Akt/PKB activity is required for Ha-Ras-mediated transformation of intestinal epithelial cells

Phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB/Akt) is thought to serve as an oncogenic signaling pathway which can be activated by Ras. The role of PI3K/Akt in Ras-mediated transformation of intestinal epithelial cells is currently not clear. Here we demonstrate that inducible expression of oncogenic Ha-Ras results in activation of PKB/Akt in rat intestinal epithelial cells (RIE-iHa-Ras), which was blocked by treatment with inhibitors of PI3K activity. The PI3K inhibitor, LY-294002, partially reversed the morphological transformation induced by Ha-Ras and resulted in a modest stimulation of apoptosis. The most pronounced phenotypic alteration following inhibition of PI3K was induction of G(1) phase cell cycle arrest. LY-294002 blocked the Ha-Ras-induced expression of cyclin D1, cyclin-dependent kinase (CDK) 2, and increased the levels of p27(kip). Both LY-294002 and wortmannin significantly reduced anchorage-independent growth of RIE-iHa-Ras cells. Forced expression of both the constitutively active forms of Raf (DeltaRaf-22W or Raf BXB) and Akt (Akt-myr) resulted in transformation of RIE cells that was not achieved by transfection with either the Raf mutant construct or Akt-myr alone. These findings delineate an important role for PI3K/Akt in Ras-mediated transformation of intestinal epithelial cells.     

Dual Inhibition of PI3K/Akt Signaling and the DNA Damage Checkpoint in p53-Deficient Cells with Strong Survival Signaling: Implications for Cancer Therapy

Natural (intrinsic) resistance of many tumor types to DNA damaging agents is closely associated with their capacity to undergo robust cell cycle arrest in G2/M. G2 arrest is regulated by the DNA damage checkpoint and by survival signaling, with a potential role of PI3K/Akt in checkpoint function. In this work, we wanted to clarify if inhibition of multiple checkpoint/survival pathways may confer better efficacy in the potentiation of genotoxic agents compared to inhibition of either pathway alone. We compared the influence of UCN-01, which affects both the DNA damage checkpoint and PI3K/Akt-mediated survival signaling, with the PI3K inhibitors wortmannin and LY294002 in p53-deficient M1 acute myeloid leukemia cells treated with the DNA damaging agent cisplatin. Our results show that direct inhibition of PI3K/Akt in G2-arrested cells by wortmannin or LY294002 strongly enhanced the cytotoxicity of cisplatin without influencing the G2 checkpoint. Unexpectedly, dual inhibition of both survival and checkpoint signaling by UCN-01, also increased the cytotoxicity of cisplatin, but to a lesser degree than wortmannin or LY294002. The differences in cytotoxicity were accompanied by differences in cell death pathways: direct inhibition of PI3K/Akt was accompanied by rapid apoptotic cell death during G2, whereas cells underwent mitotic transit and cell division followed by cell death during G1 when both checkpoint and survival signaling were inhibited. Our results elucidate a novel function for PI3K/Akt as a survival factor during DNA damage-induced G2 arrest and could have important pharmacological consequences for the application of response modulators in p53-deficient tumors with strong survival signaling.     

Oncogenic Ras Blocks Anoikis by Activation of a Novel Effector Pathway Independent of Phosphatidylinositol 3-Kinase

Activated Ras, but not Raf, causes transformation of RIE-1 rat intestinal epithelial cells, demonstrating the importance of Raf-independent effector signaling in mediating Ras transformation. To further assess the contribution of Raf-dependent and Raf-independent function in oncogenic Ras transformation, we evaluated the mechanism by which oncogenic Ras blocks suspension-induced apoptosis, or anoikis, of RIE-1 cells. We determined that oncogenic versions of H-, K-, and N-Ras, as well as the Ras-related proteins TC21 and R-Ras, protected RIE-1 cells from anoikis. Surprisingly, our analyses of Ras effector domain mutants or constitutively activated effectors indicated that activation of Raf-1, phosphatidylinositol 3-kinase (PI3K), or RalGDS alone is not sufficient to promote Ras inhibition of anoikis. Treatment of Ras-transformed cells with the U0126 MEK inhibitor caused partial reversion to an anoikis-sensitive state, indicating that extracellular signal-regulated kinase activation contributes to inhibition of anoikis. Unexpectedly, oncogenic Ras failed to activate Akt, and treatment of Ras-transformed RIE-1 cells with the LY294002 PI3K inhibitor did not affect anoikis resistance or growth in soft agar. Thus, while important for Ras transformation of fibroblasts, PI3K may not be involved in Ras transformation of RIE-1 cells. Finally, inhibition of epidermal growth factor receptor kinase activity did not overcome Ras inhibition of anoikis, indicating that this autocrine loop essential for transformation is not involved in anoikis protection. We conclude that a PI3K- and RalGEF-independent Ras effector(s) likely cooperates with Raf to confer anoikis resistance upon RIE-1 cells, thus underscoring the complex nature by which Ras transforms cells.     

Syk activation of phosphatidylinositol 3-kinase/Akt prevents HtrA2-dependent loss of X-linked inhibitor of apoptosis protein (XIAP) to promote survival of Epstein-Barr virus+ (EBV+) B cell lymphomas

B cell lymphoma survival requires tonic or ligand-independent signals through activation of Syk by the B cell receptor. The Epstein-Barr virus (EBV) protein latent membrane 2a (LMP2a), a mimic of the B cell receptor, provides constitutive survival signals for latently infected cells through Syk activation; however, the precise downstream mechanisms coordinating this survival response in EBV+ B cell lymphomas remain to be elucidated. Herein, we assess the mechanism of Syk survival signaling in EBV+ B cell lymphomas from post-transplant lymphoproliferative disorder (PTLD) to discover virally controlled therapeutic targets involved in lymphomagenesis and tumor progression. Using small molecule inhibition and siRNA strategies, we show that Syk inhibition reduces proliferation and induces apoptosis of PTLD-derived EBV+ B cell lines. Syk inhibition also reduces autocrine IL-10 production. Although Syk inhibition attenuates signaling through both the PI3K/Akt and Erk pathways, only PI3K/Akt inhibition causes apoptosis of PTLD-derived cell lines. Loss of the endogenous caspase inhibitor XIAP is observed after Syk or PI3K/Akt inhibition. The loss of XIAP and apoptosis that results from Syk or PI3K/Akt inhibition is reversed by inhibition of the mitochondrial protease HtrA2. Thus, Syk drives EBV+ B cell lymphoma survival through PI3K/Akt activation, which prevents the HtrA2-dependent loss of XIAP. Syk, Akt, and XIAP antagonists may present potential new therapeutic strategies for PTLD through targeting of EBV-driven survival signals.     

A chemical screen in diverse breast cancer cell lines reveals genetic enhancers and suppressors of sensitivity to PI3K isoform-selective inhibition

The PI3K (phosphoinositide 3-kinase) pathway regulates cell proliferation, survival and migration and is consequently of great interest for targeted cancer therapy. Using a panel of small-molecule PI3K isoform-selective inhibitors in a diverse set of breast cancer cell lines, we have demonstrated that the biochemical and biological responses were highly variable and dependent on the genetic alterations present. p110alpha inhibitors were generally effective in inhibiting the phosphorylation of PKB (protein kinase B)/Akt and S6, two downstream components of PI3K signalling, in most cell lines examined. In contrast, p110beta-selective inhibitors only reduced PKB/Akt phosphorylation in PTEN (phosphatase and tensin homologue deleted on chromosome 10) mutant cell lines, and was associated with a lesser decrease in S6 phosphorylation. PI3K inhibitors reduced cell viability by causing cell-cycle arrest in the G(1) phase, with multi-targeted inhibitors causing the most potent effects. Cells expressing mutant Ras were resistant to the cell-cycle effects of PI3K inhibition, which could be reversed using inhibitors of Ras signalling pathways. Taken together, our data indicate that these compounds, alone or in suitable combinations, may be useful as breast cancer therapeutics, when used in appropriate genetic contexts.     

Impact of oncogenic driver mutations on feedback between the PI3K and MEK pathways in cancer cells

Inhibition of the PI3K (phosphoinositide 3-kinase)/Akt/mTORC1 (mammalian target of rapamycin complex 1) and Ras/MEK [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase]/ERK pathways for cancer therapy has been pursued for over a decade with limited success. Emerging data have indicated that only discrete subsets of cancer patients have favourable responses to these inhibitors. This is due to genetic mutations that confer drug insensitivity and compensatory mechanisms. Therefore understanding of the feedback mechanisms that occur with respect to specific genetic mutations may aid identification of novel biomarkers that predict patient response. In the present paper, we show that feedback between the PI3K/Akt/mTORC1 and Ras/MEK/ERK pathways is cell-line-specific and highly dependent on the activating mutation of K-Ras or overexpression c-Met. We found that cell lines exhibited differential signalling and apoptotic responses to PD184352, a specific MEK inhibitor, and PI103, a second-generation class I PI3K inhibitor. We reveal that feedback from the PI3K/Akt/mTORC1 to the Ras/MEK/ERK pathway is present in cancer cells harbouring either K-Ras activating mutations or amplification of c-Met but not the wild-type counterparts. Moreover, we demonstrate that inhibition of protein phosphatase activity by OA (okadaic acid) restored PI103-mediated feedback in wild-type cells. Together, our results demonstrate a novel mechanism for feedback between the PI3K/Akt/mTORC1 and the Ras/MEK/ERK pathways that only occurs in K-Ras mutant and c-Met amplified cells but not the isogenic wild-type cells through a mechanism that may involve inhibition of a specific endogenous phosphatase(s) activity. We conclude that monitoring K-Ras and c-Met status are important biomarkers for determining the efficacy of PI103 and other PI3K/Akt inhibitors in cancer therapy.     

PI3K pathway inhibitor LY294002 alters Jurkat T cell biobehaviours via ERK1/2-ICBP90 mediation

Little is known about whether there is a relationshipbetweenPI3K/AKT, ERK1/2 and an inverted CCAAT box binding protein (ICBP90) in biological behaviours of tumour cells. The aim of this study was to determine thisusing Jurkat T cells. Compared to PD98059 (an ERK1/2 signaling inhibitor), DAPT (a Notch signaling inhibitor) or adriamycin (a classical anti-tumour drug), the inhibition of Jurkat T cell growth by LY294002 (a PI3K/Akt signaling inhibitor) was more obvious. LY294002 combined with adriamycin appeared to have a synergistic effect. LY294002 strongly blocked Jurkat T cells at each phase of cell cycle with a decrease of DNA content, superior to adriamycin. Consistent with these changes, the expression of phosphorylated ERK1/2 was markedly decreased in the LY294002-treated Jurkat T cells, leading to the reduction of ICBP90 production, followed by moderate attenuation of TGF-β secretion. The results suggest that deactivation of PI3K/Akt signalling can surpress Jurkat T cell growth through inhibiting cell proliferation and blocking the cell cycle. ICBP90 may mediate the PI3K/AKT-ERK1/2 signalling to regulate leukemia cell growth.     

Requirement for phosphatidylinositol-3 kinase activity during progression through S-phase and entry into mitosis

Phosphatidylinositol-3 kinase (PI3K) proteins are important regulators of cell survival and proliferation. PI3K-dependent signalling regulates cell proliferation by promoting G1- to S-phase progression during the cell cycle. However, a definitive role for PI3K at other times during the cell cycle is less clear. In these studies, we provide evidence that PI3K activity is required during DNA synthesis (S-phase) and G2-phase of the cell cycle. Inhibition of PI3K with LY294002 at the onset of S-phase caused a 4- to 5-h delay in progression through G2/M. LY294002 treatment at the end of S-phase caused an approximate 2-h delay in progression through G2/M, indicating that PI3K activity functions for both S- and G2-phase progression. The expression of constitutively activated Akt partially reversed the inhibitory effects of LY294002 on mitotic entry, which demonstrated that Akt was one PI3K target that was required during G2/M transitions. Inhibition of PI3K resulted in enhanced susceptibility of G2/M synchronized cells to undergo apoptosis in response to DNA damage as compared to asynchronous cells. Thus, similar to its role in promoting cell survival and cell cycle transitions from G1 to S phase, PI3K activity appears to promote entry into mitosis and protect against cell death during S- and G2-phase progression.     

Genetic analysis of Ras signalling pathways in cell proliferation,migration and survival

We have used mouse embryonic fibroblasts (MEFs) devoid of Ras proteins to illustrate that they are essential for proliferation and migration, but not for survival, at least in these cells. These properties are unique to the Ras subfamily of proteins because ectopic expression of other Ras‐like small GTPases, even when constitutively active, could not compensate for the absence of Ras proteins. Only constitutive activation of components of the Raf/Mek/Erk pathway was sufficient to sustain normal proliferation and migration of MEFs devoid of Ras proteins. Activation of the phosphatidylinositol 3‐kinase (PI3K)/PTEN/Akt and Ral guanine exchange factor (RalGEF)/Ral pathways, either alone or in combination, failed to induce proliferation or migration of Rasless cells, although they cooperated with Raf/Mek/Erk signalling to reproduce the full response mediated by Ras signalling. In contrast to current hypotheses, Ras signalling did not induce proliferation by inducing expression of D‐type Cyclins. Rasless MEFs had normal levels of Cyclin D1/Cdk4 and Cyclin E/Cdk2. However, these complexes were inactive. Inactivation of the pocket proteins or knock down of pRb relieved MEFs from their dependence on Ras signalling to proliferate.     

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.     

Leishmania promastigotes activate PI3K/Akt signalling to confer host cell resistance to apoptosis

Previous reports have shown that cells infected with promastigotes of some Leishmania species are resistant to the induction of apoptosis. This would suggest that either parasites elaborate factors that block signalling from apoptosis inducers or that parasites engage endogenous host signalling pathways that block apoptosis. To investigate the latter scenario, we determined whether Leishmania infection results in the activation of signalling pathways that have been shown to mediate resistance to apoptosis in other infection models. First, we showed that infection with the promastigote form of Leishmania major, Leishmania pifanoi and Leishmania amazonensis activates signalling through p38 mitogen-activated protein kinase (MAPK), NFkappaB and PI3K/Akt. Then we found that inhibition of signalling through the PI3K/Akt pathway with LY294002 and Akt IV inhibitor reversed resistance of infected bone marrow-derived macrophages and RAW 264.7 macrophages to potent inducers of apoptosis. Moreover, reduction of Akt levels with small interfering RNAs to Akt resulted in the inability of infected macrophages to resist apoptosis. Further evidence of the role of PI3K/Akt signalling in the promotion of cell survival by infected cells was obtained with the finding that Bad, which is a substrate of Akt, becomes phosphorylated during the course of infection. In contrast to the observations with PI3K/Akt signalling, inhibition of p38 MAPK signalling with SB202190 or NFkappaB signalling with wedelolactone had limited effect on parasite-induced resistance to apoptosis. We conclude that Leishmania promastigotes engage PI3K/Akt signalling, which confers to the infected cell, the capacity to resist death from activators of apoptosis.     

The small GTPase N-Ras regulates extracellular matrix synthesis,proliferation and migration in fibroblasts

In addition to their role as oncogenes, Ras GTPases are key regulators of cell function. There is a proven relationship between the signaling pathways of transforming growth factor-β1 (TGF- β1) and Ras GTPases. Each of the Ras isoforms (H, N and K) exhibits specific modulatory activity on different cellular pathways. Our purpose has been to study some of the mechanisms involved in the development of renal fibrosis, assessing the individual role of N-Ras in basal and TGF-β1-mediated extracellular matrix (ECM) synthesis, proliferation, and migration in immortalized N-Ras deficient fibroblasts (N-ras^?/?). Compared to normal counterparts, fibroblasts deficient for N-Ras exhibited higher basal activity levels of phosphatidylinositol-3-kinase (PI3K)/Akt and MEK/Erk, accompanied by upregulated collagen synthesis and diminished proliferation and migration rates. We found that the absence of N-Ras did not affect TGF-β1-induced proliferation and migration, which required PI3K/Akt but not Erk1/2 activation. Similar effector pathway dependence was found for fibronectin and collagen type I expression.Our results indicate that N-Ras might contribute to renal fibrosis through the down-regulation of ECM synthesis and up-regulation proliferation and migration modulating Akt activation. N-Ras also regulates TGF-β1-induced collagen I and fibronectin expression through Erk-independent pathways.     

Intestinal epithelial cancer cell anoikis resistance: EGFR‐mediated sustained activation of Src overrides Fak‐dependent signaling to MEK/Erk and/or PI3‐K/Akt‐1

Herein, we investigated the survival roles of Fak, Src, MEK/Erk, and PI3‐K/Akt‐1 in intestinal epithelial cancer cells (HCT116, HT29, and T84), in comparison to undifferentiated and differentiated intestinal epithelial cells (IECs). We report that: (1) cancer cells display striking anoikis resistance, as opposed to undifferentiated/differentiated IECs; (2) under anoikis conditions and consequent Fak down‐activation, cancer cells nevertheless exhibit sustained Fak–Src interactions and Src/MEK/Erk activation, unlike undifferentiated/differentiated IECs; however, HCT116 and HT29 cells exhibit a PI3‐K/Akt‐1 down‐activation, as undifferentiated/differentiated IECs, whereas T84 cells do not; (3) cancer cells require MEK/Erk for survival, as differentiated (but not undifferentiated) IECs; however, T84 cells do not require Fak and HCT116 cells do not require PI3‐K/Akt‐1, in contrast to the other cells studied; (4) Src acts as a cornerstone in Fak‐mediated signaling to MEK/Erk and PI3‐K/Akt‐1 in T84 cells, as in undifferentiated IECs, whereas PI3‐K/Akt‐1 is Src‐independent in HCT116, HT29 cells, as in differentiated IECs; and (5) EGFR activity inhibition abrogates anoikis resistance in cancer cells through a loss of Fak–Src interactions and down‐activation of Src/MEK/Erk (T84, HCT116, HT29 cells) and PI3‐K/Akt‐1 (T84 cells). Hence, despite distinctions in signaling behavior not necessarily related to undifferentiated or differentiated IECs, intestinal epithelial cancer cells commonly display an EGFR‐mediated sustained activation of Src under anoikis conditions. Furthermore, such sustained Src activation confers anoikis resistance at least in part through a consequent sustenance of Fak–Src interactions and MEK/Erk activation, thus not only overriding Fak‐mediated signaling to MEK/Erk and/or PI3‐K/Akt‐1, but also the requirement of Fak and/or PI3‐K/Akt‐1 for survival. J. Cell. Biochem. 107: 639–654, 2009. © 2009 Wiley‐Liss, Inc.     

Phosphoinositide 3-kinases p110alpha and p110beta regulate cell cycle entry, exhibiting distinct activation kinetics in G1 phase

Phosphoinositide 3-kinase (PI3K) is an early signaling molecule that regulates cell growth and cell cycle entry. PI3K is activated immediately after growth factor receptor stimulation (at the G(0)/G(1) transition) and again in late G(1). The two ubiquitous PI3K isoforms (p110alpha and p110beta) are essential during embryonic development and are thought to control cell division. Nonetheless, it is presently unknown at which point each is activated during the cell cycle and whether or not they both control S-phase entry. We found that p110alpha was activated first in G(0)/G(1), followed by a minor p110beta activity peak. In late G(1), p110alpha activation preceded that of p110beta, which showed the maximum activity at this time. p110beta activation required Ras activity, whereas p110alpha was first activated by tyrosine kinases and then further induced by active Ras. Interference with p110alpha and -beta activity diminished the activation of downstream effectors with different kinetics, with a selective action of p110alpha in blocking early G(1) events. We show that inhibition of either p110alpha or p110beta reduced cell cycle entry. These results reveal that PI3Kalpha and -beta present distinct activation requirements and kinetics in G(1) phase, with a selective action of PI3Kalpha at the G(0)/G(1) phase transition. Nevertheless, PI3Kalpha and -beta both regulate S-phase entry.     

Involvement of H- and N-Ras isoforms in transforming growth factor-beta1-induced proliferation and in collagen and fibronectin synthesis

Transforming growth factor beta1 (TGF-beta1) has a relevant role in the origin and maintenance of glomerulosclerosis and tubule-interstitial fibrosis. TGF-beta and Ras signaling pathways are closely related: TGF-beta1 overcomes Ras mitogenic effects and Ras counteracts TGF-beta signaling. Tubule-interstitial fibrosis is associated to increases in Ras, Erk, and Akt activation in a renal fibrosis model. We study the role of N- and H-Ras isoforms, and the involvement of the Ras effectors Erk and Akt, in TGF-beta1-mediated extracellular matrix (ECM) synthesis and proliferation, using embrionary fibroblasts from double knockout (KO) mice for H- and N-Ras (H-ras(-/-)/N-ras(-/-)) isoforms and from heterozygote mice (H-ras(+/-)/N-ras(+/-)). ECM synthesis is increased in basal conditions in H-ras(-/-)/N-ras(-/-) fibroblasts, this increase being higher after stimulation with TGF-beta1. TGF-beta1-induced fibroblast proliferation is smaller in H-ras(-/-)/N-ras(-/-) than in H-ras(+/-)/N-ras(+/-) fibroblasts. Erk activation is decreased in H-ras(-/-)/N-ras(-/-) fibroblasts; inhibition of Erk activation reduces fibroblast proliferation. Akt activation is higher in double KO fibroblasts than in heterozygotes; inhibition of Akt activation also inhibits ECM synthesis. We suggest that H- and N-Ras isoforms downregulate ECM synthesis, and mediate proliferation, in part through MEK/Erk activation. PI3K-Akt pathway activation may be involved in the increase in ECM synthesis observed in the absence of H- and N-Ras.     

Signaling network crosstalk in human pluripotent cells: a Smad2/3-regulated switch that controls the balance between self-renewal and differentiation

A general mechanism for how intracellular signaling pathways in human pluripotent cells are coordinated and how they maintain self-renewal remain to be elucidated. In this report, we describe a signaling mechanism where PI3K/Akt activity maintains self-renewal by restraining prodifferentiation signaling through suppression of the Raf/Mek/Erk and canonical Wnt signaling pathways. When active, PI3K/Akt establishes conditions where Activin A/Smad2,3 performs a pro-self-renewal function by activating target genes, including Nanog. When PI3K/Akt signaling is low, Wnt effectors are activated and function in conjunction with Smad2,3 to promote differentiation. The switch in Smad2,3 activity after inactivation of PI3K/Akt requires the activation of canonical Wnt signaling by Erk, which targets Gsk3β. In sum, we define a signaling framework that converges on Smad2,3 and determines its ability to regulate the balance between alternative cell states. This signaling paradigm has far-reaching implications for cell fate decisions during early embryonic development.