Role Of Phosphatidylinositol 3-kinase Research Articles

Abrogating endocrine resistance by targeting ERα and PI3K in breast cancer.

Antiestrogen therapies targeting estrogen receptor α (ER) signaling are a mainstay for patients with ER+ breast cancer. While many cancers exhibit resistance to antiestrogen therapies, a large body of clinical and experimental evidence indicates that hyperactivation of the phosphatidylinositol 3-kinase (PI3K) pathway promotes antiestrogen resistance. In addition, continued ligand-independent ER signaling in the setting of estrogen deprivation may contribute to resistance to endocrine therapy. PI3K activates several proteins which promote cell cycle progression and survival. In ER+ breast cancer cells, PI3K promotes ligand-dependent and -independent ER transcriptional activity. Models of antiestrogen-resistant breast cancer often remain sensitive to estrogen stimulation and PI3K inhibition, suggesting that clinical trials with combinations of drugs targeting both the PI3K and ER pathways are warranted. Herein, we review recent findings on the roles of PI3K and ER in antiestrogen resistance, and clinical trials testing drug combinations which target both pathways. We also discuss the need for clinical investigation of ER downregulators in combination with PI3K inhibitors.

Regulation of glucose metabolism in T cells: new insight into the role of Phosphoinositide 3-kinases

Naïve T cells are relatively quiescent cells that only require energy to prevent atrophy and for survival and migration. However, in response to developmental or extrinsic cues T cells can engage in rapid growth and robust proliferation, produce of a range of effector molecules and migrate through peripheral tissues. To meet the significantly increased metabolic demands of these activities, T cells switch from primarily metabolizing glucose to carbon dioxide through oxidative phosphorylation to utilizing glycolysis to convert glucose to lactate (termed aerobic glycolysis). This metabolic switch allows glucose to be used as a source of carbon to generate biosynthetic precursors for the production of protein, DNA, and phospholipids, and is crucial for T cells to meet metabolic demands. Phosphoinositide 3-kinases (PI3K) are a family of inositol lipid kinases linked with a broad range of cellular functions in T lymphocytes that include cell growth, proliferation, metabolism, differentiation, survival, and migration. Initial research described a critical role for PI3K signaling through Akt (also called protein kinase B) for the increased glucose uptake and glycolysis that accompanies T cell activation. This review article relates this original research with more recent data and discusses the evidence for and against a role for PI3K in regulating the metabolic switch to aerobic glycolysis in T cells.

Tannerella forsythia invasion in oral epithelial cells requires phosphoinositide 3-kinase activation and clathrin-mediated endocytosis

Tannerella forsythia, a Gram-negative anaerobe implicated in periodontitis, has been detected within human buccal epithelial cells and shown to invade oral epithelial cells in vitro. We have previously shown that this bacterium triggers host tyrosine kinase-dependent phosphorylation and actin-dependent cytoskeleton reorganization for invasion. On the bacterial side, the leucine-rich repeat cell-surface BspA protein is important for entry. The present study was undertaken to identify host signalling molecules during T. forsythia entry into human oral and cervical epithelial cells. Specifically, the roles of phosphatidylinositol 3-kinase (PI3K), Rho-family GTPases, cholesterol-rich membrane microdomains and the endocytic protein clathrin were investigated. For this purpose, cell lines were pretreated with chemical inhibitors or small interfering RNAs (siRNAs) that target PI3Ks, Rho GTPases, clathrin and cholesterol (a critical component of 'lipid rafts'), and the resulting effects on T. forsythia uptake were determined. Our studies revealed that T. forsythia entry is dependent on host PI3K signalling, and that purified BspA protein causes activation of this lipid kinase. Bacterial entry also requires the cooperation of host Rac1 GTPase. Finally, our findings indicate an important role for clathrin and cholesterol-rich lipid microdomains in the internalization process.

If you can’t beat me, join me: collaborating oncogenes circumventing senescence and causing cancer

death. Signaling routes commonly altered in cancer include the RAS-MAP Kinase and PI3 Kinase (PI3K) pathways. Activation of the first can take place at many levels, most frequently through mutations in members of the RAS and RAF families, while also further upstream receptor tyrosine kinases can be activated by mutation or amplification. PI3K pathway activation occurs largely through mutations in its catalytic subunit PIK3CA or amplification of AKT family members. Additionally, the Phosphatase and Tensin Homolog (PTEN), a phosphatase negatively regulating the PI3K pathway by dephosphorylating the lipid phosphatidylinositol-3,4,5-trisphosphate (PIP3), is inactivated in several tumor types by different mechanisms including mutation, deletion, and epigenetic silencing. Perhaps somewhat counter intuitively, although PI3K is an established downstream effector of RAS in oncogenic transformation, simultaneous mutations in RAS and in the PIK3CA ⁄ PTEN ⁄AKT pathway do occur in human cancer. Consistently, in several mouse models, they have been shown to cooperate in accelerating tumorigenesis. While it is reasonably well understood why mutant RAS and deficiencies in the pRB or p53 pathways collaborate in cancer, it is less clear what the mechanistic basis is for activating mutations at two points in a presumed single signaling pathway, like RAS and PI3K. Kennedy et al. recently reported on a molecular interplay model in which the second oncogene, acting downstream, counteracts the senescence-inducing effect of the first (Kennedy et al.,

Deletion of the p85α Regulatory Subunit of Phosphoinositide 3-Kinase in Cone Photoreceptor Cells Results in Cone Photoreceptor Degeneration

Downregulation of the retinal insulin/mTOR pathway in mouse models of retinitis pigmentosa is linked to cone cell death, which can be delayed by systemic administration of insulin. A classic survival kinase linking extracellular trophic/growth factors with intracellular antiapoptotic pathways is phosphoinositide 3-kinase (PI3K), which the authors have shown to protect rod photoreceptors from stress-induced cell death. The role of PI3K in cones was studied by conditional deletion of its p85α regulatory subunit. Mice expressing Cre recombinase in cones were bred to mice with a floxed pi3k gene encoding the p85α regulatory subunit of the PI3K and were back-crossed to ultimately generate offspring with cone-specific p85α knockout (cKO). Cre expression and cone-specific localization were confirmed by Western blot analysis and immunohistochemistry (IHC), respectively. Cone structural integrity was determined by IHC using peanut agglutinin and an M-opsin-specific antibody. Electroretinography (ERG) was used to assess rod and cone photoreceptor function. Retinal structure was examined by light and electron microscopy. An age-related cone degeneration was found in cKO mice, evidenced by a reduction in photopic ERG amplitudes and loss of cone cells. By 12 months of age, approximately 78% of cones had died, and progressive disorganization of synaptic ultrastructure was noted in surviving cone terminals in cKO retinas. Rod viability was unaffected in p85α cKO mice. The present study suggests that PI3K signaling pathway is essential for cone survival in the mouse retina.

Controlling embryonic stem cell proliferation and pluripotency: the role of PI3K- and GSK-3-dependent signalling

ESCs (embryonic stem cells) are derived from the inner cell mass of pre-implantation embryos and are pluripotent, meaning they can differentiate into all of the cells that make up the adult organism. This property of pluripotency makes ESCs attractive as a model system for studying early development and for the generation of specific cell typesfor use in regenerative medicine and drug screening. In order to harness their potential, the molecular mechanisms regulating ESC pluripotency, proliferation and differentiation (i.e. cell fate) need to be understood so that pluripotency can be maintained during expansion, while differentiation to specific lineages can be induced accurately when required. The present review focuses on the potential roles that PI3K (phosphoinositide 3-kinase) and GSK-3 (glycogen synthase kinase 3)-dependent signalling play in the co-ordination and integration of mouse ESC pluripotency and proliferation and contrast this with our understanding of their functions in human ESCs.

On the Role of Phosphatidylinositol 3-Kinase, Protein Kinase B/Akt, and Glycogen Synthase Kinase-3β in Photodynamic Injury of Crayfish Neurons and Glial Cells

Photodynamic treatment that causes intense oxidative stress and cell death is currently used in neurooncology. However, along with tumor cells, it may damage healthy neurons and glia. To study the involvement of signaling processes in photodynamic injury or protection of neurons and glia, we used crayfish mechanoreceptor consisting of a single neuron surrounded by glial cells. It was photosensitized with alumophthalocyanine Photosens. Application of specific inhibitors showed that phosphatidylinositol 3-kinase did not participate in photoinduced death of neurons and glia. Akt was involved in photoinduced necrosis but not in apoptosis of neurons and glia. Glycogen synthase kinase-3β participated in photoinduced apoptosis of glial cells and in necrosis of neurons. Therefore, phosphatidylinositol 3-kinase/protein kinase Akt/glycogen synthase kinase-3β pathway was not involved as a whole in photodynamic injury of crayfish neurons and glia but its components, Akt and glycogen synthase kinase-3β, independently and cell specifically regulated death of neurons and glial cells. According to these data, necrosis in this system was a controlled but not a non-regulated cell death mode. The obtained results may be used for the search of pharmacological agents selectively modulating death and survival of normal neurons and glial cells during photodynamic therapy of brain tumors.

Microglial MAC1 receptor and PI3K are essential in mediating β-amyloid peptide-induced microglial activation and subsequent neurotoxicity

Backgroundβ-Amyloid peptide (Aβ) is a major protein in the brain associated with Alzheimer's and Parkinson's diseases. The purpose of this study was to investigate the role of macrophage antigen-1 (MAC1) receptor, an integrin scavenger receptor in microglia, and subsequent signaling events in mediating Aβ-induced neurotoxicity. We have previously reported that NADPH oxidase (PHOX) on microglia and superoxide produced by PHOX are critical for Aβ-induced loss of dopaminergic neurons. However, the upstream signaling pathway of superoxide production remains unclear.MethodsFor the in vitro study, mesencephalic neuron-glia cultures and microglia-enriched cultures from mice deficient in the MAC1 receptor (MAC1-/-) and wild type controls were used to investigate the role of MAC1 receptor in Aβ-induced neurotoxicity and the role of phosphoinositide-3 kinase (PI3K) in the signal pathway between MAC1 receptor and PHOX. For the in vivo study, Aβ was injected into the substantia nigra of MAC1-/- mice and wild type mice to confirm the role of MAC1 receptor.ResultsWe found that Aβ-induced activation of microglia, activation of PHOX, generation of superoxide and other reactive oxygen species, and loss of dopaminergic neurons were decreased in MAC1-/- cultures compared to MAC1+/+ cultures. In MAC1-/- mice, dopaminergic neuron loss in response to Aβ injection into the substantia nigra was reduced relative to MAC1+/+ mice. Thus, MAC1 receptor-mediated PHOX activation and increased superoxide production are associated with Aβ-induced neurotoxicity. PI3K activation was one downstream step in MAC1 signaling to PHOX and played an important role in Aβ-induced neurotoxicity. In microglia-enriched cultures from MAC1-/- mice, Aβ-induced activation of PI3K (phosphorylation of target proteins and PIP3 production) was reduced relative to MAC1+/+ cultures.ConclusionsTaken together, our data demonstrate that Aβ activates MAC1 receptor to increase the activity of PI3K, which in turn phosphorylates p47phox, triggers the translocation of cytosolic subunits of PHOX to microglia membrane, increases PHOX activation and the subsequent production of superoxide and causes neurotoxicity.

103 TRANSDUCTION PATHWAYS RELATED TO GLUCOSE METABOLISM IN MALE AND FEMALE BOVINE EMBRYOS PRODUCED IN VITRO

Glucose metabolism plays an important role in energy balance control in mammalian cells and has been widely used as an indicator of embryo developmental competence. Previous studies have shown that developmental kinetic rates following IVF are lower in female than in male blastocysts, which may be related to differences in glucose consumption and metabolism. In addition, we have demonstrated that inhibition of phosphatidylinositol 3-kinase (PI3-K) with a structurally unrelated inhibitor, LY294002, suggests a negative role for PI3-K in the regulation of bovine embryo development (Aparicio et al. 2010 Reproduction 140, 83–92). The aim of this study was to determine whether PI3-K has a role in the regulation of glucose metabolism in Day 7 bovine blastocysts and to study the possible differential protein expression involved in glucose metabolism [hexokinase-I (HK-I), phosphofructokinase-1 (PFK-1), pyruvate kinase1/2 (PMK1/2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), lactate dehydrogenase A/C (LDHA/C), glucose transporter-1 (GLUT-1) and glycogen synthase kinase-3 (GSK-3A/B)] between in vitro produced male and female embryos derived from IVF with either X- or Y-sorted semen. Day 7 blastocysts derived from unsorted semen (n = 25 blastocysts per group) were incubated up to 12 h in SOF culture medium in the presence or absence of LY294002 (10 μM) and stored. Similarly, male and female Day 7 blastocysts derived from sorted semen were collected apart and stored at –80°C until proteomic analysis (Western blot analysis of proteins separated by sodium dodecyl sulfate-polyacylamide gel electrophoresis). Inhibition of PI3K significantly decreased HK-I (P < 0.01), PFK-1 (P < 0.001), GAPDH (P < 0.05), GSK-3A/B (P < 0.001), and GLUT-1 (P < 0.01) protein levels. Interestingly, protein expression of HK-1 (P < 0.001), PFK-1 (P < 0.01), PMK1/2 (P < 0.05), GAPDH (P < 0.01), and GLUT-1 (P < 0.001) was significantly higher in male compared with female blastocysts. The significant increase in the phosphorylated forms (Ser21 and Ser9) of both isoforms (GSK-3A/B) in male compared with female embryos is indicative of a higher inactivation of GSK-3A/B in males (P < 0.001). The presence of LDHA/C activity was not detected in any blastocyst group, irrespective of the gender or treatment studied. In conclusion, our data suggest that PI3K plays a major role in the regulation of glucose metabolism in bovine embryos, because pretreatment with LY294002 significantly modified the protein expression of HK-I, PFK-1, GAPDH, GSK- 3A/B, and GLUT-1, and underline the possibility of modulating glucose metabolism via the PI3K cellular pathway. The differential glycolytic metabolism between male and female blastocysts might explain the higher developmental kinetic rates in males described by other authors, because the expression of proteins involved in glycolysis and glycogenogenesis was significantly higher in male than in female in vitro-produced bovine embryos.

Overexpression of nerve growth factor in peritoneal fluid from women with endometriosis may promote neurite outgrowth in endometriotic lesions

To investigate the role of the nerve growth factor (NGF) in the endometriosis-associated innervation in the development of endometriosis-associated symptoms, 41 peritoneal fluid samples (PF) from patients with surgically and histologically proven endometriosis and 20 PF from patients with other gynecologic conditions were analyzed with Western blot and a novel in vitro model using dorsal root ganglia (DRG) to show neuronal outgrowth; endometrial cells also were analyzed. The results suggest that the PF of endometriosis patients and endometriotic lesions have neurotropic properties, because the Western blot analysis and the cell culture stainings showed NGF expression, and the neurite outgrowth of DRG treated with PF of patients with endometriosis was significantly higher than when treated with PF of patients without endometriosis. Furthermore, blocking NGF with both anti-NGF and K252a leads to a significant decrease in neurite outgrowth.

Phosphoinositide 3-kinase regulates myogenin expression at both the transcriptional and post-transcriptional level during myogenesis

It is well-established that phosphoinositide 3-kinase (PI3-kinase) regulates myogenesis by inducing transcription of myogenin, a key muscle regulatory factor, at the initiation of myoblast differentiation. In this study, we investigated the role of PI3-kinase in cells that have committed to differentiation. PI3-kinase activity increases during myogenesis, and this increase is sustained during the myogenic process; however, its function after the induction of differentiation has not been investigated. We show that LY294002, a PI3-kinase inhibitor, blocked myoblast fusion even after myogenin expression initially increased. In contrast to the inhibitory effects of LY294002 on myogenin mRNA levels during the initiation of differentiation, LY294002 blocked the accumulation of myogenin protein without affecting its mRNA level after differentiation was induced. Treatment with cycloheximide, a translation inhibitor, or actinomycin D, a transcription inhibitor, indicated that the stability of myogenin protein is lower than that of its mRNA. LY294002 inhibited the activities of several important translation factors, including eukaryotic elongation factor-2 (eEF2), by altering their phosphorylation status. In addition, LY294002 blocked the incorporation of [35S]methionine into newly synthesized proteins. Since myogenin has a relatively short half-life, LY294002-mediated inhibition of post-transcriptional processes resulted in a rapid depletion of myogenin protein. In summary, these results suggest that PI3-kinase plays an important role in regulating the expression of myogenin through post-transcriptional mechanisms after differentiation has been induced.

The Interferon-γ–induced Murine Guanylate-Binding Protein-2 Inhibits Rac Activation during Cell Spreading on Fibronectin and after Platelet-derived Growth Factor Treatment: Role for Phosphatidylinositol 3-Kinase

Exposure of cells to certain cytokines can alter how these same cells respond to later cues from other agents, such as extracellular matrix or growth factors. Interferon (IFN)-γ pre-exposure inhibits the spreading of fibroblasts on fibronectin. Expression of the IFN-γ–induced GTPase murine guanylate-binding protein-2 (mGBP-2) can phenocopy this inhibition and small interfering RNA knockdown of mGBP-2 prevents IFN-γ–mediated inhibition of cell spreading. Either IFN-γ treatment or mGBP-2 expression inhibits Rac activation during cell spreading. Rac is required for cell spreading. mGBP-2 also inhibits the activation of Akt during cell spreading on fibronectin. mGBP-2 is incorporated into a protein complex containing the catalytic subunit of phosphatidylinositol 3-kinase (PI3-K), p110. The association of mGBP-2 with p110 seems important for the inhibition of cell spreading because S52N mGBP-2, which does not incorporate into the protein complex with p110, is unable to inhibit cell spreading. PI3-K activation during cell spreading on fibronectin was inhibited in the presence of mGBP-2. Both IFN-γ and mGBP-2 also inhibit cell spreading initiated by platelet-derived growth factor treatment, which is also accompanied by inhibition of Rac activation by mGBP-2. This is the first report of a novel mechanism by which IFN-γ can alter how cells respond to subsequent extracellular signals, by the induction of mGBP-2.

Role of phosphoinositide 3-kinase β in platelet aggregation and thromboxane A2 generation mediated by Gi signalling pathways

PI3Ks (phosphoinositide 3-kinases) play a critical role in platelet functional responses. PI3Ks are activated upon P2Y12 receptor stimulation and generate pro-aggregatory signals. P2Y12 receptor has been shown to play a key role in the platelet aggregation and thromboxane A2 generation caused by co-stimulation with Gq or Gz, or super-stimulation of Gi pathways. In the present study, we evaluated the role of specific PI3K isoforms alpha, beta, gamma and delta in platelet aggregation, thromboxane A2 generation and ERK (extracellular-signal-regulated kinase) activation. Our results show that loss of the PI3K signal impaired the ability of ADP to induce platelet aggregation, ERK phosphorylation and thromboxane A2 generation. We also show that Gq plus Gi- or Gi plus Gz-mediated platelet aggregation, ERK phosphorylation and thromboxane A2 generation in human platelets was inhibited by TGX-221, a PI3Kbeta-selective inhibitor, but not by PIK75 (a PI3Kalpha inhibitor), AS252424 (a PI3Kgamma inhibitor) or IC87114 (a PI3Kdelta inhibitor). TGX-221 also showed a similar inhibitory effect on the Gi plus Gz-mediated platelet responses in platelets from P2Y1-/- mice. Finally, 2MeSADP (2-methyl-thio-ADP)-induced Akt phosphorylation was significantly inhibited in the presence of TGX-221, suggesting a critical role for PI3Kbeta in Gi-mediated signalling. Taken together, our results demonstrate that PI3Kbeta plays an important role in ADP-induced platelet aggregation. Moreover, PI3Kbeta mediates ADP-induced thromboxane A2 generation by regulating ERK phosphorylation.

Both p110α and p110β Isoforms of Phosphatidylinositol 3‐OH‐Kinase are Required for Insulin Signalling in the Hypothalamus

Both insulin and leptin action in the brain are considered to involve activation of phosphoinositide 3-kinase (PI3K), although the roles of different PI3K isoforms in insulin signalling in the hypothalamus are unknown. In the present study, we characterised the roles of these isoforms in hypothalamic insulin and leptin signalling and investigated the cross-talk of both hormones. To evaluate PI3K levels in the hypothalamus, PI3K was immunoprecipitated using an antibody directed against the p85 subunit, and then total PI3K activity was measured in the presence of novel isoform-selective pharmacological inhibitors of each isoform of PI3K. Subsequently, these inhibitors were administered into the lateral ventricle of male Sprague-Dawley rats, followed by vehicle, insulin, leptin or both hormones 45 min later. PI3K activity was determined by immunohistochemical detection of phosphorylated AKT (S473). In a separate study, the effects of the inhibitors on the anorexigenic action of insulin and leptin were determined. Hypothalamic insulin signalling was specifically mediated by the combined actions of the class Ia isoforms p110alpha and p110beta. Total hypothalamic PI3K activity was inhibited 65% by a p110alpha inhibitor, and 35% by a p110beta inhibitor, with a combination of inhibitors being equally effective as the broad-spectrum PI3K inhibitor wortmannin. Individual i.c.v. administration of p110alpha and p110beta inhibitors partly prevented insulin-induced phosphorylated AKT (S473) in the arcuate nucleus, whereas simultaneous application completely blocked insulin action. Unlike insulin, leptin did not induce phosphorylated AKT in the hypothalamus, as detected by immunohistochemistry, and the anorectic effects of leptin were not affected by pre-treatment with a combination of p110alpha and p110beta inhibitors. The enhanced anorectic effect of a combined i.c.v. application of both insulin and leptin could be prevented by pre-treatment with the combination of p110alpha and p110beta inhibitors. The data suggest that p110alpha and p110beta isoforms of PI3K are necessary to mediate insulin action in the hypothalamus. The role of PI3K in leptin action is less clear, but it may be involved by means of an insulin-dependent sensitisation of leptin action.

Abstract 1187: The role of PI3K as a biomarker

Abstract The phosphatidylinositol 3-kinase (PI3K) signaling pathway has been implicated in tumorigenesis. Evidence over the past years suggests a pivotal role for the catalytic subunit, PIK3CA, in human cancers. Receptor tyrosine kinases (RTKs) on the cell surface are key regulators of PI3K activity, and RTK antagonists have demonstrated significant anticancer effects in animal models and in patients. However, among tumor harboring activating mutations of PIK3CA, the therapeutic value of RTK antagonists may be compromised. The mutation status of PIK3CA may, therefore, serve as a potential biomarker for both prognosis and response to RTK inhibitors. Somatic mutations of the pik3ca gene occur primarily in three hot spots, E542K, E545K (exon 9) and H1047R (exon 20). These mutations are detected in a broad spectrum of cancer indications including breast, colorectal and lung. This study was designed to explore the nature of PIK3CA mutations among numerous human cancer cell lines, utilizing mostly cells with the capacity to form tumors in mice. Primers corresponding to exon 9 and 20 of human pik3ca gene were designed. Genomic DNA was harvested from over 200 lines, derived from different human cancer indications. The pik3ca locus was sequenced and its WT or mutation status was determined. Of these cells, 8% presented mutations equally distributed between exon 9 and 20. The frequency of PIK3CA mutations was particularly high among colon cancer lines, with fewer incidences among lung, breast and ovarian cancer lines. Interestingly, 50% of the PIK3CA mutated lines presented a concurrent mutation of either KRAS or BRAF. The capacity of PIK3CA mutation-bearing cell lines to respond to RTK blockers was then evaluated, and the mechanism contributing to such phenomenon was further discussed. These results stress PI3K diagnosis value and its potential role as a valid biomarker for patient susceptibility. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1187.

Aerobic exercise acutely improves insulin‐ and insulin‐like growth factor‐1‐mediated vasorelaxation in hypertensive rats

Limited information is available concerning the effects of aerobic exercise on vasorelaxation in hypertension. The aim of this study was to investigate the effects of a single bout of aerobic exercise on insulin- and insulin-like growth factor-1 (IGF-1)-induced vasorelaxation in hypertensive rats. Four-month-old spontaneously hypertensive rats were randomly divided into a sedentary group (SHR) and an exercise group (SHR+Ex) subjected to a single bout of aerobic exercise conducted by treadmill running at 21 m min(1) for 1 h. Age-matched Wistar-Kyoto rats were used as a normotensive control group (WKY). Insulin- and IGF-1-induced vasorelaxant responses in the three groups were evaluated by using isolated aortic rings, with or without endothelial denudation, in organ baths. Possible roles of phosphatidylinositol 3-kinase (PI3K) and nitric oxide synthase (NOS) involved in the NO-dependent vasorelaxation were examined by adding selective inhibitors. The role of superoxide was also clarified by adding superoxide dismutase (SOD). In addition, the endothelium-independent vascular responses to sodium nitroprusside (SNP), a NO donor, were examined. The insulin- and IGF-1-induced vasorelaxation was significantly (P < 0.05) decreased in the SHR group compared with the WKY group. This decreased response in SHR was improved by exercise. These vasorelaxant responses among the three groups became similar after endothelial denudation and pretreatment with the PI3K inhibitor, NOS inhibitor or SOD. Also, no difference among groups was found in the SNP-induced vasorelaxation. We concluded that a single bout of aerobic exercise acutely improves insulin- and IGF-1-mediated vasorelaxation in an endothelium-dependent manner in hypertensive rats.

Role of PI3K and autophagy in virus-stimulated IFN-α production by human plasmacytoid dendritic cells (136.46)

Abstract Human plasmacytoid dendritic cells (pDC) produce large amount of IFN-α in response to viral stimuli through TLR7 and TLR9. Phosphatidylinositol-3 kinase (PI3K) has been shown to be activated by TLR stimulation in many cell types and Class I and class III PI3K are essential in the regulation of autophagy. We aimed to study the role of autophagy in both the processing of virus and induction of IFN-α in human pDC, which have not been described. We investigated the effects of Class I and class III PI3K inhibitors wortmannin, 3-methyladenine (3MA) and LY294002 on IFN-α production by HSV, Flu, SeV, CpG A and HIV-MN/AT2-stimulated human pDC. pDC were pretreated with the inhibitors then stimulated with virus for 6hr for detection of intracellular IFN-α by intracellular flow cytometry or overnight for stimulation and release of IFN-α, which was detected by ELISA. Inhibition of PI3K with wortmannin, 3MA and LY294002 inhibited IFN-α production by pDC. The PI3K inhibitors did not affect endocytosis or macropinocytosis. In addition, HSV dramatically induced autophagosome double-membrane structures in pDC as visualized by TEM and LC3B aggregation as seen in imaging flow cytometry. All of the inhibitors inhibited HSV-induced LC3B aggregation in pDC. These results support key roles for PI3K and autophagy in mediating IFN-α secretion and virus detection by human pDC. Supported by AI26806.

Hepatitis C virus genotype-3a core protein enhances sterol regulatory element-binding protein-1 activity through the phosphoinositide 3-kinase-Akt-2 pathway

Hepatitis C virus genotype-3a (HCV-3a) is directly linked to the development of steatosis. We previously showed that, through sterol regulatory element binding protein-1 (SREBP-1), HCV-3a core protein upregulates the promoter activity of fatty acid synthase, a major enzyme involved in de novo lipid synthesis. In this study, we investigated whether HCV-3a core can activate SREBP-1 and studied the role of phosphoinositide 3-kinase (PI3K)-Akt-2 pathway in modulating SREBP-1 activity by HCV-3a core. To determine whether HCV-3a core could activate SREBP-1, the level of mature SREBP-1 was analysed by Western blotting. Our results showed that the level of mature SREBP-1 was enhanced by HCV-3a core protein after transient expression and in the chimeric HCV-3a core/1b replicon cells in comparison to controls. To investigate the role of the PI3K-Akt-2 pathway in SREBP-1 activation by HCV-3a core, PI3K and Akt-2 activity was inhibited by using the chemical inhibitor LY294002, a dominant-negative Akt-2 plasmid, or knockdown of Akt-2 by small hairpin RNA. Our results showed that inhibition of PI3K and Akt-2 was associated with reduced SREBP-1 activation by HCV-3a core. These results indicate a role for PI3K and Akt-2 in increasing SREBP-1 activity by HCV-3a core protein and provide a mechanism of steatosis caused by HCV.

Abstract C7: Sensitization of human colorectal cancer cells to TRAIL induced-apoptosis following inhibition of PI3K or HSP90

Abstract TRAIL (Tumor-Necrosis-Factor related apoptosis-inducing ligand) induces apoptosis by binding to the death receptors DR4 and DR5. Recombinant TRAIL or agonist antibodies against TRAIL death-receptors are now in Phase I/II clinical trials. Despite specificity for cancer cells, a percentage of tumor cells remain insensitive to TRAIL-induced apoptosis. Previous work has demonstrated that signaling through PI3K (phosphatidylinositol-3-kinase) can prevent TRAIL-induced apoptosis in different cancer cell types. Moreover, inhibition of HSP90 (Heat Shock Protein 90) by 17-AAG can sensitize colon cancer cells to TRAIL-induced apoptosis. The aim of this project is to explore the mechanism of TRAIL resistance and to focus on the role of PI3K and HSP90 signaling and the ability of PI3K or HSP90 inhibitors to reverse resistance to TRAIL-induced apoptosis in human colorectal cancer cells. We demonstrated that co-treatments with TRAIL and PI-103, a PI3K inhibitor, or 17-AAG, an HSP90 inhibitor, synergistically contributed to the apoptotic stimulus in TRAIL sensitive and resistant colorectal cancer cells through the amplification of both extrinsic and intrinsic apoptotic pathways; this effect was lower or not present in non-tumor cells, suggesting a therapeutic index. These data support the potential use of TRAIL in combination with PI3K or HSP90 inhibitors in patients with colorectal cancer who showed no response to the ligand alone. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C7.

Abstract C63: Biological characterization of GSK2126458, a novel and potent inhibitor of phosphoinositide 3-kinase and the mammalian target of rapamycin (mTOR)

Abstract The phosphoinositide 3-kinase (PI3K) pathway is among the most commonly activated pathways in human cancer. The biological role of PI3K in growth and survival of cancer cells and the prevalence of activating mutations in human cancers are well documented and a significant proportion of tumors would be predicted to benefit from inhibition of this pathway. Here we report on the characterization of the pan-PI3K pyridylsulfonamide inhibitor GSK2126458: a very potent (PI3K app Ki = 19 pM), reversible, ATP-competitive inhibitor of wild-type PI3K and the ‘hotspot’ activating mutants of p110 found in human cancer. GSK2126458 demonstrated good selectivity for the PI3K family of enzymes including the mTORC1 and mTORC2 complexes, when evaluated in a large panel of protein kinases. Consistent with potent PI3K enzyme inhibition, GSK2126458 decreased the cellular levels of phosphorylated AKT, p70S6K, PRAS40 and ERK in a concentration and time dependent manner, the IC50 for pAKT in the HCC1954 breast carcinoma cell line was 2 nM. GSK2126458 induced the nuclear translocation of the FOXO3a transcription factor in a concentration dependent manner that mechanistically appeared bimodal. Growth inhibition was time and concentration dependent with a 3 day exposure resulting in a growth IC50 (gIC50) of 9 nM in HCC1954 cells with evidence of cell death. Cell death correlated with induction of caspase 3 and 7 activity suggesting apoptosis was the mechanism of cell death. GSK2126458 had a breadth of activity for potent cell growth inhibition and induction of cell death in a variety of human cancer cells. GSK2126458 demonstrated robust, dose dependent in vivo pharmacodynamic activity as measured by inhibition of phospho-AKT in advanced BT474 breast cancer cell xenograft tumors. Inhibition of AKT phosphorylation was rapid and lasted for several hours after a single oral administration of GSK2126458. Transient increases in plasma insulin and blood glucose were observed. Evaluation of in vivo tumor growth effects of GSK2126458 in xenograft models demonstrated dose dependent tumor growth delay in several models of diverse tumor lineage and tumor regression in a breast cancer xenograft. The biological profile of GSK2126458 supports the clinical advancement of this compound and GSK2126458 has entered Phase I human clinical trials. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C63.