Growth and proliferation are normal functions of cells mediated in part via receptor tyrosine kinases such as the epidermal growth factor (EGF) receptor (EGFR). The EGFR binds the extracellular signaling ligand EGF and transduces the signal into the cell. Phosphatidylinositol 3'-kinase (PI3K) responds to EGFR activation and initiates downstream signaling cascades responsible for cell cycle entry, proliferation and inhibition of apoptosis. Cell cycle arrest is required to stop cell growth and proliferation as well as allow apoptosis, if required. The phosphatase and tensin homologue deleted on chromosome ten (PTEN) directly opposes PI3K signaling since its substrate is the PI3K product phosphatidylinositol 3,4,5-trisphosphate. PI3K is a heterodimer composed of a p85 regulatory subunit and a p110 catalytic subunit. PTEN is an essential tumor suppressor protein. Absence of PTEN has been associated with several types of cancer. <p>Our laboratory has characterized new specialized functions for the p85 protein. One function discovered was the ability of p85 to enhance PTEN lipid phosphatase activity. In this thesis PTEN activity is shown to be enhanced at least 3.5 fold in vitro by an equimolar amount of p85. <p>We performed an analysis of PTEN using seven PTEN mutants. Two types of mutants were created: i) regulatory or possible regulatory phosphorylation sites were substituted to mimic both phosphorylated and non-phosphorylated states and ii) alanine substitution of basic amino acid residues. The phosphorylation sites altered were the casein kinase 2 phosphorylation sites in the regulatory domain and tyrosine 336, a proposed regulatory phosphorylation site. Three mutants involving alanine substitution for basic amino acid residues included one mutant in the PASE domain and two more mutants in the C2 domain. It was observed that GFP-PTEN translocates to the plasma membrane upon EGF stimulation. The mimic of constitutive phosphorylation of the Casein kinase 2 sites resulted in cytoplasmic localization whereas the non-phosphorylated mimic was plasma membrane localized regardless of EGFR activation status. Neutralization of positive charge in the PASE and C2 domains seriously impeded the ability of PTEN to bind to phosphorylated phosphatidylinositol lipids and abolished the ability of the protein to translocate to the plasma membrane in response to receptor activation. Located within a cluster of positively charged lysine residues in the C2 domain is a potential phosphorylation site at tyrosine 336. The phosphorylation mimic showed decreased binding to some membrane lipids compared to the non-phosphorylated mimic. The results we generated are consistent with a current model for PTEN regulation that proposes PTEN is localized to the cytoplasm in quiescent cells and dephosphorylation of the regulatory domain occurs upon EGF stimulation allowing translocation to the plasma membrane. The model proposes that dephosphorylation of the casein kinase 2 sites unmasks regions of positive charge that interact with the anionic plasma membrane. Furthermore, the results suggested that at the plasma membrane p85 interacts with PTEN to increase lipid phosphatase activity and may be involved in targeting PTEN to the activated receptor where PI3,4,5P3 lipids are being produced.

Growth and proliferation are normal functions of cells mediated in part via receptor tyrosine kinases such as the epidermal growth factor (EGF) receptor (EGFR). The EGFR binds the extracellular signaling ligand EGF and transduces the signal into the cell. Phosphatidylinositol 3'-kinase (PI3K) responds to EGFR activation and initiates downstream signaling cascades responsible for cell cycle entry, proliferation and inhibition of apoptosis. Cell cycle arrest is required to stop cell growth and proliferation as well as allow apoptosis, if required. The phosphatase and tensin homologue deleted on chromosome ten (PTEN) directly opposes PI3K signaling since its substrate is the PI3K product phosphatidylinositol 3,4,5-trisphosphate. PI3K is a heterodimer composed of a p85 regulatory subunit and a p110 catalytic subunit. PTEN is an essential tumor suppressor protein. Absence of PTEN has been associated with several types of cancer. <p>Our laboratory has characterized new specialized functions for the p85 protein. One function discovered was the ability of p85 to enhance PTEN lipid phosphatase activity. In this thesis PTEN activity is shown to be enhanced at least 3.5 fold in vitro by an equimolar amount of p85. <p>We performed an analysis of PTEN using seven PTEN mutants. Two types of mutants were created: i) regulatory or possible regulatory phosphorylation sites were substituted to mimic both phosphorylated and non-phosphorylated states and ii) alanine substitution of basic amino acid residues. The phosphorylation sites altered were the casein kinase 2 phosphorylation sites in the regulatory domain and tyrosine 336, a proposed regulatory phosphorylation site. Three mutants involving alanine substitution for basic amino acid residues included one mutant in the PASE domain and two more mutants in the C2 domain. It was observed that GFP-PTEN translocates to the plasma membrane upon EGF stimulation. The mimic of constitutive phosphorylation of the Casein kinase 2 sites resulted in cytoplasmic localization whereas the non-phosphorylated mimic was plasma membrane localized regardless of EGFR activation status. Neutralization of positive charge in the PASE and C2 domains seriously impeded the ability of PTEN to bind to phosphorylated phosphatidylinositol lipids and abolished the ability of the protein to translocate to the plasma membrane in response to receptor activation. Located within a cluster of positively charged lysine residues in the C2 domain is a potential phosphorylation site at tyrosine 336. The phosphorylation mimic showed decreased binding to some membrane lipids compared to the non-phosphorylated mimic. The results we generated are consistent with a current model for PTEN regulation that proposes PTEN is localized to the cytoplasm in quiescent cells and dephosphorylation of the regulatory domain occurs upon EGF stimulation allowing translocation to the plasma membrane. The model proposes that dephosphorylation of the casein kinase 2 sites unmasks regions of positive charge that interact with the anionic plasma membrane. Furthermore, the results suggested that at the plasma membrane p85 interacts with PTEN to increase lipid phosphatase activity and may be involved in targeting PTEN to the activated receptor where PI3,4,5P3 lipids are being produced.

The islets of Langerhans secrets insulin in response to fluctuations of blood glucose level and efficient secretion requires extensive intra-islet communication. Secretory failure from islets is one of the hallmark in progression of type 2 diabetes.  Changes in islet structure and high levels of saturated free fatty acids may contribute to this failure. The aim of this thesis is to study the role of cell-cell interactions and palmitate on β-cells functions. To address the role of cell-cell interactions on β-cells functions MIN6 cells were cultured as monolayers and as pseudoislets. Glucose stimulated insulin secretion was higher in pseudoislets compared to monolayers. Transcript levels of mitochondrial metabolism as well glucose oxidation rate was higher in pseudoislets. Insulin receptor substrate-1 (IRS-1) phosphorylation was altered when cells were grown as pseudoislets. Proteins expression levels related to glycolysis, cellular connections and translational regulations were up-regulated in pseudoislets. We propose the superior capacity of pseudoislets compared to monolayers depend on metabolism, cell coupling, gene translation, protein turnover and differential IRS-1 phosphorylation. To address the role of palmitate on β-cells human islets were cultured in palmitate. Long term palmitate treatment decreased insulin secretion which is associated with up-regulation of suppressor of cytokine signaling-2 (SOCS2) and protein inhibitor of activated STAT-1 (PIAS1). Up-regulation of SOCS2 decreased phosphorylation of Akt at site T308, whereas PIAS1 decreased protein level of ATP- citrate lyase (ACLY) and ATP synthase subunit B (ATP5B). We propose long term palmitate treatment reduces phosphatidylinositol 3-kinase (PI3K) activity, attenuates formation of acetyl-CoA and decreases ATP synthesis which may aggravate β-cells dysfunction.

Le CD95 appartient à la famille du TNF-R. Il est capable de déclencher un signal apoptotique et joue un rôle prépondérant dans le maintien de l’homéostasie du système immunitaire et dans l’élimination de cellules infectées ou transformées. Suite à la fixation de son ligand le CD95L ou d’un anticorps agoniste, le CD95 recrute la protéine FADD, qui à son tour agrège les caspases initiatrices (i.e., caspase -8 et -10). Le complexe formé par le CD95, FADD et les caspases -8/10 est appelé DISC, pour Deah Inducing Signaling Complex. Une fois le DISC formé, l’agrégation des caspases initiatrices entraîne leur activation, et la mort de la cellule par apoptose. Ce signal médié par CD95, peut être modulé par la distribution ou l’exclusion du récepteur vis à vis des microdomaines membranaires ou radeaux lipidiques. Ces domaines de la membrane plasmique sont des structures membranaires enrichies en sphingolipides et cholestérol. La relocalisation de CD95 dans ces radeaux lipidiques permet d’amplifier le signal de mort. L’activation de la voie phosphatidylinositol 3-kinase (PI3K)/Akt, est connue pour sa capacité à protéger les cellules tumorales du signal apoptotique médié par CD95, et à induire des mouvements latéraux de CD95 à la membrane. Nos travaux montrent que l’inhibition de la voie PI3K/Akt induit i) la relocalisation du CD95 dans les microdomaines et ii) l’induction du signal apoptotique CD95 indépendamment de la présence du ligand CD95L. Ainsi, nous mettons en évidence que la voie PI3K/Akt est capable d’augmenter le seuil d’activation du signal CD95 en agissant en amont de la formation du DISC ou de l’interaction CD95/CD95L, en maintenant le récepteur exclu des microdomaines. / CD95 belongs to the TNF-R superfamily, and it triggers an apoptotic signal. CD95 plays a key role in homeostasis of the immune system and in the elimination of infected and transformed cells. Upon CD95L binding, CD95 recruits FADD, which in turn aggregates initiator caspases (i.e., caspase-8 and -10). The complex CD95, FADD and caspase-8/10 is called DISC for Death Inducing Signaling Complex. At the DISC level, caspase aggregation leads to their activation and death of the cells through apoptosis. The CD95-mediated apoptotic signal is modulated by microdomains, or lipid rafts, which are plasma membrane sub-domains enriched in sphygolipids and cholesterol. Thereby, partition of CD95 into lipid rafts promotes the apoptotic signal. Activation of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway is known to prevent the CD95-mediated apoptotic signal in malignant cells, and to control lateral mobility of CD95 at the plasma membrane. Herein, we showed that inhibition of PI3K signal induced i) the distribution of CD95 into lipid rafts and ii) the subsequent induction of the CD95-mediated apoptotic signal through a CD95L independent manner. In conclusion, we pinpointed that PI3K/Akt signaling pathway inhibits the CD95 signal by acting upstream DISC formation and even upstream the CD95-CD95L interaction through the exclusion of the death receptor from the microdomains.

Background: Radiotherapy is used in the treatment of over 50% of cancer patients and bar surgery, is the most effective cancer intervention. However, in the clinic secondary malignancies have been observed following radiotherapy and in vitro increased cell migration and invasion have been seen following radiation. The Src/FAK signalling pathway is known to play an important role in the metastatic phenotype through its involvement in cell adhesion, migration and invasion and we have previously demonstrated that radiotherapy can activate this pathway along with the phosphoinositide 3-kinase (PI3K) pathway, also associated with tumour metastases and an aggressive phenotype. Using pharmacological inhibitors, we have investigated combination approaches to evaluate whether Src and PI3K targeting is beneficial in a radiotherapy context, especially focusing on metastatic phenotype. We wished to relate pathway activation to cellular phenotype and increase understanding of the metastatic cascade, the processes involved and the signalling pathways taking the lead. Method: Using thyroid carcinoma cell lines FTC133 and 8505c the effects of Src inhibition using AZD0530, FAK inhibition using FAKi and PI3K inhibition using GDC-0941 were studied. The effects of radiotherapy alone, and in combination with the above inhibitors, were also studied. In vitro MTT, apoptosis and clonogenic assays were used to assess cell proliferation and cell survival and scratch assays, cell adhesion and cell spreading assays were used to assess the effects of the drugs on metastatic characteristics. In vivo tumour growth, survival and ex vivo clonogenics were used to measure the effects of AZD0530 and GDC-0941. Western blotting, immunofluorescence and immunohistochemistry was used to observe the effects on pathway activation and protein localisation. Results: Src and FAK inhibition reduced metastatic characteristics of thyroid carcinoma cell lines in vitro such as cell spreading and migration. FAK inhibition showed a greater effect on cell survival by MTT, clonogenic and apoptosis. In the thyroid carcinoma cell lines radiotherapy enhanced the metastatic phenotype. This was seen by enhanced activation of the Src and PI3K pathways, increased migration and invasion in vitro and enhanced tumour metastasis in vivo. By combining Src inhibition with radiation a reduction in metastatic characteristics was observed and by combining PI3K inhibition with radiotherapy radiosensitivity could be improved. With the triple combination of Src and PI3K inhibition with radiotherapy a significant reduction in cell survival was demonstrated in vitro compared to radiation alone and either inhibitor combined with radiation, with a corresponding significant reduction in tumour growth being observed in vivo. With the combination of Src and PI3K inhibition significant reductions in metastatic characteristics were also observed both in vitro and in vivo seen by a reduction in cell migration and tumour metastasis. Finally combined inhibition of the Src and PI3K pathway reduced the radiation enhanced activation of several pathways in vivo including Src and PI3K.Conclusions: Together these results suggest that the Src and PI3K pathways play a role in radiation enhanced metastatic characteristics in thyroid carcinoma and through combined inhibition of the pathway the negative effects of radiation, enhanced migration and invasion, can be inhibited and the cells can be made more radiosensitive. Full characterisation of the pathways involved in radiation induced motility and radioresistance will provide further rationale for combination therapies and provide potential for application of these therapies in the clinic.

The phosphoinositide 3-kinases (PI3Ks) are a conserved family of signal transduction enzymes that are involved in regulating cellular activation, inflammatory responses, chemotaxis, and apoptosis. We have discovered that a carbohydrate ligand, glucan, will stimulate the endogenous PI3K/Akt signaling pathway. This article reviews the current data on the role of the PI3K/Akt signaling pathway as a negative feedback mechanism or compensatory regulator of septic and inflammatory responses. Of greater importance, the data reviewed in this article suggest that modulation of the PI3K/ Akt signaling pathway can reduce the morbidity and mortality associated with septic and I/R injury. Thus, manipulation of the endogenous PI3K/Akt signaling pathway may represent a new and novel therapeutic approach to management of important diseases.

No description available.

No description available.

Cbl is a multivalent protein that interacts with a number of signaling molecules that affect cell proliferation, migration and apoptosis. Although it is a downstream effector of growth factors, cytokines and integrin signaling all of which influence bone mass, very few studies have examined the role of Cbl in osteoblast proliferation and differentiation. To examine the role(s) of Cbl in the skeletal system we have focused specifically on phosphorylation of CblY737 since it is a unique to Cbl (not present on other family members) and upon phosphorylation by Src family kinases it provides a binding site for the p85 subunit of PI3K which regulates signaling events that modulate apoptosis and survival. To determine the role of tyrosine 737 we are using CblYF/YF knock-in mice (YF) where tyrosine 737 has been substituted to phenylalanine. YF mice had increased bone volume (WT 9%; YF 14%; p= 0.05 vs WT), trabecular thickness, and trabecular numbers. Although the increased bone volume is partly attributed to the decreased bone resorption, static and dynamic parameters of bone formation indicated that numbers of osteoblasts (WT 13 N.OB/BS; YF 20 N.OB/BS; p=0.05 vs WT) and bone formation rates were also upregulated in the CblYF/YF mice. To investigate the role of osteoblast differentiation in increased bone formation, we differentiated osteoblast and assessed ALP activity and Alizarin Red S staining. Both WT and YF osteoblasts had similar levels of ALP activity and mineral deposition during differentiation. To determine if the increased numbers of osteoblasts were due to increased survival and/or proliferation, we performed in vitro experiments with calvarial osteoblasts from age-matched WT and YF pups. MTT assay and TUNEL-staining, for cell viability, showed abrogation of Cbl-PI3K interaction did not affect osteoblast survival. Interestingly, inhibition of PI3K activity with LY294002 showed comparable survival between the WT and YF osteoblasts. We next examined proliferation and found that there was a 2-fold increase in the rate of the proliferation for the YF osteoblasts. This result was further substantiated by colony forming unit assay using bone marrow stromal cells. To establish the role of extracellular factors on osteoblast increased proliferation, various growth factors were assessed (EGF, FGF, IGF, PDGF). Treatment with the growth factors has no differential effects on the WT versus YF osteoblasts. We next used conditioned media from differentiated osteoclasts and bone marrow cells to treat MC3T3-E1, preosteoblast cell line. The osteoclast media from YF osteoclasts did not increase osteoblast proliferation. However, YF bone marrow conditioned media increased proliferation of the MC3T3-E1. Cytokine assays were done to determine the factor(s) that were increased in the YF conditioned media compared to the WT conditioned media. SDF-1 was found to be increased in the YF conditioned media compared to the WT conditioned media. Taken together, this suggests that the abrogation of Cbl-PI3K interaction leads to increased bone formation due to osteoclast resorption deficiency and increased osteoblast proliferation, which may be caused in part by increased SDF-1 expression in the bone marrow niche. / Cell Biology

Yes / Background: Many urothelial carcinomas (UC) contain activating PIK3CA mutations. In telomerase-immortalized normal urothelial cells (TERT-NHUC), ectopic expression of mutant PIK3CA induces PI3K pathway activation, cell proliferation and cell migration. However, it is not clear whether advanced UC tumors are PIK3CA-dependent and whether PI3K pathway inhibition is a good therapeutic option in such cases. Methods: We used retrovirus-mediated delivery of shRNA to knock down mutant PIK3CA in UC cell lines and assessed effects on pathway activation, cell proliferation, migration and tumorigenicity. The effect of the class I PI3K inhibitor GDC-0941 was assessed in a panel of UC cell lines with a range of known molecular alterations in the PI3K pathway. Results: Specific knockdown of PIK3CA inhibited proliferation, migration, anchorage-independent growth and in vivo tumor growth of cells with PIK3CA mutations. Sensitivity to GDC-0941 was dependent on hotspot PIK3CA mutation status. Cells with rare PIK3CA mutations and co-occurring TSC1 or PTEN mutations were less sensitive. Furthermore, downstream PI3K pathway alterations in TSC1 or PTEN or co-occurring AKT1 and RAS gene mutations were associated with GDC-0941 resistance. Conclusions: Mutant PIK3CA is a potent oncogenic driver in many UC cell lines and may represent a valuable therapeutic target in advanced bladder cancer.