Prevented Akt Phosphorylation Research Articles

Antiproliferative activity of CD44 siRNA-PEI-PEG nanoparticles in glioblastoma: involvement of AKT signaling.

Background and purpose:Glioblastoma multiforme (GBM) is the most invasive type of cancer which starts inside the brain. GBM cells were found to have similar properties to glioblastoma cancer stem cells. CD44 can be used as a marker of the cancer stem cells in a subset of glioblastoma tumor cells. Recent studies showed that CD44 is involved in developing cancer cells via the protein kinase B (PKB or AKT) signaling pathway. Therefore, this study aimed to investigate the CD44 mRNA silencing effects on the glioblastoma cell cycle via AKT signaling pathway.Experimental approach:To determine CD44 expression in the samples of the patients with GBM, we used the analysis of data extracted from TCGA database. qRT-PCR and western blotting were used to evaluate the expression level of genes and proteins. Different cell cycles were evaluated by DAPI staining and flow cytometry.Findings/Results:Bioinformatics results showed that CD44 expression level in GBM tumor samples is higher than in normal samples. Effects of poly (ethylene imine)-polyethylene glycol (PEI-PEG)-loaded CD44 siRNA in cell cycle showed that CD44 silencing could inhibit cell cycle in G0-G1 phase by more than 20% compared to the negative control (P < 0.05). Furthermore, PEI-PEG-loaded CD44 siRNA reduces the expression of cyclin D1 and CKD-4. According to our findings, this structure also prevented AKT phosphorylation at Thr-308 and Ser-473.Conclusion and implications:Our results suggest that PEI-PEG-loaded CD44 siRNA may attenuate the cell cycle by suppressing AKT signaling pathway.

AKT/PKB‐Megalin Positive Feedback Loop Expands Albumin Endocytosis in Proximal Tubule Cells

Albuminuria is an independent risk factor for mortality in cardiovascular diseases and is a marker of progression to end-stage renal disease (ESRD). It is accepted that proximal tubule epithelial cells (PTECs) play a central role in the genesis of albuminuria. PTECs reabsorb 3-7g of filtered albumin daily through a receptor-mediated endocytosis being megalin one of the main receptors. Nowadays, one of the main challenges is to understand the regulatory mechanisms involved in this process. In this context, PI3K and AKT have been pointed out as a central mechanism to control albumin endocytosis in PTECs but the mechanism involved is poorly known. Here, we investigated how PI3K/AKT pathway and receptor-mediated albumin endocytosis are interconnected. The incubation of LLC-PK1 cells, a model of PTECs, with albumin 0.01 mg/mL promoted a transient AKT activation measured by its phosphorylation (pAKT) at Ser473 (S473) and by phosphorylation of GSK-3β at Ser9 (S9). Pre-incubation with Pit-Stop-2, an endocytosis inhibitor, prevented AKT phosphorylation (S473) induced by albumin. Using confocal microscopy, the increase of pAKT S473 in subapical vesicles was observed indicating that albumin endocytosis promotes endosomal AKT activation. Acute inhibition of PI3K and AKT by wortmannin (10-6M) and MK-2206 (10-5M) reduced albumin endocytosis capacity and lysosomal delivery by 50%, accessed by BSA-FITC and DQ-BSA uptake, respectively. To study megalin trafficking, LLC-PK1 cells were transfected with a truncated megalin tagged with HA, named MegT0. The inhibition of PI3K/AKT pathway reduced MegT0 surface expression what was followed by an increase in EEA1+ endosomes, but not in LAMP1+ lysosomes, leading to the reduction of megalin recycling. Conversely, we studied the possible feedback between PI3K/AKT pathway and receptor-mediated albumin endocytosis. LLC-PK1 cells were pre-incubated with albumin 0.01 mg/mL, washed, and BSA-FITC endocytosis was accessed. Albumin pre-incubation induced receptor-mediated albumin endocytosis what was abolished when PI3K/AKT pathway was inhibited. This result shows that albumin expands albumin endocytosis capacity at physiological concentration. To test this mechanism in vivo, BALB/c micewere i.v. injected with L-lysine, a known inhibitor of megalin-mediated albumin endocytosis in PTECs. L-lysine reduced pAKT S473 and albumin endocytosis, promoting albuminuria. Additionally, wortmannin and MK-2206 incubation reduced albumin endocytosis ex vivo in renal cortex obtained from BALB/c mice. In conclusion, our results suggest a positive feedback between megalin and PI3K/AKT pathway what could be correlated with the capacity of albumin reabsorption in PTECs and the genesis of albuminuria.

Stimulation of mTORC2 by integrin αIIbβ3 is required for PI3Kβ-dependent activation of Akt but is dispensable for platelet spreading on fibrinogen

Phosphatidylinositol 3 kinase (PI3K) is a major player in platelet activation and regulates thrombus formation and stabilization. The β isoform of PI3K is implicated in integrin αIIbβ3 outside-in signaling, is required for the phosphorylation of Akt, and controls efficient platelet spreading upon adhesion to fibrinogen. In this study we found that during integrin αIIbβ3 outside-in signaling PI3Kβ-dependent phosphorylation of Akt on Serine473 is mediated by the mammalian target of rapamycin complex 2 (mTORC2). The activity of mTORC2 is stimulated upon platelet adhesion to fibrinogen, as documented by increased autophosphorylation. However, mTORC2 activation downstream of integrin αIIbβ3 is PI3Kβ-independent. Inhibition of mTORC2, but not mTORC1, also prevents Akt phosphorylation of Threonine308 and affects Akt activity, resulting in the inhibition of GSK3α/β phosphorylation. Nevertheless, mTORC2 or Akt inhibition does not alter PI3Kβ-dependent platelet spreading on fibrinogen. The activation of the small GTPase Rap1b downstream of integrin αIIbβ3 is regulated by PI3Kβ but is not affected upon inhibition of either mTORC2 or Akt. Altogether, these results demonstrate for the first time the activation of mTORC2 and its involvement in Akt phosphorylation and stimulation during integrin αIIbβ3 outside-in signaling. Moreover, the results demonstrate that the mTORC2/Akt pathway is dispensable for PI3Kβ-regulated platelet spreading on fibrinogen.

PI3K inhibition reduces murine and human liver fibrogenesis in precision-cut liver slices

BackgroundLiver fibrosis results from continuous inflammation and injury. Despite its high prevalence worldwide, no approved antifibrotic therapies exist. Omipalisib is a selective inhibitor of the PI3K/mTOR pathway that controls nutrient metabolism, growth and proliferation. It has shown antifibrotic properties in vitro. While clinical trials for idiopathic pulmonary fibrosis have been initiated, an in-depth preclinical evaluation is lacking. We evaluated omipalisib’s effects on fibrogenesis using the ex vivo model of murine and human precision-cut tissue slices (PCTS). MethodsMurine and human liver and jejunum PCTS were incubated with omipalisib up to 10 μM for 48 h. PI3K pathway activation was assessed through protein kinase B (Akt) phosphorylation and antifibrotic efficacy was determined via a spectrum of fibrosis markers at the transcriptional and translational level. ResultsDuring incubation of PCTS the PI3K pathway was activated and incubation with omipalisib prevented Akt phosphorylation (IC50 = 20 and 1.5 nM for mouse and human, respectively). Viability of mouse and human liver PCTS was compromised only at the high concentration of 10 and 1–5 μM, respectively. However, viability of jejunum PCTS decreased with 0.1 (mouse) and 0.01 μM (human). Spontaneously increased fibrosis related genes and proteins were significantly and similarly suppressed in mouse and in human liver PCTS. ConclusionsOmipalisib has antifibrotic properties in ex vivo mouse and human liver PCTS, but higher concentrations showed toxicity in jejunum PCTS. While the PI3K/mTOR pathway appears to be a promising target for the treatment of liver fibrosis, PCTS revealed likely side effects in the intestine at higher doses.

Novel pharmacological inhibitors demonstrate the role of the tyrosine kinase Pyk2 in adhesion and aggregation of human platelets.

Pyk2 is a Ca2+-regulated kinase predominantly expressed in neuronal and in haematopoietic cells. Previous studies on Pyk2-null mice have demonstrated that Pyk2 plays a crucial role in platelet activation and thrombus formation, thus representing a possible target for antithrombotic therapy. Very limited information is available about the role of Pyk2 in human platelets, mainly because of the lack of specific pharmacological inhibitors. In this work, we have tested two novel Pyk2 inhibitors, PF-4594755 and PF-4520440, to validate their specificity and to investigate their ability to modulate platelet activation. Both molecules were able to efficiently block Pyk2 activity in human and mouse platelets stimulated with thrombin or with the Ca2+-ionophore. In wild-type murine platelets, PF-4594755 and PF-4520440 reduced thrombin-induced aggregation to the level observed in Pyk2 knockout platelets, but did not affect aggregation induced by GPVI stimulation. Importantly, neither compounds affected the residual thrombin-induced aggregation of Pyk2-null platelets, thus excluding possible off-target effects. In human platelets, PF-4594755 and PF-4520440 significantly reduced aggregation stimulated by thrombin, but not by the GPVI agonist convulxin. Both inhibitors reduced platelet adhesion on fibrinogen and prevented Akt phosphorylation in adherent cells, indicating that Pyk2 regulates PI3K and cell spreading downstream of integrins in human platelets. Finally, the Pyk2 inhibitors significantly inhibited thrombus formation upon blood perfusion on immobilized collagen under arterial flow rate. These results demonstrate that PF-4594755 and PF-4520440 are specific inhibitors of Pyk2 in intact platelets and allowed to reliably document that this kinase plays a relevant role in human platelet activation.

MICAL1 controls cell invasive phenotype via regulating oxidative stress in breast cancer cells.

BackgroundMolecules Interacting with CasL (MICAL1), a multidomain flavoprotein monoxygenase, is strongly involved in the mechanisms that promote cancer cell proliferation and survival. Activation of MICAL1 causes an up-regulation of reactive oxygen species (ROS) in HeLa cells. ROS can function as a signaling molecule that modulates protein phosphorylation, leading to malignant phenotypes of cancer cells such as invasion and metastasis. Herein, we tested whether MICAL1 could control cell migration and invasion through regulating ROS in breast cancer cell lines.MethodsThe effects of depletion/overexperssion of MICAL1 on cell invasion rate were measured by matrigel-based transwell assays. The contents of ROS in breast cancer cells were evaluated by CM2-DCFHDA staining and enhanced lucigenin chemiluminescence method. RAB35 activity was assessed by pulldown assay. The relationship of RAB35 and MICAL1 was evaluated by immunofluorescence, coimmunoprecipitation, immunoblotting and co-transfection techniques. Immunoblotting assays were also used to analyze Akt phosphorylation level.ResultsIn this study, we found that depletion of MICAL1 reduced cell migration and invasion as well as ROS generation. Phosphorylation of Akt was also attenuated by MICAL1 depletion. Likewise, the over-expression of MICAL1 augmented the generation of ROS, increased Akt phosphorylation, and favored invasive phenotype of breast cancer cells. Moreover, we investigated the effect of EGF signaling on MICAL1 function. We demonstrated that EGF increased RAB35 activation and activated form of RAB35 could bind to MICAL1. Silencing of RAB35 repressed ROS generation, prevented Akt phosphorylation and inhibited cell invasion in response to EGF.ConclusionsTaken together, our results provide evidence that MICAL1 plays an essential role in the activation of ROS/Akt signaling and cell invasive phenotype and identify a novel link between RAB35 and MICAL1 in regulating breast cancer cell invasion. These findings may provide a basis for designing future therapeutic strategy for blocking breast cancer metastasis.

Abstract LB-C18: First-in-Human Clinical Trial of ABTL0812, a Compound that Inhibits PI3K/Akt/mTOR Pathway by Upregulating TRIB3, in Patients with Advanced Solid Tumors

Abstract Background: ABTL0812 is a first-in-class antitumor drug whose mechanism of action relies on the inhibition of PI3K/Akt/mTOR (PAM) pathway by upregulation of TRIB3 levels, an endogenous inhibitor of Akt activity that prevents Akt phosphorylation. Preclinical studies showed good efficacy of ABTL0812 in xenograft models with high safety margin. Here we describe the First-in-Human (FiH) Phase I/Ib clinical trial of ABTL0812 in patients with advanced solid tumors (NCT02201823). Methods: ABTL0812 was dosed daily, by the oral route, in 28-day cycles. The study included a 4-cohort dose escalation, in a 3+3 dose escalating design, followed by an expansion cohort. The trial objectives were to determine safety and tolerability, to evaluate signs of efficacy, to determine drug pharmacokinetics (PK) in plasma and to analyze inhibition of Akt phosphorylation in platelets by MSD® as pharmacodynamic (PD) biomarker. Pretreatment tumor biopsies were analyzed by next generation sequencing to identify mutations in a panel of 50 cancer-related genes. Results: Fifteen patients were recruited in the escalation part and 14 patients in the expansion cohort (February 2014 to May 2015). No dose-limiting-toxicities were detected and the recommended Phase II was 1300 mg tid based on PK/PD modeling. The median number of previous chemotherapy lines was 2 (range 0-11). The profile of ABTL0812 grade 1-2 related adverse events (AEs) included asthenia, nausea/vomiting and throat burning (34%, 31% and 24% of patients, respectively). Only one case of grade 3-4 AE (elevated hepatic enzymes) appeared. ABTL0812 half-life was short (3-5 h), which supported the increase in the administration schedule from once to twice and finally to three times a day. Biomarker analysis showed inhibition of Akt phosphorylation with increasing doses, with average 90% inhibition in the expansion cohort; furthermore it correlated with C-trough plasma levels. Five patients had stable disease (SD) for at least 16 weeks: 1 endometrial cancer (62 weeks), 1 cholangiocarcinoma (35 weeks, ongoing on Sep 15th, 2015), 2 colorectal cancer (28 and 22 weeks) and 1 lung adenocarcinoma (16 weeks). The median time to progression in the expansion cohort was 11 weeks (range 1-≥35 weeks). Interestingly, the tumor biopsy from the patient with endometrial cancer had activating mutations in Akt1 (E17K) and PIK3CA (R88Q). The tumor from the patient with colorectal cancer (28 weeks SD) showed deleterious mutations in TP53 (R248W) and APC (Q1469Ter). None of these samples included major mutations in Ras. Conclusions: ABTL0812 is a PAM pathway inhibitor acting by a novel mechanism of action that involves upregulation of TRIB3 levels. The FiH Phase I/Ib study in patients with advanced solid tumors showed excellent tolerability and safety profile and demonstrated dose-dependent inhibition of Akt phosphorylation. ABTL0812 treatment induced several long term disease stabilizations (5/29), being the best responder a patient with endometrial cancer (68 weeks) with activating mutations in Akt1 and PIK3CA. Based on these data, a Phase II clinical trial in patients with endometrial cancer is planned. Citation Format: Laura Vidal, Lydia Gaba, Ivan Victoria, Marta Gil-Martin, Berta Laquente, Marc Cortal, Merce Brunet, Pilar Paredes, Mariana Gomez-Ferreria, Jose Alfon, Carles Domenech, Pedro Gascon. First-in-Human Clinical Trial of ABTL0812, a Compound that Inhibits PI3K/Akt/mTOR Pathway by Upregulating TRIB3, in Patients with Advanced Solid Tumors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-C18.

Involvement of the Na+/Ca2+ exchanger isoform 1 (NCX1) in Neuronal Growth Factor (NGF)-induced Neuronal Differentiation through Ca2+-dependent Akt Phosphorylation

NGF induces neuronal differentiation by modulating [Ca(2+)]i. However, the role of the three isoforms of the main Ca(2+)-extruding system, the Na(+)/Ca(2+) exchanger (NCX), in NGF-induced differentiation remains unexplored. We investigated whether NCX1, NCX2, and NCX3 isoforms could play a relevant role in neuronal differentiation through the modulation of [Ca(2+)]i and the Akt pathway. NGF caused progressive neurite elongation; a significant increase of the well known marker of growth cones, GAP-43; and an enhancement of endoplasmic reticulum (ER) Ca(2+) content and of Akt phosphorylation through an early activation of ERK1/2. Interestingly, during NGF-induced differentiation, the NCX1 protein level increased, NCX3 decreased, and NCX2 remained unaffected. At the same time, NCX total activity increased. Moreover, NCX1 colocalized and coimmunoprecipitated with GAP-43, and NCX1 silencing prevented NGF-induced effects on GAP-43 expression, Akt phosphorylation, and neurite outgrowth. On the other hand, the overexpression of its neuronal splicing isoform, NCX1.4, even in the absence of NGF, induced an increase in Akt phosphorylation and GAP-43 protein expression. Interestingly, tetrodotoxin-sensitive Na(+) currents and 1,3-benzenedicarboxylic acid, 4,4'-[1,4,10-trioxa-7,13-diazacyclopentadecane-7,13-diylbis(5-methoxy-6,12-benzofurandiyl)]bis-, tetrakis[(acetyloxy)methyl] ester-detected [Na(+)]i significantly increased in cells overexpressing NCX1.4 as well as ER Ca(2+) content. This latter effect was prevented by tetrodotoxin. Furthermore, either the [Ca(2+)]i chelator(1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid) (BAPTA-AM) or the PI3K inhibitor LY 294002 prevented Akt phosphorylation and GAP-43 protein expression rise in NCX1.4 overexpressing cells. Moreover, in primary cortical neurons, NCX1 silencing prevented Akt phosphorylation, GAP-43 and MAP2 overexpression, and neurite elongation. Collectively, these data show that NCX1 participates in neuronal differentiation through the modulation of ER Ca(2+) content and PI3K signaling.

DNA-PKcs is important for Akt activation and gemcitabine resistance in PANC-1 pancreatic cancer cells

Pancreatic cancer is one of the most aggressive human malignancies with extremely poor prognosis. The moderate activity of the current standard gemcitabine and gemcitabine-based regimens was due to pre-existing or acquired chemo-resistance of pancreatic cancer cells. In this study, we explored the potential role of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) in gemcitabine resistance, and studied the underlying mechanisms. We found that NU-7026 and NU-7441, two DNA-PKcs inhibitors, enhanced gemcitabine-induced cytotoxicity and apoptosis in PANC-1 pancreatic cancer cells. Meanwhile, PANC-1 cells with siRNA-knockdown of DNA-PKcs were more sensitive to gemcitabine than control PANC-1 cells. Through the co-immunoprecipitation (Co-IP) assay, we found that DNA-PKcs formed a complex with SIN1, the latter is an indispensable component of mammalian target of rapamycin (mTOR) complex 2 (mTORC2). DNA-PKcs–SIN1 complexation was required for Akt activation in PANC-1 cells, while inhibition of this complex by siRNA knockdown of DNA-PKcs/SIN1, or by DNA-PKcs inhibitors, prevented Akt phosphorylation in PANC-1 cells. Further, SIN1 siRNA-knockdown also facilitated gemcitabine-induced apoptosis in PANC-1 cells. Finally, DNA-PKcs and p-Akt expression was significantly higher in human pancreatic cancer tissues than surrounding normal tissues. Together, these results show that DNA-PKcs is important for Akt activation and gemcitabine resistance in PANC-1 pancreatic cancer cells.

PI3K inhibitors LY294002 and IC87114 reduce inflammation in carrageenan-induced paw oedema and down-regulate inflammatory gene expression in activated macrophages.

PI3K/Akt pathway is a well-characterized pathway controlling cellular processes such as proliferation, migration and survival, and its role in cancer is vastly studied. There is also evidence to suggest the involvement of this pathway in the regulation of inflammatory responses. In this study, an attempt was made to investigate the role of PI3Ks in acute inflammation in vivo using pharmacological inhibitors against PI3Ks in the carrageenan-induced paw oedema model. A non-selective PI3K inhibitor LY294002 and a PI3Kδ-selective inhibitor IC87114 were used. Both of these inhibitors reduced inflammatory oedema upon carrageenan challenge in the mouse paw. To explain this result, the effects of the two inhibitors on inflammatory gene expression were investigated in activated macrophages. LY294002 and IC87114 prevented Akt phosphorylation as expected and down-regulated the expression of inflammatory factors IL-6, MCP-1,TNFα and iNOS. These findings suggest that PI3K inhibitors could be used to attenuate inflammatory responses and that the mechanism of action behind this effect is the down-regulation of inflammatory gene expression.

ATM regulates Cdt1 stability during the unperturbed S phase to prevent re-replication

Ataxia-telangiectasia mutated (ATM) plays crucial roles in DNA damage responses, especially with regard to DNA double-strand breaks (DSBs). However, it appears that ATM can be activated not only by DSB, but also by some changes in chromatin architecture, suggesting potential ATM function in cell cycle control. Here, we found that ATM is involved in timely degradation of Cdt1, a critical replication licensing factor, during the unperturbed S phase. At least in certain cell types, degradation of p27Kip1 was also impaired by ATM inhibition. The novel ATM function for Cdt1 regulation was dependent on its kinase activity and NBS1. Indeed, we found that ATM is moderately phosphorylated at Ser1981 during the S phase. ATM silencing induced partial reduction in levels of Skp2, a component of SCFSkp2 ubiquitin ligase that controls Cdt1 degradation. Furthermore, Skp2 silencing resulted in Cdt1 stabilization like ATM inhibition. In addition, as reported previously, ATM silencing partially prevented Akt phosphorylation at Ser473, indicative of its activation, and Akt inhibition led to modest stabilization of Cdt1. Therefore, the ATM-Akt-SCFSkp2 pathway may partly contribute to the novel ATM function. Finally, ATM inhibition rendered cells hypersensitive to induction of re-replication, indicating importance for maintenance of genome stability.

IL‐1β‐mediated up‐regulation of DEC1 in human gingiva cells via the Akt pathway

Growing evidence indicates that inflammation is a contributing factor leading to cancer development. However, pathways involved in this progression are not well understood. The involvement of DEC1 in cancer prompted us to examine whether pro-inflammatory cytokine interleukin-1β (IL-1β) induces the expression of DEC1 in oral inflammation. We found that IL-1β up-regulated DEC1 and hypoxia-inducible factor-1α (HIF-1α) protein and elevated the HIF-1α-responsive gene vascular endothelial growth factor (VEGF) expression in human primary gingival cells. HIF-1α and DEC1 immunoreactivity were significantly higher in the cases of gingival inflammation. We demonstrate that IL-1β up-regulates DEC1 and HIF-1α protein through a classical inflammatory signaling pathway involving Akt. Our data strongly suggest that PI-3K-Akt is an upstream participant in IL-1β-mediated DEC1 and HIF-1α induction. This is supported by the following data: (1) IL-1β induces 473 serine phosphorylation of Akt; (2) IL-1β-mediated Akt activation occurs in a PI-3K-dependent manner, and specific inhibition of PI-3K prevents Akt phosphorylation; and (3) inhibition of Akt prevents IL-1β-mediated DEC1 and HIF-1α induction. Taken together, these results suggest that DEC1 is one of the important transcription factors in inflammation.

Abstract 3456: Nicotine and the alpha-7 nicotinic acetylcholine receptor in the regulation of response to radiotherapy

Abstract Purpose: Nicotine is a systemically available component of tobacco known to decrease the effectiveness of chemotherapy and/or radiotherapy (RT) however, the mechanisms of decreased RT response are not well characterized. The purpose of this study is to evaluate the effects of nicotine and activation of the alpha-7 nicotinic acetylcholine receptor (a7-nAChR) on regulating cell survival following RT and to identify potential mechanisms of regulation. Methods: Human lung cancer (A549, H460) and oropharyngeal cancer (FaDu) cell lines were treated with nicotine alone or in combination with nAChR inhibitors, MEK inhibitors, PI3K inhibitors, or mTOR inhibitors followed by treatment with RT to evaluate cell survival using clonogenic assays. The effects of a7-nAChR agonists on RT were also evaluated. Western blots were performed to evaluate nAChR subunit expression profiles in a broad spectrum of cancer cell lines and to evaluate the effects of nicotine (alone or with inhibitors listed above) or agonists on downstream signal transduction. Results: Nicotine administration 2 hours prior to RT increased survival in all cell lines. nAChR expression was confirmed in several cell lines including A549 and H460 cells. Inhibition of specific nAChRs in A549 and H460 cells demonstrated that the a7-nAChR prevented the effects of nicotine on RT in both cell lines. Nicotine administration increased p-ERK 1/2, p-Akt (Ser-473), mTOR (S2448), activated matrix metalloproteinase 2 (MMP2), and hypoxia inducible factor 1-alpha (HIF-1a). Inhibition of MEK prevented ERK 1/2 phosphorylation, but did not prevent the effects of nicotine on RT. Inhibition of PI3K prevented Akt phosphorylation, MMP2 activation, and HIF-1a induction with concomitant inhibition of the effects of nicotine on RT. Administration of an mTOR inhibitor (rapamycin) significantly sensitized cells to RT, but did not prevent the pro-survival effects of nicotine. Administration of selective a7-nAChR agonists (LY 2087101, PHA 54613, PNU 282987, TC 1698) demonstrated that all increased p-ERK 1/2 and p-Akt; however, only LY 2087101 and PHA 54613 increased cell survival with RT. Further analysis demonstrates that only LY 2087101 and PHA 54613 increased mTOR (S2448) while there was no change in mTOR (S2448) with PNU 282987 or TC 1698. Conclusions: Data demonstrates that activation of the a7-nAChR can decrease the effectiveness of RT. Results suggest that PI3K and mTOR may play a central role in conferring resistance to radiation by nicotine or during a7-nAChR activation. Further analyses of critical mechanisms are required to understand the adverse systemic effects of tobacco on cancer treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3456. doi:1538-7445.AM2012-3456

W1744 The Extracellular Calcium-Sensing Receptor Regulates the Proliferation of Normal Colonic Epithelial Cells and Reverses the Malignant Phenotype of Colon Cancer Cells

Background: EGFR family members are all thought to signal through the MAPK and PI3K/ Akt pathways. Upregulation of EGFR, HER2, HER3 and MAPK signaling is well documented in colorectal tumors. Similarly, PI3K activity is elevated in the vast majority of colorectal cancers and over-expression of Akt isoforms has also been detected in CRC. We previously reported that the sulfide and sulfone metabolites of the non-steroidal anti-inflammatory drug (NSAID) sulindac downregulate the expression of EGFR, inhibit EGFR signaling through the MAPK pathway, and induce apoptotic cell death in CRC cells. Goals: 1) To examine the effects of sulindac metabolites (SMs) on protein levels and phosphorylation status of the other EGFR family members, HER2 and HER3, and the downstream signaling effector, Akt and 2) To determine if downregulation of EGFR family members is required for the apoptotic effects of SMs. Results: SMs downregulated both total and phosphorylated forms of HER2 and HER3 in a manner similar to their effects on EGFR. This downregulation was seen by 12h after treatment and persisted through at least 48h. Unexpectedly bothmetabolites significantly increased Akt phosphorylation as early as 1h after treatment in all cell lines tested. SW620 CRC cells which lack EGFR and do not signal through HER2 remained sensitive to both the apoptotic effects and the pAkt stimulatory effects of SMs. siRNA treatment resulted in a 76% knockdown of HER3 expression in HT29 (EGFR+) cells and 84% in SW620 (EGFR-) cells. HER3 knockdown failed to induce significant levels of apoptosis in either cell line but it did not prevent the apoptotic effect or the pAkt induction by SMs. Pre-treatment of HT29 cells with LY294002 prevented Akt phosphorylation by SMs and potentiated the growth inhibitory effects of sulindac sulfide in this cell line. Conclusion: These results demonstrate that I) EGFR, HER2, and HER3 appear to be similarly downregulated by SMs; II) Downregulation of EGFR family members does not appear to be sufficient for the apoptotic effect of sulindacmetabolites; III) Sulindacmetabolites induce an unexpected stimulation of Akt phosphorylation through an EGFR family member-independent effect and IV) Prevention of NSAID-induced AKT phosphorylation might potentiate the growth inhibitory activity of these drugs.

Mechanisms Underlying the Anti-inflammatory Effects of the Ca2+/Calmodulin Antagonist CV-159 in Cultured Vascular Smooth Muscle Cells

CV-159 is a unique dihydropyridine Ca2+antagonist with an anti-calmodulin (CaM) action. A pathogenic feature of atherosclerosis is vascular inflammatory change. In the present study, we examined whether CV-159 exerts protective effects on smooth muscle inflammatory responses. After pretreatment of rat mesenteric arterial smooth muscle cells (SMCs) with CV-159 (0.1 – 10 μM, 30 min), TNF-α (10 ng/ml) was applied for 20 min or 24 h. CV-159 inhibited TNF (24 h)–induced vascular cell adhesion molecule (VCAM)-1 as determined by Western blotting. CV-159 inhibited TNF (20 min)–induced phosphorylation of Akt (Ser473) and NF-κB p65 (Ser536). An Akt inhibitor, LY294002, and an NF-κB inhibitor, pyrrolidine dithiocarbamate, inhibited TNF-induced VCAM-1. An antioxidant drug, N -acetyl-l-cysteine (NAC) inhibited TNF-induced VCAM-1. NAC also inhibited TNF-induced phosphorylation of Akt and NF-κB. Furthermore, CV-159 inhibited TNF-induced reactive oxygen species (ROS) production as determined fluorometrically using dichlorodihydrofluorescein diacetate. A CaM inhibitor, W-7, and a calcium/ CaM-dependent protein kinase type II inhibitor, KN93, inhibited TNF-induced VCAM-1. W-7 and KN93 inhibited TNF-induced phosphorylation of Akt but not NF-κB. The present results indicate that in vascular SMCs, CV-159 inhibits TNF-induced VCAM-1 through inhibition of NF-κB and Akt phosphorylation. CV-159 prevents NF-κB phosphorylation by inhibiting ROS, while it prevents Akt phosphorylation by inhibiting both ROS and CaM.

The anti-malaria agent artesunate inhibits expression of vascular endothelial growth factor and hypoxia-inducible factor-1α in human rheumatoid arthritis fibroblast-like synoviocyte

Increasing evidence indicates that the anti-malarial agent artemisinin and its derivatives may exert anti-angiogenic effect. In the present study, we explored the effect of artesunate, a artemisinin derivative, on TNFα- and hypoxia-induced expression of hypoxia inducible factor-1α (HIF-1α) and secretion of vascular endothelial growth factor (VEGF) and inteleukin-8 (IL-8) in human rheumatoid arthritis fibroblast-like synoviocytes (RA FLS), and further investigated the signal mechanism by which this compound modulates HIF-1α, VEGF and IL-8 expression. RA FLS obtained from patients with active rheumatoid arthritis were pretreated with artesunate, and then stimulated with TNFα and hypoxia. Production of VEGF and IL-8 was measured by ELISA. Nuclear location of HIF-1α was measured by confocal fluorescence microscopy. HIF-1α and other signal transduction proteins expression was measured by Western blot. Artesunate decreased the secretion of VEGF and IL-8 from TNFα- or hypoxia-stimulated RA FLS in a dose-dependent manner. Artesunate also inhibited TNFα- or hypoxia-induced nuclear expression and translocation of HIF-1α. We also showed that artesunate prevented Akt phosphorylation, but did not find evidence that phosphorylation of p38 and ERK was affected. TNFα- or hypoxia-induced secretion of VEGF and IL-8 and expression of HIF-1α were hampered by treatment with the PI3 kinase inhibitor LY294002, suggesting that inhibition of PI3 kinase/Akt activation might inhibit VEGF and IL-8 secretion and HIF-1α expression induced by TNFα or hypoxia. Our results suggest that artesunate inhibits angiogenic factor expression in RA FLS, and provide novel evidence that, as a low-cost agent, artesunate may have therapeutic potential for RA.

Depolarization is the trigger for PI3K/Akt activation and leads to ROS production in a model of pulmonary ischemia

We reported that ischemia or loss of shear in the lung endothelium causes endothelial plasma membrane depolarization (via KATP channel closure) that leads to NADPH oxidase activation and reactive oxygen species (ROS) production. Both high K+ induced depolarization and ischemia resulted in activation of PI3K; its inhibitor (wortmanin) prevented NADPH oxidase assembly and ROS production with ischemia. We used pulmonary microvascular endothelial cells, either wild type (WT), KATP−/−, gp91phox−/− or WT infected with dominant negative (DN) PI3K, and isolated Akt‐1 null lungs to study signal transduction with ischemia. Cells with DN PI3K and lungs with Akt1−/−, both showed the depolarization response but failed to show NADPH oxidase assembly and ROS generation. In WT cells, Akt activation (as monitored by the expression of p‐Akt) that was observed with ischemia was abolished in KATP−/− but not in gp91phox−/− cells. Blocking the ischemic depolarization by addition of cromakalim (KATP channel agonist) to gp91phox−/− cells prevented Akt phosphorylation. These results indicate that KATP induced depolarization leads to NADPH oxidase assembly and ROS generation and precedes PI3K/Akt activation. Thus, ROS which has hitherto been shown to activate PI3K/Akt in other systems lies downstream of PI3K/Akt in our model of pulmonary ischemia.

Identification of Peripherin as a Akt Substrate in Neurons

Activation of Akt-mediated signaling pathways is crucial for survival and regeneration of injured neurons. In this study, we attempted to identify novel Akt substrates by using an antibody that recognized a consensus motif phosphorylated by Akt. PC12 cells that overexpressed constitutively active Akt were used. Using two-dimensional PAGE, we identified protein spots that exhibited increased immunostaining of the antibody. Mass spectrometry revealed several major spots as the neuronal intermediate filament protein, peripherin. Using several peripherin fragments, the phosphorylation site was determined as Ser(66) in its head domain in vitro. Furthermore, a co-immunoprecipitation experiment revealed that Akt interacted with the head domain of peripherin in HEK 293T cells. An antibody against phosphorylated peripherin was raised, and induction of phosphorylated peripherin was observed not only in Akt-activated cultured cells but also in nerve-injured hypoglossal motor neurons. These results suggest that peripherin is a novel substrate for Akt in vivo and that its phosphorylation may play a role in motor nerve regeneration.

Anti-malarial agent artesunate inhibits TNF- -induced production of proinflammatory cytokines via inhibition of NF- B and PI3 kinase/Akt signal pathway in human rheumatoid arthritis fibroblast-like synoviocytes

Recent studies indicate that the anti-malarial agent artemisinin and its derivatives may exert an anti-inflammatory effect. In this study, we explored the effect of artesunate, an artemisinin derivative, on tumour necrosis factor (TNF)-alpha-induced production of interleukins, IL-1beta, IL-6 and IL-8, in human rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS), and further investigated the signal mechanism by which this compound modulates those cytokines' production. RA FLS obtained from patients with active RA were stimulated with TNF-alpha and incubated with artesunate, and IL-1beta, IL-6 and IL-8 production was measured by ELISA. DNA-binding activity and nuclear translocation of nuclear factor kappa B (NF-kappaB) were measured by a sensitive multi-well colourimetric assay and confocal fluorescence microscopy, respectively. Signal transduction proteins expression was measured by western blot. Artesunate decreased the secretion of IL-1beta, IL-6 and IL-8 from TNF-alpha-stimulated RA FLS in a dose-dependent manner. Artesunate also prevented TNF-alpha-induced nuclear NF-kappaB translocation, DNA-binding activity and gene transcriptional activity, as well as phosphorylation and degradation of IkappaBalpha, but phosphorylation of p38 mitogen-activated protein kinase, extracellular signal-regulated kinase and c-Jun N-terminal kinase were unaffected. The production of IL-1beta, IL-6 and IL-8 induced by TNF-alpha was decreased by pyrrolidine dithiocarbamate (PDTC), a chemical inhibitor of NF-kappaB. These observations suggest that artesunate inhibits production of IL-1beta, IL-6 and IL-8 through inhibition of NF-kappaB signalling pathway. We also showed that artesunate prevented Akt phosphorylation. TNF-alpha-induced production of IL-1beta, IL-6 and IL-8 was hampered by treatment with the phosphatidylinositol 3 (PI3) kinase inhibitor LY294002, suggesting that inhibition of Akt activation might inhibit IL-1beta, IL-6 and IL-8 production induced by TNF-alpha. Our results indicate that artesunate exerts an anti-inflammatory effect in RA FLS and provide the evidence that artesunate may have therapeutic potential for RA.

Protein kinase B/Akt phosphorylation of PDE3A and its role in mammalian oocyte maturation

cGMP-inhibited cAMP phosphodiesterase 3A (PDE3A) is expressed in mouse oocytes, and its function is indispensable for meiotic maturation as demonstrated by genetic ablation. Moreover, PDE3 activity is required for insulin/insulin-like growth factor-1 stimulation of Xenopus oocyte meiotic resumption. Here, we investigated the cAMP-dependent protein kinase B (PKB)/Akt regulation of PDE3A and its impact on oocyte maturation. Cell-free incubation of recombinant mouse PDE3A with PKB/Akt or cAMP-dependent protein kinase A catalytic subunits leads to phosphorylation of the PDE3A protein. Coexpression of PDE3A with constitutively activated PKB/Akt (Myr-Akt) increases PDE activity as well as its phosphorylation state. Injection of pde3a mRNA potentiates insulin-dependent maturation of Xenopus oocytes and rescues the phenotype of pde3(-/-) mouse oocytes. This effect is greatly decreased by mutation of any of the PDE3A serines 290-292 to alanine in both Xenopus and mouse. Microinjection of myr-Akt in mouse oocytes causes in vitro meiotic maturation and this effect requires PDE3A. Collectively, these data indicate that activation of PDE3A by PKB/Akt-mediated phosphorylation plays a role in the control of PDE3A activity in mammalian oocytes.