The cancer stem cell (CSC) concept is still very controversial; therefore identification and isolation of this specific population remain challenging. A variety of putative markers have been described and measurement of high aldehyde dehydrogenase (ALDH) activity has been defined as a characteristic of stem cells (SCs). In this study, a library of novel small molecules (1,4-disubstituted acetalanthraquinones, AAQs), containing an acetal group as protected aldehyde functionality, was designed with the aim of probing affinity for ALDH metabolism and demonstrating their potential as molecular fluorescent probes to identify CSCs. The AAQs were shown to be subjective to acidic hydrolysis using 2M HCl at 37ºC; however compounds containing secondary or tertiary amine functionalities in their sidechain were only partly hydrolysed at 70 ºC. Metabolism studies were conducted using cytosolic fractions from rat liver enriched in ALDHs, yeast ALDH and human recombinant ALDH1A1. Some evidence was demonstrated which linked ALDH metabolism with aldehyde functionalities of hydrolysed AAQs (HAAQs). The AAQs were shown to emit far-red fluorescence (600-750 nm). A close relationship between structure modifications and alteration of cellular localisation, with gained specificity for selected sub-cellular compartments were achieved when assessed in A549 and U-2 OS cell lines. Thermal DNA denaturation and chemosensitivity assays were used to obtain information about DNA binding properties and cytotoxicity of AAQs and HAAQ congeners. All compounds were shown to be weak-to-moderately binding to DNA, and symmetrical 1,4-disubstituted compounds were shown to be non-toxic (IC50 = 100 :M) with nonsymmetrical analogues generating IC50 values in the 1-100 :M range. No fundamental variation in the biological activity was observed when comparing AAQs with HAAQs in the A549 (+ALDH) and MCF7 (-ALDH) cell lines. A pilot investigation revealed that aberrant gene methylation was cell-type dependent for three ALDH isoforms (1A1, 2, 3A1). Decitabine treatment led to enhanced protein expression for ALDH1A1 (A549), ALDH2 (MCF7) and ALDH3A1 (A549). In contrast, the protein level was reduced for ALDH1A1 in HT29 cells after decitabine treatment. ALDH1A1, ALDH2 and ALDH3A1 were highly expressed in prostate cell lines, with expression linked to promoter methylation. In contrast, low levels of DNA methylation were found in primary prostate cancer cells and benign prostatic hyperplasia. Interestingly, ALDH1A1, considered a SC marker, was found to be expressed at low levels in CD133⁺/ α₂β₁ hi stem cell fraction and upregulated in CD133⁻/ α₂β₁ lo differentiated prostate cancer cells. In summary, the results in this thesis demonstrate the complexity and tumour type specificity of ALDH expression. This creates challenges for the development of selective probes for CSC isolation, such as the AAQs discussed in this thesis. Although inconclusive results were obtained in regard to AAQs and their potential in targeting ALDHs, selected AAQs were shown to reveal interesting biological features highlighting them as potential non-invasive cytometric probes for tracking molecular interactions in live cells.

De par leur incidence et un diagnostic tardif, les cancers du sein et du colon restent parmi les plus meurtriers en France. L’identification de marqueur précoce semble primordiale pour détecter rapidement ces maladies et ainsi améliorer la survie des patients. Le protéome de chaque stade du cancer du côlon a été identifié et quantifié par spectrométrie de masse. L’Olfactomedine-4 (OLFM4) a été définie comme un biomarqueur potentiel par sa présence dans le sérum et ses variations d’expression. Alors que cette protéine est surexprimée, au niveau du site tumoral primaire,uniquement dans les stades précoces du cancer colorectal, sa surexpression est maintenue dans le sang des patients tous stades confondus. Cette variabilité d’expression est également retrouvée dans le cancer du sein. L’OLFM4 est décrite comme un marqueur des cellules souches colorectales mais ses fonctions restent contradictoires. Selon le contexte cellulaire, nos travaux associent cette protéine sécrétée à l’établissement de propriétés de cellule souche cancéreuse comme la prolifération en faible adhésion et la formation de mammosphères. Elle facilite également le processus migratoire et la résistance à la chimiothérapie. De plus, des expériences in vivo ont confirmé le caractère protumoral de cette protéine. Nous avons également mis en évidence son implication dans la régulation de l’expression du facteur de transcription GLI1 de la voie Sonic Hedgehog et de protéines d’adhésion telle que l’E-Cadherine. Cette étude décrit le lien étroit entre l’induction d’un phénotype agressif et l’OLFM4 dont le dosage sérique permettrait une détection précoce de ces maladies. / Through their high frequencies and the lack of early diagnosis, breast and colorectal cancer remain poor prognosis’ diseases. Therefor, the identification of early markers appears as crucial. The proteomic approach isone of the potential tools to identify these biomarkers as it enables the study of tumour cell lines or tissues amples. Indeed, proteins enriched from a shotgun proteomic approach can be identified and quantified by mass spectrometry. In a previous study, we have analysed the proteome of colorectal tumour at different stages and defined Olfactomedine-4 (OLFM4) as a potential biomarker. While OLFM4 expression is increased at primary tumoral site only in non-invasive stages, we have observed that OLFM4 isover expressed in the blood of patients regardless of the cancer stage. The same analysis was made on breast cancer patients. Although OLFM4 has been described as a stem cell marker, its functions remain unclear. In this study, we found that OLFM4 confers cancer stem cell properties. It acts as a regulator of proliferation in low adhesion conditions, migration, mammosphere formation and tumor growth. These abilities could be dependent of Sonic Hedgehog signalling pathway, especially of transcriptional factor GLI1, and regulation of adhesion proteins like E-cadherin. According to the cellular background, all these features highlight a close relationship between a potential biomarker and its involvement in the acquisition of an aggressive phenotype.

Clinical management of esophageal adenocarcinoma (EAC) relies on radiation therapy, yet radioresistance is a pervasive challenge in this disease. The mechanisms of EAC radioresistance remain largely unknown due to a paucity of validated preclinical models. The present studies report on the development of seven primary xenograft models established from patient tumours. These models are used to interrogate the range of radiosensitivities and mechanisms of radioresistance in EAC tumours. We found that radiation enriches the tumour-initiating cell population in two xenograft lines tested. Furthermore, three tested xenograft lines respond to irradiation by upregulating Hedgehog transcripts, a pathway involved in stem cell maintenance and proliferation. Upregulation occurs in autocrine and paracrine patterns simultaneously, suggesting that Hedgehog signalling may have a complex role in the radioresponse of EAC tumours. These findings suggest that inhibiting stem cell pathways in combination with radiotherapy may have an important role in the clinical management of EAC.

Clinical management of esophageal adenocarcinoma (EAC) relies on radiation therapy, yet radioresistance is a pervasive challenge in this disease. The mechanisms of EAC radioresistance remain largely unknown due to a paucity of validated preclinical models. The present studies report on the development of seven primary xenograft models established from patient tumours. These models are used to interrogate the range of radiosensitivities and mechanisms of radioresistance in EAC tumours. We found that radiation enriches the tumour-initiating cell population in two xenograft lines tested. Furthermore, three tested xenograft lines respond to irradiation by upregulating Hedgehog transcripts, a pathway involved in stem cell maintenance and proliferation. Upregulation occurs in autocrine and paracrine patterns simultaneously, suggesting that Hedgehog signalling may have a complex role in the radioresponse of EAC tumours. These findings suggest that inhibiting stem cell pathways in combination with radiotherapy may have an important role in the clinical management of EAC.

Sporadic breast cancers are mainly attributable to long-term exposure to environmental factors, via a multi-year, multi-step, and multi-path process of tumorigenesis involving cumulative genetic and epigenetic alterations in the chronic carcinogenesis of breast cells from a non-cancerous stage to precancerous and cancerous stages. Epidemiologic and experimental studies have suggested that various dietary compounds like green tea and grape seed may be used as preventive agents for breast cancer control. In this research, I have developed a cellular model that mimics breast cell carcinogenesis chronically induced by cumulative exposures to low doses of environmental carcinogens. I used the chronic carcinogenesis model as a target system to investigate the activity of dietary compounds at non-cytotoxic levels in intervention of cellular carcinogenesis induced by cumulative exposures to pico-molar 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and benzo[a]pyrene (B[a]P). I used various cancer-associated properties like, reduced dependence on growth factors, anchorage-independent growth, increased cell mobility, and acinar-conformational disruption as measurable endpoints of carcinogenesis. The first part (Part-I) of this dissertation focuses on the understanding the breast cancer progression, importance of environmental carcinogens, role of diet in cancer prevention and importance of epithelial to mesenchymal transition and stem-like cells in chronic carcinogenesis. The next three parts (Part II-IV) focus on understanding the role and mechanisms of dietary compounds in prevention of carcinogenesis and stem-like cell properties. Results in part II revealed the green tea extract at bio-achievable concentration can suppress carcinogen-induced cancerous properties. In Part-III, I compared the four major catechins in green tea extract in suppressing chronic carcinogenesis and the results revealed that epicatechin gallate to be most effective. I also identified that short-term exposure to NNK and B[a]P resulted in elevation of reactive oxygen species, ERK pathway activation and induction of cell proliferation and DNA damage, which can be blocked by green tea catechins. Results in Part-IV describe the roles of properties and markers associated with stem-like cells and the epithelial to mesenchymal transition induced by chronic carcinogenesis and their suppression by green tea catechins and grape seed proanthocyanidin extract. The last section (Part-V) summarizes the findings with their importance and discusses future directions.

Stem cells and cancer are inextricably linked and many tumours, including colorectal cancers, contain a small population of self-renewing cells, referred to as cancer stem cells (CSCs), able to give rise to proliferating but progressively differentiating cells that contribute to the cellular heterogeneity typical of solid tumours. Thus, the identification of CSCs and the factors that regulate their behaviour should have a profound impact on cancer treatment. Notch signalling controls the maintenance and differentiation of stem cells in several tissues, including the intestine, where it is essential for stem cells maintenance. Based on these premises, my work was aimed at identifying and characterising the cells that express the Notch1 receptor in intestinal tumours in vivo, with the objective of getting insights into the cellular hierarchy of colon cancer cells. We found that the Notch1 receptor is expressed in rare undifferentiated tumour cells that present self-renewal and multipotency in vivo, as they indefinitely give rise to marked differentiated tumour cells and fuel tumour growth. Our analysis on the transcriptomic profile of these cells confirmed our in vivo observations that Notch1+ tumour cells represent a specific population of highly proliferative tumour cells, expressing several, but not all, known markers of normal intestinal stem cells (ISCs). Indeed, their transcriptional signature highly correlates with normal ISCs. Given that the tumour cells we characterised appear not to carry Apc mutations, we hypothesise that during the earlies steps of tumourigenesis, normal Notch1+ ISCs are engulfed within the nascent tumour (in aberrant hyperproliferative crypts) and are able to grow and expand within this new ecosystem, as they are supported by extrinsic secreted growth factors from the neighbouring mutant cells. The concept that normal ISCs might contribute to tumour expansion highlights the complications that patients can encounter during treatment, since these cells share many features with their wild-type counterparts, making therapy deleterious to normal ISCs. / Les cellules souches et le cancer sont inextricablement liés et de nombreuses tumeurs, y compris les cancers colorectaux, contiennent une petite population de cellules auto-renouvelables, appelées cellules souches cancéreuses (CSCs), capables de donner naissance à des cellules proliférantes mais progressivement différenciatrices qui contribuent à l'hétérogénéité cellulaire typique des tumeurs solides. Ainsi, l'identification des CSC et des facteurs qui régissent leur comportement devrait avoir un impact profond sur le traitement du cancer. Notch signale le contrôle le maintien et la différenciation des cellules souches dans plusieurs tissus, y compris l'intestin, où elles sont essentielles au maintien des cellules souches. Sur la base de ces prémisses, mes travaux visaient à identifier et caractériser les cellules qui expriment le récepteur Notch1 dans les tumeurs intestinales in vivo, dans le but de mieux comprendre la hiérarchie cellulaire des cellules cancéreuses du colon. Nous avons constaté que le récepteur Notch1 s'exprime dans de rares cellules tumorales indifférenciées qui se renouvellent et se multiplient in vivo, car elles donnent lieu indéfiniment à une différenciation marquée des cellules tumorales et à une croissance tumorale. Notre analyse du profil transcriptomique de ces cellules a confirmé nos observations in vivo selon lesquelles les cellules tumorales Notch1+ représentent une population spécifique de cellules tumorales hautement prolifératives, exprimant plusieurs marqueurs connus, mais pas tous, des cellules souches intestinales normales (CSI). En effet, leur signature transcriptionnelle est fortement corrélée avec les CSI normaux. Étant donné que les cellules tumorales que nous avons caractérisées ne semblent pas être porteuses de mutations Apc, nous supposons que durant les premières étapes de la tumorigénèse, les CSI normales Notch1+ sont englouties dans la tumeur naissante (dans des cryptes hyperprolifératives aberrantes) et sont capables de croître et de s'étendre dans ce nouvel écosystème, car elles sont soutenues par les facteurs de croissance extrinsèques des cellules mutantes voisines. Le concept selon lequel les CSI normaux pourraient contribuer à l'expansion tumorale met en évidence les complications que les patients peuvent rencontrer pendant le traitement, puisque ces cellules partagent de nombreuses caractéristiques avec leurs homologues de type sauvage, ce qui rend le traitement délétère pour les CSI normaux.

Head and neck squamous cell carcinomas are still challenging despite progress in the oncological treatment. Study of the molecular biology allows to deeply characterize tumor properties and to predict the prognosis for affected patients. Nowadays there are many drugs clinically tested in the group of targeted therapy medicine Experimental work comprised both in vitro and in situ assays, being performed thanks to the collaboration between a number of departments of the 1st Faculty of Medicine of the Charles University in Prague, Academy of Sciences of the Czech Republic, Institute of Hematology and Blood Transfusion and Faculty of Veterinary Medicine of the Ludwig-Maxmillian University Munich. Galectin-1 is important inductor of the myofibroblasts/cancer associated fibroblasts. These fibroblasts are regarded as negative prognostic markers thanks to their capability of invasive cancer cells induction. On the other hand, Galectin-9 is not present in the carcinoma and in the case of dysplasia, its expression indicate aberrant features together with aberrant expression of keratin 14 and 19. Except from galectins using as prognostic markers, we focused on the galectins as a therapeutics instruments as well. Presented work with mutant variants of galectin-2 proved their effect on both pharmacodynamics and...

Les glioblastomes sont les tumeurs du système nerveux central les plus fréquentes et agressives. Avec une survie médiane inférieure à 2 ans, les thérapies actuelles restent inefficaces. Cet échec pourrait être expliqué en partie par l’existence de cellules particulières, les cellules souches cancéreuses. Ces cellules ont plusieurs propriétés communes aux cellules souches, qui les rendent résistantes aux traitements des glioblastomes. Il est donc important de pouvoir les identifier et les cibler pour pouvoir éliminer totalement la tumeur. L’objectif de ce travail de thèse est de déterminer des biomarqueurs des cellules souches de glioblastomes (gCSCs). Pour cela, nous avons d’abord développé une méthode générique permettant de prédire des antigènes spécifiques de cancer à partir de données de puces d’expression. Puis, nous avons travaillé sur les gCSCs, en identifiant des biomarqueurs potentiels, puis en étudiant les modifications du signal calcium, dérégulé dans de nombreux cancers. / Glioblastoma are the most common and aggressive nervous system tumors. With a median overall survival smaller than 2 years, usual therapies remain inefficient. This failure could be explained in part by the existence of cancer stem cells. These cells share several properties with stem cells which make them resistant to glioblastoma treatments. This is why it is important to identify and target them to suppress the whole tumor.The goal of this thesis work is to identify glioblastoma stem cells (gCSCs) biomarkers. To this end, we first developed a global method predicting cancer antigens from microarray data. Then, by studying gCSCs we identified several putative biomarkers and generated insights concerning the calcium signals which are deregulated in numerous cancers.

La technologie iPSC (induced pluripotent stem cells) permet l’obtention d’une source cellulaire illimitée pour la modélisation et la thérapie de maladies génétiques, la médecine régénérative, l’étude pharmacologique et récemment la modélisation et l’étude du cancer. La leucémie myéloïde chronique (LMC) est la pathologie modèle du concept de « cellule souche cancéreuse » : des cellules souches LMC (CS-LMC) s’avèrent résistantes aux inhibiteurs de tyrosine kinases (ITK), traitement conventionnel de ce syndrome myéloprolifératif. Elles sont responsables de la persistance d’une maladie résiduelle et de rechutes lors de l’interruption du traitement. Malheureusement, leur isolement est difficile. Nous proposons dans ce travail de modéliser les CS-LMC par les iPSC LMC. Nous montrons pour la première fois dans les iPSC LMC que BCR-ABL1 réprime l’expression des facteurs clés de la pluripotence (OCT4,NANOG, le cluster miR302-367) via les kinases ERK1/2. Les ITK, en bloquant l’activité deBCR-ABL1, entraîne une hausse des niveaux d’expression de ces marqueurs. L’effet « prosouche» des ITK, découvert dans les iPSC LMC, est également observé lors du traitement de CS-LMC primaires. L’agent thérapeutique pourrait ainsi, en maintenant ou renforçant le compartiment LMC immature, participer paradoxalement à la persistance de la maladie résiduelle. Étonnamment, les ITK augmentent également l’expression des marqueurs« souches » dans les iPSC et CS hématopoïétiques normales. Ce résultat suggère un effet« pro-souche » généralisé à plusieurs types cellulaires. / IPSC (induced pluripotent stem cells) offer renewable source of biologically relevant human cells for genetic diseases modelling and therapy, regenerative medicine, pharmacological study and, recently, cancer research. The proof of « cancer stem cell concept » was established in chronic myeloid leukemia (CML) : CML stem cells (CML-SC) are resistant to treatment (tyrosine kinase inhibitors (TKI)). They are involved in residual disease persistence and relapse when treatment is discontinued in the majority of patients. We modelled CML-SC with CML iPSC. We showed, for the first time, that BCR-ABL1 acts as a repressor of key stemness markers (OCT4, NANOG, miR302-367 cluster) via ERK1/2 in human CML iPSC. By blocking BCR–ABL1 activity, TKI increase pluripotency gene expression. Interestingly, a similar pro-stemness effect was observed during CML-SC treatment. By inducing a more primitive stemness phenotype, TKI could promote residual disease and relapse. Interestingly, an increase of stemness gene expression was also observed during TKI treatment of healthy cells (iPSC and hematopoietic stem cells). These data suggest a global TKI pro-stemness effect in other stem cell types.

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