Inhibition of Phosphatidylinositol 3V-Kinase/AKTSignaling Promotes Apoptosis of Primary Effusion Lymphoma Cells

Purpose: Phosphatidylinositol 3V-kinase (PI3V-kinase) can be activated by the K1protein of Kaposi sarcoma ^ associated herpes virus (KSHV). However, the role of PI3V-kinase in KSHVassociated primary effusion lymphoma (PEL) is not known. To assess this, we studied survival and apoptosis in PEL cell lines following inhibition of PI3V-kinase. Experimental Design: Constitutive activation of several targets of PI3-kinase and apoptotic proteins were determined byWestern blot analysis using specific antibodies.We used LY294002 to block PI3V-kinase/AKTactivation and assess apoptosis by flow cytometric analysis. Results: Blocking PI3V-kinase induced apoptosis in PEL cells, including BC1, BC3, BCBL1, and HBL6, whereas BCP1was refractory to LY294002-induced apoptosis. LY294002-induced apoptosis did not seem to involve Fas/Fas-L but had an additive effect to CH11-mediated apoptosis. We also show that AKT/PKB is constitutively activated in all PELs and treatment with LY294002 causes complete dephosphorylation in all cell lines except BCP1where a residual AKT phosphorylation remained after 24 hours of treatment. FKHRandGSK3were also constitutively phosphorylated inPELs and treatmentwith LY294002 caused their dephosphorylation. Although inhibition of PI3V-kinase induced cleavage of BID in all cell lines, cytochrome c was released from themitochondria and caspase-9 and caspase-3 were activated in LY294002-induced apoptotic BC1but not in resistant BCP1. Similarly, XIAP, a target of AKT, was down-regulated after LY294002 treatment only in sensitive PEL cells. Conclusions: Our data show that the PI3V-kinase pathway plays a major role in survival of PEL cells and suggest that this cascade may be a promising target for therapeutic intervention in primary effusion lymphomas. Primary effusion lymphoma (PEL) is a subtype of nonHodgkin’s B-cell lymphoma that mainly presents in patients with advanced AIDS but is sometimes also found in HIVnegative individuals (1, 2). PEL grows as a lymphomatous effusion in body cavities and is always associated with Kaposi sarcoma–associated herpes virus (KSHV/HHV8). Most cases also show dual infection with EBV (EBV/HHV4; ref. 3). Pleural and abdominal effusions from patients with AIDS-PEL contain a number of cytokines (4, 5), which serve as autocrine growth factors for PELs. Interleukin 10 (IL-10) has been reported to serve as an autocrine growth factor for AIDS-related B-cell lymphoma (6). Recently, it has also been shown that PEL cells use viral IL-6 and IL-10 in an autocrine fashion for their survival and proliferation (4). Most PELs become resistant against the conventional chemotherapeutic agents. KSHV protein K1 has been shown to regulate a number of survival proteins. Studies have shown that K1 transgenic mice have constitutive activation of nuclear factor nB and Oct-2 as well as activation of Src-kinase Lyn (7). Furthermore, K1 protein activates phosphatidylinositol 3V-kinase (PI3V-kinase/ AKT) signaling pathway in B lymphocytes (8). AKT has been shown a target of a number of other transforming viral proteins including SV40 large and small T antigens, LMP1 and LMP2A (9–13). G protein–coupled receptor (vGPCR) of KSHV has been shown to transform cells by targeting AKT kinase (14–17). A recent study by Cannon and Cesarman has shown that vGPCR induces activation of AP1 and cAMPresponsive element-binding protein in PEL cells (18). Furthermore, KSHV infection in PEL leads to the activation of nuclear factor nB, which plays a vital role in the survival of these cells (19). Considerable evidence shows a role of PI3V-kinase/AKT signaling in oncogenic transformation and cancer progression (20). Deregulated PI3V-kinase signaling also provides attractive targets for therapy (21, 22). AKT prevents apoptosis by generating antiapoptotic signals through phosphorylation of Bad, GSK3, and caspase-9 and activation of transcriptional factors such as Forkhead (FOXO1) and nuclear factor nB (23–27). These functions of PI3V-kinase make it a potential www.aacrjournals.org Clin Cancer Res 2005;11(8) April 15, 2005 3102 Authors’Affiliations: King Fahad National Center for Children’s Cancer and Research, Biological and Medical Research, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia; and Section of Hematology-Oncology, University of Chicago, Chicago, Illinois Received 9/11/04; revised10/27/04; accepted11/18/04. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section1734 solely to indicate this fact. Requests for reprints: Shahab Uddin, King Fahad National Center for Children’s Cancer and Research, MBC #98-16, P.O. Box 3354, Riyadh 11211, Saudi Arabia. Phone: 966-1-2294444, ext. 51815; Fax: 966-1-2293671; E-mail: shahab@ kfshrc.edu.sa. F2005 American Association for Cancer Research. CancerTherapy: Preclinical Research. on June 3, 2017. © 2005 American Association for Cancer clincancerres.aacrjournals.org Downloaded from target for chemotherapeutic agents. Indeed, PI3V-kinase pathway inhibitors are already in early clinical trials. In this study, we show for the first time that the PI3V-kinase/ AKT pathway is constitutively activated in several PEL cell lines. Inhibition of PI3V-kinase activity by LY294002 not only caused dephosphorylation of basal levels of AKT, GSK3, and FKHR but also induced apoptosis in most PEL cell lines. Apoptosis occurred through release of cytochrome c from the mitochondria, activation of caspase-9 and caspase-3, and downregulation of the inhibitor of apoptosis, XIAP. Materials andMethods Cell culture. Human primary effusion lymphoma cell lines established from lymphomatous effusion from patients with body cavity– based lymphomas, BC1, BC3, BCBL1, BCP1, and HBL6 were used. Cells were cultured in RPMI 1640 supplemented with 10% (v/v) fetal bovine serum, 100 units/mL penicillin, and 100 units/mL streptomycin at 37jC in an humidified atmosphere containing 5% CO2. All PEL cell lines are infected with HHV8 and BC1 and HBL6 are also infected with EBV. These cell lines have been thoroughly characterized for immunophenotypic expression and genetic lesions (3–5). Reagents and antibodies. LY294002 and anti – caspase-9 were obtained from Calbiochem (San Diego, CA). The anti–phospho-AKT, anti–phospho-GSK3a/h, anti–phospho-FKHRL1, anti-cleaved caspase3, and anti-BID antibodies were purchased from Cell Signaling Technologies (Beverly, MA). PTEN antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti–h-actin was purchased from Abcam (Cambridge, United Kingdom). Anti–cytochrome c and anti-XIAP antibodies were obtained from R&D Systems (Minneapolis, MN). Anti–caspase-3 was purchased from BD PharMingen (San Diego, CA). The anti–poly(ADP-ribose) polymerase antibody was obtained from Zymed Lab (San Francisco, CA). Anti–Fas CH11 and ZB4 antibodies were purchased from MBL (Watertown, MA). Apoptosis. PEL cell lines were treated with LY294002 as described in figure legends. Cells were harvested and the percentage apoptosis was measured by flow cytometry after staining with flourescein-conjugated Annexin V and propidium iodide (PI; Molecular Probes, Eugene, OR) as described previously (28). We scored viable cells as those that are negative for Annexin V and PI. Percentage of apoptosis was calculated from the reduction of the number of viable cells between treated and untreated samples. The amount of necrotic cells (Annexin V negative, PI positive) was always minimal. Cell lysis and immunoblotting. After treatment in various conditions, cells were collected by centrifugation at 1,000 rpm and lysed in phosphorylation lysis buffer (0.5-1.0% Triton X-100 or 1% digitonin, 150 mmol/L NaCl, 1 mmol/L EDTA, 200 Amol/L sodium orthovanadate, 10 mmol/L sodium pyrophosphate, 100 mmol/L sodium fluoride, 1.5 mmol/L magnesium chloride, 1 mmol/L phenylmethylsulfonyl flouride, and 10 Ag/mL aprotonin; ref. 29). Protein concentrations were assessed by the Bradford assay. Equal amount of proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride membrane (Immobilon-PVDF; Millipore, Bedford, MA). Immunoblotting was done with different antibodies and developed using Enhanced Chemiluminescence Plus (Amersham, Buckinghamshire, United Kingdom). Assay for cytochrome c release. Release of cytochrome c from mitochondria was assayed as described earlier (30). Briefly, cell pellets were resuspended in 5 volumes of a hypotonic buffer [20 mmol/L HEPES/KOH (pH 7.5), 10 mmol/L KCl, 1.5 mmol/L MgCl2, 1 mmol/L EDTA, 1 mmol/L EGTA, 1 mmol/L DTT, 20 Ag/mL leopeptin, 10 Ag/mL aprotonin, and 250 mmol/L sucrose] and incubated for 15 minutes on ice. Cells were homogenized by 15 to 20 passages through a 22-gauge needle, 1.5 in. long. The lysates were centrifuged at 1,000 g for 5 minutes at 4jC to pellet nuclei and unbroken cells. Supernatants were collected and centrifuge at 12,000 g for 15 minutes. The resulting mitochondrial pellets were suspended in lysis buffer. Supernatants were transferred to new tubes and centrifuged again at 12,000 g for 15 minutes. These resulting supernatants represent cytosolic fractions. Twenty Ag of proteins from the cytosolic fraction of each sample were analyzed by immunoblotting using an anti–cytochrome