Mes travaux de recherche révèlent une nouvelle voie de signalisation qui est impliquée dans l'étape d'internalisation cellulaire. Cette voie de signalisation cellulaire nécessite une libération d'ATP ainsi que l'activation de récepteur purinergique au niveau des cellules cannibales et va aboutir à l'élimination de la cellule internalisée. Nous avons également défini au cours de ces travaux le devenir de la cellule cannibale et préciser au cours d'expérimentations in vivo l'activité oncosuppressive du cannibalisme cellulaire. Nous avons également observé ce processus au niveau de biopsies tumorales obtenues à partir de patients ayant un cancer du sein et ayant reçu un traitement néo-adjuvant et avons révélé que sa détection pouvait prédire l’efficacité d'un traitement néo-adjuvant. L’ensemble de ces résultats révèle les bases moléculaires du cannibalisme cellulaire et précise le rôle du cannibalisme cellulaire lors du développement tumoral. / My research reveals a new signaling pathway which is involved in the cellular internalization. This signaling pathway requires ATP release and purinergic receptor activation at the level of cannibal cells and will lead to the elimination of internalized cell. We also defined during this work the future of the cannibal cell and specify during experiments in vivo the tumor suppressor activity of cellular cannibalism. We also observed this process in tumor biopsies obtained from patients with breast cancer who received neoadjuvant treatment and have revealed that its detection could predict the efficacy of neoadjuvant treatment. Theses results reveal molecular bases of cellular cannibalism and indicate the role of cellular cannibalism during tumor development.

Le cannibalisme d’une cellule vivante par une autre cellule vivante représente une nouvelle modalité de mort cellulaire non autonome. Mes travaux de thèse ont permis d’identifier et de caractériser les acteurs impliqués et d’apprécier l’influence de ce processus sur le devenir de la cellule cannibale. Nous avons ainsi révélé que l’activation d’une signalisation cellulaire impliquée dans la régulation du cycle cellulaire va causer une libération d’ATP qui stimulera les récepteurs purinergiques P2Y2 de la cellule de manière autocrine. Cette étape sera suivie d’une augmentation de l’exposition de la protéine d’adhérence E-cadhérine à la membrane plasmique et de réarrangements du cytosquelettes médiés par la kinase ROCK, et permettra ainsi à une cellule vivante de cannibaliser une autre cellule vivante. Ce phénomène aussi connu sous le nom de « cellule dans une cellule » est fréquemment observé dans les biopsies tumorales. De plus, nous révélons au cours de ces travaux la capacité des cellules internalisées à être éliminées par un processus qui implique la protéine de l’autophagie ATG5 et les protéines pro-apoptotiques BAK et BAX. Ce processus est associé au déclenchement d’une instabilité génétique et d’un stress oxydatif au niveau des cellules cannibales et va déclencher la sénescence de ces cellules que nous avons appelé « entescence ». Cette nouvelle modalité d’induction de la sénescence participe à la suppression des tumeurs in vivo et semble prédire la réponse des patients aux traitements néoadjuvants anticancéreux. À l’opposé, l’échappement à l’entescence favorise la progression tumorale et est associé à une mauvaise réponse des patients aux traitements. L’ensemble de ces travaux met en lumière l’existence d’une nouvelle modalité d’induction de la sénescence cellulaire qui survient à la suite du cannibalisme cellulaire. Une meilleure compréhension des mécanismes impliqués dans son déclenchement et son exécution pourrait selon nous participer au développement de nouvelles approches thérapeutiques afin de lutter contre le cancer. / Cannibalism of live cells by other live cells is a new modality of non-autonomous cell death. This investigation led to the characterization of the molecular mechanisms implicated as well as the identification of the consequences of this process on the fate of the cannibal cell.We revealed that the activation of a signaling pathway involved in the regulation of the cell cycle can trigger a release of ATP that will stimulate the activity of the P2Y2 purinergic receptor in an autocrine manner. These events will lead to the increase of E-cadherin membrane exposition and change the organisation of the cytoskeleton in a ROCK-dependent manner, allowing this live cell to eat another live cell. This process called "cell in cell structure" is frequently observed in tumoral biopsies. Then, we revealed that the internalized cell will be eliminated by a process dependent on the autophagy protein ATG5 and the pro-apoptotic proteins BAX and BAK. These events are associated to the triggering of genomic instability and an oxidative stress in the cannibal cell leading these cells to a new senescence program that we called "entescence".This new senescence program seems to be a tumor suppressor mechanisms in vivo and is correlated to a better response of patient to neoadjuvant anticancer treatments. Moreover, escaping entescence seems to favor tumor growth and is associated to a bad response to anticancer treatments.Taken together, these results highlight the existence of a new senescence program that is initiated by cellular cannibalism. A better understanding of the molecular mechanisms regulating its initiation and its execution may lead to develop new innovative anticancer therapeutical approaches.

Microcephaly is a debilitating condition in which children are born with small brains. It can be caused by a variety of factors including maternal infection, harmful substance exposure, and genetic mutation. Cerebral cortical development is often severely disrupted in human microcephaly patients. In order to form the billions of neurons which exist in the cortex, efficient and correct neural progenitor division, differentiation, and migration are key. As the center of higher brain function in mammals, reduction in cortical mass is associated with the developmental delays that are symptomatic of microcephaly. Recently, a number of microcephaly causes have been linked to P53-mediated apoptosis of neural cells. The tumor suppressor protein P53 is upregulated in response to mitotic cycle stress, and its activation can trigger cell cycle arrest or apoptotic cell death. In microcephaly, P53 can become activated by mitotic stress and trigger apoptosis to cause the loss of cortical cell numbers that leads to microcephaly. Microcephaly has often been linked to mutations in mitotic proteins that alter neural progenitor division. However, the apical polarity complex Protein Associated with Lin-7 1 (PALS1) – known as membrane palmitoylated protein (MPP)5 in people – has recently been implicated in human microcephaly. PALS1 is integral to establishing polarity in neural progenitors. Deletion of Pals1 in mouse models has also resulted in microcephaly characterized by smaller brains and a global reduction in cortical cell numbers. Interestingly, a cellular phenomenon known as entosis can be caused by polarity disruptions in epithelial cells, and P53 activation has been shown to cause entosis in MDCK cell culture. While entosis is mainly associated with cancer cells, it is a form of competitive cell cannibalism that can eliminate unfit cells from a population. The loss of PALS1 from the developing cortex is known to result in apical polarity complex disruption and microcephaly in mouse models. However, the mechanism by which the loss of PALS1 results in cortical abrogation has yet to be determined. In Chapter 1 of this dissertation, I begin by reviewing cortical development. The normal progression of cortical cells from neural progenitors to fully differentiated neurons is explained in detail. Neural progenitor mitosis in particular is addressed in detail. Furthermore, an overview of microcephaly is provided to address the similarities between known causes of microcephaly. Next, I review the polarity complex proteins and their roles in cortical development. I compare and contrast the cortical phenotypes that have been described when each of the polarity complex proteins has been genetically deleted from the mouse cortex. I go on to review studies that have shown P53-mediated apoptosis in microcephaly in order to address the phenotypic features of microcephaly that are or are not caused by P53 activation. Finally, I provide a brief history of entosis. As a newly discovered cellular process in neural progenitors, the overview of entosis highlights what is known about cell cannibalism and the contexts in which it occurs. Following this background, I describe the experimental aims, hypotheses, and methods for this project in Chapter 2. In Chapter 3, I describe our investigation of three human patients with mutations in the Pals1 gene. One of the patients, possessing a heterozygous de novo nonsense mutation in Pals1 (or MPP5), was diagnosed with microcephaly. In order to model this patient’s phenotype, we generated a heterozygous conditional knockout of Pals1 from the entire mouse nervous system with Nestin-Cre. Through behavioral analysis of these mice, I demonstrate that they are hyperactive and blind, mimicking the microcephaly patient’s symptoms. Furthermore, via analysis of the mouse cortex, I show that heterozygous deletion of Pals1 results in severe microcephaly in mice with a global reduction in cortical cell numbers at both adult and embryonic stages. Importantly, I determine that Pals1 deletion does not result in proliferation or migration defects in the mouse cortex. Instead, loss of PALS1 results in massive apoptotic cell death that affects every cell type produced in the developing cortex. In Chapter 4, I detail our investigation into the mechanism underlying cell death in the PALS1-deficient cortex. By studying dividing neural progenitors at the apical surface in both Emx1-Cre and hGFAP-Cre drive Pals1 conditional knockout models, I demonstrate an as yet undescribed neural progenitor phenotype called entosis. As has been shown in cancer cells, neural progenitor entosis is dynamic and reliant on Rho-ROCK activity to occur. Furthermore, entosis produces observable cell-in-cell structures that persist through outer cell division and cause mitotic delay. I go on to demonstrate P53 activation in Pals1 deficient mouse cortices, and show that genetic deletion of Trp53 significantly rescues microcephaly. Trp53 deletion significantly restores all cortical cell types in addition to ameliorating entosis and mitotic length. This study suggests that P53 activation is a major mechanism by which PALS1 loss results in microcephaly. Overall, these studies show that deletion of Pals1 in mice can mimic microcephaly found in a human patient with a Pals1 mutation. Furthermore, PALS1 loss promotes P53-mediated cortical cell apoptosis. These studies provide the first description of entosis in neural progenitors, and suggest that entosis could be a mechanism for unfit cell removal in the developing cortex. Furthermore, I provide evidence that ROCK inhibition can fully rescue the presence of entosis in PALS1-deficient neural progenitors, and that genetic deletion of Trp53 significantly restores microcephaly pathology after PALS1 loss. These studies open up a field of research into the causes and effects of entosis in neural progenitors, and provide further evidence that apoptotic cell death in microcephaly is largely mediated by P53 activation. / Biomedical Sciences

L’entose est une forme de mort cellulaire non apoptotique caractérisée par l’internalisation d’une cellule cible vivante dans une cellule hôte vivante. Ce processus de cannibalisme cellulaire qui est également connu sous le nom de « cellule dans une cellule » est retrouvé dans de nombreux cancers humains. Au cours de mes travaux de thèse, nous avons développé différents modèles d’entose in vitro et avons débuté l’identification des protéines qui répriment l’entose en combinant un criblage de petits ARN interférants à une approche de microscopie confocale. Nous avons découvert que la protéine suppressive de tumeur TP53 ainsi que son isoforme Δ133TP53 bloquent le processus d’internalisation cellulaire et l’entose. La perte de l’expression de la protéine TP53 ou de Δ133TP53 entraîne une libération extracellulaire d’adénosine triphosphate ainsi que l’activation du récepteur purinergique P2Y2, deux évènements cellulaires qui aboutissent à l’internalisation d’une cellule par une autre cellule. De plus, nous avons constaté que les cellules cannibales deviennent énescentes à la suite de l’induction de la protéine p21WAF1.Mes travaux de recherche révèlent l’existence d’une nouvelle modalité d’induction de la sénescence cellulaire. De façon surprenante, nous avons également observé que l’induction de la sénescence par l’oncogène RasV12ou à la suite de stress (réplicatifouoxydatif) déclenchait le cannibalisme cellulaire, suggérant que le cannibalisme cellulaire est une caractéristique des cellules sénescentes. L’ensemble de mes travaux de recherche souligne le lien étroit qui existe entre le cannibalisme cellulaire et la senescence. / Entosis is a non-apoptotic cell death process of live internalized cell inside the host/cannibal cell. In human cancers, commonly "cell-in-cell" cytological features have been observed over the period of time. In this study we have established in vitro models of entosis and initiated the identification of entotic repressors by developing fluorescent confocal microscopy screening of small interfering RNA. We identified that TP53 and one of its isoform 133TP53 specifically inhibits the cell internalization. Loss of TP53 or 133TP53 expression increases extracellular ATP release and the consequent activation of purinergic P2Y2 receptors, which signal for engulfment. Cannibal cells activate a senescence program through p21WAF1 induction, revealing a new modality of induction of cellular senescence that can occur in the absence of TP53 or 133TP53. Senescence induced by oncogenic RasV12 and by replicative or oxidative stresses also results in cellular cannibalism, suggesting that cannibalism is a common feature of senescent cells. Altogether, our results provide evidence that cellular cannibalism and senescence are tightly linked.