La prise en charge clinique du cancer de la prostate est complexe. L’identification des éléments et altérations génétiques impliqués dans la progression de la maladie est donc capitale. Les altérations métaboliques représentent des changements clés vis-à-vis de la croissance de la cellule tumorale. Parmi ces modifications, l’augmentation du métabolisme du cholestérol est fréquemment observée au sein de différentes pathologies tumorales, dont le cancer de la prostate. En lien, des études épidémiologiques associent l’enrichissement du cholestérol avec une augmentation de l’incidence, la progression et l’agressivité de la maladie. Les récepteurs nucléaires aux oxystérols LXRs (Liver X Receptors) constituent des régulateurs majeurs de l’homéostasie du cholestérol, et différents travaux récents mettent en évidence leur potentiel anti-prolifératif et pro-apoptotique dans différentes lignées cellulaires du cancer de la prostate. En association ces travaux de thèse démontrent, dans un modèle murin d’adénocarcinome prostatique PTEN-dépendant, une activation endogène et constitutive des LXRs en réponse à une augmentation du taux de cholestérol intra-prostatique, principale source des ligands oxystérols. L’invalidation additionnelle des LXRs dans ce contexte conduit à une augmentation importante de la croissance tumorale et à la dissémination métastatique, notamment expliquée par une dérégulation de la voie du TGFβ et de la transition épithélio-mésenchymateuse. L'ensemble de ces résultats place les LXRs comme des cibles thérapeutiques prometteuses. / Clinical management of prostate cancer is complex. The identification of elements and genetic alterations involved in disease progression is therefore crucial. Metabolic alterations represent key modifications during tumor cell growth. Among these modifications, cholesterol metabolism increase is frequently observed in various tumor pathologies, including prostate cancer. Related, epidemiological studies associate cholesterol enrichment with an increase of disease incidence, progression and aggressiveness. LXRs (Liver X Receptors) belong to the nuclear receptors superfamily and represent major sensors and regulators of cholesterol homeostasis. Recent studies have demonstrated their antiproliferative and pro-apoptotic potential in various prostate cancer cell lines. In link, this thesis work demonstrate endogenous and constitutive activation of LXRs in a murine model of PTEN-dependent prostate adenocarcinoma in response to an increase of intra-prostatic cholesterol, the major source of oxysterol ligands. The additional invalidation of LXRs in this context leads to a significant increase of tumor growth and metastatic dissemination, notably explained by a TGFβ pathway deregulation, concomitant with epithelial-mesenchymal transition process increase. All of these results define LXRs as promising therapeutic targets.

La voie PI3K/AKT est impliquée dans la progression de divers cancers humains, et semble jouer un rôle majeur dans le développement de tumeurs lymphoïdes. Elle pourrait être impliquée dans la pathogénie du lymphome folliculaire (LF) par certains mécanismes non identifiés. Les travaux de thèse portent sur l'étude des anomalies de la voie PI3K/AKT dans le LF, dans le but d'apporter une nouvelle cible thérapeutique. 38 biopsies tissulaires de LF humain ont été étudiées pour une analyse mutationnelle du gène PIK3CA dans les exons 9 et 20 par séquençage. Les mêmes échantillons ont été analysés par western blot et immunohistochimie pour détecter l'expression des protéines AKT, AKT phosphorylée (pAKT), et PTEN. Deux cas de lymphadénite ont été utilisés comme témoins.Les résultats obtenus montrent que l'expression d'AKT était présente dans tous les cas de LF et lymphadénite, et 14/38 (37%) échantillons de LF et 2/2 cas de lymphadénite exprimaient pAKT. 9/38 (24%) échantillons de LF ont montré un niveau élevé d'expression de pAKT, alors que 5/38 (13%) cas de LF, et 2/2 échantillons de lymphadénite montraient un faible niveau d'expression de pAKT. L'expression de PTEN a été observée dans 30/38 (79%) cas de LF et 2/2 cas de lymphadénite, tandis que 8/38 (21%) cas ont montré une perte d'expression de PTEN. En outre, 3 cas qui expriment pAKT montrent une perte d'expression de PTEN. Aucune mutation du gène PIK3CA n'a été détectée dans les échantillons étudiés. Ces données suggèrent que la voie PI3K/AKT peut être activée dans certains cas de LF, soit en raison de la phosphorylation d'AKT, soit en raison d'une perte d'expression de PTEN, en absence de mutations de PIK3CA. / The phosphoinositide 3- kinase (PI3K) pathway is involved in the growth of various human cancers, including lymphoid malignancies. However its role in the pathogenesis of follicular lymphoma (FL) has not been yet described.The PhD work focuses on the study of alterations in the PI3K/AKT pathway in follicular lymphoma, in order to provide a new therapeutic target.To clarify this point, biopsy tissue samples from 38 human FL cases were investigated for PIK3CA somatic mutations in exons 9 and 20 using Sanger sequencing. The same samples were analyzed using western blotting and immunohistochemistry to detect expression of AKT, phosphorylated AKT (pAKT), and PTEN proteins. Two cases of benign lymphadenitis were used as controls. AKT expression was present in all FL and lymphadenitis cases. 14/38 (37%) FL and 2/2 lymphadenitis cases expressed pAKT. 9/38 (24%) FL samples showed high level of pAKT, whereas 5/38 (13%) FL cases and 2/2 benign lymphadenitis samples expressed pAKT at low level. PTEN expression was observed in 30/38 (79%) FL and 2/2 benign lymphadenitis cases, whereas 8/38 (21%) of FL cases showed loss of PTEN expression. In addition, 3 cases with positive pAKT did not express PTEN. PIK3CA mutations were not detected in any sample. These data suggest that the PI3K/AKT signaling pathway could be activated in a subset of FL cases, due to either AKT phosphorylation or PTEN downregulation, in the absence of PIK3CA mutations.

No description available.

PTEN est une protéine dotée d'une activité phosphatase qui déphosphoryle les phosphatidylinositols issus de l'activation de la phosphatidylinositol 3-kinase (PI3K). Des mutations germinales du gène PTEN ont été mises en évidence dans le syndrome de Cowden, une maladie caractérisée par le développement de polypes le long du tube digestif et associée à un risque accru de cancer. De plus, la perte d'un allèle de Pten chez la souris conduit à la formation d'hyperplasie et de dysplasie du tractus gastro-intestinal ainsi qu'à des tumeurs notamment au niveau du côlon. Ces observations suggèrent que PTEN joue un rôle important dans le tube digestif. Cependant, ses mécanismes d'action dans les cellules épithéliales intestinales sont peu connus. Nos travaux nous ont permis de mieux caractériser les rôles de PTEN dans ces cellules. Pour ce faire, nous avons d'abord analysé l'effet d'un shRNA inhibant spécifiquement l'expression de PTEN dans les cellules Caco-2/15, une lignée cancéreuse colorectale qui a la particularité de se différencier suite à l'atteinte de la confluence en adoptant un phénotype semblable aux cellules absorbantes de l'intestin grêle. La perte d'expression de PTEN stimule la prolifération de ces cellules. Cette augmentation de la prolifération semble résulter d'une diminution de l'expression de p21 et de p27 ainsi que d'une hausse des cyclines D2 et E. De plus, le shRNA contre PTEN inhibe la différenciation fonctionnelle et morphologique des Caco-2/15. Cela découle partiellement de l'inhibition de l'expression des facteurs de transcription CDX2, HNF-1? et HNF-4?. Les jonctions serrées sont également altérées dans ces cellules. En effet, une réduction importante de l'expression des claudines et une augmentation de la perméabilité transépithéliale a été observée. Une augmentation de la synthèse protéique a aussi été remarquée. De plus, nos résultats laissent également croire que PTEN pourraient jouer un rôle dans la carcinogenèse colorectale. Nous avons effectivement constaté une augmentation du potentiel tumorigénique des Caco-2/l5 exprimant le shRNA contre PTEN suite à l'injection des cellules dans des souris nues. De plus, ces cellules ont des capacités de migration et d'invasion accrues. Nous avons également constaté que les niveaux de PTEN sont plus faibles dans plusieurs lignées cancéreuses colorectales comparativement aux cellules épithéliales intestinales normales. Nous avons aussi analysé le phénotype d'une lignée de souris possédant une délétion du gène Pten exclusivement au niveau de l'épithélium intestinal, générée à l'aide du système Cre/loxP. Macroscopiquement, une organomégalie de l'intestin grêle et du côlon a été observé chez les souris déficientes pour Pten. Histologiquement, nous avons constaté une désorganisation de l'architecture épithéliale intestinale caractérisée par un allongement des villosités et par la présence d'embranchements villositaires. De plus, un épaississement important des couches musculaires a été remarqué. Il y a également une augmentation du nombre de cellules prolifératives au niveau des cryptes intestinales corrélant avec une augmentation des niveaux de ?-caténine et des cyclines D. Finalement, une augmentation du contenu protéique par cellule a également été observée ainsi qu'une activation de la voie mTOR. En conclusion, nos résultats montrent que la phosphatase PTEN est impliquée dans l'établissement de l'architecture générale de l'intestin et qu'elle contrôle la synthèse protéique, la migration, le cycle cellulaire ainsi que la différenciation des cellules épithéliales intestinales. De plus, nos résultats indiquent que la perte d'expression de PTEN pourrait influencer la progression des cancers colorectaux. [Symboles non conformes]

OBJECTIVE: Glioblastoma Multiforme (GBM), one of the most malignant forms of primary brain tumors, is characterized by its highly heterogenous genetic composition, aggressive infiltration of surrounding tissue, and resistance to current treatments. Gene expression analysis has characterized GBM into four main types, with a significant portion belonging to the Classical subtype, typified by overexpression and/or mutation of the epidermal growth factor receptor (EGFR). Also common to this subtype of GBM is the loss of crucial tumor suppressor genes Ink4A/ARF and PTEN, which contribute to the invasive nature and unregulated proliferation that underlie the GBM pathology. The high rate of tumor recurrence post treatment with surgical resection, chemotherapy, and radiation has driven the pursuit of more effective molecularly targeted therapies. This study was undertaken to determine the effects of two types of small molecule tyrosine kinase inhibitors on cells overexpressing wild-type EGFR in the context of their respective complements of tumor suppressor genes. METHODS: Several cell lines were established from mouse models of EGFR wild-type (EGFRWT) driven gliomagenesis and treated with 10 μM of type I tyrosine kinase inhibitors Gefitinib (Iressa®, Astra Zeneca), CI-1033 (Canertinib, Pfizer), or Dimethyl Sulfoxide vehicle. Cells were exposed to each drug treatment as part of a time course ranging from 0 to 24 hours and then evaluated by trypan blue exclusion and Western blot analysis for cell viability and molecular and biochemical effects respectively. RESULTS: Evaluation of cell viability indicated that CI-1033 caused a greater increase in cell death than gefitinib when compared to control treated cells regardless of the tumor suppressors lost. Gefitinib was found to cause cell death only in cells expressing the PTEN tumor suppressor whereas CI-1033 showed similar levels of cell death for cells deficient in Ink4A/ARF or both Ink4A/ARF and PTEN tumor suppressors. Western blot analysis revealed that CI-1033 more effectively inhibited EGFR compared to gefitinib. Treatment with both gefitinib and CI-1033 effectively blocked phosphorylation of EGFR, but this effect was less pronounced with gefitinib treatment. Further analysis of downstream signaling molecules showed a greater presence of cleaved caspase 3, a hallmark of apoptosis, in gefitinib treated cells expressing PTEN than in those cells treated with CI-1033. Cells deficient in both Ink4A/ARF and PTEN did not demonstrate any induction of cleaved caspase 3 following either treatment. CONCLUSIONS: Based on the significant differences in cell viability between treatments, CI-1033 is an overall more effective inhibitor of EGFRWT expressing cells lacking PTEN, while gefitinib and CI-1033 were found to be similarly effective in cells expressing PTEN. The results of western blot analysis indicate that total and irreversible EGFR inhibition may be necessary to induce cell death in a manner that effectively terminates downstream cell signaling. It is likely that CI-1033, unlike gefitinib, induces apoptosis in a caspase-independent manner, which may be one of the many differences in downstream effects produced by these two drugs. Further research is necessary to determine the extent to which each inhibitor shuts down proliferative cell signaling pathways such as PI3K-AKT and MEK-ERK signaling pathways downstream of EGFR. Overall, these data indicate that genotype plays an important role in the determination of therapeutic response and may aid in the evaluation of clinical prognoses.

PTEN (phosphatase and tensin homolog deleted on chromosome ten) is a potent tumor suppressor. PTEN’s tumor suppressor action is rooted in its phosphatase function on the lipid substrate phosphatidylinositol-(3,4,5)-trisphosphate (PI(3,4,5)P3). PTEN’s enzymatic activity is specific for the third position of the inositol headgroup. PI(3,4,5)P3 is a second messenger that is a part of the PI3K-Akt pathway, and its dysregulation leads to constitutively activated AKT. The result of AKT activation is cell cycle progression, motility, cell growth, and proliferation, and consequently, overaction leads to neoplastic growth and tumorigenesis. PTEN antagonizes this pathway by regulating PI(3,4,5)P3 population through its phosphatase activity which produces the lipid PI(4,5)P2 (phosphatidylinositol-(4,5)-bisphosphate). A result of PTEN’s function is that its activity must be localized at the PM (plasma membrane) since this is where its substrate resides. Additionally, the mole percent of the phosphoinositide family of lipids is small. From highest percent composition to lowest the phosphoinositide species in the PM rank as PI(4,5)P2 (~2%), PI(4)P (~1%), and PI(3,4,5)P3 (~0.02%). For PTEN to turn over its substrate, it must first translocate from the cytosol to the PM and then search through the plasma membrane for this rare but high in demand lipid. This is at the center of the scarcity paradox. This work explores how PTEN may overcome this paradox by using its multiple lipid binding domains to interact with multiple lipid partners to efficiently localize it toward a region with a high probability of having PI(3,4,5)P3. This hypothesis is tested using two kinetic methodologies. First, we use pre- steady state stopped-flow spectrometry to determine the rates that govern PTEN-lipid binding. Second, we use single-molecule total internal reflectance fluorescence (smTIRF) microscopy to resolve the diffusion coefficients and dwell times of bound PTEN on SLBs supported lipid bilayers (SLBs). We test PTEN against various lipid compositions to determine how the bilayer structure in addition to the chemistry of the lipid influences the enzyme’s binding. These compositions include PI(4,5)P2, PI phosphatidylinositol (PI), phosphatidylserine (PS), PI(4,5)P2/PI and PI(4,5)P2/PS. In addition to this kinetic work, we will also present a novel model membrane platform that takes advantage of a microfluidic device to develop lateral lipid gradients in SLBs. This microfluidic platform, in the future, will allow for the investigation of the dynamic behavior of proteins interacting with lipids but with a bilayer that has a structure recapitulating polarized membranes like in chemotaxing cells.

Very few cancer patients are cured through drug therapy alone, with the majority exhibiting acquired resistance. To date, most studies of therapeutic escape have focused upon tumor-intrinsic mechanisms of drug resistance with little attention paid to the role of normal host cells in preventing complete tumor eradication. In the present study we implicate co-operative bi-directional signaling between melanoma cells and fibroblasts in the generation of a pro-survival niche that mediates drug resistance. Mass-spectrometry based phosphoproteomics was used to show that BRAF inhibition and chemotherapy drugs enhanced the survival of both melanoma cells and fibroblasts through the induction of fibronectin (FN)/integrin α5β1 signaling. Immunohistochemical staining confirmed the induction of FN in mouse xenografts and human melanoma specimens following BRAF inhibitor treatment. Adhesion to FN amplified the adaptive EGFR, HER3 and c-MET receptor tyrosine kinase (RTK) signals required for PI3K/AKT/Mcl-1-mediated melanoma cell survival when BRAF was inhibited. At the same time, BRAF inhibition led, directly and indirectly, to the paracrine release of HGF and neuregulin from fibroblasts, with TGF-β release from the melanoma cells increasing both fibroblast differentiation and survival. Although dual inhibition of RTKs and BRAF did not reverse host-mediated resistance, therapeutic escape was overcome through combined BRAF/PI3K inhibition, suggesting the PI3K/AKT pathway to be a common signaling vulnerability in microenvironment-mediated drug resistance. Our work suggests that durable responses to targeted therapies will only be achieved through dual targeting of the tumor and the adaptive host responses to therapy. These findings are especially important for a cancer such as melanoma, where as few as one cell can repopulate the entire tumor in vivo.

Activated receptor tyrosine kinase (RTK), such as the epidermal growth factor (EGF) receptor (EGFR) and the platelet-derived growth factor (PDGF) receptor (PDGFR), recruit downstream signalling proteins, including phosphatidylinositol 3-kinase (PI3K). PI3K, composed of a regulatory p85 subunit and a catalytic p110 subunit, phosphorylates phosphatidylinositol 4,5-bisphosphate at the 3 position to generate phosphatidylinositol 3,4,5-trisphosphate. This lipid second messenger activates Akt, which promotes cell growth, cell cycle entry and progression, as well as cell survival and cellular migration. PTEN, a tumor suppressor protein, dephosphorylates phosphatidylinositol 3,4,5-trisphosphate at the 3 position, turning off Akt signalling. PTEN contains a C-terminal PDZ binding motif that binds to the PDZ2 domain of MAGI-2, a scaffolding protein that localizes signalling molecules to the plasma membrane. MAGI-2 has ten domains that potentially mediate multiple protein-protein interactions simultaneously. A PTEN associated-complex (PAC) has been described and may contain MAGI-2, PTEN and p85. The PAC is hypothesized to form at the plasma membrane at appropriate sites for PTEN to gain access to its lipid substrates, since the binding of PTEN to MAGI-2 has been shown to enhance the suppression of PI3K-mediated Akt signalling. In order to better understand the role of the PAC in attenuation of the Akt signalling pathway, regions of the MAGI-2 scaffolding protein were mapped to identify the interactions taking place in the PAC. MAGI-2, and its individual domains, were expressed as GST fusion proteins. These were immobilized onto beads and allowed to bind to cellular proteins including PTEN, p85, PDGFR and EGFR using a GST pull-down experiment. The proteins bound to GST-MAGI-2 were identified using an immunoblot analysis. In vitro pull-down experiments revealed that MAGI-2 PDZ2 and PDZ5 domains bind to PTEN, and both MAGI-2 WW domains were shown to bind to p85. EGFR and PDGFR did not bind to the PDZ domains of MAGI-2 under the conditions studied. In order to study protein-protein interactions in cells, immunoprecipitation assays were also performed. Full length MAGI-2 was expressed tagged to a Myc epitope. This was used in immunoprecipitation assays to determine if MAGI-2 could co-immunoprecipitate with proteins involved in the Akt signalling pathway, such as PTEN, p85, PDGFR and EGFR. MAGI-2 can co-immunoprecipitate with PTEN upon 5 min EGF stimulation however, this result was inconclusive because replicate experiments did not verify this initial observation. MAGI-2 does not co-immunoprecipitate with the EGFR nor p85, under the conditions tested. We examined for these interactions after 5 min of growth factor stimulation and more experiments that test different time points after growth factor stimulation would reveal if these interactions are present at shorter time points. MAGI-2 has been shown to bind to PTEN and p85 in vitro and therefore has the potential to regulate the attenuation of the PI3K/Akt signalling pathway in response to activated EGFR and/or PDGFR.

Activated receptor tyrosine kinase (RTK), such as the epidermal growth factor (EGF) receptor (EGFR) and the platelet-derived growth factor (PDGF) receptor (PDGFR), recruit downstream signalling proteins, including phosphatidylinositol 3-kinase (PI3K). PI3K, composed of a regulatory p85 subunit and a catalytic p110 subunit, phosphorylates phosphatidylinositol 4,5-bisphosphate at the 3 position to generate phosphatidylinositol 3,4,5-trisphosphate. This lipid second messenger activates Akt, which promotes cell growth, cell cycle entry and progression, as well as cell survival and cellular migration. PTEN, a tumor suppressor protein, dephosphorylates phosphatidylinositol 3,4,5-trisphosphate at the 3 position, turning off Akt signalling. PTEN contains a C-terminal PDZ binding motif that binds to the PDZ2 domain of MAGI-2, a scaffolding protein that localizes signalling molecules to the plasma membrane. MAGI-2 has ten domains that potentially mediate multiple protein-protein interactions simultaneously. A PTEN associated-complex (PAC) has been described and may contain MAGI-2, PTEN and p85. The PAC is hypothesized to form at the plasma membrane at appropriate sites for PTEN to gain access to its lipid substrates, since the binding of PTEN to MAGI-2 has been shown to enhance the suppression of PI3K-mediated Akt signalling. In order to better understand the role of the PAC in attenuation of the Akt signalling pathway, regions of the MAGI-2 scaffolding protein were mapped to identify the interactions taking place in the PAC. MAGI-2, and its individual domains, were expressed as GST fusion proteins. These were immobilized onto beads and allowed to bind to cellular proteins including PTEN, p85, PDGFR and EGFR using a GST pull-down experiment. The proteins bound to GST-MAGI-2 were identified using an immunoblot analysis. In vitro pull-down experiments revealed that MAGI-2 PDZ2 and PDZ5 domains bind to PTEN, and both MAGI-2 WW domains were shown to bind to p85. EGFR and PDGFR did not bind to the PDZ domains of MAGI-2 under the conditions studied. In order to study protein-protein interactions in cells, immunoprecipitation assays were also performed. Full length MAGI-2 was expressed tagged to a Myc epitope. This was used in immunoprecipitation assays to determine if MAGI-2 could co-immunoprecipitate with proteins involved in the Akt signalling pathway, such as PTEN, p85, PDGFR and EGFR. MAGI-2 can co-immunoprecipitate with PTEN upon 5 min EGF stimulation however, this result was inconclusive because replicate experiments did not verify this initial observation. MAGI-2 does not co-immunoprecipitate with the EGFR nor p85, under the conditions tested. We examined for these interactions after 5 min of growth factor stimulation and more experiments that test different time points after growth factor stimulation would reveal if these interactions are present at shorter time points. MAGI-2 has been shown to bind to PTEN and p85 in vitro and therefore has the potential to regulate the attenuation of the PI3K/Akt signalling pathway in response to activated EGFR and/or PDGFR.

<p>Regulation of cell growth is controlled by a variety of factors, including a number of oncogenes and tumor suppressors. PTEN is an inositol phosphatase that regulates cell growth by hydrolyzing the phospholipid products of PI3K. PTEN is mutated in a number of cancers, leading to its characterization as an important tumor suppressor. Recent data indicate that PTEN may also perform important functions that are independent of its phosphatase activity, most notably within the nucleus. Studies in this thesis addressed a novel role for PTEN in the regulation of the cellular response to hypoxia.</p> <p>PTEN overexpression significantly increased hypoxic gene expression independent of its catalytic activity, while shRNA-mediated silencing of PTEN significantly inhibited hypoxia-mediated HRE-luciferase activity. Nuclear-localized PTEN was more effective in promoting HRE activity than nuclear-excluded PTEN. These results suggested a scaffolding function of PTEN in the hypoxic nucleus. To identify specific gene targets regulated by PTEN in hypoxia, a custom oligo-array consisting of 46 hypoxia-responsive genes was utilized following both gain- and loss-of- PTEN function. Based on real-time quantitative results, PTEN positively regulated genes involved in metabolism (PFKFB3, PFKFB4, ALDOA, PGK-1), oxygen supply (VEGFA, EPO), cell growth (Tgf-a, TERT, cyclin D1, BNIP3), motility (E-cadherin) and transcription (DEC2). A single missense mutation at isoleucine 224 (I224M) of PTEN, however, abrogated the ability of PTEN to regulate the hypoxia response without affecting its lipid phosphatase activity. PTEN has previously been shown to bind to the co-activator p300 and to affect p53 acetylation and stabilization. As p300 is also a co-activator for the HIF proteins, we hypothesized that PTEN's association with p300 would promote the HIF/p300 complex to positively regulate hypoxic gene transcription. Overexpression of PTEN-WT extended the half-life of p300 and histone acetyltransferase activity of p300 in hypoxia, while overexpression of PTEN-I224M or PTEN silencing decreased both. In vivo, these effects resulted in a significant increase in hypoxic area in PTEN-null tumors compared to tumors expressing endogenous levels of PTEN, suggesting an inability to mount a hypoxia response necessary for revascularization of the tissue. PTEN's effect on p300 extended to other functions of p300 outside of the hypoxia response, most notably p300's role in p53 stability and p53-mediated gene transcription. Overexpression of PTEN resulted in an increase in p53 reporter activity following DNA damage (mitomycin C treatment). PTEN silencing or overexpression of PTEN-I224M resulted in abrogation of these effects. Taken together, these findings demonstrate that PTEN is required for the hypoxia response and they suggest that PTEN acts as a scaffold for p300 and the HIF machinery in the hypoxic nucleus independent of its canonical lipid phosphatase activity. These results may have important implications for the treatment of tumors in which PTEN is lost or mutated. The potential use of PTEN-I224M as a therapeutic is also discussed</p> / Dissertation