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iv Abstract Crosstalk of integrin and mTOR signalling is an essential process that monitors cellular interaction with extracellular matrix and transmits these inputs to cell growth signalling. Although adhesion status of the cell monitored by integrin signalling is clearly important for regulation of cellular growth, a little is known about the crosstalk of integrin and mTOR signalling. In this study, we employed two different approaches to describe and elucidate character of this crosstalk. p130Cas is an adaptor protein phosphorylated by Src kinase and focal adhesion kinase upon integrin ligand binding and implicated in cell adhesion, motility and survival in both Src-transformed and untransformed cells. Recently, p130Cas was also described in cellular pathology, mainly by its ability to stimulate cell invasion and metastasis. In this study, we described that p130Cas affects mTOR signalling in Src-transformed cells. Substrate domain of p130Cas was found to be indispensable for this effect and is also responsible for serum-induced activation of mTOR signalling. In addition, we prepared cell lines overexpressing various Rheb protein versions and characterized them in context of mTOR signalling, integrin signalling and cell cycle progression. Interestingly, a cell line overexpressing constitutively active...

Central Nervous System (CNS) injury may lead to irreversible damage to cognitive and motor abilities when injured. This is due to the inability of axons to regenerate. This thesis focuses on two methods of promoting axonal regeneration: microtubule stabilization and upregulation of the intrinsic growth capacity of the neuron via the mechanistic target of rapamycin (mTOR) pathway. Both have shown promising results in potentially being a therapeutic treatment for CNS trauma. This research seeks to (1) test a combinatorial method of axonal regeneration utilizing both methods simultaneously and (2) compare microtubule stabilization and upregulation of the mTOR pathway as neuronal regeneration methods. Aim 1 serves to test the combinatorial treatment of Taxol, a microtubule stabilizer, and cRheb transfection, which upregulates the mTOR pathway, on neuronal cell cultures. Cells were cultured in either a growth-promoting substrate or a mix of growth-promoting and growth-inhibitory substrates. The results of this study revealed combinatorial treatment of 2DIV Taxol application with cRheb transfection as a promising treatment that yielded significantly greater axonal outgrowth than either treatment alone. Aim 2 serves to compare the two established methods of axonal regeneration in the scientific community. Based off of a meta-analysis, results of this aim indicate upregulation of mTOR is more effective at promoting axonal regeneration than microtubule stabilization.

MTOR est une sérine/thréonine kinase appartenant au complexe mTORC1 (mTOR Complexe 1), un régulateur clé de la traduction. Ce complexe joue un rôle au sein de la LTP (Potentialisation à Long Terme), une forme de plasticité synaptique qui requiert la synthèse de nouvelles protéines pour renforcer la transmission synaptique. La première partie de ma thèse porte sur les mécanismes de régulation de la voie mTORC1 dans les neurones. Dans les cellules non neuronales, cette voie de signalisation est classiquement régulée par deux voies distinctes. D’une part, les acides aminés induisent le recrutement du complexe mTORC1 à la membrane des endo-lysosomes où la protéine Rheb est enrichie et favorisent ainsi l’activation de mTORC1. D’autre part, les facteurs de croissance activent mTORC1 en stimulant la voie PI3K/Akt/TSC/Rheb. Nos résultats indiquent que les neurones sont capables d’ "utiliser" le mécanisme responsable de la translocation de mTORC1 en réponse à la supplémentation en acides aminés pour coupler l’induction de la plasticité synaptique à l’activation de mTORC1. En effet, les récepteurs NMDA et le BDNF, deux acteurs centraux de la LTP, augmentent le recrutement de mTORC1 à la membrane des endo-lysosomes même en absence d’acides aminés, et activent mTORC1. Par des stratégies induisant la translocation de mTORC1 à la membrane des endo-lysosomes, nous avons montré que ce mécanisme est important pour l’activation de mTORC1 mais n’est pas suffisant : il faut également une activation de la protéine Rheb. Le second aspect de mon projet porte sur la régulation de mTORC1 dans le cadre de la maladie d’Alzheimer, une maladie neurodégénérative caractérisée par une perte progressive de la mémoire. Les déficits cognitifs s’accompagnent d’un dysfonctionnement progressif des synapses suivi par la perte neuronale, tous deux causés par une accumulation anormale du peptide amyloïde β (Aβ). Les données de la littérature montrent que les oligomères toxiques du peptide Aβ (AβO) inhibent la plasticité synaptique dans les stades précoces de la maladie. Cependant, les mécanismes restent obscurs. Plusieurs études mettent en évidence une altération de la voie mTORC1. Nos résultats montrent que les AβO inhibent le recrutement de mTORC1 à la membrane des endo-lysosomes. Ce mécanisme est rétabli par une inhibition pharmacologique de l’AMPK. Ainsi, ces données indiquent que les AβO inhibent l’adressage de mTORC1 aux compartiments endo-lysosomaux via l’AMPK. Cela aurait pour conséquence une inhibition de la synthèse protéique décrite dans la littérature et contribuerait ainsi au dysfonctionnement synaptique. / MTOR is a serine/threonine kinase that belongs to mTORC1 (mTOR complex 1), a key regulator of translation. This complex is involved in LTP (Long Term Potentiation), a form of synaptic plasticity requiring new protein synthesis to reinforce synaptic transmission. The first part of my thesis investigates the mechanism of mTORC1’s regulation in neurons. In non-neuronal cells, mTORC1 pathway is commonly activated by two distinct pathways. On the one hand, amino acids induce mTORC1 recruitment to the membrane of endo-lysosomes where Rheb is enriched and can thus promote mTORC1 activation. On the other hand, growth factors activate mTORC1 via the PI3K/Akt/TSC/Rheb pathway. Our results indicate that neurons are capable of “using” amino acid-induced translocation of mTORC1 to connect synaptic plasticity induction to mTORC1 activation. Indeed, NMDA receptors and BDNF, two main actors of synaptic plasticity, increase mTORC1 recruitment to the membrane of endo-lysosomes even in the absence of amino acids, and activate mTORC1. Using strategies targeting mTORC1 to endo-lysosomes, we show that this mechanism promotes activation of mTORC1 but is not sufficient: Rheb activation is also required. The second part of my project is focused on the regulation of mTORC1 in Alzheimer’s disease, a neurodegenerative pathology characterized by a progressive memory loss. Cognitive deficits are widely believed to result from a progressive dysfunction of synapses, followed by a loss of neurons, both caused by an abnormal accumulation of the amyloid β peptide (Aβ). Data from others show that toxic Aβ oligomers (AβOs) inhibit synaptic plasticity at early stages of the disease. However, the mechanisms remain poorly understood. Several studies indicate an alteration of the mTORC1 pathway. Our results show that AβOs inhibit mTORC1 recruitment to the membrane of endo-lysosomes and that this effect can be rescued by a pharmacological inhibition of AMPK. Thus our data indicate that AβOs inhibit mTORC1 translocation to endo-lysosomal compartments via AMPK. This could lead to the impairment of protein synthesis reported in other studies and thus alter synaptic function.

The tumor suppressor TP53 is the most frequently altered gene in human cancers. The growth-promoting complex, mTORC1 plays a part of the oncogenic profile caused by dysfunctional p53. mTORC1 sits downstream of AMPK and other crucial tumor suppressors/oncogenes, PTEN, LKB1, and Akt. The antifolate pemetrexed was found by this laboratory to activate AMPK via the inhibition of the enzyme AICART in de novo purine synthesis. This work presents a mechanism of mTORC1 activation with p53 loss, as well as of mTORC1 inhibition by pemetrexed-induced AMPK. We have found that mTORC1 activity was substantially upregulated by the loss or mutation of p53. This activation involves the loss of TSC2 from lysosomal membranes, the site of mTORC1 activation by Rheb. We demonstrate that loss of lysosomal TSC2 increased the levels of lysosomal Rheb. Control of mTORC1 was restored by overexpression of TSC2, which correlated with decreased lysosomal Rheb. Surprisingly, pemetrexed-activated AMPK did not phosphorylate TSC2 because of an accumulation of nonfunctional p53, and a subsequent decrease in TSC2 mRNA. Accordingly, lysosomal TSC2 decreased, however, the levels of lysosomal Rheb decreased. Future studies will question whether the robust Raptor phosphorylation by pemetrexed is involved in this decrease in lysosomal Rheb. AMPK activation by pemetrexed also significantly increased the translocation of AMPK to the nucleus, and we will explore the function of this nuclear AMPK. Overall, these findings present a mechanism involved in the oncogenic signaling of mTORC1 with loss of p53 and offer insight into how pemetrexed reinstates control.

New drug discoveries and new approaches towards diagnosis and treatment have improved cancer therapeutics remarkably. One of the most influential and effective discoveries in the field of cancer therapeutics was antimetabolites, such as the antifolates. The interest in antifolates increased as some of the antifolates showed responses in cancers, such as mesothelioma, leukemia, and breast cancers. When pemetrexed (PTX) was discovered, our laboratory had established that the primary mechanism of action of pemetrexed is to inhibit thymidylate 22 synthase (TS) (E. Taylor et al., 1992). Preclinical studies have shown that PTX has a broad range of antitumor activity in human and murine models of cancer (Adjei, 2000; Adjei, 2004; S. Chattopadhyay, Moran, & Goldman, 2007; Miller et al., 2000). Accordingly, in February 2004, the FDA issued first-line treatment approval for pemetrexed in malignant pleural mesothelioma and in 2008 for first line treatment for locally advanced or metastatic NSCLC (reviewed in (Rollins & Lindley, 2005). As an antifolate this level of therapeutic activity of PTX against lung cancers was surprising and atypical (Hazarika, White, Johnson, & Pazdur, 2004). This led us to the question whether the effects of pemetrexed on other folate-dependent targets could explain the clinical activity of the drug. Our lab showed that, in addition to inhibiting thymidylate synthase, PTX also inhibits aminoimidazolecarboxamide ribonucleotide formyltransferase (AICART), the second folate-dependent enzyme of de novo purine synthesis. Inhibition of AICART leads to massive accumulation of its substrate 5-amino-4-imidazolecarboxamide ribonucleotide (ZMP), causing activation of AMP-dependent kinase (AMPK), which ultimately leads to suppression of mTORC1 signaling, a central regulator of cell growth and proliferation. This secondary mechanism could explain the unusual activity of PTX against mesothelioma and lung cancers. The large proportion of lung cancers are either null or mutant for p53 function. Therefore, this thesis focused on defining what the role of p53 is in the PTX-mediated AMPK activation and mTORC1 inhibition and how the loss of p53 affects mTORC1 signaling. These two questions proved to be interlinked. Chapter 2 investigates this relationship in detail. We found that, upon loss of p53, mTORC1 signaling is enhanced to a significant degree in colon carcinoma and lung cancer cell lines. Clearly, this observation required explanation. We found that the major factors responsible for these differences in mTORC1 activity upon loss of p53 23 were lower levels of two p53 target genes Tuberin (TSC2) and sestrin2. Immunoprecipitation studies of mTORC1 complexes from p53 wt and p53 null cells revealed quite interesting differences in the components of the mTORC1 complex. Immunoprecipitates from p53 null cells had higher levels of mTOR and lower levels of TSC2 and PRAS40 bound to raptor. This suggested that, in comparision to p53 competent cells, p53 null cells have more mTORC1 complex with enhanced activity due to decreased interaction of TSC2 and PRAS40, both of which are inhibitors of mTORC1. These observations explained the higher mTORC1 in p53 null cells and laid the foundation for determining the role of p53 in PTX-activated AMPK and mTORC1 inhibition. In the experiments described in Chapter 3, we found that PTX-mediated AMPK activation inhibited mTORC1 regardless of the p53 status in colon carcinoma cells. This suggested that mTORC1 inhibition by PTX was either independent of p53 mediated negative regulation of mTORC1 or was somewhere bypassing it. Therefore, we compared the effects of PTX with the classic AMPK activator aminoimidazolecarboxamide ribonucleoside (AICAR). In spite of a common mechanism of AMPK activation, namely, expansion of cellular ZMP levels, signaling from AMPK activated by PTX or AICAR were quite different. PTX-activated AMPK phosphorylated the mTORC1 component Raptor but not tuberin (TSC2), whereas AICARactivated AMPK phosphorylated both the targets. This differential behavior of two AMPK activators was due to differential behavior of p53 under these two treatments. Both, AICAR and PTX treatment led to increase in p53 levels but the p53 that accumulated after AICAR treatment was transcriptionally active while the p53 that accumulated after PTX treatment was not. Transcription of p53 targets, including TSC2 and sestrin2, was activated in AICAR- but not in PTX-treated cells. In the absence of p53 function, TSC2 was deficient and mTORC1 activity 24 enhanced, but Raptor phosphorylation by AMPK following PTX was robust and independent of both p53 and TSC2. Therefore we concluded that p53 deficiency suppresses TSC2 and upregulates mTORC1, but AMPK-phosphorylation of Raptor after pemetrexed treatment was sufficient to suppress mTORC1, even in TSC2 deficiency. This suggested pemetrexed as a drug for treatment of Tuberous Sclerosis, a genetic disease caused by functional inactivity of TSC1 or TSC2 due to point mutations in these genes. Mutation of p53 is one of the most common genetic alterations in human cancers and tumors. Cancers that express mutant p53 tend to be more aggressive, resistant to chemotherapy and show worse prognosis then p53-null tumors (Elledge et al., 1993; Olivier et al., 2006). This tumor-promoting activity of mutant p53 has been correlated with acquired and novel transcriptional activities of mutant p53. It has been shown that mutp53 can activate the transcription of cell growth promoting genes, such as, NFκB2, PCNA, MDR1, Axl, EGFR, hTERT, and HSP70, which are not usually transcriptional targets of wt p53. Interestingly, we found that whereas DNA damaging drugs enhance the acquired oncogenic transcriptional activities of mutp53, PTX interferes with this transcription activation. We also found in Chapter 4 that PTX can limit or block the DNA damaging drug-mediated increment of transcriptional activation of mutp53. This suggests that blockade of transcriptional activation of mutp53 by pemetrexed may provide an additional therapeutic benefit in mutp53 bearing cancers. As discussed in Chapter Three, although pemetrexed (with TdR) increases the levels of p53 and its binding to the promoter of its target gene, p21, this p53 is transcriptionally inactive. In order to understand the mechanism of the pemetrexed-mediated transcriptional defect of wt p53, we studied the PTX-mediated signaling towards ATM and ATR and their effects on their substrates Chk2 and Chk1, respectively. These studies suggested that the difference between 25 signaling under AICAR treatment and PTX treatment was that, unlike PTX, AICAR treatment was leading to DNA damage, followed by Chk2 phosphorylation at Thr68. We found there were three major differences between AICAR and pemetrexed (+ TdR) mediated signaling: AICAR caused DNA damage, followed by ATM mediated phosphorylation of Chk2 at Thr68 and phosphorylation of p53 at Ser15 all of which lead to activation of p53 transcriptional activity, events which do not take place under PTX treatment. Studies aimed at understanding the effects of PTX on wt and mutp53 transcriptional activities are discussed in detail in Chapters Three and Four of this dissertation. Overall, we concluded that PTX interferes with the transcription activity of wild type as well as gain-of-function mutant p53. The blockade of DNA damaging agent-mediated enhancement of mutp53 transcription activity by PTX, suggests the clinical relevance of PTX in carcinomas with mutp53. We suggest that this could be one of the contributing factors in the effects of PTX against human lung cancers.