Mcl-1 is a key regulator of apoptosis resistance in Chlamydia trachomatis-infected cells

Chlamydia are obligate intracellular bacteria that cause variety of human diseases. Host cells infected with Chlamydia are protected against many different apoptotic stimuli. The induction of apoptosis resistance is thought to be an important immune escape mechanism allowing Chlamydia to replicate inside the host cell. Infection with C. trachomatis activates the Raf/MEK/ERK pathway and the PI3K/AKT pathway. Here we show that inhibition of these two pathways by chemical inhibitors sensitized C. trachomatis infected cells to granzyme B-mediated cell death. Infection leads to the Raf/MEK/ERK-mediated up-regulation and PI3K-dependent stabilization of the anti-apoptotic Bcl-2 family member Mcl-1. Consistently, interfering with Mcl-1 up-regulation sensitized infected cells for apoptosis induced via the TNF receptor, DNA damage, granzyme B and stress. Our data suggest that Mcl-1 up-regulation is primarily required to maintain apoptosis resistance in C. trachomatis-infected cells.     

The phosphoinositide 3-OH kinase/AKT2 pathway as a critical target for farnesyltransferase inhibitor-induced apoptosis

Farnesyltransferase inhibitors (FTIs) represent a novel class of anticancer drugs that exhibit a remarkable ability to inhibit malignant transformation without toxicity to normal cells. However, the mechanism by which FTIs inhibit tumor growth is not well understood. Here, we demonstrate that FTI-277 inhibits phosphatidylinositol 3-OH kinase (PI 3-kinase)/AKT2-mediated growth factor- and adhesion-dependent survival pathways and induces apoptosis in human cancer cells that overexpress AKT2. Furthermore, overexpression of AKT2, but not oncogenic H-Ras, sensitizes NIH 3T3 cells to FTI-277, and a high serum level prevents FTI-277-induced apoptosis in H-Ras- but not AKT2-transformed NIH 3T3 cells. A constitutively active form of AKT2 rescues human cancer cells from FTI-277-induced apoptosis. FTI-277 inhibits insulin-like growth factor 1-induced PI 3-kinase and AKT2 activation and subsequent phosphorylation of the proapoptotic protein BAD. Integrin-dependent activation of AKT2 is also blocked by FTI-277. Thus, a mechanism for FTI inhibition of human tumor growth is by inducing apoptosis through inhibition of PI 3-kinase/AKT2-mediated cell survival and adhesion pathway.     

Identification of nonsteroidal anti-inflammatory drug-activated gene (NAG-1) as a novel downstream target of phosphatidylinositol 3-kinase/AKT/GSK-3beta pathway

The signaling pathway of phosphatidylinositol 3-kinase (PI3K)/AKT, which is involved in cell survival, proliferation, and growth, has become a major focus in targeting cancer therapeutics. Nonsteroidal anti-inflammatory drug-activated gene (NAG-1) was previously identified as a gene induced by several anti-tumorigenic compounds including nonsteroidal anti-inflammatory drugs, peroxisome proliferator-activated receptor gamma ligands, and dietary compounds. NAG-1 has been shown to exhibit anti-tumorigenic and/or pro-apoptotic activities in vivo and in vitro. In this report, we showed a PI3K/AKT/glycogen synthase kinase-3beta (GSK-3beta) pathway regulates NAG-1 expression in human colorectal cancer cells as assessed by the inhibition of PI3K, AKT, and GSK-3beta. PI3K inhibition by LY294002 showed an increase in NAG-1 protein and mRNA expression, and 1l-6-hydroxymethyl-chiro-inositol 2(R)-2-O-methyl-3-O-octadecylcarbonate (AKT inhibitor) also induced NAG-1 expression. LY294002 caused increased apoptosis, cell cycle, and cell growth arrest in HCT-116 cells. Inhibition of GSK-3beta, which is negatively regulated by AKT, using AR-A014418 and lithium chloride completely abolished LY294002-induced NAG-1 expression as well as the NAG-1 promoter activity. Furthermore, the down-regulation of GSK-3 gene using small interference RNA resulted in a decline of the NAG-1 expression in the presence of LY294002. These data suggest that expression of NAG-1 is regulated by PI3K/AKT/GSK-3beta pathway in HCT-116 cells and may provide a further understanding of the important role of PI3K/AKT/GSK-3beta pathway in tumorigenesis.     

Mammalian 14-3-3beta associates with the Chlamydia trachomatis inclusion membrane via its interaction with IncG

Chlamydiae replicate intracellularly within a vacuole that is modified early in infection to become fusogenic with a subset of exocytic vesicles. We have recently identified four chlamydial inclusion membrane proteins, IncD-G, whose expression is detected within the first 2 h after internalization. To gain a better understanding of how these Inc proteins function, a yeast two-hybrid screen was employed to identify interacting host proteins. One protein, 14-3-3beta, was identified that interacted specifically with IncG. The interaction between 14-3-3beta and IncG was confirmed in infected HeLa cells by indirect immunofluorescence microscopy and interaction with a GFP-14-3-3beta fusion protein. 14-3-3 proteins are phosphoserine-binding proteins. Immunoprecipitation studies with [32P]-orthophosphate-labelled cells demonstrated that IncG is phosphorylated in both chlamydia-infected HeLa cells and in yeast cells expressing IncG. Site-directed mutagenesis of predicted 14-3-3 phosphorylation sites demonstrated that IncG binds to 14-3-3beta via a conserved 14-3-3-binding motif (RS164RS166F). Finally, indirect immunofluorescence demonstrated that 14-3-3beta interacts with Chlamydia trachomatis inclusions but not C. psittaci or C. pneumoniae inclusions. 14-3-3beta is the first eukaryotic protein found to interact with the chlamydial inclusion; however, its unique role in C. trachomatis pathogenesis remains to be determined.     

Apoptosis suppression by Raf-1 and MEK1 requires MEK- and phosphatidylinositol 3-kinase-dependent signals

Two Ras effector pathways leading to the activation of Raf-1 and phosphatidylinositol 3-kinase (PI3K) have been implicated in the survival signaling by the interleukin 3 (IL-3) receptor. Analysis of apoptosis suppression by Raf-1 demonstrated the requirement for mitochondrial translocation of the kinase in this process. This could be achieved either by overexpression of the antiapoptotic protein Bcl-2 or by targeting Raf-1 to the mitochondria via fusion to the mitochondrial protein Mas p70. Mitochondrially active Raf-1 is unable to activate extracellular signal-related kinase 1 (ERK1) and ERK2 but suppresses cell death by inactivating the proapoptotic Bcl-2 family member BAD. However, genetic and biochemical data also have suggested a role for the Raf-1 effector module MEK-ERK in apoptosis suppression. We thus tested for MEK requirement in cell survival signaling using the interleukin 3 (IL-3)-dependent cell line 32D. MEK is essential for survival and growth in the presence of IL-3. Upon growth factor withdrawal the expression of constitutively active MEK1 mutants significantly delays the onset of apoptosis, whereas the presence of a dominant negative mutant accelerates cell death. Survival signaling by MEK most likely results from the activation of ERKs since expression of a constitutively active form of ERK2 was as effective in protecting NIH 3T3 fibroblasts against doxorubicin-induced cell death as oncogenic MEK. The survival effect of activated MEK in 32D cells is achieved by both MEK- and PI3K-dependent mechanisms and results in the activation of PI3K and in the phosphorylation of AKT. MEK and PI3K dependence is also observed in 32D cells protected from apoptosis by oncogenic Raf-1. Additionally, we also could extend these findings to the IL-3-dependent pro-B-cell line BaF3, suggesting that recruitment of MEK is a common mechanism for survival signaling by activated Raf. Requirement for the PI3K effector AKT in this process is further demonstrated by the inhibitory effect of a dominant negative AKT mutant on Raf-1-induced cell survival. Moreover, a constitutively active form of AKT synergizes with Raf-1 in apoptosis suppression. In summary these data strongly suggest a Raf effector pathway for cell survival that is mediated by MEK and AKT.     

Basic fibroblast growth factor inhibits radiation-induced apoptosis of HUVECs. I. The PI3K/AKT pathway and induction of phosphorylation of BAD

Radiation-induced endothelial cell apoptosis is involved in the development of many radiation injuries, including radiation-induced skin ulcers. The proangiogenic growth factors basic fibroblast growth factor (bFGF, NUDT6) and VEGF enhance endothelial cell survival. In the present study, we used primary cultured human umbilical vein endothelial cells (HUVECs) irradiated with (60)Co gamma rays to explore the effects of bFGF on radiation-induced apoptosis of HUVECs and its signaling pathways. We found that bFGF inhibited radiation-induced apoptosis of HUVECs, and that the effect was mediated by the PI3K/AKT pathway. This pathway was activated by exposure of irradiated HUVECs to bFGF, involving phosphorylation of FGFR, PI3K and AKT. The survival-enhancing effect of bFGF was abrogated by wortmannin and LY294002. Transfection of a dominant-negative mutant of AKT completely blocked the anti-apoptosis effect of bFGF in irradiated HUVECs. We also found evidence for the first time that bFGF induced BAD phosphorylation in the gamma-irradiated HUVECs. These results showed that the PI3K/AKT pathway participated in the bFGF-induced modulation of the survival of irradiated HUVECs. Activation of the PI3K/AKT pathway plays an important role in bFGF-induced endothelial cell survival in the treatment of radiation-induced skin ulcers.     

Anhuienoside C inhibits human ovarian cancer cell growth by inducing apoptosis,suppression of cell migration and invasion,and targeting PI3K/AKT/mTOR signaling pathway

The present study was initiated to examine the anticancer effects of Anhuienoside C (AC) against ovarian cancer and postulates the possible molecular mechanism of its action. 3-[4,5-Dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide assay was implemented for determination of the effects of AC on cell viability of the ovarian cancer OVACAR-3 cell line. To study cellular morphology, phase contrast microscopy was performed. Apoptosis was examined via acridine orange/ethidium bromide used staining assays. Flow cytometry was used to check the different phases of the cell cycle. Cell migration and invasion assays were performed via transwell chamber assay. The effects of AC on expression of phosphoinositide 3-kinases (PI3K), protein kinase B (AKT), and mammalian target of rapamycin (mTOR) protein in ovarian cell were assessed using western blotting assay. The results indicated that the cell proliferation rate lowered in AC-treated OVACAR-3 cells as compared to the untreated controls in a dose-dependent manner. Cell morphology changed substantially by the exposure to AC and remained dose dependent. These morphological changes were indicative of apoptotic cell death. Apoptosis analysis showed dose-dependent increase of apoptosis. The cell migration and invasion of OVACAR-3 cells was reduced to a minimum by AC in a dose-dependent manner. Finally, western blotting assay showed blocking of PI3K/AKT/mTOR signaling pathway with increasing AC doses. Taking all together, AC is a potential ovarian cancer inhibitor. It induces its anti-ovarian cancer effects via induction of apoptosis, delaying cell migration and invasion, and blocking PI3K/AKT/mTOR signaling pathway.

     

Silencing of S-phase kinase-associated protein 2 enhances radiosensitivity of esophageal cancer cells through inhibition of PI3K/AKT signaling pathway

We investigated the effect of S-phase kinase-associated protein 2 (SKP2) on radiosensitivity of esophageal cancer (EC) cells. Expression of SKP2, PI3K, AKT, Bcl-2 and Bax were assayed in EC. EC cells were transfected with SKP2-siRNA/IGF-1 to detect expression of SKP2, PI3K, AKT, Bcl-2 and Bax. At last, the radiosensitivity of cells in different doses of X (0, 2, 4, 6, 8 Gy) irradiation and cell apoptosis were also detected. EC cells displayed a higher positive expression rate of SKP2, elevated mRNA and protein expression of SKP2, PI3K, AKT, Bcl-2 and Bax, as well as higher extent of PI3K and AKT phosphorylation. SKP2 silencing downregulated mRNA and protein expression of PI3K, AKT and Bcl-2 but increased p27 protein expression, and inhibited the cell survival rate while promoting cell apoptosis. Taken together, silencing SKP2 can inhibit the PI3K/AKT signaling pathway, thereby increasing the radiosensitivity of EC cells.     

Modulation of MICA on the surface of Chlamydia trachomatis-infected endocervical epithelial cells promotes NK cell-mediated killing

Chlamydia trachomatis serovars D-K are obligate intracellular bacteria that have tropism for the columnar epithelial cells of the genital tract. Chlamydia trachomatis infection has been reported to induce modifications in immune cell ligand expression on epithelial host cells. In this study, we used an in vitro infection model that resulted in a partial infection of C. trachomatis-exposed primary-like immortalized endocervical epithelial cells (A2EN). Using this model, we demonstrated that expression of the natural killer (NK) cell activating ligand, MHC class I-related protein A (MICA), was upregulated on C. trachomatis-infected, but not on noninfected bystander cells. MICA upregulation was concomitant with MHC class I downregulation and impacted the susceptibility of C. trachomatis-infected cells to NK cell activity. The specificity of MICA upregulation was reflected by a higher cytolytic activity of an NK cell line (NK92MI) against C. trachomatis-infected cells compared with uninfected control cells. Significantly, data also indicated that NK cells exerted a partial, but incomplete sterilizing effect on C. trachomatis as shown by the reduction in recoverable inclusion forming units (IFU) when cocultured with C. trachomatis-infected cells. Taken together, our data suggest that NK cells may play a significant role in the ability of the host to counter C. trachomatis infection.     

CTLA-4 Activation of Phosphatidylinositol 3-Kinase (PI 3-K) and Protein Kinase B (PKB/AKT) Sustains T-Cell Anergy without Cell Death

The balance of T-cell proliferation, anergy and apoptosis is central to immune function. In this regard, co-receptor CTLA-4 is needed for the induction of anergy and tolerance. One central question concerns the mechanism by which CTLA-4 can induce T-cell non-responsiveness without a concurrent induction of antigen induced cell death (AICD). In this study, we show that CTLA-4 activation of the phosphatidylinositol 3-kinase (PI 3-K) and protein kinase B (PKB/AKT) sustains T-cell anergy without cell death. CTLA-4 ligation induced PI 3K activation as evidenced by the phosphorylation of PKB/AKT that in turn inactivated GSK-3. The level of activation was similar to that observed with CD28. CTLA-4 induced PI 3K and AKT activation also led to phosphorylation of the pro-apoptotic factor BAD as well as the up-regulation of BcL-XL. In keeping with this, CD3/CTLA-4 co-ligation prevented apoptosis under the same conditions where T-cell non-responsiveness was induced. This effect was PI 3K and PKB/AKT dependent since inhibition of these enzymes under conditions of anti-CD3/CTLA-4 co-ligation resulted in cell death. Our findings therefore define a mechanism by which CTLA-4 can induce anergy (and possibly peripheral tolerance) by preventing the induction of cell death.     

ROS accumulation contributes to abamectin‐induced apoptosis and autophagy via the inactivation of PI3K/AKT/mTOR pathway in TM3 Leydig cells

Abamectin (ABA) as one of the worldwide used compounds in agriculture has raised safety concerns on nontarget organism toxicity. However, the study of male reproductive system damage caused by ABA remains unclear. Our aim is to investigate the effect of ABA‐induced cytotoxicity in TM3 Leydig cells and their underlying mechanisms. ABA inhibits TM3 cell viability and proliferation via cell cycle arrested in the G0/G1 phase. In addition, ABA‐induced mitochondrial depolarization leads to an imbalance in Bcl‐2 family expression, causing caspase‐dependent apoptosis in TM3 cells. The increased ratio of cells expression LC3 protein and LC3‐II to LC3‐I indicated the activation of autophagy potentially. Further experiments revealed ABA treatment reduced phosphatidylinositol 3‐kinase (PI3K), protein kinase B (AKT) phosphorylation, and mammalian target of rapamycin (mTOR) phosphorylation. Pretreatment with a PI3K/AKT inhibitor, LY294002, mimicked the ABA‐mediated effects on cytotoxicity. Pretreatment with a PI3K/AKT agonist, insulin‐like growth factor‐1, reversed the effects of ABA. ABA caused the accumulation of intracellular reactive oxygen species (ROS) by increased intensity of the ROS indicator. However, N‐acetylcysteine as ROS scavengers inhibited ABA‐induced apoptosis and autophagy and reversed these ABA‐mediated effects on PI3K/AKT/mTOR pathway. On the basis of the above results, it is suggested that ABA exposure induces apoptosis and autophagy in TM3 cells by ROS accumulation to mediate PI3K/AKT/mTOR signaling pathway suppression.     

Wnt3a regulates proliferation,apoptosis and function of pancreatic NIT‐1 beta cells via activation of IRS2/PI3K signaling

Wnt‐signaling pathway is implicated in pancreatic development and functional regulation of mature beta‐cells. Wnt3a/Wnt pathway activation expands islet cell mass in vitro by increasing proliferation and decreasing apoptosis of beta‐cells, thereby enhancing its function. However, the signaling pathways that mediate these effects remain unknown. By using a clonal beta‐cell line (NIT‐1), we examined the role of IRS2/PI3K in the mediation of Wnt3a‐stimulated beta‐cell growth. Real‐time PCR and Western blot were employed to investigate the activity of Wnt/β‐catenin and IRS2/PI3K signaling. Proliferation of NIT‐1 cells was assessed by BrdU incorporation, and apoptosis was quantitatively determined by TUNEL and flow cytometry (FCM). Dkk1, an inhibitor of Wnt signaling, and wortmannin, an inhibitor of PI3K, were also used. Results showed that Wnt3a rapidly activated Wnt/β‐catenin signaling, promoted IRS2 expression and Akt phosphorylation in NIT‐1 cells. These effects were completely abrogated by Dkk1 or partially eliminated by wortmannin. Wnt3a also promoted NIT‐1 cell proliferation, inhibited cytokine‐induced beta‐cell apoptosis, and increased insulin secretion. Both of these effects were also eliminated by Dkk1 or wortmannin. Our results demonstrated that Wnt3a regulates proliferation, apoptosis and enhances function of pancreatic NIT‐1 beta cells via activation of Wnt/β‐catenin signaling, involving crosstalk with IRS2/PI3K signaling, with the effect of Wnt signaling on beta‐cells also being IRS2/PI3K/AKT dependent. J. Cell. Biochem. 114: 1488–1497, 2013. © 2013 Wiley Periodicals, Inc.     

LY294002 and Rapamycin promote coxsackievirus-induced cytopathic effect and apoptosis via inhibition of PI3K/AKT/mTOR signaling pathway

Coxsackievirus B3 (CVB3) is a common human pathogen for acute myocarditis, pancreatitis, non-septic meningitis, and encephalitis; it induces a direct cytopathic effect (CPE) and apoptosis on infected cells. The Phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT/PKB)/mammalian target of Rapamycin (mTOR) signaling pathway regulates several cellular processes and it is one of the most important pathways in human networks. However, the effect and mechanism of PI3K/AKT/mTOR signaling pathway in CVB3 infected cells are poorly understood. In this study, we demonstrate that inhibition of PI3K/AKT/mTOR signaling pathway increased CVB3-induced CPE and apoptosis in HeLa cells. The activity of downstream targets of PI3K and mTOR is attenuated after CVB3 infection and inhibitors of PI3K and mTOR made their activity to decrease more significantly. We further show that LY294002 and Rapamycin, the inhibitor of PI3K and mTOR respectively, promote CVB3-induced CPE and apoptosis. Taken together, these data illustrate a new and imperative role for PI3K/AKT/mTOR signaling in CVB3 infection in HeLa cells and suggest an useful approach for the therapy of CVB3 infection.     

14-3-3 proteins regulate glycogen synthase 3beta phosphorylation and inhibit cardiomyocyte hypertrophy

14-3-3 proteins are dimeric phophoserine-binding molecules that participate in important cellular processes such as cell proliferation, cell-cycle control and the stress response. In this work, we report that several isoforms of 14-3-3s are expressed in neonatal rat cardiomyocytes. To understand their function, we utilized a general 14-3-3 peptide inhibitor, R18, to disrupt 14-3-3 functions in cardiomyocytes. Cardiomyocytes infected with adenovirus-expressing YFP-R18 (AdR18) exhibited markedly increased protein synthesis and atrial natriuretic peptide production and potentiated the responses to norepinephrine stimulation. This response was blocked by the pretreatment with LY294002, a phosphoinositide 3-kinase (PI3K) inhibitor. Consistent with a role of PI3K in the R18 effect, R18 induced phosphorylation of a protein cloned from the vakt oncogene of retrovirus AKT8 (Akt - also called protein kinase B, PKB) at Ser473 and glycogen synthase 3beta (GSK3beta) at Ser9, but not extracellular signal-regulated kinase 1/2 (ERK1/2). AdR18-induced PKB and GSK3beta phosphorylation was completely blocked by LY294002. In addition, a member of the nuclear factor of activated T cells (NFAT) family, NFAT3, was converted into faster mobility forms and translocated into the nucleus upon the treatment of AdR18. These results suggest that 14-3-3s inhibits cardiomyocytes hypertrophy through regulation of the PI3K/PKB/GSK3beta and NFAT pathway.     

Cancer Associated Fibroblast-Derived Hepatocyte Growth Factor Inhibits the Paclitaxel-Induced Apoptosis of Lung Cancer A549 Cells by Up-Regulating the PI3K/Akt and GRP78 Signaling on a Microfluidic Platform

Tumor stroma and growth factors provide a survival environment to tumor cells and can modulate their chemoresistance by dysregulating several signal pathways. In this study, we fabricated a three-dimensional (3D) microfluidic chip using polydimethylsiloxane (PDMS) to investigate the impact of hepatocyte growth factor (HGF) from cancer-associated fibroblasts (CAF) on the Met/PI3K/AKT activation, glucose regulatory protein (GRP78) expression and the paclitaxel-induced A549 cell apoptosis. With a concentration gradient generator, the assembled chip was able to reconstruct a tumor microenvironment in vitro. We found high levels of HGF in the supernatants of CAF and the CAF matrix from the supernatants of activated HFL1 fibroblasts or HGF enhanced the levels of Met, PI3K and AKT phosphorylation and GRP78 expression in A549 cells cultured in a 3D cell chamber, which was abrogated by anti-HGF. Inhibition of Met attenuated the CAF matrix-enhanced PI3K/AKT phosphorylation and GRP78 expression while inhibition of PI3K reduced GRP78 expression, but not Met phosphorylation in A549 cells. Inhibition of GRP78 failed to modulate the CAF matrix-enhanced Met/PI3K/AKT phosphorylation in A549 cells. Furthermore, inhibition of PI3K or GRP78 enhanced spontaneous and paclitaxel-induced A549 cell apoptosis. Moreover, treatment with the CAF matrix inhibited spontaneous and medium or high dose of paclitaxel-induced A549 cell apoptosis. Inhibition of PI3K or GRP78 attenuated the CAF matrix-mediated inhibition on paclitaxel-induced A549 cell apoptosis. Our data indicated that HGF in the CAF matrix activated the Met/PI3K/AKT and up-regulated GRP78 expression, promoting chemoresistance to paclitaxel-mediated apoptosis in A549 cells. Our findings suggest that the microfluidic system may represent an ideal platform for signaling research and drug screening.     

Regulation of neuron survival through an intersectin-phosphoinositide 3'-kinase C2beta-AKT pathway

While endocytosis attenuates signals from plasma membrane receptors, recent studies suggest that endocytosis also serves as a platform for the compartmentalized activation of cellular signaling pathways. Intersectin (ITSN) is a multidomain scaffolding protein that regulates endocytosis and has the potential to regulate various biochemical pathways through its multiple, modular domains. To address the biological importance of ITSN in regulating cellular signaling pathways versus in endocytosis, we have stably silenced ITSN expression in neuronal cells by using short hairpin RNAs. Decreasing ITSN expression dramatically increased apoptosis in both neuroblastoma cells and primary cortical neurons. Surprisingly, the loss of ITSN did not lead to major defects in the endocytic pathway. Yeast two-hybrid analysis identified class II phosphoinositide 3'-kinase C2beta (PI3K-C2beta) as an ITSN binding protein, suggesting that ITSN may regulate a PI3K-C2beta-AKT survival pathway. ITSN associated with PI3K-C2beta on a subset of endomembrane vesicles and enhanced both basal and growth factor-stimulated PI3K-C2beta activity, resulting in AKT activation. The use of pharmacological inhibitors, dominant negatives, and rescue experiments revealed that PI3K-C2beta and AKT were epistatic to ITSN. This study represents the first demonstration that ITSN, independent of its role in endocytosis, regulates a critical cellular signaling pathway necessary for cell survival.     

Blockade of fatty acid synthase induces ubiquitination and degradation of phosphoinositide-3-kinase signaling proteins in ovarian cancer

Aberrations within the phosphoinositide-3-kinase (PI3K) pathway occur in greater than 45% of ovarian carcinomas. The PI3K cascade transmits signals from ErbB receptors downstream to S6 and 4EBP1, which are involved in protein biosynthesis. Many ovarian carcinomas reveal hyperactivation of ErbB1 (epidermal growth factor receptor) or ErbB2 (HER2/neu). Unfortunately, the benefit of anti-ErbB drugs is yet rather limited in ovarian carcinomas. Thus, novel targeting strategies are needed for ovarian carcinomas. The lipogenic enzyme fatty acid synthase (FASN) is overexpressed in approximately 80% of ovarian carcinomas. It stimulates cell growth and signifies poor prognosis. FASN inhibition impedes (ErbB) membrane receptor signaling and sensitizes cells against anti-ErbB drugs. Here, we show that the FASN inhibitor C75 and FASN-targeting siRNAs abrogate growth, induce apoptosis, and downregulate phosphorylation/expression of the PI3K effectors AKT, mTOR, p70S6K, S6, and 4EBP1. In contrast, FASN inhibition impairs expression but only weakly affects phosphorylation of ERK1/2 mitogen-activated protein kinases in ovarian carcinoma cells. Cycloheximide-mediated blockade of protein translation reveals that C75- or FASN siRNA-induced shutdown of FASN accelerates decomposition of signaling proteins. This effect is caused by C75- or FASN siRNA-dependent stimulation of ubiquitination followed by lysosomal-autophagosomal proteolysis. In contrast, PI3K inhibitor LY294002 blocks phosphorylation but does not reduce expression/stability of PI3K effectors. Forced expression of hyperactive (HA) AKT1, unlike HA-MEK1, impairs the growth-inhibitory action of C75. We provide first evidence that the anticancer action of FASN inhibitors is at least partially mediated by drug-dependent proteolysis of PI3K effectors. FASN is a promising cancer target, whose inhibition not only abrogates lipogenesis, which is indispensable for cancer growth, but also downregulates oncogenic PI3K signaling.     

Dissociation of apoptosis from proliferation, protein kinase B activation, and BAD phosphorylation in interleukin-3-mediated phosphoinositide 3-kinase signaling

Interleukin-3 (IL-3) acts as both a growth and survival factor for many hemopoietic cells. IL-3 treatment of responsive cells leads to the rapid and transient activation of Class IA phosphoinositide-3-kinases (PI3Ks) and the serine/threonine kinase Akt/protein kinase B (PKB) and phosphorylation of BAD. Each of these molecules has been implicated in anti-apoptotic signaling in a wide range of cells. Using regulated expression of dominant-negative p85 (Deltap85) in stably transfected IL-3-dependent BaF/3 cells, we have specifically investigated the role of class IA PI3K in IL-3 signaling. The major functional consequence of Deltap85 expression in these cells is a highly reproducible, dramatic reduction in IL-3-induced proliferation. Expression of Deltap85 reduces IL-3-induced PKB phosphorylation and activation and phosphorylation of BAD dramatically, to levels seen in unstimulated cells. Despite these reductions, the levels of apoptosis observed in the same cells are very low and do not account for the reduction in IL-3-dependent proliferation we observe. These results show that Deltap85 inhibits both PKB activity and BAD phosphorylation without significantly affecting levels of apoptosis, suggesting that there are targets other than PKB and BAD that can transmit survival signals in these cells. Our data indicate that the prime target for PI3K action in IL-3 signaling is at the level of regulation of proliferation.