The Hippo signalling pathway is an evolutionarily conserved kinase cascade responsible for the cell proliferation, tissue growth and apoptosis during development and its dysregulation contributes to tumourigenesis. This signalling pathway was initially discovered in Drosophila and soon after that, it was shown to be highly conserved in mammals. The core Lats kinases of this tumour suppressive pathway phosphorylate and inhibit the downstream transcriptional co-activators YAP and TAZ, which are implicated in various cancers. Latest reports revealed various E3 ubiquitin ligases to negatively regulate the Hippo pathway through ubiquitination, yet few deubiquitinating enzymes have been described. In the present study, we report USP9X deubiquitinating enzyme as an essential regulator of the central components of this pathway. USP9X interacted strongly with Lats2 kinase and to a lesser extent with WW45, Kibra and Angiomotin family proteins. The knockdown of USP9X resulted in notable downregulation and destabilisation of Lats kinase and to lesser extents WW45, Kibra and Amot. This resulted in enhanced nuclear localisation of YAP and TAZ accompanied with activation of their target genes, CTGF and CYR61. USP9X was shown to stabilise Hippo components through its deubiquitinating activity. USP9X enzyme defective mutant lost the activity to stabilise Lats2, WW45, Kibra and Angiomotins through deubiquitination, leading to their ubiquitination. In the absence of USP9X, cells exhibited epithelial to mesenchymal transition phenotype and additionally gained anchorage-independent growth in soft agar. Moreover, USP9X knockdown disrupted acinar organisation of breast cells in three-dimensional acini cultures. In addition, YAP/TAZ target gene activation in USP9X knockdown cells could be rescued by knockdown of YAP, TAZ and TEAD2. Lastly, USP9X protein expression showed a positive correlation with Lats kinases, but negative correlation with YAP/TAZ in pancreatic cancer tissues as well as pancreatic and breast cancer cell lines. The results strongly indicate that USP9X cooperates with Lats2 and other important Hippo components to suppress tumour growth.

Cell based therapy using stem cell derived cardiomyocytes, has emerged as a potential therapeutic approach for cardiac diseases such as myocardial infarction and heart failure. Induced pluripotent stem cells (iPS cells) could be an ideal source of cardiomyocytes (iPS-CM). Challenges facing cell therapy include the high number of viable cells needed to survive in pathological conditions. The Hippo signalling pathway has been described as a key pathway involved in regulating cardiomyocyte proliferation and survival in both embryonic and adult hearts. We hypothesise that modification of the Hippo pathway will enhance the efficiency of iPS-CM generation and will increase iPS-CM survival and viability in pathological conditions. Skin fibroblasts were reprogrammed to iPS cells and then differentiated to cardiomyocytes. The Hippo signalling pathway was modified by genetic ablation of MST1, a major upstream regulator of the Hippo pathway, or by overexpressing YAP, the main downstream effector of the pathway. Cell proliferation was analysed using an EdU incorporation assay and staining for cytokinesis markers Ki67 and phospho-histone H3. Cell death and viability were analysed by measuring caspase 3/7 and MTT activity and by trypan blue staining in both normal and hypoxic conditions (CoCl2 treatment). Analysis of cell proliferation shows that genetic ablation of Mst1 leads to significantly increased proliferation (+12±1.5% P < 0.001), survival and viability (+20±4.3% P < 0.001) of iPS cells in both normal and hypoxic (CoCl2 treatment) conditions compared to controls. In addition, overexpression of YAP, which is normally inhibited by upstream Hippo pathway components, and overexpression of mutated constitutively active form of YAP (S127A) increases cell proliferation in iPS-CM compared to control iPS-CM as shown with EdU assay (46±2.60% P < 0.01) and Ki67 staining (4.9±0.9% P < 0.001). Overexpression of YAP leads to up regulation of genes associated with inhibition of apoptosis and promotion of cell proliferation. Preliminary studies show mouse iPS-CM are retained in the myocardium following intra-cardiac injection and do not cause any adverse effects confirmed with histological, echocardiography and electrocardiogram analysis. In conclusion targeting the Hippo pathway in iPS cells and iPS-CM significantly increases proliferation and survival in both normal and hypoxic conditions. Therefore, modulation of the Hippo pathway could become a new strategy to enhance the therapeutic potential of iPS-CM.

Indiana University-Purdue University Indianapolis (IUPUI) / During gestation, the maternal liver undergoes various adaptive changes to cope with the in-creasing physiological and metabolic demands from both maternal and fetal compartments. Among these changes are robust growth and changes in transcriptome profile. However, how these events happen, and other aspects of this physiological phenomenon remains unexplored. Therefore, we aimed at further understanding how maternal liver responds to pregnancy. We used BrdU labeling combined with a virus-based tracing approach to quantify the percentage of maternal hepatocytes undergoing DNA synthesis and division over the course of gestation in mice. We found that ~50% maternal hepatocytes entered S-phase but, unexpectedly, did not undergo cytokinesis. This strongly suggests that maternal hepatocytes in fact undergo endoreplication instead of hyperplasia, as believed previously. Pericentral Axin2+ hepatocytes were reported to behave as liver stem cells responsible for liver homeostasis and turnover. We generated an in vivo fate-tracing mouse model to monitor the behavior of these cells in the maternal liver. Our results showed that they did not proliferate during pregnancy, homeostasis, and following par-tial hepatectomy. Curiously, we uncovered that, hepatocytes exhibit developmental phenotypes at mRNA level pre-pregnancy and at both mRNA and protein level during pregnancy. In the non-pregnant state, hepatocytes reserved mRNA expression of liver progenitor marker genes Cd133 and Afp, which are localized in the nuclei, without protein translation. During gestation, maternal hepatocytes displayed cytoplasmic translocation of Cd133 and Afp transcripts, con-comitant with corresponding protein expression. Overall, all maternal hepatocytes became CD133+, and a subset of them express AFP. Addi-tionally, in non-pregnant livers, mRNA of Epcam, another liver progenitor marker, was ex-pressed within majority of hepatocytes, whereas its protein was solely translated in the pericen-tral region. In contrast, by end-gestation, EPCAM protein expression switched to the periportal region. These observations indicate that maternal hepatocytes exhibit heterogeneous develop-mental phenotypes, partially resembling fetal hepatocytes. It is intriguing why mature hepato-cytes dedifferentiate into a progenitor state in response to pregnancy. AFP is considered to be produced primarily from fetal liver and thus is used to evaluate fetal development health. A potential clinical relevance of our data is that we identified maternal liver as a new source of AFP. The hippo signaling pathway has been shown to potently control liver growth and hepato-cyte heterogenicity. Surprisingly, we found that pregnancy neither altered the expression nor activities of the components of this pathway and its effector YAP1/TAZ. This finding indicates that pregnancy-induced maternal liver growth is not driven by hippo-YAP1 pathway. However, we demonstrate that the presence of YAP1 is essential for CD133 protein expression in mater-nal hepatocytes. Collectively, we revealed that, as pregnancy advances, maternal hepatocytes likely undergo endoreplication and display developmental phenotypes. Mechanistically, YAP1 dictates the expression of CD133, contributing to the pregnancy-dependent phenotypic changes of maternal hepatocytes.

La voie Hippo est une voie conservée impliquée dans la croissance des tissus et la suppression de tumeurs. Des études ont démontré son implication dans le développement des cancers chez l'homme. Cette cascade contrôle l'activité du co-activateur transcriptionnel Yorkie (Yki) chez la drosophile et de la protéine YAP (Yes Associated Protein) chez les mammifères. En raison de l'épissage alternatif de leur transcrits, les protéines Yki et YAP existent sous deux isoformes contenant un domaine WW (Yki1/YAP1) ou deux (Yki2/YAP2). Puisque les domaines WW sont essentiels pour l’interaction avec des partenaires spécifiques, l’inclusion alternative de ce domaine dans la protéine Yki/YAP peut remodeler leur réseau d’interaction et donc leur activité. La régulation et les conséquences fonctionnelles de l’épissage alternatif de yki / YAP in vivo sont inconnues.Dans le cadre de ce doctorat, nous avons constaté que la déplétion du facteur d’épissage B52 chez la drosophile réduit l’inclusion de l’exon alternatif dans l’ARNm de yki et favorise l’expression de l’isoforme Yki1 aux dépens de l’isoforme Yki2. La déplétion en B52 dans l'aile réduit la croissance et l'activité de Yki. Nous montrons que l'isoforme Yki1 est une version atténuée de la protéine Yki qui peut entrer en concurrence avec l'isoforme Yki2 dans le noyau. Pour déterminer le rôle de l’épissage alternatif de yki in vivo et l'importance de l'isoforme courte Yki1, nous avons abrogé cet épissage en utilisant la technologie CRISPR/Cas9 et avons créé des mouches capables d'exprimer uniquement l'isoforme Yki2. Ces mouches yki2only sont viables mais présentent un phénotype aléatoire d’ailes asymétriques. Cette augmentation de l'«asymétrie fluctuante», qui traduit une déviation par rapport au développement normal, suggère que l’épissage alternatif de yki est crucial pour la stabilité développementale. Ces résultats mettent en évidence un nouveau niveau de modulation de la voie Hippo via l’épissage alternatif de yki.L'inclusion alternative du deuxième domaine WW est une caractéristique conservée entre Yki et YAP. Cela conforte l'idée que les isoformes Yki1 et YAP1 ont une fonction importante in vivo et que l'épissage alternatif de yki/YAP est un mécanisme conservé de contrôle de la voie Hippo. Cette étude ouvre de nouvelles perspectives pour la modulation de la voie Hippo dans les cellules cancéreuses en modifiant l’épissage alternatif de YAP. / The Hippo pathway is a conserved pathway involved in tissue growth and tumor suppression. Studies have demonstrated its significance in the development of human cancers. This cascade controls the activity of the transcription co-activator Yorkie (Yki) in flies and Yes-associated protein (YAP) in mammals. Due to Alternative Splicing (AS), both Yki and YAP proteins exist as two isoforms containing one (Yki1/YAP1) or two (Yki2/YAP2) WW domains. Since WW domains are essential for interaction with specific partners, the alternative inclusion of this domain in Yki/YAP protein may remodel their interaction network and therefore their activity. The regulation and functional consequences of AS of yki/YAP in vivo are unknown.In this Ph.D. project, we identified that depletion of splicing factor B52 in Drosophila lowers inclusion of the alternative exon in yki mRNAs and favors the expression of Yki1 isoform at the expense of the Yki2 isoform. B52 depletion in the wing reduces growth and Yki activity. We demonstrate that Yki1 isoform is an attenuated version of Yki protein that can compete with Yki2 isoform in the nucleus. To ascertain the role of yki AS in vivo and the importance of short isoform Yki1, we abrogated this splicing by using CRISPR/Cas9 technology and created flies that can express Yki2 isoform only. yki2only flies are viable but display a random phenotype of asymmetric wing size. This rise in “fluctuating asymmetry” that is the consequence of subtle deviation from normal development, suggests that AS of yki is crucial for the development robustness. Taking together, these results highlight a new layer of modulation of Hippo pathway via AS of yki.Alternative inclusion of the second WW domain is a conserved feature between Yki and YAP. This further supports the idea that Yki1 and YAP1 isoforms have an important function in vivo and that AS of yki/YAP is a conserved mechanism of control of the Hippo pathway. This study opens up new perspectives for modulation of the Hippo pathway in cancer cells by altering YAP AS.

The Hippo tumor suppressor pathway is a highly conserved signaling pathway that regulates cell proliferation, differentiation, and organ size. Activation of the Hippo pathway leads to the phosphorylation and cytoplasmic sequestration of the pro-growth transcriptional co-activators YAP/TAZ; by contrast, impairment of the Hippo pathway enables YAP/TAZ to enter the nucleus where they bind to the TEAD transcription factors and induce the expression of genes involved in cell proliferation. Functional impairment of the Hippo pathway, and subsequent hyperactivation of YAP/TAZ, is common in many human malignancies, including melanoma. Recently, small molecule inhibitors that disrupt YAP/TAZ-TEAD binding, and thus reduce oncogenic transcriptional signaling have been discovered, but their efficacy in preventing cancer cell growth has not yet been well characterized. Moreover, no simple biomarker has been identified that can predict sensitivity to such inhibitors. We hypothesized that cells in which YAP/TAZ are enriched in the nucleus relative to the cytoplasm, indicative of an impaired Hippo pathway, would be more susceptible to TEAD inhibition. This would provide a useful biomarker to identify cancer cell lines most likely to respond to TEAD inhibition. We therefore developed and validated an automated quantification method to score nuclear:cytoplasmic YAP/TAZ localization in melanoma cell lines. This enabled us to identify “Hippo-On” and “Hippo-Off” signatures. We then treated these lines with the TEAD inhibitor MGH-CP1 and performed cell viability assays. Results from these data demonstrated that cell lines that have greater nuclear localization of YAP/TAZ are more susceptible to MGH-CP1 inhibition, suggesting that YAP/TAZ nuclear localization may be a biomarker to identify candidates for TEAD inhibitor treatment.

Indiana University-Purdue University Indianapolis (IUPUI) / Hepatic fibrosis is a cellular repair mechanism that is initiated upon prolonged damage to the liver, resulting in an accumulation of excess extracellular matrix. This eventually leads to the formation of scar tissue, which disrupts the hepatic architecture and causes liver dysfunction. Hepatic stellate cells (HSCs) play a major role in hepatic fibrosis. However, the molecular mechanisms remain incompletely understood. Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ or WWTR1), key players of the Hippo pathway, have been implicated in the liver fibrosis, but the HSC-specific functions of YAP and TAZ are largely unclear. Here we have identified Sirtuin 6 (SIRT6), an NAD+ dependent deacetylase, as a key epigenetic regulator in the protection against hepatic fibrosis by suppressing the YAP/TAZ activity. SIRT6 has been previously implicated in the regulation of the canonical transforming growth factor β (TGFβ)-SMAD3 pathway. This study has revealed the significant contribution of the non-canonical pathways including the Hippo pathway to the development of hepatic fibrosis. HSC-specific Sirt6 deficient mice developed severe fibrosis when fed a high-fat-cholesterol-cholate diet compared to their wild-type counterparts. YAP became more active in the SIRT6-deficient HSCs. Expression of the YAP/TAZ downstream genes like CTGF, CYR61 and ANKRD1 were elevated in the SIRT6-deficient HSCs. Biochemical and mutagenic analyses have revealed that SIRT6 deacetylates YAP and TAZ at key lysine residues and reprograms the composition of the TEA domain transcription factor complex to suppress the YAP/TAZ function in the hepatic fibrogenesis.

No description available.

The paralogous multi-functional adaptor proteins YAP and TAZ are nuclear effectors of the Hippo pathway, a central regulator of developmental organ size control, tissue homeostasis and tumour suppression. YAP/TAZ target the TEAD transcription factor family to promote cell survival and inhibit apoptosis. TEAD proteins contain a DNAbinding domain and a YAP/TAZ interaction domain. PCR analysis of medaka fish TEAD cDNA revealed the presence of alternative TEAD splice-forms with variations at the C-terminus of the DNA-binding domain. Structural analysis indicated the YAPbinding domain of TEAD proteins is folded and globular. NMR spectroscopy showed that the TEAD binding domain of YAP does not contain secondary structure. YAP and TAZ both contain WW domains, which are small protein-protein interaction modules. Two YAP isoforms are known, YAP1 and YAP2 that contain one and two WW domains, respectively. To date, only a single WW isoform of TAZ has been described. PCR analysis of medaka TAZ cDNA identified both single WW and tandem WW isoforms of TAZ. NMR spectroscopy was used to characterise structural, conformational, and peptide binding features of the tandem WW domains from YAP and TAZ. The YAP WW2 solution structure confirms that the domain has the canonical anti-parallel β-sheet WW fold. WW1 of YAP and both WW domains of TAZ undergo conformational exchange. The region linking the two WW domains is flexible and allows interaction of both WW domains with peptides containing single and dual PPxY binding motifs. In addition to YAP and TAZ, tandem WW domains are also present in the core and upstream Hippo pathway proteins Salvador and Kibra. Both proteins contain one atypical WW domain; the tandem WW domains of these two proteins are unstable. Understanding structure and function of Hippo pathway components could contribute to drug development and will also contribute to knowledge of protein folding and interactions.

Les cellules épithéliales sont des cellules polarisées qui forment l’un des types cellulaires le plus abondant dans le corps humain. Leur polarité apico-basale (A/B) est établie et maintenue par la ségrégation asymétrique de protéines adaptatrices hautement conservées. Cette polarité est essentielle pour de nombreuses fonctions cellulaires clés comme l’adhésion (jonctions intercellulaires) ou la signalisation et la prolifération par la localisation et la concentration des complexes de signalisation. Durant la cancérogénèse, un grand nombre de ces processus est dérégulé aboutissant à la sur-prolifération, la migration et/ou l’invasion des cellules cancéreuses. Une meilleure compréhension des mécanismes à l’origine de ces processus est indispensable pour trouver de nouvelles cibles thérapeutiques pour le traitement du cancer. Dans l’équipe, nous sommes particulièrement intéressés par les protéines adaptatrices à domaines PDZ (domaine de liaison protéine-protéine). De par leur structure modulaire et la diversité de leurs partenaires, ces protéines adaptatrices sont impliquées dans la régulation de très nombreuses fonctions et fournissent des plateformes où différents processus peuvent être intégrés. Durant mon doctorat, j’ai étudié deux protéines adaptatrices dans le système animal modèle Drosophila melanogaster, Bbg et Magi, impliquées dans deux processus cellulaires essentiels : la dynamique des jonctions et la prolifération. Grâce aux molécules d’adhésion, les cellules non seulement restent cohésives dans un tissu, mais c’est aussi à ce niveau qu’elles peuvent obtenir une information concernant la densité cellulaire d'un tissu. Cette information est alors relayée au cytosquelette d’actine via des protéines adaptatrices spécialisées dans le but de réguler la prolifération et la voie Hippo. Cependant, le contrôle de la voie de signalisation Hippo par certaines protéines adaptatrices et par le cytosquelette d’actine n'est que partiellement compris à ce jour. Dans le laboratoire, nous étudions notamment le rôle d'une nouvelle protéine adaptatrice apicale nommée Big Bang (Bbg) dans le disque d’aile de la drosophile. Nous nous sommes intéressés à Bbg car c’est une cible de la voie Notch chez la drosophile et son homologue humain PDZD2 (pour PDZ domain-containing 2 protein) est sur-exprimé dans plusieurs cancers (sein et prostate).Mes résultats montrent que Bbg est un nouveau régulateur du cytosquelette d’actine et de la voie Hippo. Une étude détaillée de la fonction de Bbg et de ses partenaires permet de mieux comprendre les relations existantes entre dynamique de l’actine et prolifération. Bbg induit une accumulation d’actine filamenteuse en augmentant l’activité d’Enabled et la phosphorylation de Myosin Light Chain (MLC). Cette régulation résulte en une augmentation de l’activité de Yorkie, effecteur final de la voie Hippo, pour soutenir la prolifération cellulaire.La régulation des jonctions adhérentes est une étape cruciale lors de l’évolution d'une tumeur solide. Malgré les récentes avancées dans le domaine, de nombreux aspects clés de la dynamique des jonctions restent peu caractérisés.Dans le laboratoire, nous recherchons de nouveaux régulateurs de jonctions et grâce au modèle de remodelage des AJs lors du développement de l’œil de pupe de drosophile. Nous avons identifié Magi en tant que protéine adaptatrice recrutant le complexe formé de RASSF8 et ASPP. Magi régule le recrutement de Bazooka à la membrane, le dépôt d’E-Cadhérine et des Caténines et finalement le remodelage des jonctions pendant la morphogénèse. J’ai identifié Echinoid, une protéine de type immunoglobuline impliquée dans l’adhésion cellulaire et la régulation de la voie Hippo, comme un nouveau partenaire responsable du recrutement de Magi aux futures zones de jonctions. / Epithelial cells are polarised cells that form one of the most abundant cell types in the human body. Their apico-basal (A/B) polarity is established and maintained by the asymmetric segregation of highly conserved scaffold proteins. Proper A/B polarity is critical for many key cellular functions such as intercellular junctions and therefore adhesion, or signalling and proliferation by localising and concentrating signalling complexes. During carcinogenesis, many of these processes are mis-regulated leading to the over-proliferation, migration and/or invasion of cancer cells. A better understanding of the mechanisms underlying these processes is really needed to find new therapeutic targets in cancer treatment.In the team, we are particularly interested in scaffold proteins with PDZ domains (protein-protein interaction domains). Due to their modular structure, the high number of interactions they engage in, and the variety of their binding partners, these scaffold proteins are implicated in the regulation of many key cellular functions and processes. During my PhD, I have studied two scaffold proteins in the Drosophila melanogaster animal model, Bbg and Magi, which are involved in two important cell processes: adherens junctions (AJs) dynamic and cell proliferation.Through adhesion molecules, epithelial cells not only remain cohesive, but can also sense cellular density in a tissue and relay this information through dedicated scaffolds to the actin cytoskeleton to ultimately regulate the Hippo pathway and proliferation. However, many aspects of the control of Hippo signalling by apical scaffolds and the actin cytoskeleton are still poorly understood. In the laboratory, we are interested in the study of a new conserved apical scaffold, Big Bang (Bbg). Bbg is a new and quite unknown protein expressed in a variety of Drosophila epithelia, and appears as a potential Notch target in Drosophila. Its’ human homolog called PDZD2 (PDZ domain-containing 2 protein) has been shown to be over-expressed in several types of cancers (breast and prostate cancers).My results show that Bbg is a new regulator of the actin cytoskeleton and of the Hippo pathway in Drosophila. A detailed study of Bbg function and of its associated partners have helped to better understand the intricate relationships between actin dynamics and proliferation. My results suggest that Bbg promotes accumulation of filamentous actin (F-Actin) through the increase of the activity of Enabled (Ena) and the phosphorylation of the molecular motor Myosin Light Chain (MLC). This regulation leads to the increase of Yorkie activity, the final effector of the Hippo pathway, to promote cell proliferation.The regulation of adhesion, and in particular of Adherens Junctions (AJs), is a critical step during the evolution of solid tumours. A better understanding of how these structures are regulated will provide valuable insights into different phases of the disease. Despite the recent advances, many key aspects of AJ dynamics remain poorly understood. In the laboratory, we are interested in the identification of new AJs regulators. Using the remodelling of AJs during the development of the Drosophila pupal eye as a model, we have identified Magi as a scaffold recruiting a complex formed by RASSF8 and ASPP, regulating Bazooka membrane recruitment, E-Cadherin and catenins deposition, and ultimately AJs remodelling during morphogenesis. I uncovered Echinoid, an immunoglobulin-like protein involved in cell adhesion and in Hippo pathway regulation, as a new binding partner responsible for the recruitment of Magi at future AJ sites.

L’AICAR (5-Aminoimidazole-4-carboxamide ribonucleotide) est un intermédiaire de la voie de biosynthèse des purines. A des concentrations importantes, ce métabolite a un effet cytotoxique sur les cellules cancéreuses aneuploïdes, c’est-à-dire contenant un nombre anormal de chromosome. Or,90% des tumeurs solides sont aneuploïdes. Les mécanismes responsables de cette cytotoxicité doivent donc être mieux étudiés pour une utilisation éventuelle en thérapie anti-cancéreuse.Dans la littérature, l’effet de l’AICAR est expliqué par son rôle mimétique de l’AMP sur l’AMPK.Cependant, certaines données de la littérature et du laboratoire laissent penser que l’inhibition de la croissance par l’AICAR peut impliquer plusieurs types de mécanismes dont certains sont dépendantsde l’AMPK et d’autres indépendants. L’identification des cibles de l’AICAR alternatives à l’AMPK estdonc nécessaire pour une meilleure compréhension de ses effets.Dans ce projet, j’ai pu confirmer la présence d’autres cibles de l’AICAR indépendantes de l’AMPKet responsables de son effet cytotoxique. Grâce à une approche transcriptomique, j’ai montré un effetde l’AICAR sur l’expression et l’activation de LATS1 et LATS2 (large tumor suppressor 1 and 2). Ces protéines kinases fond partie du core enzymatique de la voie HIPPO, dont le rôle en cancérologie est fondamental. Les effecteurs finaux de cette voie sont YAP et TAZ, deux cofacteurs de transcription,aussi régulés par l’AICAR. J’ai pu montrer que la cytotoxicité de l’AICAR est due en partie à l’activation de cette voie. Depuis la découverte récente de la voie HIPPO, de nombreuses études visent à identifier des molécules permettant l’inhibition directe de cette voie. L’AICAR s’avère être une molécule puissante dans le cadre d’une thérapie anticancéreuse ciblant la voie HIPPO. / AICAR (5-Aminoimidazole-4-carboxamide ribonucleotide) is an intermediate of the purine biosynthesis pathway. At high concentrations, this metabolite has a cytotoxic effect on aneuploid cancer cells that is cells containing an abnormal chromosome number. However, 90% of solid tumorsare aneuploid. The mechanisms responsible for this cytotoxicity should be better studied for possible use in anti-cancer therapy.In the literature, the effect of AICAR is explained by its AMP mimetic role on the AMPK. However,some literature and laboratory data suggest that AICAR growth inhibition may involve several types of mechanisms, some of which are dependent and other independent of AMPK. Therefore, the identification of AMPK alternative targets is necessary for a better understanding the AICAR effects. In this project, I was able to confirm the presence of other AICAR targets independent of AMPK and responsible for its cytotoxic effect. Using a transcriptomic approach, I showed an effect of AICAR on the expression and activation of LATS1 and LATS2 (large tumor suppressor 1 and 2). These proteinkinases form part of the enzymatic nucleus of the HIPPO pathway, whose role in oncology is fundamental. The effectors of this pathway are YAP and TAZ, two transcription cofactors, also regulated by the AICAR. I have been able to show that the cytotoxicity of AICAR is due to the activation of this pathway. Since the recent discovery of the HIPPO pathway, numerous studies aim to identify molecules allowing direct inhibition of this pathway. AICAR has proven to be a potent molecule in anticancer therapy which goal is targeting the HIPPO pathway.