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

The dual specificity protein/lipid kinase, phosphoinositide 3-kinase (PI3K), promotes growth factor-mediated cell survival and is frequently deregulated in cancer. However, in contrast to canonical lipid-kinase functions, the role of PI3K protein kinase activity in regulating cell survival is unknown. We have employed a novel approach to purify and pharmacologically profile protein kinases from primary human acute myeloid leukemia (AML) cells that phosphorylate serine residues in the cytoplasmic portion of cytokine receptors to promote hemopoietic cell survival. We have isolated a kinase activity that is able to directly phosphorylate Ser585 in the cytoplasmic domain of the interleukin 3 (IL-3) and granulocyte macrophage colony stimulating factor (GM-CSF) receptors and shown it to be PI3K. Physiological concentrations of cytokine in the picomolar range were sufficient for activating the protein kinase activity of PI3K leading to Ser585 phosphorylation and hemopoietic cell survival but did not activate PI3K lipid kinase signaling or promote proliferation. Blockade of PI3K lipid signaling by expression of the pleckstrin homology of Akt1 had no significant impact on the ability of picomolar concentrations of cytokine to promote hemopoietic cell survival. Furthermore, inducible expression of a mutant form of PI3K that is defective in lipid kinase activity but retains protein kinase activity was able to promote Ser585 phosphorylation and hemopoietic cell survival in the absence of cytokine. Blockade of p110α by RNA interference or multiple independent PI3K inhibitors not only blocked Ser585 phosphorylation in cytokine-dependent cells and primary human AML blasts, but also resulted in a block in survival signaling and cell death. Our findings demonstrate a new role for the protein kinase activity of PI3K in phosphorylating the cytoplasmic tail of the GM-CSF and IL-3 receptors to selectively regulate cell survival highlighting the importance of targeting such pathways in cancer.     

Cytokine-induced protein kinase B activation and Bad phosphorylation do not correlate with cell survival of hemopoietic cells.

Activation of phosphoinositide-3 kinases (PI3Ks), their downstream target protein kinase B (PKB), and phosphorylation of Bad have all been implicated in survival signaling in many systems. However, it is not known whether these events are sufficient or necessary to universally prevent apoptosis. To address this issue, we have used three different factor-dependent hemopoietic cell lines, MC/9, BaF/3, and factor-dependent (FD)-6, which respond to a range of cytokines, to investigate the relationship between PI3K, PKB, and Bad activity with survival. The cytokines IL-3, IL-4, stem cell factor (SCF), GM-CSF, and insulin all induced the rapid and transient activation of PKB in responsive cell lines. In all cases, cytokine-induced PKB activation was sensitive to inhibition by the PI3K inhibitor, LY294002. However, dual phosphorylation of the proapoptotic protein Bad was found not to correlate with PKB activation. In addition, we observed cell-type-specific differences in the ability of the same cytokine to induce Bad phosphorylation. Whereas IL-4 induced low levels of dual phosphorylation of Bad in FD-6, it was unable to in MC/9 or BaF/3. Insulin, which was the most potent inducer of PKB in FD-6, induced barely detectable Bad phosphorylation. In addition, the ability of a particular cytokine to induce PKB activity did not correlate with its ability to promote cell survival and/or proliferation. These data demonstrate that, in hemopoietic cells, activation of PKB does not automatically confer a survival signal or result in phosphorylation of Bad, implying that other survival pathways must be involved.     

Reciprocal regulation of Notch and PI3K/Akt signalling in T-ALL cells in vitro

Notch signalling plays an important role in hematopoiesis and in the pathogenesis of T-ALL. Notch is known to interact with Ras and PTEN/PI3K (phosphoinositide-3 kinase)/Akt pathways. We investigated the interaction of Notch with these pathways and the possible reciprocal regulation of these signalling systems in T-ALL cells in vitro. Our analyses indicate that the PI3K/Akt pathway is constitutively active in the four T-ALL cell lines tested. Akt phosphorylation was not altered by the sequestration of growth factors, that is, Akt activation seems to be less dependent on but not completely independent of growth factors, possibly being not subject to negative feedback regulation. PTEN expression was not detected in 3/4 cell lines tested, suggesting the loss of PTEN-mediated Akt activation. Inhibition of the PI3K/Akt pathway arrests growth and enhances apoptosis, but with no modulation of expression of Bax-alpha and Bcl-2 proteins. We analysed the relationship between Notch-1 and the PI3K/Akt signalling and show that inhibition of the Akt pathway changes Notch expression; Notch-1 protein decreased in all the cell lines upon treatment with the inhibitor. Our studies strongly suggest that Notch signalling interacts with PI3K/Akt signalling and further that this occurs in the absence of PTEN expression. The consequences of this to the signalling outcome are yet unclear, but we have uncovered a significant inverse relationship between Notch and PI3K/Akt pathway, which leads us to postulate the operation of a reciprocal regulatory loop between Notch and Ras-PI3K/Akt in the pathogenesis of T-ALL.     

Localization of Fas/CD95 into the lipid rafts on down-modulation of the phosphatidylinositol 3-kinase signaling pathway

Activation of the phosphatidylinositol 3-kinase (PI3K) signaling pathway is known to protect tumor cells from apoptosis and more specifically from the Fas-mediated apoptotic signal. The antitumoral agent edelfosine sensitizes leukemic cells to death by inducing the redistribution of the apoptotic receptor Fas into plasma membrane subdomains called lipid rafts. Herein, we show that inhibition of the PI3K signal by edelfosine triggers a Fas-mediated apoptotic signal independently of the Fas/FasL interaction. Furthermore, similarly to edelfosine, blockade of the PI3K activity, using specific inhibitors LY294002 and wortmannin, leads to the clustering of Fas whose supramolecular complex is colocalized within the lipid rafts. These findings indicate that the antitumoral agent edelfosine down-modulates the PI3K signal to sensitize tumor cells to death through the redistribution of Fas into large platform of membrane rafts.     

Varicella-zoster virus requires a functional PI3K/Akt/GSK-3alpha/beta signaling cascade for efficient replication

Successful replication of Varicella-zoster virus (VZV) relies upon strategies to counteract host defense mechanisms. This can be achieved by modulating host cell signaling pathways, which regulate apoptosis and cell survival. The Akt cascade is crucial for the regulation of cell survival since it controls factors such as Bad, FOXO1, mTor and GSK-3alpha/beta. These factors are involved in the regulation of cell death, cell cycle and translation. Here, we report i) that the VZV infection of MeWo cells caused a 9 to 18-fold increased phosphorylation of Akt. This phosphorylation was independent from PI3K inasmuch as the PI3K phosphorylation pattern differed strongly from the one of Akt. Bad, FOXO1 and mTor showed also variations in their phosphorylation patterns: phosphorylation of Bad (ser-136) decreased during the infection while phosphorylation of ser-2448 of mTor and of ser-256 of FOXO1 increased. The phosphorylation of GSK-3alpha/beta remained relatively stable during the infection. ii) Inhibition of PI3K, Akt or GSK-3alpha/beta prior to infection resulted in a severe decline of viral replication. The inhibition of Akt resulted also in an increased apoptotic response. iii) Transfection studies using plasmids coding for functional or inactive VZV protein kinases, pORFs 47 and 66, demonstrated an increase in Akt phosphorylation. Infection of MeWo cells with VZVDelta47 and VZVDelta66 resulted in a decline of Akt and GSK-3alpha/beta phosphorylation. These results suggest i) an essential role of PI3K/Akt/GSK-3alpha/beta signaling for a successful replication of VZV and ii) a key function of VZV kinases pORFs 47 and 66 to activate this pathway.     

ANG II induces apoptosis of human vascular smooth muscle via extrinsic pathway involving inhibition of Akt phosphorylation and increased FasL expression

In addition to well-documented vascular growth-promoting effects, ANG II exerts proapoptotic effects that are poorly understood. IGF-1 is a potent survival factor for human vascular smooth muscle cells (hVSMC), and its antiapoptotic effects are mediated via the IGF-1 receptor (IGF-1R) through a signaling pathway involving phosphatidylinositol 3-kinase and Akt. We hypothesized that there would be cross talk between ANG II proapoptotic effects and IGF-1 survival effects in hVSMC. To investigate ANG II-induced apoptosis and the potential involvement of IGF-1, we exposed quiescent and nonquiescent hVSMC to ANG II. ANG II induced apoptosis only in nonquiescent cells but stimulated hypertrophy in quiescent cells. ANG II-induced apoptosis was characterized by marked inhibition of Akt phosphorylation and stimulation of membrane Fas ligand (FasL) expression, caspase-8 activation, and a reduction in soluble FasL expression. Adenovirally mediated overexpression of Akt rescued hVSMC from ANG II-induced apoptosis. IGF-1R activation increased Akt phosphorylation and soluble FasL expression, and these effects were completely blocked by coincubating hVSMC with ANG II. In conclusion, ANG II-induced apoptosis of hVSMC is characterized by marked inhibition of Akt phosphorylation and stimulation of an extrinsic cell death signaling pathway via upregulation of membrane FasL expression, caspase-8 activation, and a reduction in soluble FasL expression. Furthermore, ANG II antagonizes the antiapoptotic effect of IGF-1 by blocking its ability to increase Akt phosphorylation and soluble FasL. These findings provide novel insights into ANG II-induced apoptotic signaling and have significant implication for understanding ANG II-induced remodeling in hypertension and atherosclerosis.     

Scutellarin exerts protective effects against atherosclerosis in rats by regulating the Hippo–FOXO3A and PI3K/AKT signaling pathways

Atherosclerosis (AS), a progressive disorder, is one of the tough challenges in the clinic. Scutellarin, an extract from Herba Erigerontis, is found to have oxygen-free radicals scavenging effects and antioxidant effects. In this study, we aimed to investigate the anti-AS effects of scutellarin is related to controlling the Hippo–FOXO3A and PI3K/AKT signal pathway. To establish an AS model, the rats in the scutellarin and model groups were intraperitoneally injected with vitamin D ₃ and then fed a high-fat diet for 12 weeks. In addition, in vitro angiotensin II-induced apoptosis of human aortic endothelial cells (HAECs) were used to establish models. Scutellarin significantly reduced blood lipid levels and increased antioxidase levels in both models. Additionally, scutellarin inhibited reactive oxygen species generation and apoptosis in HAECs. The impaired vascular barrier function was restored by using scutellarin in AS rats and in HAECs cells characterized by inhibiting mammalian sterile-20-like kinases 1 (Mst1) phosphorylation, Yes-associated protein (YAP) phosphorylation, forkhead box O3A (FOXO3A) phosphorylation at serine 207, nuclear translocation of FOXO3A, and upregulating protein expression of AKT and FOXO3A phosphorylation at serine 253. Scutellarin significantly reduced Bcl-2 interacting mediator of cell death (Bim), caspase-3, APO-1, CD95 (Fas), and Bax: Bcl-2-associated X (Bax) levels and activated Bcl-2: B-cell lymphoma-2 (Bcl-2). Scutellarin also significantly inhibited the expression of Mst1, YAP, FOXO3A at the messenger RNA level. When Mst1 was overexpressed or phosphoinositide 3-kinases suppressed, the effects of scutellarin were significantly blocked. In conclusion, the results of the present study suggest that scutellarin exerts protective effects against AS by inhibiting endothelial cell injury and apoptosis by regulating the Hippo–FOXO3A and PI3K/AKT signal pathways.     

Role of dephosphorylation of FOXO1 on apoptosis induced by wortmannin for non-Hodgkin’s lymphoma cells

The aim of this study was to ascertain the relationship between the phosphorylation of FOXO1 and the apoptosis and the proliferation of lymphoma cells so as to further clarify the cellular biology and pathogenesis of the disease. The lymphoma cells Namalwa and Jurkat were treated with PI3K inhibitor wortmannin or etoposide alone or wortmannin plus etoposide with different schedule. The inhibition rates of lymphoma cell growth were examined by XTT assay. Apoptosis were detected by flow cytometry. The expression of p-Akt, p-FOXO1, FOXO1, bim were determined by western blot analysis. Wortmannin induced apoptosis of Jurkat cells and Namalwa cells and inhibited their survival effectively. The rate of growth inhibition and apoptosis of lymphoma cells induced by wortmannin plus etoposide were higher than those induced by etoposide alone. After treated with wortmannin, phosphorylation of FOXO1 remarkably reduced and bim increased. The dephosphorylation of FOXO1 inhibited proliferation of Jurkat cells and Namalwa cells, promoted their apoptosis and sensitized Non-Hodgkin lymphoma cells to etoposide. Bim activated by FOXO1 promoted cells apoptosis.     

A Genome-Wide RNAi Screen Identifies FOXO4 as a Metastasis-Suppressor through Counteracting PI3K/AKT Signal Pathway in Prostate Cancer

Activation of the PI3K/AKT signal pathway is a known driving force for the progression to castration-recurrent prostate cancer (CR-CaP), which constitutes the major lethal phenotype of CaP. Here, we identify using a genomic shRNA screen the PI3K/AKT-inactivating downstream target, FOXO4, as a potential CaP metastasis suppressor. FOXO4 protein levels inversely correlate with the invasive potential of a panel of human CaP cell lines, with decreased mRNA levels correlating with increased incidence of clinical metastasis. Knockdown (KD) of FOXO4 in human LNCaP cells causes increased invasion in vitro and lymph node (LN) metastasis in vivo without affecting indices of proliferation or apoptosis. Increased Matrigel invasiveness was found by KD of FOXO1 but not FOXO3. Comparison of differentially expressed genes affected by FOXO4-KD in LNCaP cells in culture, in primary tumors and in LN metastases identified a panel of upregulated genes, including PIP, CAMK2N1, PLA2G16 and PGC, which, if knocked down by siRNA, could decrease the increased invasiveness associated with FOXO4 deficiency. Although only some of these genes encode FOXO promoter binding sites, they are all RUNX2-inducible, and RUNX2 binding to the PIP promoter is increased in FOXO4-KD cells. Indeed, the forced expression of FOXO4 reversed the increased invasiveness of LNCaP/shFOXO4 cells; the forced expression of FOXO4 did not alter RUNX2 protein levels, yet it decreased RUNX2 binding to the PIP promoter, resulting in PIP downregulation. Finally, there was a correlation between FOXO4, but not FOXO1 or FOXO3, downregulation and decreased metastasis-free survival in human CaP patients. Our data strongly suggest that increased PI3K/AKT-mediated metastatic invasiveness in CaP is associated with FOXO4 loss, and that mechanisms to induce FOXO4 re-expression might suppress CaP metastatic aggressiveness.