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Ras Signaling Influences Permissiveness of Malignant Peripheral Nerve Sheath Tumor Cells to Oncolytic Herpes

2008; Elsevier BV; Volume: 173; Issue: 6 Linguagem: Inglês

10.2353/ajpath.2008.080376

1525-2191

Faris Farassati, Weihong Pan, Farnaz Yamoutpour, Susann Henke, Mark Piedra, Silke Frahm, Said Al-Tawil, Wells I. Mangrum, Luis F. Parada, Samuel D. Rabkin, Robert L. Martuza, Armin Kurtz,

Neuroblastoma Research and Treatments

Lack of expression of neurofibromin in neurofibromatosis 1 and its lethal derivative, malignant peripheral nerve sheath tumors (MPNSTs), is thought to result in the overactivation of the Ras signaling pathway. Our previous studies have shown that cells with overactivation in the Ras pathway are more permissive to infection with herpes simplex virus 1 and its mutant version R3616. In this study, we show that among five different mouse MPNST cell lines, only the ones with elevated levels of Ras signaling are highly permissive to infection with oncolytic herpes G207. Specific inhibitors of the Ras, ERK, and JNK pathways all reduced the synthesis of viral proteins in MPNST cells. The cell lines that contained lower levels of Ras and decreased activation of downstream signaling components underwent an enhancement in apoptosis upon exposure to G207. Additionally, mouse SW10 Schwann cells were able to become infected by parental herpes but were found to be resistant to G207. The immortalization of these cell lines with the expression of SV40 large T antigen increased the levels of Ras activation and permissiveness to oncolytic herpes. A Ras/Raf kinase inhibitor reduced the synthesis of both herpes simplex virus-1 and G207 proteins in SW10 cells. The results of this study, therefore, introduce Ras signaling as a divergent turning point for the response of MPNST cells to an assault by oncolytic herpes. Lack of expression of neurofibromin in neurofibromatosis 1 and its lethal derivative, malignant peripheral nerve sheath tumors (MPNSTs), is thought to result in the overactivation of the Ras signaling pathway. Our previous studies have shown that cells with overactivation in the Ras pathway are more permissive to infection with herpes simplex virus 1 and its mutant version R3616. In this study, we show that among five different mouse MPNST cell lines, only the ones with elevated levels of Ras signaling are highly permissive to infection with oncolytic herpes G207. Specific inhibitors of the Ras, ERK, and JNK pathways all reduced the synthesis of viral proteins in MPNST cells. The cell lines that contained lower levels of Ras and decreased activation of downstream signaling components underwent an enhancement in apoptosis upon exposure to G207. Additionally, mouse SW10 Schwann cells were able to become infected by parental herpes but were found to be resistant to G207. The immortalization of these cell lines with the expression of SV40 large T antigen increased the levels of Ras activation and permissiveness to oncolytic herpes. A Ras/Raf kinase inhibitor reduced the synthesis of both herpes simplex virus-1 and G207 proteins in SW10 cells. The results of this study, therefore, introduce Ras signaling as a divergent turning point for the response of MPNST cells to an assault by oncolytic herpes. The Ras signal transduction pathway acts as a central hub through which a variety of cell functions are controlled. 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4: 4Crossref PubMed Scopus (9) Google Scholar Malignant peripheral nerve sheath tumors (MPNSTs) arise from plexiform neurofibromas in 10 to 15% of NF1 patients. MPNST is considered the main cause of mortality in adult NF1 patients, with only 34 to 52% of patients surviving for 5 years.25Woodruff JM Pathology of tumors of the peripheral nerve sheath in type 1 neurofibromatosis.Am J Med Genet. 1999; 89: 23-30Crossref PubMed Scopus (156) Google Scholar The molecular events involved in the malignant transformation of benign neurofibromas to MPNST are poorly defined. It has been postulated that one of the main tumor-causing effects of mutations in the tumor suppressor nf1 gene is the subsequent activation of Ras.26Harrisingh MC Lloyd AC Ras/Raf/ERK signalling and NF1.Cell Cycle. 2004; 3: 1255-1258Crossref PubMed Scopus (50) Google Scholar, 27Der SD Yang YL Weissmann C Williams BR A double-stranded RNA-activated protein kinase-dependent pathway mediating stress-induced apoptosis.Proc Natl Acad Sci USA. 1997; 94: 3279-3283Crossref PubMed Scopus (365) Google Scholar, 28Rasmussen SA Friedman JM NF1 gene and neurofibromatosis 1.Am J Epidemiol. 2000; 151: 33-40Crossref PubMed Scopus (312) Google Scholar, 29Bajenaru ML Donahoe J Corral T Reilly KM Brophy S Pellicer A Gutmann DH Neurofibromatosis 1 (NF1) heterozygosity results in a cell-autonomous growth advantage for astrocytes.Glia. 2001; 33: 314-323Crossref PubMed Scopus (67) Google Scholar, 30Sherman LS Atit R Rosenbaum T Cox AD Ratner N Single cell Ras-GTP analysis reveals altered Ras activity in a subpopulation of neurofibroma Schwann cells but not fibroblasts.J Biol Chem. 2000; 275: 30740-30745Crossref PubMed Scopus (112) Google Scholar Inactivation of both copies of the nf1 gene has been demonstrated in benign human neurofibromas and has been shown to cause tumors in murine models.31Serra E Puig S Otero D Gaona A Kruyer H Ars E Estivill X Lazaro C Confirmation of a double-hit model for the NF1 gene in benign neurofibromas.Am J Hum Genet. 1997; 61: 512-519Abstract Full Text PDF PubMed Scopus (190) Google Scholar Loss of heterozygosity of nf1 and p53 has frequently been observed in human MPNST.32Menon AG Anderson KM Riccardi VM Chung RY Whaley JM Yandell DW Farmer GE Freiman RN Lee JK Li FP Chromosome 17p deletions and p53 gene mutations associated with the formation of malignant neurofibrosarcomas in von Recklinghausen neurofibromatosis.Proc Natl Acad Sci USA. 1990; 87: 5435-5439Crossref PubMed Scopus (337) Google Scholar, 33Legius E Dierick H Wu R Hall BK Marynen P Cassiman JJ Glover TW TP53 mutations are frequent in malignant NF1 tumors.Genes Chromosom Cancer. 1994; 10: 250-255Crossref PubMed Scopus (149) Google Scholar, 34Rey JA Pestana A Bello MJ Cytogenetics and molecular genetics of nervous system tumors.Oncol Res. 1992; 4: 321-331PubMed Google Scholar Recombinant mouse strains (NP mice), which harbor inactivated nf1 and p53 alleles (cis-nf1+/−:p53+/−) demonstrate the cumulative effects of loss of both nf1 and p53 genes in the etiology of MPNST.35Vogel KS Klesse LJ Velasco-Miguel S Meyers K Rushing EJ Parada LF Mouse tumor model for neurofibromatosis type 1.Science. 1999; 286: 2176-2179Crossref PubMed Scopus (291) Google Scholar, 36Cichowski K Shih TS Schmitt E Santiago S Reilly K McLaughlin ME Bronson RT Jacks T Mouse models of tumor development in neurofibromatosis type 1.Science. 1999; 286: 2172-2176Crossref PubMed Scopus (374) Google Scholar To understand the extent of the influence of nf1-mutations on the outcome of Ras signaling, we have examined the activation of this pathway in cells isolated from distinct MPNST tumors of NP mice35Vogel KS Klesse LJ Velasco-Miguel S Meyers K Rushing EJ Parada LF Mouse tumor model for neurofibromatosis type 1.Science. 1999; 286: 2176-2179Crossref PubMed Scopus (291) Google Scholar that exhibit loss of heterozygosity for both nf1 and p53 genes but no mutations in Ras gene. The results of our study show that although all of the studied MPNST cell lines originated from a cis-nf1+/−:p53+/− background, overactivity in Ras signaling was only observed in two of the five cell lines. Activation patterns of downstream Ras-signaling pathways follow the overactivity seen for Ras: cell lines with higher Ras-GTP levels exhibit elevated levels of kinase activity for extracellular signal-related kinase (ERK), Jun amino-terminal kinase (JNK), p38-kinase, and phosphatidylinositol 3-kinase (PI3K). In the next step, we linked the overall Ras-signaling portfolio of MPNST cells to biological characteristics that may influence the outcome of therapy. The apoptotic capabilities of MPNST cells and their permissiveness to G207, an oncolytic version of herpes simplex virus-1 (HSV-1),37Mineta T Rabkin SD Yazaki T Hunter WD Martuza RL Attenuated multi-mutated herpes simplex virus-1 for the treatment of malignant gliomas.Nat Med. 1995; 1: 938-943Crossref PubMed Scopus (706) Google Scholar, 38Markert JM Medlock MD Rabkin SD Gillespie GY Todo T Hunter WD Palmer CA Feigenbaum F Tornatore C Tufaro F Martuza RL Conditionally replicating herpes simplex virus mutant. G207 for the treatment of malignant glioma: results of a phase I trial.Gene Ther. 2000; 7: 867-874Crossref PubMed Scopus (840) Google Scholar was studied. G207 has been explored for its therapeutic efficacy in a murine model of MPNST.39Mashour GA Moulding HD Chahlavi A Khan GA Rabkin SD Martuza RL Driever PH Kurtz A Therapeutic efficacy of G207 in a novel peripheral nerve sheath tumor model.Exp Neurol. 2001; 169: 64-71Crossref PubMed Scopus (18) Google Scholar Upon infection with G207, MPNST cells with increased Ras signaling exhibited higher viral permissiveness than cell lines with lower levels of Ras signaling. The cells with lower Ras signaling underwent apoptosis after exposure to G207. This finding is in agreement with our previous studies in which Ras signaling was correlated to the permissiveness of cells to herpesvirus40Farassati F Lee PW Ras signalling pathway: a gateway for HSV-1 infection.Sci World J. 2003; 3: 533-535Crossref Scopus (7) Google Scholar, 41Farassati F Yang AD Lee PW Oncogenes in Ras signalling pathway dictate host-cell permissiveness to herpes simplex virus 1.Nat Cell Biol. 2001; 3: 745-750Crossref PubMed Scopus (199) Google Scholar and other viruses such as reovirus.42Coffey MC Strong JE Forsyth PA Lee PW Reovirus therapy of tumors with activated Ras pathway.Science. 1998; 282: 1332-1334Crossref PubMed Scopus (657) Google Scholar, 43Norman KL Coffey MC Hirasawa K Demetrick DJ Nishikawa SG DiFrancesco LM Strong JE Lee PW Reovirus oncolysis of human breast cancer.Hum Gene Ther. 2002; 13: 641-652Crossref PubMed Scopus (142) Google Scholar, 44Strong JE Coffey MC Tang D Sabinin P Lee PW The molecular basis of viral oncolysis: usurpation of the Ras signaling pathway by reovirus.EMBO J. 1998; 17: 3351-3362Crossref PubMed Scopus (469) Google Scholar For MPNST cells with both high- and low-Ras signaling, a significant decrease in proliferation and invasiveness was observed. Further, we have observed that untransformed Schwann cells (with low-Ras activation) remain nonpermissive to G207 infection. However, these cells become more permissive to infection upon conditional immortalization (by expression of SV40 large T antigen), which induces the Ras-Raf pathway. Blockers of Raf, Ras, ERK, and JNK all inhibit the production of viral proteins. The results of this study, therefore, not only expand our fundamental understanding about the regulation of Ras signaling in MPNST cells but also correlate such information with the responsiveness of MPNST tumor cells to oncolytic HSV as a model for linking signaling characteristics and therapeutic outcomes. Mouse MPNST cells (6IE4, 37-3-18, 35-1-2, 38-2-18, and 32-5-24-12 cells) were originally isolated from different mouse MPNSTs.35Vogel KS Klesse LJ Velasco-Miguel S Meyers K Rushing EJ Parada LF Mouse tumor model for neurofibromatosis type 1.Science. 1999; 286: 2176-2179Crossref PubMed Scopus (291) Google Scholar Mouse Schwann cells, SW10, were purchased from American Type Culture Collection, Rockville, MD (American Type Culture Collection number CRL-2766). All cells were maintained in Dulbecco's modified Eagle's medium, 10% fetal bovine serum, and additional antibiotics. G207 was prepared as described previously37Mineta T Rabkin SD Yazaki T Hunter WD Martuza RL Attenuated multi-mutated herpes simplex virus-1 for the treatment of malignant gliomas.Nat Med. 1995; 1: 938-943Crossref PubMed Scopus (706) Google Scholar or provided by MediGene, Inc., San Diego, CA. HSV-1(F) virus was a gift from Dr. Bernard Roizman (University of Chicago, Chicago, IL). Ras activation assay were performed in accordance to the manufacturer's (Upstate, Lake Placid, NY) instruction. Briefly, cells grown in 10-cm tissue culture dishes were lysed at 75 to 80% confluency in magnesium lysis buffer. After determination of protein concentration, 10 μl of Ras assay reagent (Raf-1 binding domain-agarose) was added to 200 μg of total cell protein in 200 μl of magnesium lysis buffer. After a period of rocking at 4°C, the activated (GTP) form of Ras bound to the agarose beads was collected by centrifugation, washed, boiled in 2× Laemmli reducing sample buffer (Bio-Rad, Hercules, CA) and loaded on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel (Bio-Rad). The proteins were transferred to a nitrocellulose membrane; blocked with Tris-buffered saline/0.2%Tween/5% milk (TBST-MLK) and incubated with Pan-Ras antibody (1:1000). The membrane was then washed with TBST for 10 minutes each and then incubated with sheep anti-mouse horseradish peroxidase-conjugated IgG (1:2000; Amersham, Arlington Heights, IL). Bands were detected using LumiGLO chemiluminescent reagent peroxide (Cell Signaling, Beverly, MA). Nonradioactive Ras effector assays were performed in accordance with the manufacturer's (Cell Signaling) instructions. Briefly, cells grown in 10-cm tissue culture dishes were lysed at 75 to 80% confluency with 300 μl of cell lysis buffer. Immobilized antibody-bead slurry capable of binding to the phosphorylated forms of ERK, JUN, AKT or p38-kinase was then added to 200 μg of total cell protein in 200 μl of cell lysis buffer. The mixture was incubated with gentle rocking overnight at 4°C then collected, washed, and introduced to an in vitro kinase reaction in presence of ATP and a special buffer. The levels of phosphorylated ELK, JUN, ATF2, and AKT were then assayed by Western blotting using antibodies directed against phosphorylated forms of these proteins. Annexin-V labeling was performed according to the manufacturer's (Calbiochem, La Jolla, CA) recommendations for the conventional Annexin-V binding protocol. Briefly, cells were washed in phosphate-buffered saline (PBS) with gentle centrifuge (1000 × g for 5 minutes at room temperature) and resuspended in 0.5 ml of cold 1× binding buffer with the addition of 1.25 μl of Annexin V-fluorescein isothiocyanate (FITC). After incubation for 15 minutes, cells were centrifuged, supernatant was removed, and cells were resuspended in 0.5 ml of cold 1× binding buffer. In the next step, 10 μl of propidium iodide were added and samples were placed on ice in the dark and then analyzed by a flow cytometer using an argon 488-nm argon ion laser source. TUNEL assay was also performed according to the recommendations by the manufacturer (Chemicon, Temecula, CA). Briefly, cells were fixed in paraformaldehyde and alcohol and then incubated at −20°C overnight. After rehydration of cells in Tris-buffered saline, they were permeabilized by proteinase K, equilibrated by terminal deoxynucleotidyl transferase (TdT) buffer, and labeled by TdT reaction mixture/enzyme at 37°C for 1.5 hours in the dark. The reaction was then stopped by washing with Tris-buffered saline and cells were analyzed by a flow cytometer using an argon 488-nm argon ion laser source. MPNST and Schwann cells were grown in eight-well slide chambers (Falcon, San Jose, CA) and infected with HSV-1 (strain F) or G207 or mock-infected. At different times after infection, cells were fixed in acetone (100%) for 10 minutes and then left at room temperature to dry before incubation with a fluorescein-labeled mouse monoclonal antibody against HSV-1 gC antigen (Labvision, Fremont, CA) for 30 minutes at 37°C. The slides were washed with distilled water, dried, and mounted in 90% glycerol containing 0.1% phenylenediamine, and viewed with an Axiophot microscope (Carl Zeiss, Thornwood, NY) on which a Carl Zeiss camera was mounted. Pictures were captured by an attached computer and processed with appropriate software. Different cells were lysed with a single detergent lysis buffer [50 mmol/L Tris (pH 8.0), 150 mmol/L NaCl, 0.02% sodium azide, 100 μg/ml phenylmethyl sulfonyl fluoride, 1 μg/ml aprotinin, and 1% Triton X-100], normalized for the amount of total protein and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis using a Bio-Rad minicell protein-II system (using precast 10% discontinuous gels) followed by electroblotting onto nitrocellulose paper. The membrane was then washed and incubated with a primary rabbit antibody against all HSV-1 antigens (DAKO, Carpinteria, CA), followed by the horseradish peroxidase-conjugated secondary antibody. After extensive washing, the blot was exposed to Lumigel (Viagen, Tempa, FL) detection solution and subjected to autoradiography. Uninfected 37-3-18 cells were exposed to Ras and its downstream specific inhibitors overnight at the following concentrations: AG-14178 at 0.5 μmol/L, L-744,832 at 60 μmol/L, PD98059 at 20 μmol/L, and SP600125 at 25 μmol/L. Schwann cells were exposed to Raf1 kinase inhibitor-II. L-744,832 and Raf1 kinase inhibitor-II were obtained from Calbiochem. AG14178, PD98059, and SP600125 were obtained from Biosource (Camarillo, CA). Plaque titration was performed with the purpose of evaluating the yield of progeny virus at 24 hours after infection. Briefly, the supernatant from infected cells were serially diluted from 1/10 to 1/108. A volume of 300 μl from each dilution was added to duplicate wells (of six-well plates) containing Vero cells at ∼75% confluency after removal of the existing media and rinsing the cells with PBS. Cells were then incubated at 37°C until development of cytopathic effects in the form of plaques, which usually occurs within 2 to 3 days. At this point, a monolayer was fixed with methanol and stained with Giemsa solution for 10 minutes. The number of clear plaques was then determined by calculating the average number of plaques per well for each dilution and the volume used to infect each well. To evaluate cell invasiveness, a commercial kit was used (BD Biosciences, San Jose, CA). Briefly, cells (50,000 control or test cells) were introduced into Matrigel-coated inserts fitting 24-well plates. As cells invaded through the layer of Matrigel, the fraction of invaded cells were detected by staining with crystal violet and quantifying by spectroscopy. Invaded cells were fixed with 5% paraformaldehyde and stained with a 5% solution of crystal violet and then photographed to obtain a visual representation of their density. The cells were then solubilized in a 3% detergent (Nonidet P-40) solution, and the absorbance was measured by spectrophotometry at 590 nm. The cell proliferation assay was performed using a kit (Chemicon) according to the manufacturer's instructions. The assay is based on the cleavage of the tetrazolium salt WST-1 to formazan by cellular mitochondrial dehydrogenases. Expansion in the number of viable cells results in an increase in the overall activity of the mitochondrial dehydrogenases resulting in an increase in the amount of formazan dye formed. Briefly, 104 cells per well were seeded in a 96-well microplate in volume of 100 μl per well. At different times, 10 μl of WST-1/ECS solution was added to each well. The plates were incubated for 4 hours in standard culture conditions. The plates were then shaken thoroughly for 1 minute and absorbance was measured at 480 nm. Results are reported as means ± SD. Student's t-test was used to analyze statistical differences between groups. α level was set at 0.05. Ras activation stimulates a variety of effector pathways including ERK, JNK, p38 kinase, and PI3K (Figure 1A).1Downward J Targeting RAS signalling pathways in cancer therapy.Nat Rev Cancer. 2003; 3: 11-22Crossref PubMed Scopus (2580) Google Scholar Because the Ras-GAP function of neurofibromin is lost in the MPNST cell lines used in this study, we were interested in elucidating the effects of such Ras-deregulating genetic abnormalities on Ras activation and signaling. To achieve this, the amount of Ras-GTP was determined using an affinity precipitation assay effectively differentiating between active and inactive forms of Ras. As is shown in Figure 1B, among the five studied mouse MPNST cell lines, only 6IE4 and 37-3-18 showed elevated levels of Ras-GTP as compared with the other three MPNST cell lines (35-1-2, 38-2-18, and 32-5-24-12). The activated levels of Ras (H-, K-, and N-Ras) were determined under growth conditions in the presence of 10% fetal bovine serum. We also evaluated the amount of the active (phosphorylated) form of ERK (P-ERK) and found that at equal total enzyme levels, P-ERK levels were higher in 6IE4 and 37-3-18 as compared with the other three MPNST cell lines. JNK and p38-kinase pathways are known as stress-activated MAPK pathways.45Kyosseva SV Mitogen-activated protein kinase signaling.Int Rev Neurobiol. 2004; 59: 201-220Crossref PubMed Scopus (192) Google Scholar We evaluated the amount of the phosphorylated forms of the JNK substrate, JUN, and the p38-kinase substrate, ATF-2, in lysates of MPNST cells after in vitro kinase reactions. As shown in Figure 1B, increased activities for both pathways were detected in 6IE4 and 37-3-18 as compared to other MPNST cell lines in the absence of external stress stimuli. Notably, the cell line 38-2-18 also showed elevated levels of JNK pathway activity, independent of elevated Ras-GTP. The PI3K pathway is a downstream effector of Ras with significant anti-apoptotic effects that can greatly influence cell survival.46Downward J PI 3-kinase, Akt and cell survival.Semin Cell Dev Biol. 2004; 15: 177-182Crossref PubMed Scopus (685) Google Scholar We determined the level of PI3K activity in MPNST cell lines by measuring the amount of GSK3α/β phosphorylated on serines 9 or 21 by the phosphorylated form of AKT, a downstream substrate in the PI3K pathway. Once again, the highest levels of activity were observed for cell lines 6IE4 and 37-3-18 cells, as compared to the other cell lines. Increased levels of Ras signaling can enhance cell resistance to apoptosis by activation of the PI3K pathway and its multiple effectors47Rodriguez-Viciana P Warne PH Dhand R Vanhaesebroeck B Gout I Fry MJ Waterfield MD Downward J Phosphatidylinositol-3-OH kinase as a direct target of Ras.Nature. 1994; 370: 527-532Crossref PubMed Scopus (1740) Google Scholar such as 3-phosphoinositide-dependent protein kinase 1 (PDK1) and protein kinase-B (PKB/AKT).48Rodriguez-Viciana P Downward J Ras activation of phosphatidylinositol 3-kinase and Akt.Methods Enzymol. 2001; 333: 37-44Crossref PubMed Scopus (18) Google Scholar Therefore, MPNST cells with higher levels of Ras-GTP and AKT activation may be characterized by increased resistance to pro-apoptotic signals. To investigate this, we exposed MPNST cells to UV irradiation (10 mJ/cm2), which triggers p53-independent apoptosis.49Al-Mohanna MA Al-Khodairy FM Krezolek Z Bertilsson PA Al-Houssein KA Aboussekhra A p53 is dispensable for UV-induced cell cycle arrest at late G(1) in mammalian cells.Carcinogenesis. 2001; 22: 573-578Crossref PubMed Scopus (27) Google Scholar, 50Kumlin T Heikkinen P Kosma VM Alhonen L Janne J Juutilainen J p53-independent apoptosis in UV-irradiated mouse skin: possible inhibition by 50 Hz magnetic fields.Radiat Environ Biophys. 2002; 41: 155-158Crossref PubMed Scopus (15) Google Scholar Early induction of apoptosis was evaluated by performing the Annexin-V assay. The percentage (mean ± SD) of apoptotic 6IE4 and 37-3-18 cells (high-Ras group) was approximately three times lower than the other three MPNST cell lines with low-Ras-GTP and AKT activity levels (∼4.5% versus ∼13%; Figure 2A). Figure 2B shows a representation of the Annexin-V labeling for apoptotic cells obtained for cell lines 38-2-18 and 37-3-18. The basal level of apoptosis in the absence of any controllable insult was found to be ∼2% in multiple experiments for all MPNST cell lines (data not shown). G207 is a recombinant oncolytic HSV-1 that has a deletion in both copies of the viral γ134.5 gene and interruption of the viral UL39 gene coding for ICP6, the large subunit of HSV ribonucleotide reductase.37Mineta T Rabkin SD Yazaki T Hunter WD Martuza RL Attenuated multi-mutated herpes simplex virus-1 for the treatment of malignant gliomas.N