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Lipid Raft-Mediated Akt Signaling As a Therapeutic Target in Mantle Cell Lymphoma

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Lipid Raft-Mediated Akt Signaling As a Therapeutic Target in Mantle Cell Lymphoma OPEN Citation: Blood Cancer Journal (2013) 3, e118; doi:10.1038/bcj.2013.15 & 2013 Macmillan Publishers Limited All rights reserved 2044-5385/13 www.nature.com/bcj ORIGINAL ARTICLE Lipid raft-mediated Akt signaling as a therapeutic target in mantle cell lymphoma M Reis-Sobreiro1, G Roue´ 2, A Moros2, C Gajate1, J de la Iglesia-Vicente1, D Colomer2 and F Mollinedo1 Recent evidence shows that lipid raft membrane domains modulate both cell survival and death. Here, we have found that the phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathway is present in the lipid rafts of mantle cell lymphoma (MCL) cells, and this location seems to be critical for full activation and MCL cell survival. The antitumor lipids (ATLs) edelfosine and perifosine target rafts, and we found that ATLs exerted in vitro and in vivo antitumor activity against MCL cells by displacing Akt as well as key regulatory kinases p-PDK1 (phosphatidylinositol-dependent protein kinase 1), PI3K and mTOR (mammalian TOR) from lipid rafts. This raft reorganization led to Akt dephosphorylation, while proapoptotic Fas/CD95 death receptor was recruited into rafts. Raft integrity was critical for Ser473 Akt phosphorylation. ATL-induced apoptosis appeared to correlate with the basal Akt phosphorylation status in MCL cell lines and primary cultures, and could be potentiated by the PI3K inhibitor wortmannin, or inhibited by the Akt activator pervanadate. Classical Akt inhibitors induced apoptosis in MCL cells. Microenvironmental stimuli, such as CD40 ligation or stromal cell contact, did not prevent ATL-induced apoptosis in MCL cell lines and patient-derived cells. These results highlight the role of raft-mediated PI3K/Akt signaling in MCL cell survival and chemotherapy, thus becoming a new target for MCL treatment. Blood Cancer Journal (2013) 3, e118; doi:10.1038/bcj.2013.15; published online 31 May 2013 Keywords: mantle cell lymphoma; lipid rafts; PI3K/Akt signaling; Akt phosphorylation; apoptosis; synthetic antitumor lipids INTRODUCTION phosphatidylinositol-3,4,5-trisphosphate, which binds to Akt PH Mantle cell lymphoma (MCL) is a B cell-derived neoplasia that domain and phosphatidylinositol-dependent protein kinase 1 constitutes B6% of all non-Hodgkin lymphomas, and is char- (PDK1), recruiting them to the membrane. Once in the membrane, 8 acterized by the chromosomal translocation t(11;14)(q13;q32), Akt is phosphorylated at two key residues, Thr308 and Ser473. leading to aberrant overexpression of cyclin D1.1,2 In addition to Thr308 site is localized in the activation loop and is this initial oncogenic event, MCL may carry a high number of phosphorylated by PDK1, whereas Ser473 is localized in the secondary chromosomal and molecular alterations that influence C-terminal hydrophobic motif and is phosphorylated by PDK2.8 the aggressive behavior of this neoplasm and a poor survival Mammalian TOR (mTOR) has been suggested as the main outcome.1,2 Although conventional chemotherapy induces high- candidate for PDK2, but DNA-PK, ILK or even Akt itself through remission rates, relapse within a few years is common, autophosphorylation have also been proposed to act as PDK2.8,9 contributing to a median survival of 5–7 years.3 mTOR forms part of two different complexes, mTOR complex 1 Several signaling pathways are deregulated in MCL cells (mTORC1) and mTORC2, which exert their actions by regulating a including the constitutive activation of the phosphatidylinositol- number of important proteins.10 mTORC2 acts upstream and is 3-kinase (PI3K)/Akt pathway, which mediates the effects of a suggested to phosphorylate Akt, whereas mTORC1 acts variety of extracellular signals and is crucial for the maintenance downstream of Akt.10 and proliferation of MCL cells,4,5 thus attracting great interest as a Insulin-like growth factor 1 (IGF-1)-mediated activation of Akt possible therapeutic target. PI3K is activated by a wide range of has been reported to be dependent on lipid rafts.11,12 Rafts are tyrosine kinase growth factor receptors, and is the major activator membrane microdomains highly enriched in cholesterol and of Akt, having a central role in fundamental biological processes sphingolipids, which act as scaffolds for signaling pathways in the including cell growth, proliferation, migration and survival, cell membrane,13 and growing evidence shows their potential as through phosphorylation of a plethora of substrates.4,6 Akt is a therapeutic targets in cancer therapy.14–23 An endogenous raft- protein kinase that belongs to the AGC family of serine/threonine resident Akt has been reported in LNCaP human prostate cells that kinases and has three conserved domains, namely: pleckstrin showed a different substrate affinity as compared with non-raft homology domain (PH), which binds phosphoinositides with Akt, suggesting the presence of distinct Akt populations with high affinity, as well as catalytic and regulatory domains.7 differential signaling behavior.24 Akt suppresses apoptosis by direct phosphorylation of The synthetic antitumor lipids (ATLs) are a family of compounds, proapoptotic proteins such as Bad and pro-caspase-9.7 PI3K phos- initially synthesized as ether phospholipids and widely referred phorylates phosphatidylinositol-4,5-bisphosphate to generate to as alkyl-lysophospholipid analogs, which are able to selectively 1Instituto de Biologı´a Molecular y Celular del Ca´ncer, Centro de Investigacio´ndelCa´ncer, CSIC-Universidad de Salamanca, Campus Miguel de Unamuno, Salamanca, Spain and 2Hematopathology Unit, Department of Pathology, Hospital Clı´nic, Institut d’Investigacions Biome`diques August Pi i Sunyer (IDIBAPS), University of Barcelona, Barcelona, Spain. Correspondence: Dr F Mollinedo, Instituto de Biologı´a Molecular y Celular del Ca´ncer, Centro de Investigacio´ndelCa´ncer, CSIC-Universidad de Salamanca, Campus Miguel de Unamuno, Salamanca E-37007, Spain. E-mail: [email protected] Received 7 September 2012; revised 18 February 2013; accepted 20 March 2013 Lipid raft-mediated PI3K/Akt route in MCL therapy M Reis-Sobreiro et al 2 induce apoptosis in tumor cells.18,21,25–29 These ATLs include the of at least 80%, as assessed by flow cytometry detection of CD19 þ /CD5 þ ether lipid edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3- cells from whole peripheral blood mononuclear cells. phosphocholine) as well as perifosine (octadecyl-(1,1-dimethyl- piperidinio-4-yl)-phosphate), which accumulate in lipid rafts,14,19,30 Apoptosis assay promote co-capping of Fas/CD95 death receptor and rafts,19,31,32 and inhibit PI3K/Akt survival signaling.32–34 Edelfosine shows Quantitation of apoptotic cells was calculated by flow cytometry as the 35,36 percentage of cells in the sub-G0/G1 region (hypodiploidy) in cell cycle affinity for cholesterol likely due to geometry compensation of analysis, as previously described.39 the ‘cone shape’ of sterols and the ‘inverted cone shape’ of 36 To analyze apoptosis by Annexin V labeling in MCL patient-derived edelfosine that leads to a stable bilayer. In vivo and in vitro samples and cell lines, 5 Â 105 cells were incubated for 24 h with the experimental approaches have shown that ATLs selectively kill MCL indicated agents. Cells were then washed in Annexin V-binding buffer and cells as well as additional hematological cancer cells, including incubated in 50 ml Annexin V-binding buffer with allophycocyanin- patient-derived primary cancer cells, by a lipid raft-dependent conjugated anti-CD3 and phycoerythrin-conjugated anti-CD19 antibodies mechanism.18,21,29 (Becton Dickinson, San Jose, CA, USA) for 10 min in the dark. Cells were In this work, we provide evidence that MCL cell survival then diluted with Annexin V-binding buffer to a volume of 150 ml and depends on raft-mediated Akt activation for survival. We show incubated with 1 ml FITC-labeled Annexin V (Bender MedSystems, Vienna, Austria) for 15 min in the dark. A total of 10 000 stained cells were then here that ATLs inhibit PI3K/Akt signaling by displacing Akt and key analyzed by flow cytometry on a Becton Dickinson fluorescence-activated enzyme regulators from lipid rafts, leading to Akt dephosphoryla- cell sorting (FACS) Calibur flow cytometer using CellQuest software tion and apoptosis. In contrast, Fas/CD95 death receptor was (Becton Dickinson). recruited to rafts upon ATL treatment. Our data suggest that raft environment is essential for Ser473 Akt phosphorylation in MCL cells. ATL oral treatment inhibited MCL tumor growth in Cytofluorimetric analysis of mitochondrial transmembrane xenograft animal models. Apoptosis induced by either edelfosine potential and generation of reactive oxygen species or perifosine was not blocked by tumor microenvironmental To evaluate mitochondrial transmembrane potential (DCm) disruption and the generation of reactive oxygen species, cells (106/ml) were incubated in stimuli in MCL primary cultures and cell lines. These results 0 highlight the importance of raft-mediated PI3K/Akt targeting in phosphate-buffered saline (PBS) with 20 nM 3,3 -dihexyloxacarbocyanine iodide [DiOC (3)] (green fluorescence) (Molecular Probes, Leiden, The MCL therapy. 6 Netherlands) and 2 mM dihydroethidine (red fluorescence after oxidation) (Sigma, St Louis, MO, USA) for 40 min at 37 1C, followed by analysis on a FACSCalibur flow cytometer, as previously described.39 MATERIALS AND METHODS Reagents Western Blot Edelfosine (Inkeysa, Barcelona, Spain) and perifosine (Zentaris, Frankfurt, 6 Cells (4–5 10 ) were lysed with 60 ml lysis buffer (200 mM HEPES, pH 7.5, Germany) were prepared as 2-mM stock solutions in culture medium. Anti- Â CD40 immunoglobulin was a kind gift from Francisco Lozano (Immunology 10 mM EGTA, 40 mM b-glycerophosphate, 1% (v/v) NP-40, 25 mM MgCl2, Department, Hospital Clı´nic-IDIBAPS, Barcelona, Spain). Caspase inhibitor 2mM sodium orthovanadate, 1 mM DTT) supplemented with protease benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (z-VAD-fmk) inhibitors (1 mM phenylmethanesulfonyl fluoride, 20 mg/ml aprotinin and (Enzo Life Sciences, Lausen, Switzerland) was prepared in dimethyl sulfoxide 20 mg/ml leupeptin). Proteins (40 mg) were run on SDS-polyacrylamide gels under reducing conditions, transferred to polyvinylidene fluoride mem- as a 100-mM stock solution.
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