Folic acid is a B vitamin involved in DNA CpG methylation. Mandated dietary fortification has led to a subsequent increase in blood folate concentration which has been correlated to a simultaneous spike in colorectal cancer incidence in Canada and the US. Several human colorectal cancer cell lines were cultivated under low (0 mg/L), standard (4 mg/L), and high (16 mg/L) folate conditions for seven days, then assessed for DNA methyltransferase1 protein expression, changes in DNA methylation, and ability to generate colonospheres in culture. Low folic acid levels generally led to reduced DNMT1 protein expression, CpG hypomethylation, and reduced colonosphere yield. High folic acid levels led to increased DNMT1 protein expression, CpG hypermethylation, and maintained colonosphere yield. This data demonstrates that varying levels of folic acid in vitro can influence the methylation status and cancer stem cell self-renewal ability of human colorectal cancer cells. / Canadian Cancer Society

Objective: MS-275, a phase trialed histone deacetylase inhibitor will be characterized for its ability reduce neuroblastoma (NB) viability and to target the tumor stem cell (TSC) population in neuroblastoma. Methods: Ability of MS-275 to reduce NB growth is characterized using a tumorigenic NB N-type cell line that has high differentiation potential. TSC enriched side population from NB and a reference teratocarcinoma cell line was analyzed as a model of TSC. The potential of MS-275 to modulate functional characteristics and markers of TSC was also investigated. Results: MS-275 induces a G1 cell cycle arrest, the intrinsic apoptosis pathway in NB and can potentially differentiate NB into a more terminal phenotype. NB TSC-like population is reduced following MS-275 treatment by the targeting of their self-renewal and drug pumping ability. Conclusions: By targeting both the NB and its TSC population, MS-275 has therapeutic potential for neuroblastoma. This warrants further in-vivo investigations.

Ampliar nosso conhecimento sobre a biologia do carcinoma espinocelular bucal é fundamental para o desenvolvimento de novas estratégias terapêuticas e para melhorar a sobrevida dos pacientes acometidos por essa patologia. Para tanto, compreender as contribuições do microambiente tumoral à carcinogênese é muito importante. Um característica importante a ser avaliada do microambiente tumoral é a acidificação do meio extracelular. Considerando que o pH ácido tumoral está relacionado à maior agressividade da lesão, o objetivo deste projeto é estudar os efeitos de um microambiente ácido na biologia de células de carcinoma espinocelular bucal. Para tanto, foram realizadas duas revisões da literatura, nas quais foram avaliados os seguintes temas: influência da acidez extracelular na invasão e migração; e os mecanismos moleculares envolvidos na resistência ao tratamento quimioterápico, induzida pela acidez do microambiente tumoral. Tais revisões embasaram a construção do terceiro artigo desta tese, o qual se propôs a comparar o comportamento de células linhagens de carcinoma espinocelular bucal (SCC-4 e SCC-9) e queratinócitos (HaCat) cultivadas em meio de cultura de pH 6.8 com células mantidas em meio neutro pH 7.4. As células foram expostas de forma contínua ou intermitente ao pH 6.8 e a adaptação das células foi avaliada pelo ensaio clonogênico. Além disso, as células foram avaliadas quanto à sua capacidade migratória pelo ensaio de cicatrização de feridas e de time lapse. A expressão gênica relacionada à indiferenciação e pluripotência foi investigada por PCR em tempo real com os marcadores Bmi-1 e CD44, assim como pelo ensaio de orosferas. A resistência desses grupos de células ao tratamento anti-câncer foi avaliada pelo ensaio de viabilidade celular da sulforodamina B após o tratamento com Cisplatina. Para a análise estatística, inicialmente foi realizada a distribuição dos dados, seguido da comparação estatística dos grupos utilizando, para distribuição normal, os testes ANOVA e ANOVA de duas vias. Toda a análise foi realizada no programa GraphPad Prism 5.0 e o nível de significância considerado foi de p< 0.05.Observamos que ambas as linhagens mudaram sua morfologia para um aspecto mesenquimal. Ao avaliar o perfil migratório observou-se que as células SCC-9 apresentaram maior capacidade de migração após a exposição ao pH6.8. O aumento da migração celular pode ser causado pela indução da transição epitélio-mesênquima, visto que observamos o aumento da expressão de N-caderina (SCC-4:p<0.05) concomitante à diminuição de E-caderina (SCC-4: p<0.05). A exposição à acidez também provocou, em ambas as linhagens, aumento da capacidade de formar orosferas em placa de baixa aderência, denotando um fenótipo pluripotente (SCC-4: p=0.007/ SCC-9: p= 0.1202). Tal resultado foi reforçado com aumento da expressão gênica do marcador de célula-tronco tumoral CD44 (p= 0.0325). na linhagem SCC-4. No entanto, observamos diminuição da expressão de Bmi-1(p=0.0572) em relação ao controle. A resistência à Cisplatina aumentou nos casos de exposição contínua à acidez (SCC-4: p<0,05). O recondicionamento em meio neutro reverteu a sensibilidade celular (SCC-4: p>0,05). Concluímos que a acidez extracelular no carcinoma espinocelular bucal aumenta a capacidade de migração, induz o fenótipo de células tronco-tumorais e aumenta a resistência a quimioterápicos. / To expand our knowledge about the biology of oral squamous cell carcinoma is crucial for the development of new therapeutic strategies and to improve the survival of the patients affected by this pathology. Therefore, understanding the contributions of the tumor microenvironment to carcinogenesis is very important. An important feature to be evaluated of the tumor microenvironment is the acidification of the extracellular medium. Considering that acidic pH is related to the greater aggressiveness of the tumor, the aim of this study is to analyze the effects of an acidic microenvironment on the biology of oral squamous cell carcinoma cells. We realized two reviews of the literature, in which the following themes were evaluated: the influence of extracellular acidity on the invasion and migration; and the molecular mechanisms involved in the resistance to chemotherapeutic treatment, induced by the acidity of the tumor microenvironment. These revisions helped to construct the third article of this thesis, which proposed to compare the behavior of squamous cell carcinoma lines (SCC-4 and SCC-9) and keratinocytes (HaCat), cultured under pH 6.8 was compared to cells maintained at pH 7.4. For the statistical analysis, the data distribution was initially performed, followed by the statistical comparison of the groups using, for normal distribution, ANOVA and ANOVA two-way tests. All analysis was performed in the GraphPad Prism 5.0 program and the level of significance considered was p <0.05.After continuous or intermittent exposure to pH 6.8, cell adaptation was assessed by the clonogenic assay. In addition, the migratory capacity of the cells was evaluated by the wound healing and time-lapse assays. The gene expression related to undifferentiation and pluripotency was assessed by real-time PCR analysis of the Bmi-1 and CD44 markers, as well as by the orosphere assay. The resistance of these cell groups to anti-cancer treatment was assessed by the sulforhodamine B cell viability assay after treatment with Cisplatin. We observed that both cell lines changed their morphology to a mesenchymal aspect. When assessed the migratory profile, it was observed that SCC-9 cells showed higher migration capacity after exposure to pH6.8. Increased cell migration may be caused by the induction of the epithelial-mesenchymal transition, as we observed increased N-cadherin (SCC-4:p<0.05) expression concomitant with decreased E-cadherin (SCC-4: p<0.05). Exposure to acidity also led to increased ability to form orospheres on low-attachment dishes, denoting a pluripotent phenotype in both strains (SCC-4: p=0.007/ SCC-9: p= 0.1202). This result was reinforced by increased expression of the CD44 (p= 0.0325) tumor cell marker in the SCC- 4 cells. However, we observed a decrease in the expression of Bmi-1(p=0.0572) in relation to the control. Resistance to Cisplatin increased when cells were continuously exposed to acidity(SCC-4: p<0,05). Neutral reconditioning reversed cell sensitivity (SCC-4: p>0,05). We conclude that extracellular acidity in oral squamous cell carcinoma increases the migration capacity, induces the cancer stem cell phenotype and increases resistance to chemotherapy.

Ampliar nosso conhecimento sobre a biologia do carcinoma espinocelular bucal é fundamental para o desenvolvimento de novas estratégias terapêuticas e para melhorar a sobrevida dos pacientes acometidos por essa patologia. Para tanto, compreender as contribuições do microambiente tumoral à carcinogênese é muito importante. Um característica importante a ser avaliada do microambiente tumoral é a acidificação do meio extracelular. Considerando que o pH ácido tumoral está relacionado à maior agressividade da lesão, o objetivo deste projeto é estudar os efeitos de um microambiente ácido na biologia de células de carcinoma espinocelular bucal. Para tanto, foram realizadas duas revisões da literatura, nas quais foram avaliados os seguintes temas: influência da acidez extracelular na invasão e migração; e os mecanismos moleculares envolvidos na resistência ao tratamento quimioterápico, induzida pela acidez do microambiente tumoral. Tais revisões embasaram a construção do terceiro artigo desta tese, o qual se propôs a comparar o comportamento de células linhagens de carcinoma espinocelular bucal (SCC-4 e SCC-9) e queratinócitos (HaCat) cultivadas em meio de cultura de pH 6.8 com células mantidas em meio neutro pH 7.4. As células foram expostas de forma contínua ou intermitente ao pH 6.8 e a adaptação das células foi avaliada pelo ensaio clonogênico. Além disso, as células foram avaliadas quanto à sua capacidade migratória pelo ensaio de cicatrização de feridas e de time lapse. A expressão gênica relacionada à indiferenciação e pluripotência foi investigada por PCR em tempo real com os marcadores Bmi-1 e CD44, assim como pelo ensaio de orosferas. A resistência desses grupos de células ao tratamento anti-câncer foi avaliada pelo ensaio de viabilidade celular da sulforodamina B após o tratamento com Cisplatina. Para a análise estatística, inicialmente foi realizada a distribuição dos dados, seguido da comparação estatística dos grupos utilizando, para distribuição normal, os testes ANOVA e ANOVA de duas vias. Toda a análise foi realizada no programa GraphPad Prism 5.0 e o nível de significância considerado foi de p< 0.05.Observamos que ambas as linhagens mudaram sua morfologia para um aspecto mesenquimal. Ao avaliar o perfil migratório observou-se que as células SCC-9 apresentaram maior capacidade de migração após a exposição ao pH6.8. O aumento da migração celular pode ser causado pela indução da transição epitélio-mesênquima, visto que observamos o aumento da expressão de N-caderina (SCC-4:p<0.05) concomitante à diminuição de E-caderina (SCC-4: p<0.05). A exposição à acidez também provocou, em ambas as linhagens, aumento da capacidade de formar orosferas em placa de baixa aderência, denotando um fenótipo pluripotente (SCC-4: p=0.007/ SCC-9: p= 0.1202). Tal resultado foi reforçado com aumento da expressão gênica do marcador de célula-tronco tumoral CD44 (p= 0.0325). na linhagem SCC-4. No entanto, observamos diminuição da expressão de Bmi-1(p=0.0572) em relação ao controle. A resistência à Cisplatina aumentou nos casos de exposição contínua à acidez (SCC-4: p<0,05). O recondicionamento em meio neutro reverteu a sensibilidade celular (SCC-4: p>0,05). Concluímos que a acidez extracelular no carcinoma espinocelular bucal aumenta a capacidade de migração, induz o fenótipo de células tronco-tumorais e aumenta a resistência a quimioterápicos. / To expand our knowledge about the biology of oral squamous cell carcinoma is crucial for the development of new therapeutic strategies and to improve the survival of the patients affected by this pathology. Therefore, understanding the contributions of the tumor microenvironment to carcinogenesis is very important. An important feature to be evaluated of the tumor microenvironment is the acidification of the extracellular medium. Considering that acidic pH is related to the greater aggressiveness of the tumor, the aim of this study is to analyze the effects of an acidic microenvironment on the biology of oral squamous cell carcinoma cells. We realized two reviews of the literature, in which the following themes were evaluated: the influence of extracellular acidity on the invasion and migration; and the molecular mechanisms involved in the resistance to chemotherapeutic treatment, induced by the acidity of the tumor microenvironment. These revisions helped to construct the third article of this thesis, which proposed to compare the behavior of squamous cell carcinoma lines (SCC-4 and SCC-9) and keratinocytes (HaCat), cultured under pH 6.8 was compared to cells maintained at pH 7.4. For the statistical analysis, the data distribution was initially performed, followed by the statistical comparison of the groups using, for normal distribution, ANOVA and ANOVA two-way tests. All analysis was performed in the GraphPad Prism 5.0 program and the level of significance considered was p <0.05.After continuous or intermittent exposure to pH 6.8, cell adaptation was assessed by the clonogenic assay. In addition, the migratory capacity of the cells was evaluated by the wound healing and time-lapse assays. The gene expression related to undifferentiation and pluripotency was assessed by real-time PCR analysis of the Bmi-1 and CD44 markers, as well as by the orosphere assay. The resistance of these cell groups to anti-cancer treatment was assessed by the sulforhodamine B cell viability assay after treatment with Cisplatin. We observed that both cell lines changed their morphology to a mesenchymal aspect. When assessed the migratory profile, it was observed that SCC-9 cells showed higher migration capacity after exposure to pH6.8. Increased cell migration may be caused by the induction of the epithelial-mesenchymal transition, as we observed increased N-cadherin (SCC-4:p<0.05) expression concomitant with decreased E-cadherin (SCC-4: p<0.05). Exposure to acidity also led to increased ability to form orospheres on low-attachment dishes, denoting a pluripotent phenotype in both strains (SCC-4: p=0.007/ SCC-9: p= 0.1202). This result was reinforced by increased expression of the CD44 (p= 0.0325) tumor cell marker in the SCC- 4 cells. However, we observed a decrease in the expression of Bmi-1(p=0.0572) in relation to the control. Resistance to Cisplatin increased when cells were continuously exposed to acidity(SCC-4: p<0,05). Neutral reconditioning reversed cell sensitivity (SCC-4: p>0,05). We conclude that extracellular acidity in oral squamous cell carcinoma increases the migration capacity, induces the cancer stem cell phenotype and increases resistance to chemotherapy.

Thrombosis and colorectal cancer have a bi-directional relationship. The presence of a colorectal malignancy results in an increased risk of developing a thrombosis and the presence of a thrombosis results in a worse cancer prognosis. The physiology causing this is at present unclear but it is proposed that proteins from the tissue factor (TF) pathway may be the instigator of this bi-directional relationship. The in-vitro studies have shown that in colorectal cancer TF impairs that action of colorectal cancer stem cells as demonstrated by reduced cancer sphere formation and also lower expression of the stem cell marker ALDH. The ability for a colorectal cell to avoid anoikis is impaired by a reduced TF level. Proliferation is affected by the level of expression of TF with a significant increase in proliferation with additional expression of TF. The increase in proliferation is further increased by the presence of TF’s ligand factor VIIa. Paradoxically reduced expression of TF also increases colorectal cancer expression. The ERK1/2 pathway offers a possible method by which TF and factor VIIa may exert their proliferative effects. In the prospective clinical cohort study (CHAMPion) abnormal expression of TF pathway proteins (TF, PAR1, PAR2 and thrombin) by both malignant epithelial and cancer associated stromal cells has been demonstrated. The stromal expression was independent of the epithelial expression and was only in stroma in close contact (0.1mm) with epithelial cells suggesting that the TF pathway proteins may have a role in stromal/epithelial communication. There was no link between the expression of TF pathway proteins and clinicopathological markers of a poor prognosis. The plasma expression of markers of TF pathway activation did not demonstrate any role as a biomarker for colorectal cancer or prognosis. The CHAMPion study has demonstrated that 7% of patients undergoing surgery for colorectal cancer have asymptomatic pre-operative DVTs present. A further 6% who were DVT free pre-operatively developed a DVT in the peri-operative period despite receiving venous thromboprophylaxis in line with current national guidelines. Pre-operative d-dimer may have the potential to identify those patients at risk of a post-operative VTE.This thesis establishes the role that TF has in promoting proliferation and anoikis resistance. It also confirms the abnormal expression of TF pathway proteins by colorectal cancer epithelial cells and for the first time demonstrates abnormal expression by the cancer associated stroma. The interaction between the stroma and epithelial cells, combined with the cellular effects of TF suggests that targeting this interaction may have a therapeutic role. The incidence of DVTs pre-operatively suggests that screening patients for the asymptomatic presence of a DVT may have an impact on their clinical outcome. The development of DVTs despite prophylaxis suggests that the level of anticoagulation is insufficient and current guidelines need to be revisited.

La récidive tumorale est l'un des principaux obstacles à surmonter à l'avenir pour améliorer la survie globale des patients atteints de cancer du côlon (CCR). Les échecs thérapeutiques observés chez les patients sont compatibles avec une accumulation de cellules souches cancéreuses (CSCs) résistantes aux médicaments. Dans cette étude, nous démontrons que le récepteur nucléaire PXR (NR1I2) agit comme un régulateur important de la chimiorésistance des CSCs coliques et de leur capacité à initier la rechute tumorale après traitement. Nous avons d'abord montré que l'expression de PXR augmente avec celle de certains marqueurs des CSCs dans des cellules cancéreuses de patients CCR traitées par chimiothérapies. Nous avons constaté que PXR est préférentiellement exprimé dans les CSCs coliques et qu'il contribue à l'enrichissement des CSCs après chimiothérapies in vitro et in vivo. Par des approches de transcriptomiques, nous avons observé qu'au sein des CSCs coliques, PXR contrôle l'expression d'un large réseau de gènes marqueurs des CSCs coliques, ainsi que des gènes impliqués dans la résistance aux médicaments ou à l'apoptose, ou impliqués dans la dissémination métastatique. Enfin, l'inhibition de PXR par interférence à ARN diminue la survie et auto-renouvèlement des cellules souches cancéreuses du côlon in vitro, ainsi que leur capacité à résister à la chimiothérapie après xénogreffes, conduisant à des retards importants de rechute tumorale après traitements par chimiothérapies in vivo. Cette étude suggère fortement que l'inhibition ciblée de PXR peut représenter une stratégie de traitement néo-adjuvant afin de diminuer la résistance aux médicaments et la récidive des patients CCR via la sensibilisation des cellules souches cancéreuses aux chimiothérapies classiques. / Tumor recurrence is one of the major obstacles to overcome in the future to improve overall survival of patients with colon cancer. High rates and patterns of therapeutic failure seen in patients are consistent with a steady accumulation of drug-resistant cancer stem cells (CSCs). Here, we demonstrate that the nuclear receptor PXR (NR1I2) acts as a key regulator of colon CSC chemoresistance and of their ability to generate post-treatment tumor relapse. We first determined that the enrichment of PXR paralleled that of CSC markers upon treatment of colon cancer cells with standard of care chemotherapy. We found that PXR was highly expressed in colorectal cancer cells displaying CSC markers and function and that it was instrumental for the emergence of CSCs following chemotherapy in vitro and in vivo. mRNA profiling experiments in colon CSCs indicated that PXR transcriptionally controls a large network of genes including markers of stemness, genes involved in resistance to drug/apoptosis or migration/invasion. Finally, PXR down-regulation altered the survival and self-renewal of colon CSCs in vitro and hampered their capacity to resist chemotherapy in vivo, leading to significant delays of post-chemotherapy tumor relapse. This study strongly suggests that targeting PXR may represent a novel treatment strategy to prevent drug resistance and recurrence through the sensitization of CSCs to standard chemotherapy. Taken together, our data strongly suggest that PXR plays an instrumental role in the so-called "intrinsic" pan-resistance of CSCs against therapy.

Complementary and Alternative Medicine (CAM) is popularly used among breast cancer patients to improve their quality of life. CAM includes dietary supplements such as Active Hexose Correlated Compound (AHCC®), a cultured mushroom extract shown to positively influence the immune system and cancer. Breast cancer recurrence is believed to be caused by cancer stem cells (CSCs). We postulated that AHCC impacts CSCs epigenetically by targeting miRNA pathways. The effects of AHCC on mammosphere growth were observed in MDA-MB-231, MCF-7, and 4T1 cells. Profiling, RT2-qPCR, and western blot analyses were performed to determine AHCC influence on miRNAs and Tenascin C protein in TNBC cell line MDA-MB-231. Balb/c mice were gavaged with AHCC to examine tumorigenesis effects. Our results demonstrated that AHCC reduced mammosphere growth and cell migration, and upregulated tumor suppressor miR-335 expression in different biological settings. Inhibition of miR-335 increased Tenascin C expression. Consequently, AHCC may influence BCSCs through miRNA pathways.

L’objectif de ce travail était de démontrer que les cellules de glioblastomes sont capables de se différencier et de se dédifférencier en fonction de leur environnement, d’explorer les mécanismes biologiques qui sous-tendent ces transitions, et d’évaluer in vivo les capacités de différenciation à distance des CIG par les CIG-miR-302-367 via la sécrétion de microvésicules. A partir de plusieurs glioblastomes fraichement réséqués, nous avons caractérisé les cellules tumorales sur le plan phénotypique et fonctionnel pour l’état souche et différencié. Nous avons extraits et analysés les microvésicules des milieux de culture de 2 lignées de CIG-miR-302-367. Selon les principes de la thérapie cellulaire, des co-injections de CIG+CIG-miR-302-367 ont été réalisées dans le cerveau des souris. La majorité des cellules tumorales avaient un phénotype et étaient fonctionnellement différenciées. Après 48 heures de culture en milieu EGF, elles acquéraient les propriétés souches phénotypiques et fonctionnelles. Ce processus de dédifférenciation était réversible en 4 jours de culture en milieu sérum et inhibé par l’adjonction dans le milieu EGF d’un anti-EGFR (cétuximab), suggérant un rôle primordial de la voie EGF/EGFR/ERK. Les microvésicules produites par les CIG-miR-302-367 ont permis une baisse significative de la tumorigénicité des CIG in vivo, et une augmentation de la survie des souris. Le concept de plasticité cellulaire remet en cause les dogmes établis sur la hiérarchie tumorale unidirectionnelle. La déplétion tumorale en CIG, en les forçant à se différencier, est une stratégie thérapeutique innovante, qui peut s’envisager par une approche de thérapie cellulaire. / There is great interest but little understanding in how cancer stem cells arise. Here we show that tumor cells exhibiting stem-like properties and expression of stemness(CD133) and pluripotency markers (SOX2, NANOG, OCT4), can arise from differentiated tumor cells that are isolated from human glioblastomas. These cells could transit from a more differentiated state that cannot self-renew to a self-renewing stem-like state upon EGF/EGFR signaling. This dedifferentiation process induced expression of pluripotency markers, and restored clonal and tumorigenic properties as well as resistance to temozolomide, the chemotherapy of reference. EGF/EGFR signaling including ERK activation was crucial for this cellular reprogramming. Interestingly, expression of pluripotency markers occurred before the cells re-entered the cell cycle, demonstrating that the cells have the capacity to change and reprogram before the cell division starts. Our findings support a model of tumor homeostasis in which tumor cells driven by environmental cues such as EGF, can spontaneously acquire stem-like properties contributing thus to the enrichment in tumor propagating cells.

The role of vascular endothelial growth factor (VEGF) signaling in cancer is well-known in the context of angiogenesis but is also important in the functional regulation of tumor cells themselves. Notably, autocrine VEGF signaling mediated by its co-receptors called neuropilins (NRPs) appears be essential for sustaining the proliferation and survival of cancer stem cells (CSCs), which are implicated in mediating tumor growth, progression and drug resistance. Therefore, the first half of this thesis focuses on the mechanism of VEGF-NRP-mediated support of CSCs. Aberrant activity of the Hippo pathway effector YAP and TAZ are associated with breast CSCs and have been shown to confer stem cell-like properties. I found that VEGF-NRP2 signaling contributes to the activation of YAP/TAZ in various breast cancer cells, which mediates a positive feedback loop that promotes mammosphere formation. VEGF-NRP2 signaling activated the GTPase Rac1, which inhibited the Hippo kinase LATS, which enabled the activity of YAP/TAZ. In complex with the transcription factor TEAD, TAZ then bound and repressed the promoter of the gene encoding the Rac GTPase-activating protein (Rac GAP) β2-chimaerin. By activating GTP hydrolysis, Rac GAPs effectively turn off Rac signaling; hence, YAP/TAZ-mediated repression of β2-chimaerin sustained Rac1 activity in CSCs. Depletion of β2-chimaerin in non-CSCs increased Rac1 activity, YAP/TAZ activation and mammosphere formation. Analysis of breast cancer patients revealed an inverse correlation between β2-chimaerin and TAZ expression in tumors. These findings highlight an unexpected role for β2-chimaerin in a feedforward loop of YAP/TAZ activation and the acquisition of CSC properties. Given that CSCs have been implicated in therapy resistance and are enriched in triple negative breast cancer (TNBC), which exhibits VEGF-NRP2 signaling, the second half of this thesis focuses on understanding the mechanism by which VEGF-NRP2 contributes to the chemoresistance of TNBC. I discovered that VEGF-NRP2 promote homologous recombination (HR) in BRCA1 wild-type TNBC cells by contributing to the expression and function of Rad51, an essential enzyme in the HR pathway that mediates efficient DNA double strand break repair. Mechanistically, I found that VEGF-NRP2 stimulates YAP/TAZ-dependent Rad51 expression and that Rad51 is a direct YAP/TAZ-TEAD transcriptional target. I also discovered that VEGF-NRP2-YAP/TAZ signaling contributes to the resistance of TNBC cells to chemotherapy and that Rad51 rescues the defects in DNA repair upon inhibition of either VEGF-NRP2 or YAP/TAZ in response to cisplatin. These findings reveal novel roles for VEGF-NRP2 and YAP/TAZ in DNA repair and they indicate a unified mechanism involving VEGF-NRP2, YAP/TAZ and Rad51 that contributes to resistance to platinum chemotherapy. In summary, this thesis provides novel insight into the roles of autocrine VEGF-NRP2 signaling in breast CSC function and therapy resistance and provides rationale in inhibiting NRP2 for platinum-resistant tumors that are dependent on YAP/TAZ activation.

Ce travail s’inscrit dans la caractérisation des cellules radiorésistantes dans les cancers du sein, à l’origine des rechutes après traitement. Si de nombreuses données de la littérature indiquent un lien étroit entre la présence des « Cellules Souches Cancéreuses » (CSC) et la résistance aux traitements anticancéreux, la radiorésistance « intrinsèque » des CSC ainsi que les mécanismes impliqués restent encore mal définis.L’équipe du Dr. Weinberg a développé un modèle cellulaire in vitro permettant la génération de CSC mammaires humaines, « CD24-CD44+ », à la suite d’une transition épithélio-mésenchymateuse (TEM) induite par une exposition au TGF-β1. Basée sur l’utilisation de cellules mammaires humaines saines qui ont été ensuite immortalisées puis transformées, ce modèle a permis d’approfondir la compréhension de nombreux processus tumoraux tels que la TEM.Ainsi, dans ce contexte, mon projet de thèse a consisté à utiliser ce modèle afin de comparer la radiosensibilité des cellules « CD24-CD44+ » à celle des cellules « CD24+CD44- ». Le choix du modèle repose sur le fait qu’il permet la comparaison de cellules à deux stades différents de la progression tumorale.Après un rappel des connaissances nécessaires à la compréhension de ce travail, le manuscrit présente dans un premier temps la caractérisation du modèle cellulaire utilisée puis la réponse à l’irradiation des cellules du modèle. L’étude de la réponse à l’irradiation a permis de décrire le phénotype de radiorésistance des cellules « CD24-CD44+ » à travers un faible blocage en G2/M, une faible proportion de cellules polyploïdes ainsi qu’une capacité accrue à donner une descendance après irradiation (10 Gy).Ensuite, l’implication des mécanismes de mort apoptotique des cellules « CD24-CD44+ » dans le phénotype de radiorésistance a été montré. Ainsi, la mort cellulaire réduite retrouvée dans les « CD24-CD44+ » est liée à une plus faible activation des voies apoptotiques.Dans un dernier temps, l’identification d’une signature transcriptionnelle de gènes de détoxification lors de la caractérisation des cellules de notre modèle a permis de mettre en évidence la modulation radioinduite de deux de ces gènes, SOD2 et HMOX1, après stress radioinduit dans les cellules « CD24-CD44+ ». / This works aims at characterizing radioresistant cells within human breast cancer development that is responsible for treatment failure and cancer recurrences. Although the literature is flourishing with papers tightly linking the presence of "Cancer Stem Cells" to cancer treatment resistance, the intrinsic radioresistance of those "CSC" and the mechanism involved have yet to be established.Dr. Weinberg and his team have developed an in vitro cell model that produces mammary tumor cells noted « CD24-CD44+ » after an epithelial-to-mesenchymal transition (EMT) induced by TGF-β1. This model is based on healthy human mammary cells that have been immortalized and transformed.Within this context, my Ph.D. project has focused on using this new model in order to compare the radiosensibility of two cell lines: the « CD24-CD44+ » cells and the « CD24+CD44- » one. Underlying this choice is the fact this model allows for a comparison of two cellular populations at distinct stage of the tumor’s development.This work has shed light on apoptotic and detoxification mechanism involved in the radio resistant behavior of the « CD24-CD44+ » cells. After a brief introduction of key concepts required to the understanding of this work, this manuscript will begin by presenting the characterization of the chosen model, then a study of the radiation response that enabled a first description of the « CD24-CD44+ » cell radioresistant phenotype through a mild stop at the G2/M stage of the cell cycle, the presence of polypoid cells and a high progeny generation ability after exposure to radiation.Furthermore, this works shows implications of apoptotic mechanism of « CD24-CD44+ » cells with a radioresistance phenotype. Hence, we were able to show that reduced cell death observed for the « CD24-CD44+ » cells is linked to a lower activation of apoptotic pathways.Finally, the last part will present detoxification mechanism involved in « CD24-CD44+ » radioresistance phenotype, showing an altered transcriptional signature of two detoxication genes SOD2 and HMOX1 after exposure to radiation.