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Ras-MAPK signalling regulates key cellular processes such as proliferation, differentiation and survival. Unsurprisingly, mutations in RAS genes are now recognized as potent oncogenic drivers. However, disruption of this pathway during development is associated with a family of disorders termed the Rasopathies. Shared clinical features include cutaneous, neurological and cardiac anomalies. At the outset of this study, the genetic etiology of three neurocutaneous disorders, microcephaly-capillary malformation syndrome (MIC-CAP), encephalocraniocutaneous lipomatosis (ECCL) and PHACE (Posterior fossa malformations, facial Hemangiomas, cerebral Arterial anomalies, Cardiovascular defects and Eye abnormalities) syndrome had not yet been established. This thesis identifies mutations in STAM-binding protein (STAMBP) in a cohort of individuals with MIC-CAP syndrome using whole-exome sequencing (WES). This gene encodes a deubiquitinating isopeptidase that regulates cell surface receptor-mediated endocytosis and sorting. Cell lines of individuals with MIC-CAP show reduced STAMBP expression, associated with accumulation of ubiquitinated protein aggregates, increased apoptosis and constitutive activation of the Ras-MAPK and PI3K-AKT pathways. WES also enabled the identification of post-zygotic mutations within the tyrosine kinase domain of fibroblast growth factor receptor 1 (FGFR1) in individuals with ECCL. Fibroblasts from affected individuals showed increased phosphorylation of the FGFRs consistent with receptor activation as well as insensitive signal transduction through the Ras-MAPK pathway. Neurocutaneous syndromes can feature striking vascular lesions such as the cerebral vasculopathy and large segmented facials hemangiomas seen in PHACE syndrome. The asymmetric and patchy vascular malformations coupled with a sporadic incidence and absence of familial recurrence suggested that PHACE might be caused by post-zygotic mutations. Interrogation of a discordant sib-pair using copy number analysis and WES did not identify causative mutations indicating the need for a comprehensive and targeted –omic approach to elucidate the molecular mechanism of this syndrome. Taken together, these findings expand the spectrum of the Rasopathies while providing novel pathomechanistic insights into the regulation of cellular proliferation and survival during development.

Normal cellular behaviour in multicellular organisms is achieved by tight control of signaling pathway networks. The mitogen-activated protein kinase (MAPK) signaling cascade is one of these signaling networks, that when deregulated can lead to cellular transformation. Activation of the RAS-RAF-MEK-MAPK (ERK) signal transduction pathway or the SAPK2/p38 pathway results in the activation of mitogen- and stress-activated protein kinases 1 and 2 (MSK1/2). Subsequently, MSKs go on to phosphorylate histone H3 at Ser10 and Ser28.Here, we demonstrate that the activities of ERK and MSK1, but not p38, are elevated in Hras-transformed cells (Ciras-3) relative to these activities in the parental 10T1⁄2 cells. Analyses of the subcellular distribution of MSK1 showed that the H3 kinase was similarly distributed in Ciras-3 and 10T1/2 cells, with most MSK1 being present in the nucleus. In contrast to many other chromatin modifying enzymes, MSK1 was loosely bound in the nucleus and was not a component of the nuclear matrix. Our results provide evidence that oncogene-mediated activation of the RAS-MAPK signal transduction pathway elevates the activity of MSK1, resulting in the increased steady-state levels of phosphorylated H3, which may contribute to the chromatin decondensation and aberrant gene expression observed in oncogene-transformed cells. Furthermore, upon activation of the ERK and p38 MAPK pathways, the MSK1/2- mediated nucleosomal response, including H3 phosphorylation at serine 28 or 10, is coupled with the induction of immediate-early gene transcription. The outcome of this response, varying with the stimuli and cellular contexts, ranges from neoplastic transformation to neuronal synaptic plasticity. Here, we used sequential co-immunoprecipitation assays and chromatin immunoprecipitation (ChIP) assays on mouse fibroblast 10T1/2, Ciras-3 and MSK1 knockdown 10T1/2 cells to show that H3 serine 28 and 10 phosphorylation leads to promoter remodeling. MSK1, in complexes with phospho-serine adaptor 14-3-3 proteins and BRG1 (the ATPase subunit of the SWI/SNF remodeler) is recruited to the promoter of target genes by transcription factors such as ELK-1 or NFκB. Following MSK1-mediated H3 phosphorylation, BRG1 associates with the promoter of target genes via 14-3-3 proteins, which act as scaffolds. The recruited SWI/SNF remodels nucleosomes at the promoter of immediate-early genes enabling the binding of transcription factors like JUN and the onset of transcription. Since RAS-MAPK activated MSKs mediate H3 phosphorylation that is required for expression of various immediate-early gene products involved in cellular transformation, inhibition of MSK activity may be a therapeutic target that could be exploited in cancers with upregulated RAS-MAPK signaling.

Normal cellular behaviour in multicellular organisms is achieved by tight control of signaling pathway networks. The mitogen-activated protein kinase (MAPK) signaling cascade is one of these signaling networks, that when deregulated can lead to cellular transformation. Activation of the RAS-RAF-MEK-MAPK (ERK) signal transduction pathway or the SAPK2/p38 pathway results in the activation of mitogen- and stress-activated protein kinases 1 and 2 (MSK1/2). Subsequently, MSKs go on to phosphorylate histone H3 at Ser10 and Ser28.Here, we demonstrate that the activities of ERK and MSK1, but not p38, are elevated in Hras-transformed cells (Ciras-3) relative to these activities in the parental 10T1⁄2 cells. Analyses of the subcellular distribution of MSK1 showed that the H3 kinase was similarly distributed in Ciras-3 and 10T1/2 cells, with most MSK1 being present in the nucleus. In contrast to many other chromatin modifying enzymes, MSK1 was loosely bound in the nucleus and was not a component of the nuclear matrix. Our results provide evidence that oncogene-mediated activation of the RAS-MAPK signal transduction pathway elevates the activity of MSK1, resulting in the increased steady-state levels of phosphorylated H3, which may contribute to the chromatin decondensation and aberrant gene expression observed in oncogene-transformed cells. Furthermore, upon activation of the ERK and p38 MAPK pathways, the MSK1/2- mediated nucleosomal response, including H3 phosphorylation at serine 28 or 10, is coupled with the induction of immediate-early gene transcription. The outcome of this response, varying with the stimuli and cellular contexts, ranges from neoplastic transformation to neuronal synaptic plasticity. Here, we used sequential co-immunoprecipitation assays and chromatin immunoprecipitation (ChIP) assays on mouse fibroblast 10T1/2, Ciras-3 and MSK1 knockdown 10T1/2 cells to show that H3 serine 28 and 10 phosphorylation leads to promoter remodeling. MSK1, in complexes with phospho-serine adaptor 14-3-3 proteins and BRG1 (the ATPase subunit of the SWI/SNF remodeler) is recruited to the promoter of target genes by transcription factors such as ELK-1 or NFκB. Following MSK1-mediated H3 phosphorylation, BRG1 associates with the promoter of target genes via 14-3-3 proteins, which act as scaffolds. The recruited SWI/SNF remodels nucleosomes at the promoter of immediate-early genes enabling the binding of transcription factors like JUN and the onset of transcription. Since RAS-MAPK activated MSKs mediate H3 phosphorylation that is required for expression of various immediate-early gene products involved in cellular transformation, inhibition of MSK activity may be a therapeutic target that could be exploited in cancers with upregulated RAS-MAPK signaling.

Le profil d'expression aberrant des micro(mi)ARN est une caractéristique typique de nombreux cancers, dont le carcinome hépatocellulaire (CHC), une tumeur hépatique maligne primaire qui se classe seconde dans le monde en termes de mortalité par cancer. Notre équipe a récemment montré la baisse d’expression de miR-4510 dans des échantillons de patients atteints de CHC et son activité « suppresseur de tumeur ». L'analyse de données protéomiques recueillies à partir de cellules Huh7 transfectées par miR-4510 a révélé une diminution importante de plusieurs oncogènes, dont la sérine / thréonine protéine kinase RAF1. J’ai également découvert que le taux de protéine RAF1 était significativement surexprimé chez les patients atteints de CHC. Le rôle de RAF1 et de miR-4510 dans le CHC étant mal compris, j’ai étudié la fonction du couple RAF1/miR-4510 dans la tumorigenèse du foie. Mes analyses ont montré que miR-4510 régule négativement les taux de protéine RAF1 et d'ARNm. Une analyse par le système de double fluorescence-FunREG a révélé que miR-4510 interagit directement avec la région 3’ non-traduite de l’ARN de RAF1 via un site unique. La déplétion de RAF1 dans deux lignées tumorales de CHC par miR-4510 ou ARN interférant désactive leur caractère tumorigène in vitro et in vivo. Collectivement, mes données suggèrent que miR-4510 participe à la carcinogenèse du foie via son action directe sur RAF1 et la régulation de la voie MAPK/ERK. En conclusion, mon étude soutient l’hypothèse selon laquelle un traitement à base de miR-4510 pourrait être efficace pour traiter les patients atteints de CHC de type avancé ou réfractaire à la chimiothérapie. / Aberrant micro(mi)RNA expression signature is a hallmark of many cancers including hepatocellular carcinoma (HCC), a primary malignant liver disease which ranks second in cancer mortality worldwide. Our team previously reported the downregulation of miR-4510 in HCC samples and identified this miRNA as a strong tumor suppressor in liver. Proteomic data analysis collected from Huh7 cells transfected by miR-4510 showed a significant decrease of multiple oncogenes including RAF1 serine/threonine protein kinase. I also found that RAF1 protein level is significantly increased in HCC patients. The role of RAF1 and miR-4510 in HCC being poorly understood, I studied the function of RAF1/miR-4510 pair in tumorigenesis of the liver. My results showed that miR-4510 overexpression significantly decreases both RAF1 protein and mRNA levels and inhibits MAPK/ERK signaling. The dual fluorescence-FunREG assay revealed that miR-4510 directly interacts with RAF1 3’-untranslated region through a unique site. Silencing of RAF1 in two hepatic cell lines by miR-4510 or a specific small interfering RNA suppressed important tumorigenic features (proliferation, migration….) both in vitro and in vivo. Collectively, my data suggest that miR-4510 participates in liver carcinogenesis through RAF1 targeting and MAPK/ERK signaling inactivation. In addition, my study suggests that miR-4510-based therapy may represent a promising strategy to treat patients with advanced or refractory HCC.

Comprendre comment les facteurs génétiques et environnementaux interagissent au cours du développement est une question fondamentale en biologie. Je me suis intéressée à cette question en utilisant le réseau de développement de la vulve du nématode C. elegans comme système modèle. L’objectif de mon projet était une étude quantitative de la modulation par l’environnement des voies de signalisation impliquées dans ce processus telles que, Ras, Delta-Notch et Wnt. J’ai tout d’abord analysé comment un facteur environnemental spécifique (la carence nutritionnelle) modifie les activités et les interactions entre les voies de signalisation sous-jacentes au développement vulvaire chez C. elegans. J’ai ainsi mis en évidence que l’augmentation de l’induction vulvaire par la carence passe par une augmentation de l’activité de la voie Ras et est indépendante de la voie Wnt. Cet effet de l’environnement est assuré par la détection de la diminution de l’apport en nutriments, probablement par l’action de la voie TOR, et affecte l’induction vulvaire en parallèle ou en amont du récepteur à l’EGF. J’ai ensuite examiné la sensibilité environnementale du système de développement de la vulve de Caenorhabditis dans une perspective évolutive et ce, grâce à l'analyse comparative de différents isolats. J’ai pu observer que l’exposition à des températures extrêmes induit des variants et des défauts de manière fortement dépendante de la souche et de l’espèce. L’occurrence de certains défauts développementaux induits par la température révèlent en outre que certaines cellules précurseurs de la vulve et les voies de signalisation associées présentent une sensibilité environnementale différente. / How genetic and environmental factors interact during development is a key question in current biology, yet little is known about how molecular and cellular processes integrate environmental information. In my PhD research I aimed to address this problem using the network of C. elegans vulval signalling pathways as a model system. The principal objective of my project was to quantitatively examine how involved major signalling pathways, EGF-Ras-MAPK, Wnt and Delta-Notch, are modulated by specific environmental signals. First, I analysed how a specific environmental factor (starvation) alters activities and interplay of signalling pathways underlying C. elegans vulval cell fate patterning. I found that starvation consistently increased vulval induction through upregulation of the EGF-Ras-MAPK pathway activity independent of the Wnt pathway. This environmental effect is mediated by internal sensing of nutrient deprivation, likely acting through the TOR pathway, and affects vulval induction at the level or upstream of the EGF receptor. Second, I examined the environmental sensitivity of the Caenorhabditis vulval developmental system from an evolutionary perspective through comparative analysis of different C. elegans and C. briggsae isolates. I found that extreme temperature induced diverse developmental variants and defects, which were strongly genotype- and species-dependent. The occurrence of certain developmental defects induced by temperature extremes further revealed that vulval precursor cells and associated fates differ in temperature sensitivity, and this cell-specific sensitivity shows evolutionary variation.

Les léiomyosarcomes (LMS) sont des tumeurs d’origine mésenchymateuse caractérisées par une différenciation musculaire lisse. La voie de signalisation PI3K/mTOR (qui contrôle la prolifération et la survie cellulaire) joue un rôle majeur dans le développement de ces tumeurs. De nos jours, cette voie est devenue une cible thérapeutique majeure en oncologie. Cette étude est la première qui évalue le bénéfice thérapeutique de l’inhibition de la voie PI3K/mTOR pour des patients atteint de LMS. Nous avons mis en évidence qu’une double inhibition de PI3K et mTOR est associée à une activité antitumorale supérieure à celle observée avec une inhibition de PI3K ou mTOR seule. Nous avons également montré que l’inhibition de la voie PI3K/mTOR est associée à une activation paradoxale de la voie MAPK et qu’un ciblage concomitant de cette voie est associé à une synergie antitumorale in vitro et in vivo. Afin de caractériser les mécanismes de résistance secondaire à l’inhibition de la voie PI3K/mTOR, nous avons développé in vitro et in vivo un modèle de résistance secondaire à l’inhibiteur double cible PI3K/mTOR. Nous avons notamment détecté une sous-population de cellules résistantes à l’inhibiteur et ayant des caractéristiques proches de celles des cellules souches. Nous avons mis en évidence que l’inhibition pharmacologique d’EZH2, une protéine cruciale du complexe Polycomb, permet de restaurer la sensibilité des modèles résistants. Ces résultats apportent de nouvelles perspectives thérapeutiques pour les patients atteints de LMS. / Leiomyosarcomas (LMS) are tumors of mesenchymal origin characterized by a smooth cell differentiation. The PI3K/mTOR pathway has been shown to play a crucial role in the tumorigenesis of LMS. Several agents targeting this pathway are under clinical development for the treatment of solid tumors and hematological malignancies. We report here the first study evaluating its potential therapeutic benefit for patients with LMS. We have demonstrated that dual inhibition of PI3K and mTOR is associated with more effective antitumor activity than agents targeting PI3K or mTOR only. We have also shown that PI3K and mTOR inhibition is associated with a paradoxal activation of the MAPK pathway and that combined treatment with MEK inhibitor resulted in synergistic antitumor activity in vitro and in vivo. Moreover, we developed in vitro and in vivo resistant model to dual PI3K/mTOR inhibitor. Interestingly, we have found that a cancer stem cell-like subpopulation may be involved in treatment resistance. We have shown that pharmacological inhibition of EZH2, a crucial protein of the Polycomb complex, is able to reverse dual PI3K/mTOR inhibitor resistance in vitro and in vivo. These results provide new therapeutic strategies for patients with LMS.

The introduction of tyrosine kinase inhibitors (TKI) targeting the epidermal growth factor receptor (EGFR) inhibitors to the clinic has resulted in an improvement in the treatment of non small cell lung cancer (NSCLC). However, many patients treated with EGFR TKIs do not respond to therapy. The burden of failed treatment is largely placed on the healthcare field, limiting the effectiveness of EGFR TKIs. Furthermore, responses are hindered by the emergence of resistance. Thus, two questions must be addressed to achieve maximum benefit of EGFR inhibitors: How can patients who will benefit from EGFR TKIs be selected a priori? How can patients who respond achieve maximal benefit? To answer these questions, two hypotheses were formed. First, the EGFR-dependent phenotype, which is displayed by the tumors cells of those patients who respond clinically to EGFR TKIs, can be captured by genomic profiling of NSCLC cell lines stratified by sensitivity to EGFR TKIs. This gene signature may be used to predict the outcome of EGFR TKI therapy in unknown samples. Secondly, the predictive signature of response to EGFR TKI could provide insights into the underlying biology of the phenotype of EGFR-dependency. This information could be exploited to identify inhibitors which could be combined with EGFR inhibitors to elicit a greater effect, thereby minimizing resistance. The work herein describes the testing of these hypotheses. Pharmacogenomics was utilized to define a signature of EGFR-dependency which effectively predicted response to EGFR TKI in vitro and in vivo. Furthermore, the signature was analyzed by bioinformatic approaches to identify the RAS/MAPK pathway as a candidate target in EGFR-dependent NSCLC. The RAS/MAPK pathway regulates expression and activation of EGF-like ligands. Furthermore, the RAS/MAPK pathway modulates EGFR stability in the EGFR-dependent phenotype. Further biochemical analyses demonstrated that the RAS/MAPK pathway mediates proliferation and survival of EGFR-dependent NSCLC cells. Finally, combinatorial treatment of EGFR-dependent NSCLC cell lines with small molecules targeting EGFR and the RAS/MAPK pathway yielded cytotoxic synergy. Thus, we have used pharmacogenomics methods to potentially improve NSCLC treatment by developing a method of predicting response and identifying an additional target to combine with EGFR TKIs to maximize responses.

Ovarian carcinoma afflicts over 22,000 women each year with a 5 year survival rate of only 18% for stage IV patients [23]. Current treatment options are limited due to high rates of drug resistance and recurrence. Further, the identity of "precursor lesions" which give rise to various subclasses of epithelial ovarian cancer has been evasive. This is due to discovery of the cancer at already an advanced stage. Interestingly, endometriosis a benign but invasive gynecological disease has been described as a "precursor lesion" in the development of specific subtypes of ovarian cancer. Endometriotic cyst development involves the accumulation of "old blood" components including iron-rich heme. Published evidence implicates excess iron that is involved in the transformation of normal surface epithelial cells inducing morphological characteristics of clear cell ovarian cancer cells [13, 34]. Due to excess iron in endometriotic cysts, this essential element may play a transformative role in the development of clear cell ovarian cancer and possibly other subtypes [13, 35-38]. Further, studies show increased risk of developing ovarian cancer, particularly clear cell and endometrioid ovarian subtypes, in patients diagnosed with endometriosis [36, 37, 39, 40] . This thesis aims to initiate an investigation regarding the contribution of iron and endometriotic lesions in the development and progression of specific subtypes of epithelial ovarian cancers. Since there is a lack of well-validated and characterized endometriotic cell lines that could be used for endometriosis studies, we sought to develop an immortalized cell line for future endometriotic in vitro and in vivo studies. Thus, in Chapter 3 we present our efforts in developing a novel life-span extended epithelial endometriotic cell line. The cells were derived from the endometriotic tissue of a patient with endometriosis. We describe our attempts at immortalization and the characterization of this endometriotic cell line in relation to previously reported/available endometrial/endometriotic cell lines. In Chapter 4 we investigated the role of iron in modulating functional aspects of various gynecological cell lines. Although our expectation was that iron could transform normal ovarian surface epithelial cells (OSE) to a carcinoma-like phenotype, we instead discovered that ovarian cell lines containing Ras mutations (or with H-Ras overexpression) responded to iron (presented as ferric ammonium citrate (FAC)) with a reduced growth response. Further treatment with iron induced an apoptotic/necrotic death response in the Ras mutated HEY ovarian carcinoma cell line. Interestingly, we identified that iron induced autophagic activation in all ovarian cell lines investigate, although autophagy contributed only modestly to the cell death event. Furthermore, we noted that iron activated the MAPK pathway and its inhibition (via U0126, a MAPK inhibitor) allowed survival of cells. In Chapter 5, we briefly explore the role of iron in ovarian cell types growing under anchorage-independent conditions. We found that the cell lines displayed increased cleaved PARP and apoptosis when placed under these conditions. Treatment with iron led to a reduction in cleaved PARP suggesting that iron promotes cell survival in anchorage-independent conditions. Further, inhibition of autophagy via chloroquine led to increased cleaved PARP suggesting that autophagy may mediate a protective role against anchorage-independent apoptotic response In Chapter 6, we attempted to elucidate the downstream mechanism following Ras/MAPK activation in response to iron. This study identified several signaling pathways including that involved in translational control, iron metabolism, as well as mitochondrial function. The inhibition of the iron regulatory and translation control pathway did not significantly lead to rescue of iron-induced cell death of Ras mutated/overexpressed cells. However, we noted mitochondrial stress and damage including altered expression of mitochondrial markers (TOM20/TOM70, outer membrane transporters) which occurred concurrently with iron-induced cell death. The inhibition of iron import into mitochondria using a calcium uniporter channel inhibitor (Ru360) led to a marked reversal of the cell death response. Collectively, these studies suggest that increased mitochondrial permeabilization may be responsible for the observed iron-induced cell death response. Overall, the studies presented in this thesis have revealed novel responses to iron in the gynecological cell types investigated. We initially sought to understand the role of iron in precursor lesions which included the development of a novel life-span extended epithelial endometriotic cell type. Remarkably, our findings revealed a Ras driven sensitivity to excess iron. Treatment with iron caused decreased cell growth and increased cell death in cell types containing Ras mutation/overexpression. Further, we found that the mechanism leading to the iron-induced cell death events was mediated via the MAPK pathway. We then determined that the cell death response was associated with mitochondrial permeabilization. Loss of mitochondrial integrity occurred in Ras sensitive cell lines and inhibition of iron import into the mitochondria (via the calcium uniporter channel inhibitor, Ru360) led to reversal of this response. We show herein the cellular response of excess iron and its potential implication in ovarian cancer research.

Noonan spectrum disorders belong to the RASopathies, a group of clinically related developmental disorders caused by dysregulation of the RAS-MAPK pathway. This thesis describes genetic and clinical investigations of six families with Noonan spectrum disorders. In the first family, the index patient presented with severe Noonan syndrome (NS) and multiple café-au-lait (CAL) spots, while four additional family members displayed multiple CAL spots only. Genetic analysis of four RAS-MAPK genes revealed a de novo PTPN11 mutation and a paternally inherited NF1 mutation, which could explain the atypically severe NS, but not the CAL spots trait in the family. The co-occurrence of two mutations was also present in another patient with a severe/complex NS-like phenotype. Genetic analysis of nine RASopathy-associated genes identified a de novo SHOC2 mutation and a maternally inherited PTPN11 mutation. The latter was also identified in her brother. Both the mother and the brother displayed mild phenotypes of NS. The results from these studies suggest that an additive effect of co-occurring mutations contributes to severe/complex NS phenotypes. The inherent difficulty in diagnosing Noonan spectrum disorders is evident in families with neurofibromatosis-Noonan syndrome (NFNS). An analysis of nine RASopathy-associated genes in a five-generation family with NFNS revealed a novel NF1 mutation in all affected family members. Notably, this family was initially diagnosed with NS and CAL spots. The clinical overlap between NS and NFNS was further demonstrated in three additional NFNS families. An analysis of twelve RASopathy-associated genes revealed three different NF1 mutations, all segregating with the disorder in each family. These mutations have been reported in patients with NF1, but have, to our knowledge, not been associated with NFNS previously. Together, these findings support the notion that NFNS is a variant of NF1. Due to the clinical overlap between NS and NFNS, we propose screening for NF1 mutations in NS patients negative for mutations in NS-associated genes, preferentially when CAL spots are present. In conclusion, this thesis suggests that co-occurrence of mutations or modifying loci in the RAS-MAPK pathway contributes to the clinical variability observed within Noonan spectrum disorders and further demonstrates the importance of accurate genetic diagnosis.