Targeting phosphoinositide-3-kinase pathway in biliary tract cancers: A remedial route?

Biliary tract cancers (BTC) are aggressive tumours with a low survival rate. At the advent of the genomic era, various genetic mutations in cell signalling pathways have been incriminated in carcinogenesis. Genomic analysis studies have connected main components of the phosphoinositide-3-kinase (PI3K) signalling pathway to BTC. PI3K pathway playing a central role in cell signalling and being deregulated in various tumours has been studied as a target for chemotherapy. Novel compounds have also been identified in preclinical trials that specifically target the PI3K pathway in BTCs, but these studies have not accelerated to clinical use. These novel compounds can be examined in upcoming studies to validate them as potential therapeutic agents, as further research is required to combat the growing need for adjuvant chemotherapy to successfully battle this tumour type. Furthermore, these molecules could also be used along with gemcitabine, cisplatin and 5-fluorouracil to improve sensitivity of the tumour tissue to chemotherapy. This review focuses on the basics of PI3K signalling, genetic alterations of this pathway in BTCs and current advancement in targeting this pathway in BTCs. It emphasizes the need for gene-based drug screening in BTC. It may reveal various novel targets and drugs for amelioration of survival in patients with BTC and serve as a stepping stone for further research.     

Activated Phosphatidylinositol 3-Kinase and Akt Kinase Promote Survival of Superior Cervical Neurons

The signaling pathways that mediate the ability of NGF to support survival of dependent neurons are not yet completely clear. However previous work has shown that the c-Jun pathway is activated after NGF withdrawal, and blocking this pathway blocks neuronal cell death. In this paper we show that over-expression in sympathetic neurons of phosphatidylinositol (PI) 3-kinase or its downstream effector Akt kinase blocks cell death after NGF withdrawal, in spite of the fact that the c-Jun pathway is activated. Yet, neither the PI 3-kinase inhibitor LY294002 nor a dominant negative PI 3-kinase cause sympathetic neurons to die if they are maintained in NGF. Thus, although NGF may regulate multiple pathways involved in neuronal survival, stimulation of the PI 3-kinase pathway is sufficient to allow cells to survive in the absence of this factor.     

Phosphatidylinositol 3′-kinase signalling supports cell height in established epithelial monolayers

Cell–cell interactions influence epithelial morphogenesis through an interplay between cell adhesion, trafficking and the cytoskeleton. These cellular processes are coordinated, often by cell signals found at cell–cell contacts. One such contact-based signal is the phosphatidylinositol 3′-kinase (PI3-kinase; PI3K) pathway. PI3-kinase is best understood for its role in mitogenic signalling, where it regulates cell survival, proliferation and differentiation. Its precise morphogenetic impacts in epithelia are, in contrast, less well-understood. Using phosphoinositide-specific biosensors we confirmed that E-cadherin-based cell–cell contacts are enriched in PIP₃, the principal product of PI3-kinase. We then used pharmacologic inhibitors to assess the morphogenetic impact of PI3-kinase in MDCK and MCF7 monolayers. We found that inhibiting PI3-kinase caused a reduction in epithelial cell height that was reversible upon removal of the drugs. This was not attributable to changes in E-cadherin expression or homophilic adhesion. Nor were there detectable changes in cell polarity. While Myosin II has been implicated in regulating keratinocyte height, we found no effect of PI3-kinase inhibition on apparent Myosin II activity; nor did direct inhibition of Myosin II alter epithelial height. Instead, in pursuing signalling pathways downstream of PI3-kinase we found that blocking Rac signalling, but not mTOR, reduced epithelial cell height, as did PI3-kinase inhibition. Overall, our findings suggest that PI3-kinase exerts a major morphogenetic impact in simple cultured epithelia through preservation of cell height. This is independent of potential effects on adhesion or polarity, but may occur through PI3-kinase-stimulated Rac signaling.     

Hepatocyte growth factor induced human trophoblast motility involves phosphatidylinositol-3-kinase,mitogen-activated protein kinase,and inducible nitric oxide synthase

Hepatocyte growth factor (HGF) increases human trophoblast motility and invasion, an effect which is abrogated when inducible nitric oxide synthase (iNOS) is inhibited. In this study we have investigated the pathways involved in the regulation of trophoblast motility. Both basal and HGF-stimulated motility of the extravillous trophoblast cell line, SGHPL-4, were inhibited in a dose-dependent manner by the phosphatidylinositol-3-kinase (PI3-kinase) inhibitor, LY294002. HGF-stimulated iNOS expression was also inhibited by LY294002 and direct activation of PI3-kinase, using the peptide 740Y-P, led to an increase in iNOS expression and cell motility. Pretreatment with rapamycin, which acts at a point distal to PI3-kinase activation, also inhibited basal and HGF-stimulated motility. Inhibition of the p42/p44 mitogen activated protein kinase (MAPK) pathway but not the p38 MAPK pathway had significant inhibitory effects on HGF-stimulated but not basal trophoblast motility. Inhibition of p42/p44 MAPK also inhibited HGF-induced iNOS expression. This data demonstrate that the PI3-kinase signaling pathway is involved in basal trophoblast motility and that both MAPK and PI3-kinase signaling pathways are important in HGF-stimulated motility and iNOS expression.     

CT-1 mediated cardioprotection against ischaemic re-oxygenation injury is mediated by PI3 kinase, Akt and MEK1/2 pathways.

Cardiotrophin-1 protects cardiac myocytes from ischaemic re-oxygenation (IR) injury. CT-1 activates MEK1/2,p42/44MAPK as well as the phosphatidylinositol (PI) 3-OH kinase (PI3) protein kinase B (PKB/Akt) pathway. In this study we investigate the signalling pathways that mediate the anti-apoptotic cell survival effect of CT-1 in IR. Dominant negative gene based inhibitors of MEK1/2, PI3-kinase and Akt inhibited CT-1 mediated cardioprotection in re-oxygenation as did chemical inhibitors of the PI3-kinase pathway. Hence the PI3-kinase/Akt pathway is required in addition to MEK1/2 to mediate CT-1 cardioprotection in IR.     

Insulin activates caspase-3 by a phosphatidylinositol 3'-kinase-dependent pathway

Activation of the caspase proteases by c-Jun N-terminal kinase 1 (JNK1) has been proposed as a mechanism of apoptotic cell death. Here we report that insulin activates caspase-3 by a pathway requiring phosphatidylinositol 3'-kinase (PI3-kinase). JNK1 assays demonstrated that insulin treatment of myeloma cells induced 3-fold activation of JNK1. Inhibition of PI3-kinase with wortmannin and LY294002 blocked insulin-dependent activation of JNK1. Caspase assays demonstrated that insulin increased caspase-3 activity 3-fold and that inhibition of PI3-kinase blocked this effect. Cell death was doubled by insulin and was due to a 3-fold increase in apoptosis of cells in the G1/G0 phase of the cell cycle. Inhibition of PI3-kinase completely blocked this effect. Finally, inhibition of caspase-3 with benzyloxycarbonyl-Asp-2,6-dichlorobenzoyloxymethylketone blocked cell death due to insulin. Taken together, these findings indicate that insulin activates caspase-3 by a PI3-kinase-dependent pathway resulting in increased apoptosis and cell death.     

Erythroid cells rendered erythropoietin independent by infection with Friend spleen focus-forming virus show constitutive activation of phosphatidylinositol 3-kinase and Akt kinase: involvement of insulin receptor substrate-related adapter proteins

The erythroleukemia-inducing Friend spleen focus-forming virus (SFFV) encodes a unique envelope glycoprotein which allows erythroid cells to proliferate and differentiate in the absence of erythropoietin (Epo). In an effort to understand how SFFV causes Epo independence, we have been examining erythroid cells rendered factor independent by SFFV infection for constitutive activation of signal-transducing molecules. Previous studies from our laboratory showed that various signal-transducing molecules known to be activated by Epo, including Stat proteins and components of the Raf-1/MAP kinase pathway, are constitutively activated in SFFV-infected erythroid cells in the absence of Epo. Since another signal transduction pathway involving activation of phosphatidylinositol 3-kinase (PI 3-kinase) after Epo stimulation plays an important role in erythroid cell proliferation and differentiation, we carried out studies to determine if this pathway was also activated in SFFV-infected cells in the absence of Epo. Our studies show that PI 3-kinase is constitutively activated in erythroid cells rendered factor independent by infection with SFFV and that PI 3-kinase activity, but not Epo receptor tyrosine phosphorylation, is required for the proliferation of these cells in the absence of Epo. We further show that in SFFV-infected erythroid cells grown in the absence of Epo, PI 3-kinase associates with the insulin receptor substrate (IRS)-related adapter molecules IRS-2, Gab1, and Gab2, which are constitutively tyrosine phosphorylated in SFFV-infected cells. Finally, Akt, a protein kinase that is one of the downstream effectors of PI 3-kinase, and SHIP, a lipid phosphatase that is important for Akt activation through PI 3-kinase, are both tyrosine phosphorylated in SFFV-infected cells grown in the absence of Epo. Our results indicate that induction of Epo independence by SFFV requires the activation of PI 3-kinase and suggest that constitutive activation of this kinase in SFFV-infected cells may occur primarily through interaction of PI 3-kinase with constitutively phosphorylated IRS-related adapter molecules.     

Substrate specificity of alpha(v)beta(3) integrin-mediated cell migration and phosphatidylinositol 3-kinase/AKT pathway activation

The alpha(v)beta(3) integrin has been shown to bind several ligands, including osteopontin and vitronectin. Its role in modulating cell migration and downstream signaling pathways in response to specific extracellular matrix ligands has been investigated in this study. Highly invasive prostate cancer PC3 cells that constitutively express alpha(v)beta(3) adhere and migrate on osteopontin and vitronectin in an alpha(v)beta(3)-dependent manner. However, exogenous expression of alpha(v)beta(3) in noninvasive prostate cancer LNCaP (beta(3)-LNCaP) cells mediates adhesion and migration on vitronectin but not on osteopontin. Activation of alpha(v)beta(3) by epidermal growth factor stimulation is required to mediate adhesion to osteopontin but is not sufficient to support migration on this substrate. We show that alpha(v)beta(3)-mediated cell migration requires activation of the phosphatidylinositol 3-kinase (PI 3-kinase)/protein kinase B (PKB/AKT) pathway since wortmannin, a PI 3-kinase inhibitor, prevents PC3 cell migration on both osteopontin and vitronectin; furthermore, alpha(v)beta(3) engagement by osteopontin and vitronectin activates the PI 3-kinase/AKT pathway. Migration of beta(3)-LNCaP cells on vitronectin also occurs through activation of the PI 3-kinase pathway; however, AKT phosphorylation is not increased upon engagement by osteopontin. Furthermore, phosphorylation of focal adhesion kinase (FAK), known to support cell migration in beta(3)-LNCaP cells, is detected on both substrates. Thus, in PC3 cells, alpha(v)beta(3) mediates cell migration and PI 3-kinase/AKT pathway activation on vitronectin and osteopontin; in beta(3)-LNCaP cells, alpha(v)beta(3) mediates cell migration and PI 3-kinase/AKT pathway activation on vitronectin, whereas adhesion to osteopontin does not support alpha(v)beta(3)-mediated cell migration and PI 3-kinase/AKT pathway activation. We conclude therefore that alpha(v)beta(3) exists in multiple functional states that can bind either selectively vitronectin or both vitronectin and osteopontin and that can differentially activate cell migration and intracellular signaling pathways in a ligand-specific manner.     

Convergence of signaling pathways on the activation of ERK in B cells

The B cell receptor (BCR) initiates three major signaling pathways: the Ras pathway, which leads to extracellular signal-regulated kinase (ERK) activation; the phospholipase C-gamma pathway, which causes calcium mobilization; and the phosphoinositide 3-kinase (PI 3-kinase) pathway. These combine to induce different biological responses depending on the context of the BCR signal. Both the Ras and PI 3-kinase pathways are important for B cell development and activation. Several model systems show evidence of cross-regulation between these pathways. Here we demonstrate through the use of PI 3-kinase inhibitors and a dominant-negative PI 3-kinase construct that the BCR-induced phosphorylation and activation of ERK is dependent on PI 3-kinase. PI 3-kinase feeds into the Ras signaling cascade at multiple points, both upstream and downstream of Ras. We also show that ERK activation is dependent on phospholipase C-gamma, in keeping with its dependence on calcium mobilization. Last, the activation of PI 3-kinase itself is completely dependent on Ras. We conclude that the PI 3-kinase and Ras signaling cascades are intimately connected in B cells and that the activation of ERK is a signal integration point, since it requires simultaneous input from all three major signaling pathways.     

Regulation of TRAIL expression by the phosphatidylinositol 3-kinase/Akt/GSK-3 pathway in human colon cancer cells

The intestinal mucosa is a rapidly-renewing tissue characterized by cell proliferation, differentiation, and eventual apoptosis with progression up the vertical gut axis. The inhibition of phosphatidylinositol (PI) 3-kinase by specific chemical inhibitors or overexpression of the lipid phosphatase PTEN enhances enterocyte-like differentiation in human colon cancer cell models of intestinal differentiation. In this report, we examined the role of PI 3-kinase inhibition in the regulation of apoptotic gene expression in human colon cancer cell lines HT29, HCT-116, and Caco-2. Inhibition of PI 3-kinase with the chemical inhibitor wortmannin increased TNF-related apoptosis-inducing ligand (TRAIL; Apo2) mRNA and protein expression. Similarly, overexpression of the tumor suppressor protein PTEN, an antagonist of PI 3-kinase signaling, resulted in the increased expression of TRAIL. Activation of PI 3-kinase by pretreatment with IGF-1, a gut trophic factor, markedly attenuated the induction of TRAIL by wortmannin. Moreover, overexpression of active Akt, a downstream target of PI 3-kinase, or inhibition of GSK-3, a downstream target of active Akt, completely blocked the induction of TRAIL by wortmannin. Consistent with findings that TRAIL is induced by agents that enhance intestinal cell differentiation, TRAIL expression was specifically localized to the differentiated cells of the colon and small bowel. Adenovirus-mediated overexpression of TRAIL increased DNA fragmentation of HCT-116 cells, demonstrating the functional activity of TRAIL induction. Taken together, our findings demonstrate induction of the TRAIL by inhibition of PI 3-kinase in colon cancer cell lines. These results identify TRAIL, a novel TNF family member, as a downstream target of the PI 3-kinase/Akt/GSK-3 pathway and may have important implications for better understanding the role of the PI 3-kinase pathway in intestinal cell homeostasis.     

Ceramide regulates lipopolysaccharide-induced phosphatidylinositol 3-kinase and Akt activity in human alveolar macrophages.

The phosphatidylinositol (PI) 3-kinase pathway is an important regulator of cell survival. In human alveolar macrophages, we found that LPS activates PI 3-kinase and its downstream effector, Akt. LPS exposure of alveolar macrophages also results in the generation of ceramide. Because ceramide exposure induces apoptosis in other cell types and the PI 3-kinase pathway is known to inhibit apoptosis, we determined the relationship between LPS-induced ceramide and PI 3-kinase activation in alveolar macrophages. We found that ceramide exposure activated PI 3-kinase and Akt. When we blocked LPS-induced ceramide with the inhibitor D609, we blocked LPS-induced PI 3-kinase and Akt activation. Evaluating cell survival after ceramide or LPS exposure, we found that blocking PI 3-kinase induced a significant increase in cell death. Because these effects of PI 3-kinase inhibition were more pronounced in ceramide- vs LPS-treated alveolar macrophages, we also evaluated NF-kappaB, which has also been linked to cell survival. We found that LPS, to a greater degree than ceramide, induced NF-kappaB translocation to the nucleus. As a composite, these studies suggest that the effects of ceramide exposure in alveolar macrophages may be very different from the effects described for other cell types. We believe that LPS induction of ceramide results in PI 3-kinase activation and represents a novel effector mechanism that promotes survival of human alveolar macrophages in the setting of pulmonary sepsis.     

Identification of Akt pathway inhibitors using redistribution screening on the FLIPR and the IN Cell 3000 analyzer

The PI3-kinase/Akt pathway is an important cell survival pathway that is deregulated in the majority of human cancers. Despite the apparent druggability of several kinases in the pathway, no specific catalytic inhibitors have been reported in the literature. The authors describe the development of a fluorometric imaging plate reader (FLIPR)-based Akt1 translocation assay to discover inhibitors of Akt1 activation. Screening of a diverse chemical library of 45,000 compounds resulted in identification of several classes of Akt1 translocation inhibitors. Using a combination of classical in vitro assays and translocation assays directed at different steps of the Akt pathway, the mechanisms of action of 2 selected chemical classes were further defined. Protein translocation assays emerge as powerful tools for hit identification and characterization.     

Cell Activation-Induced Phosphoinositide 3-Kinase Alpha/Beta Dimerization Regulates PTEN Activity

The phosphoinositide 3-kinase (PI3K)/PTEN (phosphatase and tensin homolog) pathway is one of the central routes that enhances cell survival, division, and migration, and it is frequently deregulated in cancer. PI3K catalyzes formation of phosphatidylinositol 3,4,5-triphosphate [PI(3,4,5)P₃] after cell activation; PTEN subsequently reduces these lipids to basal levels. Activation of the ubiquitous p110α isoform precedes that of p110β at several points during the cell cycle. We studied the potential connections between p110α and p110β activation, and we show that cell stimulation promotes p110α and p110β association, demonstrating oligomerization of PI3K catalytic subunits within cells. Cell stimulation also promoted PTEN incorporation into this complex, which was necessary for PTEN activation. Our results show that PI3Ks dimerize in vivo and that PI3K and PTEN activities modulate each other in a complex that controls cell PI(3,4,5)P₃ levels.     

Phosphatidylinositol 3-kinase and protein kinase D1 specifically cooperate to negatively regulate the insulin-like growth factor signaling pathway

Insulin receptor substrate-1 (IRS-1) is a key protein in the insulin-like growth factor (IGF) signaling whose tyrosine phosphorylation by the type 1 IGF receptor is necessary for the recruitment and activation of the downstream effectors. Through the analysis of cross-talks occurring between different tyrosine kinase receptor-dependent signaling pathways, we investigated how two growth factors [epidermal growth factor (EGF) and fibroblast growth factor (FGF)] could modulate the IGF-I-induced IRS-1 tyrosine phosphorylation and its downstream signaling. EGF and FGF inhibited IGF-I-stimulated tyrosine phosphorylation of IRS-1 and the subsequent IGF-I-induced phosphatidylinositol 3-kinase (PI 3-kinase) activity. These EGF- and FGF-inhibitory effects were dependent on both PI 3-kinase and protein kinase D1 (PKD1) signaling pathways but independent on the extracellular signal-regulated kinase (ERK) pathway. PKD1, which was activated independently of the PI 3-kinase pathway, associated with IRS-1 in response to EGF or FGF. Unlike PI 3-kinase, PKD1 did not mediate the EGF- or FGF-induced-IRS-1 serine 307 phosphorylation which was described to inhibit IRS-1. Interestingly, specific inhibition of either PI 3-kinase or PKD1 totally impaired EGF- or FGF-induced inhibition of IGF-I-stimulated IRS-1 tyrosine phosphorylation. This indicated that serine 307 phosphorylation of IRS-1 is not sufficient per se to inhibit the IGF signaling pathway and demonstrated for the first time that the negative regulation of IRS-1 requires the coordinated action of PI 3-kinase and PKD1. This further suggests that PKD1 may be an attractive target for innovative strategies that target the IGF signaling pathway.     

Temporal Changes in PTEN and mTORC2 Regulation of Hematopoietic Stem Cell Self-Renewal and Leukemia Suppression

Pten deletion from adult mouse hematopoietic cells activates the PI3-kinase pathway, inducing hematopoietic stem cell (HSC) proliferation, HSC depletion, and leukemogenesis. Pten is also mutated in human leukemias, but rarely in early childhood leukemias. We hypothesized that this reflects developmental changes in PI3-kinase pathway regulation. Here we show that Rictor deletion prevents leukemogenesis and HSC depletion after Pten deletion in adult mice, implicating mTORC2 activation in these processes. However, Rictor deletion had little effect on the function of normal HSCs. Moreover, Pten deletion from neonatal HSCs did not activate the PI3-kinase pathway or promote HSC proliferation, HSC depletion, or leukemogenesis. Pten is therefore required in adult, but not neonatal, HSCs to negatively regulate mTORC2 signaling. This demonstrates that some critical tumor suppressor mechanisms in adult cells are not required by neonatal cells. Developmental changes in key signaling pathways therefore confer temporal changes upon stem cell self-renewal and tumor suppressor mechanisms.     

Induction of apoptosis by stomach cancer-associated protein-tyrosine phosphatase-1

Stomach cancer-associated protein-tyrosine phosphatase-1 (SAP-1), a transmembrane-type protein-tyrosine phosphatase, is thought to inhibit integrin signaling by mediating the dephosphorylation of focal adhesion-associated proteins. Adenovirus-mediated overexpression of wild-type SAP-1, but not that of a catalytically inactive mutant of this enzyme, has now been shown to induce apoptosis in NIH 3T3 fibroblasts. This effect of SAP-1 was dependent on cellular caspase activities and was preceded by inactivation of two serine-threonine protein kinases, Akt and integrin-linked kinase (ILK), both of which function downstream of phosphoinositide (PI) 3-kinase to promote cell survival. Coexpression of constitutively active forms of PI 3-kinase or Akt (which fully restored Akt and ILK activities) resulted in partial inhibition of SAP-1-induced cell death. Furthermore, expression of a dominant negative mutant of PI 3-kinase did not induce cell death as efficiently as did SAP-1, although this mutant inhibited Akt and ILK activities more effectively than did SAP-1. Overexpression of SAP-1 had no substantial effect on Ras activity. These results suggest that SAP-1 induces apoptotic cell death by at least two distinct mechanisms: inhibition of cell survival signaling mediated by PI 3-kinase, Akt, and ILK and activation of a caspase-dependent proapoptotic pathway.     

Cyclin-dependent kinase-5 is involved in neuregulin-dependent activation of phosphatidylinositol 3-kinase and Akt activity mediating neuronal survival

The phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway plays an important role in mediating survival signals in wide variety of neurons and cells. Recent studies show that Akt also regulates metabolic pathways to regulate cell survival. In this study, we reported that cyclin-dependent kinase-5 (Cdk5) regulates Akt activity and cell survival through the neuregulin-mediated PI 3-kinase signaling pathway. We found that brain extracts of Cdk5-/-mice display a lower PI 3-kinase activity and phosphorylation of Akt compared with that in wild type mice. Moreover, we demonstrated that Cdk5 phosphorylated Ser-1176 in the neuregulin receptor ErbB2 and phosphorylated Thr-871 and Ser-1120 in the ErbB3 receptor. We identified the Ser-1120 sequence RSRSPR in ErbB3 as a novel phosphorylation consensus sequence of Cdk5. Finally, we found that Cdk5 activity is involved in neuregulin-induced Akt activity and neuregulin-mediated neuronal survival. These findings suggest that Cdk5 may exert a key role in promoting neuronal survival by regulating Akt activity through the neuregulin/PI 3-kinase signaling pathway.     

Recruitment of phosphoinositide 3-kinase defines a positive contribution of tyrosine kinase signaling to E-cadherin function

Classical cadherin adhesion molecules can function as adhesion-activated cell-signaling receptors. One key target for cadherin signaling is the lipid kinase phosphoinositide (PI) 3-kinase, which is recruited to cell-cell contacts and activated by E-cadherin. In this study, we sought to identify upstream factors necessary for E-cadherin to activate PI 3-kinase signaling. We found that inhibition of tyrosine kinase signaling blocked recruitment of PI 3-kinase to E-cadherin contacts and abolished the ability of E-cadherin to activate PI 3-kinase signaling. Tyrosine kinase inhibitors further perturbed several parameters of cadherin function, including cell adhesion and the ability of cells to productively extend nascent cadherin-adhesive contacts. Notably, the functional effects of tyrosine kinase blockade were rescued by expression of a constitutively active form of PI 3-kinase that restores PI 3-kinase signaling. Finally, using dominant negative Src mutants and Src-null cells, we identified Src as one key upstream kinase in the E-cadherin/PI 3-kinase-signaling pathway. Taken together, our findings indicate that tyrosine kinase activity, notably Src signaling, can contribute positively to cadherin function by supporting E-cadherin signaling to PI 3-kinase.