 were evaluated in gastric mucosae of non-infected or H. pylori-infected patients. They were upregulated in infected mucosae and were associated to pathology. Hippo pathway regulation by H. pylori infection has biphasic kinetics and is CagA-dependent. Early in infection, H. pylori transiently triggered YAP1 expression and co-transcriptional activity, along with LATS2. This period of Hippo pathway inactivity is followed by a progressive activation, sustained by LATS2 accumulation and inhibitory YAP1Ser127-phosphorylation. LATS2 siRNA-mediated repression accelerated the acquisition of the EMT-phenotype upon infection, the up-regulation of EMT-markers ZEB1 and Snail1, and the decrease of the epithelial miR-200. H. pylori-induced CD44 upregulation, invasion and sphere-forming capacity were further enhanced upon LATS2 knockdown, suggesting that LATS2 restricts the EMT and CSC-like phenotype in hGECs upon H. pylori infection. Inhibition of either LATS2 or YAP1 reduced the expression of both proteins, revealing a positive feedback loop. In tissue sections of GC, LATS2 and YAP1 were heterogeneous and co-expressed. The positive correlation between LATS2 and YAP1 was confirmed in the 38 hGECs of the CCLE. The expression of CTGF and CYR61 was also strongly correlated to LATS2, suggesting that LATS2 could also be a YAP1/TEAD target gene.hGECs of the CCLE. The expression of CTGF and CYR61 was also strongly correlated to LATS2, suggesting that LATS2 could also be a YAP1/TEAD target gene.Verteporfin (VP) disrupts the YAP1/TEAD interaction inhibiting its transcriptional activity. In vitro, using hGECs and cells from patient derived primary tumor xenogratfs (PDXs), we showed that treatment with VP decreased cell growth, expression of YAP1/TAZ/TEAD target genes, TEAD-luciferase reporter activity and sphere-forming capacity. The activity of VP was tested in vivo, by peritumoral injection in a model of subcutaneous graft of hGECs (MKN45 and MKN74) and PDX (GC10) in NGS mice. Tumor growth was followed and a decrease was observed. Tumor weight measurement, IHC analysis (CD44, ALDH and Ki67), and CSCs were decreased in treated tumors. These results show the CSC-inhibitory activity of VP both in vitro and in vivo.We showed for the first time that the LATS2/YAP1/TEAD axis is early activated during the carcinogenesis process induced by chronic H. pylori infection and controls the subsequent EMT and CSC-like features. Targeting the Hippo pathway efficiently prevented tumor growth in a PDX model, highlighting the potential of its inhibition to be implemented in gastric cancer therapy.

Elevated level of lipogenic enzymes and overall lipogenesis have been reported in a wide variety of cancers and blocking the lipogenic pathway by chemical inhibitors or RNA interference causes tumor cell death by apoptosis which provides a strong rationale for targeting lipogenic pathway for the treatment and prevention of cancer however the exact role of lipogenesis as a cause, facilitator or consequence is not yet clearly understood. Therefore in this dissertation research, we set up to determine the mechanism of tumor cell death by inhibiting lipogenesis and to determine the role of increased lipogenesis in the breast cancer progression. In the first part of this study, we investigated the status of fatty acid synthase (FAS) gene which is regarded as the key lipogenic gene in fatty acid biosynthetic pathway and is responsible for the synthesis of lipid molecules by facilitating the condensation reaction between acetyl-CoA and malonyl-CoA in the presence of NADPH. We observed that normal breast epithelial cells MCF10A cells have very low level of FAS expression whereas breast cancer cell lines MCF7, MDA MB231 and MDA MB231 LM have significant overexpression. Next, we observed the similar trend of FAS overexpression in breast cancer stem-like cells (CSCs) isolated from the MCF7, MDA MB231 and MDA MB231 LM cell lines using cell surface markers (CD24-/CD44+/ESA+). These cells were previously transplanted into the mammary fat pad of nude mice and the results of our limiting dilution analysis indicate that CSCs had a significantly higher ability of forming breast cancer in the injected animals which explains our rationale to use CSCs in our research. In order to exploit this lipogenic pathway for the treatment and chemoprevention of breast cancer, we then examined the effects of resveratrol on breast cancer cells. Resveratrol is a natural polyphenolic compound and has been shown to exhibit cardio-protective as well as anti-neoplastic effects on various types of cancers. However, the exact mechanism of its anti-tumor effect is not clearly defined. We observed that resveratrol significantly reduced the cell viability by inducing apoptosis in parental cells as well as in CSCs. Resveratrol also inhibited mammosphere formation which is an inherent property of CSCs. This inhibitory effect of resveratrol is accompanied by a significant reduction in lipid synthesis which is caused by the down-regulation of the FAS gene followed by up-regulation of pro-apoptotic genes, DAPK2 and BNIP3. The activation of apoptotic pathway in the cancer stem-like cells was suppressed by FAS overexpression suggesting that resveratrol-induced apoptosis is indeed through the modulation of FAS-mediated cell survival signaling. Importantly, resveratrol was able to significantly suppress the growth of CSC in an animal model of human breast cancer xenograft without showing apparental toxicity. Taken together, our results indicate that resveratrol is capable of inducing apoptosis in the CSCs through suppression of lipogenesis by modulating FAS expression, which highlights a novel mechanism of anti-tumor effect of resveratrol. Taken together, our results indicate that resveratrol is capable of inducing apoptosis in the cancer stem-like cells through suppression of lipogenesis by modulating FAS expression, which highlights a novel mechanism of anti-tumor effect of resveratrol. In the second part of research, we tried to determine the role of elevated level of lipogenesis in normal to ductal carcinoma in situ (DCIS) progression. For this, we first analyzed the expression profile of various lipogenic genes using an expression microarray and found that CSCs from DCIS.com showed significantly higher level of ATP-citrate lyase (ACLY), acetyl-CoA carboxylase (ACC) and FAS than the normal non-tumorigenic stem-like cells obtained from MCF10A. The result was also confirmed by qRT-PCR and Western blot as well as in clinical specimens of DCIS by immunohistochemistry. In the next step, we detected that SREBP1, the master regulator of lipogenic genes, is also upregulated in DCIS and further identified that SREBP1 regulates the co-ordinate expression of ACLY, ACC and FAS ultimately resulting in the elevation of lipogenesis. In order to determine the role of SREBP1 overexpression in normal to DCIS transition, we overexpressed the SREBP1 in MCF10A cells which induced a significant increase in the downstream key lipogenic genes ACLY, ACC1 and FAS which resulted in the clear upregulation of total lipid content in the cells. Furthermore, we found that this elevation of lipogenesis in MCF10A-SREBP1 stem-like cells confers proliferative advantage as well as a significant increase in mammosphere forming ability and anchorage independent growth (3D culture). Thus, our results showed a possibility that increased lipogenesis in normal stem-like cells may be responsible for providing oncogenic transformation properties which can be confirmed at least in our in vitro model. We then examined the effects of resveratrol on CSCs sorted from DCIS.com. We found that resveratrol decreased the cell viability and increased apoptosis by reducing the total lipid content by inhibiting the expression of SREBP1 and downstream lipogenic genes. Resveratrol also hindered the stemness of the DCIS CSCs by inhibiting its mammosphere forming ability. When DCIS CSCs were transplanted into mammary fat pad of nude mice which were on resveratrol treatment, we observed that resveratrol significantly suppressed the formation of DCIS by downregulating lipogenic genes and by upregulating pro-apoptotic genes, DAPK2 and BNIP3. Collectively, our results indicate that lipogenic genes SREBP1 co-ordinately regulates the overexpression of ACLY, ACC1 and FAS in DCIS CSCs at an early stage of breast tumorigenesis and thus confer proliferative and survival advantages. Anti-growth effect of resveratrol on DCIS CSCs also provides us with a strong rationale to use this agent for chemo-prevention against DCIS.

Pancreatic neuroendocrine tumours (PNETs) are the most common neuroendocrine tumours diagnosed in humans and dogs. Due to the highly heterogeneous nature of these tumours, definitive data are still lacking over the molecular mechanisms involved in their cancerous behaviour. This study focused on insulinoma (INS), as it is the most commonly diagnosed PNET in human and veterinary oncology. INS is an insulin-producing tumour that causes a hypoglycaemic syndrome related to the excessive insulin production. In humans, it is often a small benign neoplasm readily curable by surgical resection whereas, in dogs, INS is often malignant. Despite current treatment modalities, malignant canine and human INS have a poor prognosis as patients tend to develop metastases in liver and lymph nodes that do not respond to current therapies. From a comparative oncology perspective, the close resemblance of canine and human malignant INS makes canine INS an interesting study model for human INS. Cancer stem cells (CSCs) are critical for the engraftment and chemoresistance of many tumours. Although CSCs have been isolated from a range of solid tumours, a comprehensive characterisation of INS CSCs has not yet been reported. In this study, it was confirmed that INS CSCs can be enriched and are potential targets for novel INS therapies. Highly invasive and tumourigenic human and canine INS CSCs were successfully isolated and exhibited greater resistance to chemotherapy, which may play a significant role in the poor prognosis of this disease. To date, the mechanisms by which tumours spread and the clinical causes of chemoresistance remain only partially understood. Here, RNA-sequencing analysis was performed over a small set of canine INS tumour samples in order to identify mechanisms involved in INS carcinogenesis through different stages of the disease. Preliminary data showed that distinct gene profiles characterised early and late stage of canine INS. Interestingly, differential gene expression and gene pathways analysis, highlighted that sets of genes involved in pancreatic embryogenesis and insulin secretion were overexpressed in canine primary INS lesions compared with normal pancreas. The Notch pathway is fundamental in pancreatic embryogenesis and it has been previously associated with carcinogenesis of neuroendocrine tumours and with the CSC phenotype. Protein analysis showed that the Notch pathway is activated in both human and canine INS CSCs, particularly when treated with chemotherapy, indicating that the Notch pathway may be involved in chemoresistance. Additionally, it was demonstrated that inhibition of the Notch pathway decreased INS CSCs' survival and chemoresistance, both in vitro and in vivo. These findings provide preclinical evidence that anti-Notch therapy may improve outcomes for patients with malignant INS.

Platinum-based therapy is the most often used chemotherapeutic agent to treat advanced cases of head and neck cancers. However, only a small fraction of the patient population responds to cisplatin, with a median survival time of less than a year. Currently, there is a lack of clinically employable molecular characterization of the disease beyond HPV status to classify patients who would respond favorably to platinum-based therapy. In this regard, CD24 expression level appears to be a significant molecular phenotype of cisplatin resistance in laryngeal carcinoma. This study demonstrates that CD24 expression level in HNSCC has a linear relationship with cisplatin resistance, and it affects the transcription of critical apoptotic, stem, and drug resistance genes. The knockdown of the CD24 transcript reduces tumor growth rate and increases the overall cisplatin sensitivity in mice xenograft experiments. A retrospective analysis of a cohort of 25 HNSCC patient tumor samples suggests that CD24-high tumors go on to show an unfavorable response to cisplatin treatment. Overall, based on the strength of further clinical analysis, CD24 presents a strong rationale to be utilized as a predictive indicator to stratify head and neck cancer patients for platinum-based therapy. This study also provides a rationale for using CD24 as a therapeutic adjuvant target along with standard cisplatin therapy in head and neck cancers.