Phosphoinositide 3-kinase Protein Kinase Research Articles

Screening and identification of lncRNAs in preadipocyte differentiation in sheep

Studies of preadipocyte differentiation and fat deposition in sheep have mainly focused on functional genes, and with no emphasis placed on the role that long non-coding RNAs (lncRNAs) may have on the activity of those genes. Here, the expression profile of lncRNAs in ovine preadipocyte differentiation was investigated and the differentially expressed lncRNAs were screened on day 0 (D0), day 2(D2) and day 8(D8) of ovine preadipocyte differentiation, with their target genes being predicted. The competing endogenous RNA (ceRNA) regulatory network was constructed by GO and KEGG enrichment analysis for functional annotation, and some differentially expressed lncRNAs were randomly selected to verify the RNA-Seq results by RT-qPCR. In the study, a total of 2517 novel lncRNAs and 3943 known lncRNAs were identified from ovine preadipocytes at the three stages of differentiation, with the highest proportion being intergenic lncRNAs. A total of 3455 lncRNAs were expressed at all three stages of preadipocyte differentiation, while 214, 226 and 228 lncRNAs were uniquely expressed at day 0, day 2 and day 8, respectively. By comparing the expression of the lncRNAs between the three stages of differentiation stages, a total of 405, 272 and 359 differentially expressed lncRNAs were found in D0-vs-D2, D0-vs-D8, and D2-vs-D8, respectively. Functional analysis revealed that the differentially expressed lncRNAs were enriched in signaling pathways related to ovine preadipocyte differentiation, such as mitogen-activated protein kinase (MAPK) pathway, the phosphoinositide 3-kinase protein kinase B (PI3K-Akt) pathway, and the transforming growth factor beta (TGF-β) pathway. In summary, lncRNAs from preadipocytes at different stages of differentiation in sheep were identified and screened using RNA-Seq technology, and the regulatory mechanisms of lncRNAs in preadipocyte differentiation and lipid deposition were explored. This study provides a theoretical reference for revealing the roles of lncRNAs in ovine preadipocyte differentiation and also offers a theoretical basis for further understanding the regulatory mechanisms of ovine preadipocyte differentiation.

Evaluation of Saudi Juniperus procera Extracts Cytotoxicity and Regulatory Mechanisms of Tumorigenesis Against Two Breast Cancer Cell Lines

Background Breast cancer poses a major threat to female health worldwide. One of the medicinal plants used in traditional Saudi medicine is the Juniperus procera (Arar) plant, which is used to cure various illnesses, including cancer. Objectives The cytotoxic properties of Saudi J. procera extracts and their regulatory mechanisms against tumorigenesis in breast cancer were investigated. Methods The antioxidant capacities of the fruit and leaf extracts were detected by the 2,2′-azino-bis 3-ethylbenzothiazoline-6-sulfonic acid (ABTS) assay and 2,2-diphenyl-1-1picrylhydrazyl (DPPH) assay. The cell viability of the extracts was determined by 3-(4,5-dimethylthiazoyl)-2,5-diphenyl-tetrazolium bromide assay. The cell migration was detected by a wound healing assay. The cell cycle and cell apoptosis were analyzed by flow cytometry. The reactive oxygen species (ROS) was evaluated using a DCFDA-cellular (the cell permeant reagent 2',7' –dichlorofluorescein diacetate) reactive oxygen species detection assay. The protein and gene expression levels were estimated by western blotting and Reverse transcription polymerase chain reaction (RT-PCR), respectively. Results Phytochemical analysis revealed a high content of phenolic and flavonoids in fruit and leaf extracts. Relative to leaf extract, the fruit extract showed higher antioxidant activities and exhibited more potent cytotoxic effects against two breast cancer cell lines: MCF-7 and MDA-MB-231. The fruit extract arrested the cell cycle of the tested cancer cell lines in S phase and G1 phase, and downregulated PI3K (phosphoinositide 3-kinase-protein kinase)/Akt (protein kinase B)/mTOR (Mammalian target of rapamycin) pathway. It also suppressed aromatase expression in MCF-7 but upregulated it in MDA-MB-231. Conclusion J procera fruit and leaf extracts have significant antitumor activities against MCF-7 and MDA-MB-231 breast cancer cell lines.

Targeting KRAS Mutations

Targeting KRAS Mutations

Peripheral Blood Gene Expression Profile of Infants with Atopic Dermatitis

To enhance the understanding of molecular mechanisms and mine previously unidentified biomarkers of pediatric atopic dermatitis, PBMC gene expression profiles were generated by RNA sequencing in infants with atopic dermatitis and age-matched controls. A total of 178 significantly differentially expressed genes (DEGs) (115 upregulations and 63 downregulations) were seen, compared with those in healthy controls. The DEGs identified included IL1β, TNF, TREM1, IL18R1, and IL18RAP. DEGs were validated by real-time RT- qPCR in a larger number of samples from PBMCs of infants with atopic dermatitis aged <12 months. Using the DAVID (Database for Annotation, Visualization and Integrated Discovery) database, functional and pathway enrichment analyses of DEGs were performed. Gene ontology enrichment analysis showed that DEGs were associated with immune responses, inflammatory responses, regulation of immune responses, and platelet activation. Pathway analysis indicated that DEGs were enriched in cytokine‒cytokine receptor interaction, immunoregulatory interactions between lymphoid and nonlymphoid cells, hematopoietic cell lineage, phosphoinositide 3-kinase‒protein kinase B signaling pathway, NK cell‒mediated cytotoxicity, and platelet activation. Furthermore, the protein‒protein interaction network was predicted using the STRING (Search Tool for the Retrieval of Interacting Genes/Proteins) database and visualized with Cytoscape software. Finally, on the basis of the protein‒protein interaction network, 18 hub genes were selected, and two significant modules were obtained. In conclusion, this study sheds light on the molecular mechanisms of pediatric atopic dermatitis and may provide diagnostic biomarkers and therapeutic targets.

Quantitative Proteomic Analysis of Zearalenone Exposure on Uterine Development in Weaned Gilts.

The aim of this study was to explore the effect of zearalenone (ZEA) exposure on uterine development in weaned gilts by quantitative proteome analysis with tandem mass spectrometry tags (TMT). A total of 16 healthy weaned gilts were randomly divided into control (basal diet) and ZEA3.0 treatments groups (basal diet supplemented with 3.0 mg/kg ZEA). Results showed that vulva size and uterine development index were increased (p < 0.05), whereas serum follicle stimulation hormone, luteinizing hormone and gonadotropin-releasing hormone were decreased in gilts fed the ZEA diet (p < 0.05). ZEA, α-zearalenol (α-ZOL) and β-zearalenol (β-ZOL) were detected in the uteri of gilts fed a 3.0 mg/kg ZEA diet (p < 0.05). The relative protein expression levels of creatine kinase M-type (CKM), atriopeptidase (MME) and myeloperoxidase (MPO) were up-regulated (p < 0.05), whereas aldehyde dehydrogenase 1 family member (ALDH1A2), secretogranin-1 (CHGB) and SURP and G-patch domain containing 1 (SUGP1) were down-regulated (p < 0.05) in the ZEA3.0 group by western blot, which indicated that the proteomics data were dependable. In addition, the functions of differentially expressed proteins (DEPs) mainly involved the cellular process, biological regulation and metabolic process in the biological process category. Some important signaling pathways were changed in the ZEA3.0 group, such as extracellular matrix (ECM)-receptor interaction, focal adhesion and the phosphoinositide 3-kinase–protein kinase B (PI3K-AKT) signaling pathway (p < 0.01). This study sheds new light on the molecular mechanism of ZEA in the uterine development of gilts.

Effect of IL-35 on apoptosis, adhesion, migration, and activation of eosinophils in allergic rhinitis.

Eosinophils play critical roles in the development of allergic rhinitis (AR) by releasing toxic substance. Interleukin-35 (IL-35), a newly identified anti-inflammatory cytokine, had potent inhibitive role for eosinophil infiltration in allergic disease. However, the direct effect of IL-35 on eosinophil was not clear. Twenty AR children and sixteen controls were recruited. The correlation between IL-35 protein expression and blood eosinophil counts and activation was analyzed. The effect of IL-35 on eosinophil apoptosis and adhesion was analyzed by flow cytometry. Transwell system was used for the migration assay. The eosinophil cationicprotein (ECP) from supernatant of eosinophils after IL-35stimulation was detected by enzyme-linked immunosorbent assay kits. The IL-35 protein levels were negatively correlated with eosinophil counts (p<.01) and ECP concentration (p<.01) in AR children. IL-35 promotes apoptosis and inhibits adhesion, migration, and activation of eosinophils. Moreover, the mRNA expression of IL-12 receptor β2 and glycoprotein 130 were significantly enhanced by eosinophils after IL-35stimulation. The apoptosis induced by IL-35 was mediated by phosphoinositide 3-kinase (PI3K) pathway. IL-35 inhibits adhesion of eosinophils through extracellular regulated proteinkinases (ERK) and PI3K pathways. The eosinophil chemotaxis and activation affected by IL-35 were mediated by PI3K and p38mitogen-activated proteinkinase (MAPK) pathways. Our results confirmed that IL-35 played inhibitive roles in apoptosis, adhesion, migration, and activation of eosinophils in AR, implying that IL-35may be used as treatment target in future.

Involvement of the α/β Isoform of p38 MAP Kinase in Chemotactic Responses of Human Eosinophils to Eotaxin (CCL11) and RANTES (CCL5)

Eosinophils are the principal effector cells for allergic inflammation in a variety of diseases, in which they contribute to tissue damage and remodelling processes via the secretion of cytotoxic granular proteins and cytokines. The intracellular mechanisms that control the activation, recruitment and survival of eosinophils are fundamental in understanding these disease processes. Phosphoinositide 3-kinase (PI3K) has been shown previously to be essential for eosinophil chemotactic responses to some stimuli but not others. Human blood neutrophils have been shown to utilize two antagonistic signalling pathways for chemotaxis: PI3K and p38 mitogen-activated protein kinase (p38 MAPK). In the present study, the role of p38 MAPK in chemotactic responses of an eosinophil-differentiated myeloid leukaemia cell line (EOL-1) and human peripheral blood eosinophils to a range of stimuli - platelet-activating factor (PAF), eotaxin 1 (CCL11), RANTES (CCL5), interleukin 8 (IL8, CXCL8) and IL16 - was explored through the use of the p38 MAPK α/β isoform inhibitor, SB 203580. SB 203580 caused significant inhibition of chemotactic responses of both EOL-1 cells and blood eosinophils to eotaxin 1 and RANTES (≥75% inhibition at 1 μM SB 203580, p<0.01) but had no effect on the migration induced by PAF and IL16 (<25%) and little or no effect on responses to IL8. Responses to PAF - but not eotaxin - have been shown previously to be suppressed by PI3K inhibition. The complementary pattern of inhibition observed in the present study provides evidence that distinct PI3K-dependent and p38 MAPK-dependent chemoattractants may also exist for eosinophils.

Combination therapy with potent PI3K and MAPK inhibitors overcomes adaptive kinome resistance to single agents in preclinical models of glioblastoma.

Glioblastoma (GBM) is the most common and aggressive primary brain tumor. Prognosis remains poor despite multimodal therapy. Developing alternative treatments is essential. Drugs targeting kinases within the phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) effectors of receptor tyrosine kinase (RTK) signaling represent promising candidates. We previously developed a non-germline genetically engineered mouse model of GBM in which PI3K and MAPK are activated via Pten deletion and KrasG12D in immortalized astrocytes. Using this model, we examined the influence of drug potency on target inhibition, alternate pathway activation, efficacy, and synergism of single agent and combination therapy with inhibitors of these 2 pathways. Efficacy was then examined in GBM patient-derived xenografts (PDX) in vitro and in vivo. PI3K and mitogen-activated protein kinase kinase (MEK) inhibitor potency was directly associated with target inhibition, alternate RTK effector activation, and efficacy in mutant murine astrocytes in vitro. The kinomes of GBM PDX and tumor samples were heterogeneous, with a subset of the latter harboring MAPK hyperactivation. Dual PI3K/MEK inhibitor treatment overcame alternate effector activation, was synergistic in vitro, and was more effective than single agent therapy in subcutaneous murine allografts. However, efficacy in orthotopic allografts was minimal. This was likely due to dose-limiting toxicity and incomplete target inhibition. Drug potency influences PI3K/MEK inhibitor-induced target inhibition, adaptive kinome reprogramming, efficacy, and synergy. Our findings suggest that combination therapies with highly potent, brain-penetrant kinase inhibitors will be required to improve patient outcomes.

C-Met as a Target for Personalized Therapy.

MET and its ligand HGF are involved in many biological processes, both physiological and pathological, making this signaling pathway an attractive therapeutic target in oncology. Downstream signaling effects are transmitted via mitogen-activated protein kinase (MAPK), PI3K (phosphoinositide 3-kinase protein kinase B)/AKT, signal transducer and activator of transcription proteins (STAT), and nuclear factor-κB. The final output of the terminal effector components of these pathways is activation of cytoplasmic and nuclear processes leading to increases in cell proliferation, survival, mobilization and invasive capacity. In addition to its role as an oncogenic driver, increasing evidence implicates MET as a common mechanism of resistance to targeted therapies including EGFR and VEGFR inhibitors. In the present review, we summarize the current knowledge on the role of the HGF-MET signaling pathway in cancer and its therapeutic targeting (HGF activation inhibitors, HGF inhibitors, MET antagonists and selective/nonselective MET kinase inhibitors). Recent advances in understanding the role of this pathway in the resistance to current anticancer strategies used in lung, kidney and pancreatic cancer are discussed.

Dietary Arginine Supplementation of Mice Alters the Microbial Population and Activates Intestinal Innate Immunity

Currently, little is known about the function of arginine in the homeostasis of the intestinal immune system. This study was conducted to test the hypothesis that dietary arginine supplementation may alter intestinal microbiota and innate immunity in mice. Mice were fed a basal diet (containing 0.93% L-arginine; grams per gram) or the basal diet supplemented with 0.5% L-arginine for 14 d. We studied the composition of intestinal microbiota, the activation of innate immunity, and the expression of toll-like receptors (Tlr s), proinflammatory cytokines, and antimicrobials in the jejunum, ileum, or colon of mice. Signal transduction pathway activation in the jejunum and ileum, including TLR4–nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), mitogen-activated protein kinase (MAPK), and phosphoinositide-3 kinase (PI3K)/PI3K–protein kinase B (Akt), was analyzed by Western blotting. Quantitative polymerase chain reaction analysis revealed that arginine supplementation induced (P < 0.05) a shift in the Firmicutes-to-Bacteroidetes ratio to favor Bacteroidetes in the jejunum (0.33 ± 0.04 vs. 1.0 ± 0.22) and ileum (0.20 ± 0.08 vs. 1.0 ± 0.27) compared with the control group. This finding coincided with greater (P < 0.05) activation of the innate immune system, including TLR signaling, as well as expression of proinflammatory cytokines, ​secretory immunoglobulin A, mucins, and Paneth antimicrobials in the jejunum and ileum. Finally, arginine supplementation reduced (P < 0.05) expression of the proteins for NF-κB, MAPK, and PI3K–Akt signaling pathways but activated (P < 0.05) p38 and c-Jun N-terminal protein kinase in the jejunum and the ileum, respectively. Collectively, dietary arginine supplementation of mice changes the intestinal microbiota, contributing to the activation of intestinal innate immunity through NF-κB, MAPK, and PI3K–phosphorylated Akt signaling pathways.

PI3K pathway dependencies in endometrioid endometrial cancer cell lines.

Endometrioid endometrial cancers (EEC) frequently harbor coexisting mutations in phosphoinositide 3-kinase (PI3K) pathway genes, including PTEN, PIK3CA, PIK3R1, and KRAS. We sought to define the genetic determinants of PI3K pathway inhibitor response in EEC cells, and whether PTEN-mutant EEC cell lines rely on p110β signaling for survival. Twenty-four human EEC cell lines were characterized for their mutation profile and activation state of PI3K and mitogen-activated protein kinase (MAPK) signaling pathway proteins. Cells were treated with pan-class I PI3K, p110α, and p110β isoform-specific, allosteric mTOR, mTOR kinase, dual PI3K/mTOR, mitogen-activated protein/extracellular signal-regulated kinase (MEK), and RAF inhibitors. RNA interference (RNAi) was used to assess effects of KRAS silencing in EEC cells. EEC cell lines harboring PIK3CA and PTEN mutations were selectively sensitive to the pan-class I PI3K inhibitor GDC-0941 and allosteric mTOR inhibitor temsirolimus, respectively. Subsets of EEC cells with concurrent PIK3CA and/or PTEN and KRAS mutations were sensitive to PI3K pathway inhibition, and only 2 of 6 KRAS-mutant cell lines showed response to MEK inhibition. KRAS RNAi silencing did not induce apoptosis in KRAS-mutant EEC cells. PTEN-mutant EEC cell lines were resistant to the p110β inhibitors GSK2636771 and AZD6482, and only in combination with the p110α selective inhibitor A66 was a decrease in cell viability observed. Targeted pan-PI3K and mTOR inhibition in EEC cells may be most effective in PIK3CA- and PTEN-mutant tumors, respectively, even in a subset of EECs concurrently harboring KRAS mutations. Inhibition of p110β alone may not be sufficient to sensitize PTEN-mutant EEC cells and combination with other targeted agents may be required.

C3orf58 , a Novel Paracrine Protein, Stimulates Cardiomyocyte Cell-Cycle Progression Through the PI3K–AKT–CDK7 Pathway

The regenerative capacity of the heart is markedly diminished shortly after birth, coinciding with overall withdrawal of cardiomyocytes from cell cycle. Consequently, the adult mammalian heart has limited capacity to regenerate after injury. The discovery of factors that can induce cardiomyocyte proliferation is, therefore, of high interest and has been the focus of extensive investigation throughout the past years. We have recently identified C3orf58 as a novel hypoxia and Akt induced stem cell factor (HASF) secreted from mesenchymal stem cells, which can promote cardiac repair through cytoprotective mechanisms. Here, we tested the hypothesis that HASF can also contribute to cardiac regeneration by stimulating cardiomyocyte division and proliferation. Neonatal ventricular cardiomyocytes were stimulated in culture for 7 days with purified recombinant HASF protein. Compared with control untreated cells, HASF-treated neonatal cardiomyocytes exhibited 60% increase in DNA synthesis as measured by bromodeoxyuridine incorporation. These results were confirmed by immunofluorescence confocal microscopy showing a 50% to 100% increase in the number of cardiomyocytes in the mitotic and cytokinesis phases. Importantly, in vivo cardiac overexpression of HASF in a transgenic mouse model resulted in enhanced level of DNA synthesis and cytokinesis in neonatal and adult cardiomyocytes. These proliferative effects were modulated by a phosphoinositide 3-kinase-protein kinase B-cycle-dependent kinase 7 pathway as revealed by the use of phosphoinositide 3-kinase -pathway-specific inhibitors and silencing of the Cdk7 gene. Our studies support the hypothesis that HASF induces cardiomyocyte proliferation via a phosphoinositide 3-kinase-protein kinase B-cycle-dependent kinase 7 pathway. The implications of this finding may be significant for cardiac regeneration biology and therapeutics.

Monoclonal antibody against the ectodomain of E-cadherin (DECMA-1) suppresses breast carcinogenesis: involvement of the HER/PI3K/Akt/mTOR and IAP pathways.

Although targeted therapies against HER2 have been one of the most successful therapeutic strategies for breast cancer, patients eventually developed acquired resistance from compensatory upregulation of alternate HERs and mitogen-activated protein kinase-phosphoinositide 3-kinase (PI3K)/Akt/mTOR signaling. As we and others have shown that the soluble ectodomain fragment of E-cadherin exerts prooncogenic effects via HER1/2-mediated binding and activation of downstream prosurvival pathways, we explored whether targeting this ectodomain [DECMA-1 monoclonal antibody (mAb)] was effective in the treatment of HER2-positive (HER2(+)) breast cancers. MMTV-PyMT transgenic mice and HER2(+)/E-cadherin-positive MCF-7 and BT474 trastuzumab-resistant (TtzmR) cells were treated with the DECMA-1 mAb. Antitumor responses were assessed by bromodeoxyuridine incorporation, apoptosis, and necrosis. The underlying intracellular prooncogenic pathways were explored using subcellular fractionation, immunoprecipitation, fluorescence microscopy, and immunoblotting. Treatment with DECMA-1 mAb significantly delayed tumor onset and attenuated tumor burden in MMTV-PyMT mice by reducing tumor cell proliferation and inducing apoptosis without any detectable cytotoxicity to mice or end-organs. In vitro treatment of MCF-7 and BT474 TtzmR cells reduced proliferation and induced cancer cell apoptosis. Importantly, this inhibition of breast tumorigenesis was due to concomitant downregulation, via ubiquitin-mediated degradation through the lysosome and proteasome pathways, of all HER family members, components of downstream PI3K/Akt/mTOR prosurvival signaling and suppression of inhibitor of apoptosis proteins. Our results establish that the E-cadherin ectodomain-specific mAb DECMA-1 inhibits Ecad(+)/HER2(+) breast cancers by hindering tumor growth and inducing apoptosis via downregulation of key oncogenic pathways involved in trastuzumab resistance, thereby establishing a novel therapeutic platform for the treatment of HER2(+) breast cancers.

Protein Kinase Activity of Phosphoinositide 3-Kinase Regulates Cytokine-Dependent Cell Survival

Author SummaryThe ability of cells to survive in the absence of proliferation (cell division), differentiation (cell maturation) or activation allows tissues to maintain cell populations that are poised for rapid responses to damage, infections, or other physiological demands. While this “survival-only” response is fundamental to all physiological processes, the underlying mechanisms are not understood. Many growth factors are potent regulators of cell survival through their ability to bind specific cell surface receptors, which in turn activate specialized enzymes called kinases. Phosphoinositide 3-kinase (PI3K) is a dual specificity kinase that is known to be involved in cell survival and malignant transformation, and it is able to phosphorylate both lipid and protein substrates. While the PI3K lipid kinase activity has been extensively studied, the functional significance of its protein kinase activity remains unclear. Here we show that PI3K protein kinase activity can directly phosphorylate growth factor receptors on human hematopoietic (blood) cells to promote a “survival-only” response. We further show that the protein kinase activity of PI3K can be hijacked to result in uncontrolled growth factor receptor phosphorylation and the deregulated survival of leukemic cells. Our studies provide the first evidence that the protein kinase activity of PI3K can control cell survival and that this activity may be deregulated in cancer.

Depolarization of Mitochondria in Endothelial Cells Promotes Cerebral Artery Vasodilation by Activation of Nitric Oxide Synthase

Mitochondrial depolarization after ATP-sensitive potassium channel activation has been shown to induce cerebral vasodilation by the generation of calcium sparks in smooth muscle. It is unclear, however, whether mitochondrial depolarization in endothelial cells is capable of promoting vasodilation by releasing vasoactive factors. Therefore, we studied the effect of endothelial mitochondrial depolarization by mitochondrial ATP-sensitive potassium channel activators, BMS-191095 (BMS) and diazoxide, on endothelium-dependent vasodilation. Diameter studies in isolated rat cerebral arteries showed BMS- and diazoxide-induced vasodilations that were diminished by endothelial denudation. Mitochondrial depolarization-induced vasodilation was reduced by inhibition of mitochondrial ATP-sensitive potassium channels, phosphoinositide-3 kinase, or nitric oxide synthase. Scavenging of reactive oxygen species, however, diminished vasodilation induced by diazoxide, but not by BMS. Fluorescence studies in cultured rat brain microvascular endothelial cells showed that BMS elicited mitochondrial depolarization and enhanced nitric oxide production; diazoxide exhibited largely similar effects, but unlike BMS, increased mitochondrial reactive oxygen species production. Measurements of intracellular calcium ([Ca(2+)]i) in cultured rat brain microvascular endothelial cells and arteries showed that both diazoxide and BMS increased endothelial [Ca(2+)]i. Western blot analyses revealed increased phosphorylation of protein kinase B and endothelial nitric oxide synthase (eNOS) by BMS and diazoxide. Increased phosphorylation of eNOS by diazoxide was abolished by phosphoinositide-3 kinase inhibition. Electron spin resonance spectroscopy confirmed vascular nitric oxide generation in response to diazoxide and BMS. Pharmacological depolarization of endothelial mitochondria promotes activation of eNOS by dual pathways involving increased [Ca(2+)]i as well as by phosphoinositide-3 kinase-protein kinase B-induced eNOS phosphorylation. Both mitochondrial reactive oxygen species-dependent and -independent mechanisms mediate activation of eNOS by endothelial mitochondrial depolarization.

Opposing Effects of Androgen Deprivation and Targeted Therapy on Prostate Cancer Prevention

Prostate cancer is an ideal target for chemoprevention. To date, chemoprevention clinical trials with 5α-reductase inhibitors have yielded encouraging yet ultimately confounding results. Using a preclinical mouse model of high-grade prostatic intraepithelial neoplasia (HG-PIN) induced by PTEN loss, we observed unprecedented deteriorating effects of androgen deprivation, in which surgical castration or MDV3100 treatment accelerated disease progression of the otherwise stable HG-PIN to invasive castration-resistant prostate cancer (CRPC). As an alternative, targeting the phosphoinositide 3-kinase (PI3K) signaling pathway via either genetic ablation of genes encoding PI3K components or pharmacologic inhibition of the PI3K pathway reversed the PTEN loss-induced HG-PIN phenotype. Finally, concurrent inhibition of the PI3K and mitogen-activated protein kinase (MAPK) pathways was effective in blocking the growth of PTEN-null CRPC. Together, these data have revealed the potential adverse effects of antiandrogen chemoprevention in certain genetic contexts (such as PTEN loss) while showing the promise of targeted therapy in the clinical management of this complex and prevalent disease. Chemoprevention with antiandrogen therapies is attractive for prostate cancer, given its prevalence and established hormonally mediated pathogenesis. However, because PTEN loss has been found in 9% to 45% of HG-PIN in the clinic, the current findings suggest that patients with PTEN-deficient prostate tumors might be better treated with PI3K-targeted therapies.

PI3Kγ inhibition reduces blood pressure by a vasorelaxant Akt/L-type calcium channel mechanism

The lipid and protein kinase phosphoinositide 3-kinase γ (PI3Kγ) is abundantly expressed in inflammatory cells and in the cardiovascular tissue. In recent years, its role in inflammation and in cardiac function and remodelling has been unravelled, highlighting the beneficial effects of its pharmacological inhibition. Furthermore, a role for PI3Kγ in the regulation of vascular tone has been emphasized. However, the impact of this signalling in the control of blood pressure is still poorly understood. Our study investigated the effect of a selective inhibition of PI3Kγ, obtained by using two independent small molecules, on blood pressure. Moreover, we dissected the molecular mechanisms involved in control of contraction of resistance arteries by PI3Kγ. We showed that inhibition of PI3Kγ reduced blood pressure in normotensive and hypertensive mice in a concentration-dependent fashion. This effect was dependent on enhanced vasodilatation, documented in vivo by decreased peripheral vascular resistance, and ex vivo by vasorelaxing effects on isolated resistance vessels. The vasorelaxation induced by PI3Kγ inhibition relied on blunted pressure-induced Akt phosphorylation and a myogenic contractile response. Molecular insights revealed that PI3Kγ inhibition affected smooth muscle L-type calcium channel current density and calcium influx by impairing plasma membrane translocation of the α1C L-type calcium channel subunit responsible for channel open-state probability. Overall our findings suggest that PI3Kγ inhibition could be a novel tool to modulate calcium influx in vascular smooth muscle cells, thus relaxing resistance arteries and lowering blood pressure.

PIKing the Right Patient

HER2 amplification and PIK3CA mutation were validated as biomarkers for sensitivity to the single-agent phosphoinositide 3-kinase (PI3K) inhibitor, GDC-0941, in breast cancer models. A novel expression profile was developed to identify other breast cancers sensitive to PI3K inhibitors. These expression studies highlighted feedback networks connecting TORC1, PI3K, and mitogen-activated protein kinase (MAPK) pathways, and underscored the potential for combination therapies.

Abstract C62: Identification of GSK2126458, a highly potent inhibitor of phosphoinositide 3-kinase (PI3K) and the mammalian target of rapamycin (mTOR)

Abstract Phosphoinositide 3-kinase (PI3K) is a critical regulator of cell growth and transformation and its signaling pathway is one of the most commonly mutated pathways in human cancer. The mammalian target of rapamycin (mTOR), a class IV PI3K protein kinase, is also a central regulator of cell growth, and mTOR inhibitors are believed to augment the antiproliferative efficacy of the PI3K/AKT pathway. GSK1059615, our first PI3K clinical compound, progressed to a dose escalation study in patients with refractory malignancies. Following the discovery of GSK1059615, we sought to identify a second inhibitor with improved potency, selectivity, and pharmacokinetics. Key to our approach to achieving the desired levels of PI3K activity was to pursue structure-based design utilizing crystallography of the more amenable PI3K as a surrogate protein. Following a chemistry lead optimization effort, the pyridylsulfonamide GSK2126458 was identified as a highly potent, orally bioavailable, pan-PI3K and mTOR inhibitor (PI3K app Ki = 19 pM; mTORC1 app Ki = 180 pM; mTORC2 app Ki = 300 pM). Consistent with potent PI3K and mTORC2 enzyme inhibition, GSK2126458 decreased cellular levels of phosphorylated AKT (BT474 pAKT IC50 = 180 pM) and inhibited cell proliferation in a large panel of cancer cell lines (e.g. BT474 growth IC50 = 2 nM). GSK2126458 showed good exposure in four pre-clinical animal species and exhibited in vivo activity in both pharmacodynamic and tumor growth efficacy models. GSK2126458 is being evaluated currently in human clinical trials for the treatment of cancer. The discovery, design, and optimization of GSK2126458 and related analogs will be presented. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C62.

AKT Mediates Platelet Activation by Stimulating Nitric Oxide Synthesis and cGMP Elevation.

Phosphoinositide 3-kinase (PI3-K) and Akt play important roles in platelet activation. However, the downstream mechanisms for their roles are unclear. We have recently shown that nitric oxide (NO) synthase 3 (NOS3) and cGMP-dependent protein kinase stimulate platelet secretion and aggregation. Here we show that PI3-K-mediated Akt activation plays a critical role in agonist-stimulated platelet NO synthesis and cGMP elevation. Agonist-induced elevation of NO and cGMP was inhibited by Akt inhibitors and reduced in Akt-1 knockout platelets. Akt-1 knockout or Akt inhibitor-treated platelets showed reduced platelet secretion and aggregation in response to low concentrations of agonists, which can be reversed by low concentrations of sodium nitroprusside (SNP) or cGMP analogs. Similarly, PI3-K inhibitors diminished elevation of cGMP and also inhibited platelet secretion and the second-wave platelet aggregation, which was also partially reversed by cGMP analogs and by SNP. These results indicate that the NO-cGMP pathway is an important downstream mechanism mediating PI3-K and Akt signals leading to platelet secretion and aggregation. Conversely, the PI3-K-Akt pathway is a major upstream mechanism responsible for activating the NO-cGMP pathway. Thus, this study delineates a novel platelet activation pathway involving sequential activation of PI3-K, Akt, NOS3, sGC, and cGMP-dependent protein kinase.