ORIGINAL ARTICLE
Appropriate Modulation of Autophagy Sensitizes Malignant Peripheral Nerve Sheath Tumor Cells to Treatment With Imatinib Mesylate Munehiro Okano, MD, Naoki Sakata, MD, Satoshi Ueda, MD, and Tsukasa Takemura, MD
Summary: Malignant peripheral nerve sheath tumor (MPNST), very rare in childhood, is a highly aggressive soft-tissue tumor. We experienced a case of a 7-year-old boy with MPNST who was treated with imatinib mesylate (imatinib) after the identification of platelet-derived growth factor receptor expression in his tumor. We were unable to observe clinical benefits of imatinib in this patient. Therefore, cellular reactions of imatinib were investigated in vitro using 3 MPNST cell lines. Imatinib induced cytotoxicity in vitro with variable IC50 values (11.7 to > 30 mM). Induction of apoptosis was not a pivotal mechanism in the inhibitory effects. We found that the treatment of MPNST cell lines with imatinib induced autophagy. Suppression of the initiation of autophagy by 3-methyladenine or small interfering RNA (siRNA) against beclin-1 attenuated the imatinib-mediated cytotoxicity. In contrast, blocking the formation of autophagosomes or the development of autolysosomes using siRNA against microtubule-associated protein light chain 3B, bafilomycin A1, chloroquine, or an MEK1/2 inhibitor (U0126) enhanced the imatinib-induced cytotoxicity in MPNST cells. Our data showed that the imatinibmediated autophagy can function as a cytotoxic mechanism and that appropriate modulation of autophagy may sensitize MPNST cells to imatinib, which in turn may be a novel therapeutic strategy for MPNST. Key Words: malignant peripheral nerve sheath tumor, imatinib mesylate, platelet-derived growth factor receptor, autophagy
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alignant peripheral nerve sheath tumor (MPNST) is very rare in the pediatric age group and involves a highly aggressive soft-tissue tumor arising either from neurofibromatosis type-1 (NF1) or de novo from peripheral nerves. The overall 5-year survival rate has been reported to be approximately 30% because of resistance to conventional therapy.1–4 We experienced a case of a 7-year-old NF1 patient with MPNST and a tumor refractory to conventional therapy. Therefore, we sought to develop a novel therapeutic strategy based on investigation of the tumor biology. Recently, platelet-derived growth factor receptor (PDGFR) and its ligands have been suggested to be involved in malignant transformation and progression of MPNST.5–8 PDGFR is a kinase targeted by imatinib Received for publication August 5, 2012; accepted August 26, 2013. From the Department of Pediatrics, Kinki University Faculty of Medicine, Osaka, Japan. The authors declare no conflict of interest. Reprints: Naoki Sakata, MD, Department of Pediatrics, Kinki University Faculty of Medicine, 377-2 Onohigashi Osakasayama, Osaka 589-8511, Japan (e-mail: [email protected]). Supplemental Digital Content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s Website, www.jpho-online.com. Copyright r 2013 by Lippincott Williams & Wilkins
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mesylate (imatinib), which provides great benefits to patients with gastrointestinal stromal tumors and chronic myelogenous leukemia.9,10 Evidence that imatinib inhibits the cellular activity of several receptor kinases has led to the investigation of its use in various human cancers including MPNST.11–13 Therefore, we treated our patient with imatinib on the basis of the pathologic findings of PDGFR-a and PDGFR-b expression in the tumor. However, imatinib produced limited clinical effects. As cellular reactions after the treatment of MPNST cells with imatinib are not completely understood, it may be difficult to use new approaches for enhancement of the cytotoxicity of imatinib. Macroautophagy (referred to hereafter as autophagy) is a process by which proteins and organelles are degraded and materials are recycled in response to stress. Membranes called phagophores, also known as isolation membranes, elongate and form autophagosomes, which are double membrane– bound structures that sequester cytoplasmic organelles. The outer membrane of an autophagosome fuses with an endosome and a lysosome, resulting in formation of an autolysosome, and the internal materials are degraded. This process has been shown to be mediated by a highly organized and hierarchical team of autophagy-related (Atg) proteins.14 As there is emerging evidence that autophagy plays a crucial role in the regulation of malignant cell survival,15 it is recognized as a target for novel therapeutic strategies in cancer.16,17 In this study, we investigated the cellular reactions of imatinib in vitro using MPNST cell lines. The present report is the first to indicate that autophagy is associated with imatinib-induced cytotoxicity and that appropriate modulation of autophagy is important for sensitizing MPNST to imatinib.
MATERIALS AND METHODS Cell Lines and Culture YST-1, an MPNST cell line established by Nagashima et al,18 was kindly provided by the Cell Resource Center for Biomedical Research, Tohoku University, and cultured in RPMI 1640 with 10% heat-inactivated fetal calf serum (FCS) from Invitrogen (Karlsruhe, Germany). HS-PSS and TABLE 1. Summary of Expression of Imatinib-sensitive Tyrosine Kinase Receptors PDGF-a PDGF-b c-Kit c-Abl
YST-1
HS-PSS
HS-Sch-2
+ + + +
+ + – +
– + + –
PDGF indicates platelet-derived growth factor.
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A
B
STI 48 h
C 0
116 kDa
5
10
STI 72 h 20
0
5
10
20 μM PARP
89 kDa
cleaved PARP
17.4 kDa
cytochrome c cleaved caspase-3
17.4 kDa
β-actin
Cell viability
D
FIGURE 1. Imatinib induced cytotoxicity in MPNST cell lines but apoptosis was partially involved. A, YST-1, HS-PSS, or HS-Sch-2 cells were incubated with various concentrations of imatinib (STI) for 72 hours, and cell viability was analyzed by MTT assay. The results are presented as mean ± SE. Similar results were obtained from 3 independent experiments. Paired t test analysis comparing untreated cells versus imatinibtreated cells demonstrated a paired value of P < 0.05 (*); P < 0.05 (**). B, YST-1 cells were incubated with imatinib for 48 hours, and cell cycle analysis was carried out according to the Materials and methods section. *Significant differences from untreated controls (P < 0.05); NS, a difference that was not significant using paired t test analysis. C, YST-1 cells were incubated with various concentrations of imatinib for the indicated time, and cytochrome c, cleaved PARP, and cleaved caspase-3 were examined by Western blotting. D, YST-1 cells were incubated with zVAD-fmk (20 mM) and imatinib with or without zVAD-fmk for 72 hours, and cell viability was then examined by MTT assay. The control sample contained DMSO as a vehicle. The results are presented as mean ± SE. Similar results were obtained from 3 independent experiments.
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HS-Sch-2, provided by the RIKEN BioResource Center, were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% heat-inactivated FCS. The cells were cultured in RPMI 1640 or DMEM with 1% FCS when evaluating cell viability by MTT assay.
Reagents Antimicrotubule-associated protein light chain 3B (LC3B), b-actin, p62SQSTM1, poly(ADP-ribose) polymerase (PARP), cytochrome c, ERK1/2, phospho-ERK1/2, and beclin-1 antibodies were from Cell Signaling Technologies (Beverly, MA). Anticleaved caspase-3 antibody was purchased from Trevigen (Gaithersburg, MD). 3methyladenine (3-MA), pepstatin A, E-64d, chloroquine diphosphate (CQ), and bafilomycin A1 were purchased from Sigma Aldrich (St Louis, MO). SP600125, SB203580, and U0126 were purchased from Merck Chemicals (Darmstadt, Germany). Imatinib mesylate was kindly provided by Novartis Pharma AG (Basel, Switzerland). Imatinib was dissolved in distilled water at a concentration of 10 mM. Small interfering RNA (siRNA) against LC3B was purchased from Qiagen (Hilden, Germany) and siRNA against beclin-1 and a negative control were purchased from Cell Signaling Technology. The caspase inhibitor, z-Val-Ala-Asp (Ome)-CH2F (zVAD-fmk), was purchased from the Peptide Institute (Osaka, Japan). 3-MA was dissolved in phosphate-buffered saline (PBS) and CQ in distilled water. Other reagents were dissolved in dimethyl sulfoxide (DMSO).
MTT Assay Cells were cultured in 96-well culture plates and incubated with various reagents for 72 hours. Cell viability was analyzed using an MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide) cell proliferation assay kit (Cayman Chemical Company, MI) following the manufacturer’s instructions. All samples were assayed in triplicate, and the mean value for each experiment was calculated.
Flow Cytometry For cell cycle analysis, incubated cells were fixed in 70% ethanol and then incubated in ribonuclease A solution
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at 371C to remove RNA. After staining with propidium iodide, the cells were subjected to fluorescence-activated cell sorting. Formation of acidic vesicular organelles (AVOs), a morphologic characteristic of autophagy, was quantitated by acridine orange staining.19 Cells were stained with acridine orange (0.5 mg/mL; Dojindo, Kumamoto, Japan) for 15 minutes, removed from trypsin, and collected in PBS. Green (510 to 530 nm, FL1) and red (650 nm, FL3) fluorescence emission was measured by flow cytometry. AVOs were detected using a fluorescence microscope (Nikon Eclipse E600; Nikon Corp., Tokyo, Japan).
Western Blot Cells were incubated for the indicated times with the indicated concentration of reagents and subsequently lysed in lysis buffer. Immunoblotting was performed as in our previous study.20 The lysates were adjusted to the expression of b-actin.
Electron Microscopy HS-Sch-2 cells were treated with or without 20 mM imatinib for 48 hours at 371C. Cells were fixed in ice-cold 2.5% electron microscopy–grade glutaraldehyde in PBS overnight at 41C. Fixed samples were washed 3 times in PBS, and then postfixed in 1% osmium tetroxide containing 0.1% potassium ferricyanide for 1 hour. After 3 PBS washes, samples were dehydrated through a graded series of 30% to 100% ethanol, and then embedded in epoxy resin according to the standard procedure. Ultrathin (70 nm) sections were collected and stained with 2% uranyl acetate in 50% methanol for 10 minutes, followed by staining with 1% lead citrate for 7 minutes. Sections were imaged using a JH-7100 transmission electron microscope (Hitachi, Japan).
siRNA-mediated Knockdown Knockdown of siRNA was performed according to the manufacturer’s instructions. Before the experiment, we tested the efficacy of transfection with various conditions using 3 MPNST cell lines which were used in the study. To assess the effects of the siRNA-mediated knockdown of beclin-1 or LC3B, transfected cells were subjected to Western blot probed with anti-beclin-1 or LC3B antibodies.
FIGURE 2. Imatinib induced autophagy in MPNST cell lines. A, HS-Sch-2 or YST-1 cells were incubated with imatinib (STI) at the indicated concentrations from 24 to 72 hours, as indicated. Total cell lysates were resolved by SDS-PAGE and immunoblotted with antiLC3 or anti-b-actin antibodies. B, Electron microscopic assessment of autophagy formation. Untreated HS-Sch-2 cells (a); HS-Sch-2 cells were treated with imatinib (20 mM) for 60 hours. Autophagosomes (black arrowheads), autolysosomes (white arrowheads) (b); high magnification. Autophagosomes (white arrowheads), autolysosomes (black arrowheads) (c); high magnification. Double-membrane autophagosome sequestering a damaged mitochondrion (d). M indicates mitochondrion; N, nucleus. C, HS-Sch-2 cells were pretreated for 60 minutes with pepstatin A (100 mM) and E-64d (10 mM) and subsequently treated for 72 hours with imatinib (30 mM) in the continuous presence or absence of inhibitors, as indicated. DMSO as a vehicle was added in the indicated samples because pepstatin A and E-64d were dissolved in DMSO. Total cell lysates were resolved by SDS-PAGE and immunoblotted with anti-LC3 or anti-b-actin antibodies. The signals for the bands corresponding to LC3B-II and b-actin were quantitated by ImageJ software (NIH) and data are expressed as ratios of LC3B-II to b-actin. Similar results were obtained in two other independent experiments as well as the one shown. D, HS-Sch-2 cells were pretreated for 60 minutes with 3-MA (1 mM) or bafilomycin A1 (BFA, 3 nM), and were subsequently treated for 72 hours with imatinib (30 mM) in the continuous presence or absence of inhibitors, as indicated. DMSO as a vehicle was added in the indicated samples. Total cell lysates were resolved by SDS-PAGE and immunoblotted with anti-LC3 or anti-b-actin antibodies. The signals for the bands corresponding to LC3B-II and b-actin were quantitated using ImageJ software and data are expressed as ratios of LC3B-II to b-actin. Similar results were obtained in 2 other independent experiments and in the one shown. E, HS-Sch-2 cells were treated for 48 hours with various concentrations of imatinib, as indicated. Total cell lysates were resolved by SDS-PAGE and immunoblotted with anti-p62SQSTM1 or anti-b-actin antibodies. Similar results were obtained in 2 other independent experiments and in the one shown. F, YST-1 or HS-Sch-2 cells were treated with various concentrations of imatinib for 48 hours and then stained with acridine orange for quantitation of the formation of acidic vesicular organelles (AVOs). An increase in AVO formation was accompanied by an increase in FL3 fluorescence, reflecting induction of autophagy (right panel). AVOs were observed by fluorescence microscopy (left panel, a magnification of 200).
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Statistical Analysis
RESULTS
Data are presented as mean ± SE. Paired Student t test (2-tailed) was used for comparison between 2 groups. ANOVA and Turkey-Kramer test were used for multiple comparisons. Differences were considered statistically significant when P was 10 mM imatinib to induce an inhibitory effect in MPNST cells,7,8 glioma cells,36 and r
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neuroectodermal tumor cells,37 which implies that the inhibitory effect of imatinib does not depend on the inhibition of tyrosine kinases, such as PDGFR or c-kit, because lower concentrations of imatinib (0.1 to 1.0 mM) are sufficient to inhibit the phosphorylation of PDGFR or c-kit.37 However, in vitro experiments with uncertain factors in the FCS medium may influence the effects of imatinib; for example, a-1 acid glycoprotein, one of the plasma proteins, is known to bind to imatinib with high affinity and inhibit imatinib activity in vitro and in vivo.38 Therefore, we reduced the concentration of FCS to 1% when measuring cell viability after exposure to imatinib. The sensitivity to imatinib varied among the 3 MPNST cell lines used in this study, which might be dependent on the expression of imatinib-sensitive receptors. YST-1, the most sensitive cell line, expressed PDGFR-a, PDGFR-b, c-abl, and c-kit, and the next most sensitive cell line, HS-PSS, expressed PDGFR-a, PDGFR-b, and c-abl but not c-kit. HS-Sch-2 cell line that expressed PDGFR-b and c-kit was less sensitive (Table 1). Bond et al12 performed a pharmacokinetic study in pediatric patients and showed that the trough plasma imatinib concentration obtained on day 8 with administration of 440 mg/m2/d was 6.1 mM (median; range, 0.5 to 21.4 mM), suggesting that the inhibitory effect of imatinib may be clinically demonstrated in patients with MPNST if some type of enhancement is present. Little is known about the signaling pathways that regulate imatinib-mediated autophagy. We demonstrated here that ERK was phosphorylated after treatment with imatinib, and U0126, an MEK inhibitor, inhibited the autophagic machinery. Previous works reported evidence that the ERK signaling pathway is involved in the induction of autophagy.24,33,39–42 In particular, the ERKMEK signaling pathway played a crucial role in the
Modulation of Autophagy for Sensitizing MPNST Cells
development of autophagosomes to functional autolysosomes, and MEK inhibitor did not impede the initiation of autophagy,24,39 which is consistent with our data that the presence of U0126 abolished the upregulation of LC3B-II or the formation of AVOs, resulting in the augmentation of imatinib-mediated cytotoxicity. Interestingly, treatment with imatinib still induced the conversion of LC3B-I to LCB-II in beclin-1 siRNA-transfected YST-1 cells (Fig. 4B, right middle panel), suggesting that the accumulation of autophagosome after treatment with imatinib is not dependent on the expression of beclin-1 in YST-1. Furthermore, we detected the phosphorylation of ERK1/2 after treatment with imatinib in the beclin-1 siRNA-transfected YST-1 cells (see Figure, Supplemental Digital Content 1, http://links.lww.com/JPHO/ A54), indicating that the MEK/ERK activation may be independent of the pathway involved in beclin-1 (Fig. 6). Pharmacological inhibition of ERK1/2 resulted in the dysregulation of mitochondrial membrane potential followed by the release of cytochrome c in MPNST cells. In YST-1 cells, it subsequently activated the apoptosis pathway with the enhancement of imatinib-mediated cytotoxicity because there is crosstalk between the induction of apoptosis and autophagy.42 In contrast, in HS-Sch-2 cells, in which the late stage of autophagy is impeded, nonapoptotic cell death enhanced without the activation of apoptosis because HS-Sch-2 cells have a p53 mutation.29 Treatment with bafilomycin A1 or chloroquine resulted in the accumulation of LC3B-II and augmentation of imatinib-induced cytotoxicity. It has been reported that inhibition of imatinib-induced autophagy by bafilomycin A1 enhanced apoptosis associated with mitochondrial permeabilization and release of cathepsin D from mitochondria,43 which is consistent with the data in this study from YST-1 cells. Recently, Ghadimi et al44 reported that
Imatinib mesylate
(YST-1) PI3 kinase Class III
MEK/ERK activation
Beclin-1 lysosome X bafilomycin A1 X chloroquine
Protective effects autophagosome
X LC3B X MEK inhibitor
Cell death
autolysosome
Degradation of damaged organelles
X
Cell death
Activation of apoptosis or Enhancememt of non-apoptotic cell death FIGURE 6. Appropriate inhibition of autophagy mediated by imatinib-enhanced cytotoxicity in MPNST. r
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treatment with dual PI3K/mTOR inhibitors induced autophagy and suppressed MPNST xenograft growth, and the additional administration of chloroquine, as an autophagy inhibitor, enhanced these inhibitor-induced suppression of tumor growth by inducing apoptosis. Chloroquine represents a promising drug for cotreatment with chemotherapeutic agents, and several clinical trials of it are underway.35 In conclusion, imatinib may be an appropriate candidate for novel therapeutic strategies because MPNST expresses imatinib-sensitive tyrosine kinase receptors. We demonstrated that imatinib mediated autophagy in MPNST cells, which was associated with the cytotoxicity of imatinib. However, the subsequent autophagic machinery, formation of autophagosome and autolysosome in which degradation process, may play an important role of the imatinib resistance. We have provided evidence that impeding this process by genetic and pharmacological approaches sensitized MPNST cells to imatinib, resulting in the activation of apoptosis or nonapoptotic cell death pathways (Fig. 6). Thus, appropriate autophagy modulation is very important for cancer treatment because the induction of autophagy has a double-edged sword function. In order for us to develop successful autophagy-modulating strategies against cancer, we need to understand better how the roles of autophagy differ depending on the tumor cell type and genetic factors, and we need to determine how specific pathways of autophagy are activated or inhibited by the various anticancer therapies. Further research will be required for the development of new combinatorial therapeutic strategies that will hopefully sensitize tumor cells to anticancer therapy.
nerve sheath tumors, and its implications for imatinib sensitivity. Carcinogenesis. 2006;27:664–671. Aoki M, Nabeshima K, Koga K, et al. Imatinib mesylate inhibits cell invasion of malignant peripheral nerve sheath tumor induced by platelet-derived growth factor-BB. Lab Invest. 2007;87:767–779. Demetri GD, von Mehren M, Blanke CD, et al. Efficacy and safety of imatinib mesylate in advanced gastrointestinal stromal tumors. N Engl J Med. 2002;347:472–480. Druker BJ, Talpaz M, Resta DJ, et al. Efficacy and safety of a specific inhibitor of the BCR-ABL tyrosine kinase in chronic myeloid leukemia. N Engl J Med. 2001;344:1031–1037. Heinrich MC, Joensuu H, Demetri GD, et al. Phase II, open-label study evaluating the activity of imatinib in treating life-threatening malignancies known to be associated with imatinib-sensitive tyrosine kinases. Clin Cancer Res. 2008;14: 2717–2725. Bond M, Bernstein ML, Pappo A, et al. A phase II study of imatinib mesylate in children with refractory or relapsed solid tumors: a Children’s Oncology Group study. Pediatr Blood Cancer. 2008;50:254–258. Chugh R, Wathen JK, Maki RG, et al. Phase II multicenter trial of imatinib in 10 histologic subtypes of sarcoma using a Bayesian hierarchical statistical model. J Clin Oncol. 2009; 27:3148–3153. Klionsky DJ, Emr SD. Autophagy as a regulated pathway of cellular degradation. Science. 2000;290:1717–1721. Kondo Y, Kanzawa T, Sawaya R, et al. The role of autophagy in cancer development and response to therapy. Nat Rev Cancer. 2005;5:726–734. Dalby KN, Tekedereli I, Lopez Berestein G, et al. Targeting the prodeath and prosurvival functions of autophagy as novel therapeutic strategies in cancer. Autophagy. 2010;6:322–329. Kimmelman AC. The dynamic nature of autophagy in cancer. Genes Dev. 2011;25:1999–2010. Nagashima Y, Ohaki Y, Tanaka Y, et al. Establishment of an epithelioid malignant schwannoma cell line (YST-1). Virchows Arch B Cell Pathol Incl Mol Pathol. 1990;59:321–327. Paglin S, Hollister T, Delohery T, et al. A novel response of cancer cells to radiation involves autophagy and formation of acidic vesicles. Cancer Res. 2001;61:439–444. Sakata N, Patel HR, Terada N, et al. Selective activation of c-Jun kinase mitogen-activated protein kinase by CD40 on human B cells. J Biol Chem. 1995;270:30823–30828. Kabeya Y, Mizushima N, Ueno T, et al. LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J. 2000;19:5720–5728. Bjorkoy G, Lamark T, Johansen T. p62/SQSTM1: a missing link between protein aggregates and the autophagy machinery. Autophagy. 2006;2:138–139. Corcelle EA, Puustinen P, Jaattela M. Apoptosis and autophagy: targeting autophagy signalling in cancer cells— “trick or treats”? FEBS J. 2009;276:6084–6096. Corcelle E, Djerbi N, Mari M, et al. Control of the autophagy maturation step by the MAPK ERK and p38: lessons from environmental carcinogens. Autophagy. 2007;3:57–59. Sun T, Li D, Wang L, et al. c-Jun NH2-terminal kinase activation is essential for up-regulation of LC3 during ceramide-induced autophagy in human nasopharyngeal carcinoma cells. J Transl Med. 2011;9:161–170. Vazquez Martin A, Oliveras Ferraros C, Menendez JA. Autophagy facilitates the development of breast cancer resistance to the anti-HER2 monoclonal antibody trastuzumab. PLoS One. 2009;4:e6251. Chen N, Karantza V. Autophagy as a therapeutic target in cancer. Cancer Biol Ther. 2011;11:157–168. Solomon VR, Lee H. Chloroquine and its analogs: a new promise of an old drug for effective and safe cancer therapies. Eur J Pharmacol. 2009;625:220–233. Sonobe H, Takeuchi T, Furihata M, et al. A new human malignant peripheral nerve sheath tumour-cell line, HS-sch-2, harbouring p53 point mutation. Int J Oncol. 2000;17:347–352.
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ACKNOWLEDGMENTS The authors thank Shinji Kurashimo (Kinki University Life Science Research Institution) for technical support during flow cytometry analysis, Yoshitaka Horiuchi (Kinki University Life Science Research Institution) for technical support for imaging by transmission electron microscopy, and Masatomo Kimura (Department of Pathology, Kinki University Faculty of Medicine) and Mikiko Aoki (Department of Pathology, Fukuoka University School of Medicine) for analyzing the expression of PDGFR in the tumor samples.
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Appropriate Modulation of Autophagy Sensitizes Malignant Peripheral Nerve Sheath Tumor Cells to Treatment With Imatinib Mesylate Munehiro Okano, MD, Naoki Sakata, MD, Satoshi Ueda, MD, and Tsukasa Takemura, MD
Summary: Malignant peripheral nerve sheath tumor (MPNST), very rare in childhood, is a highly aggressive soft-tissue tumor. We experienced a case of a 7-year-old boy with MPNST who was treated with imatinib mesylate (imatinib) after the identification of platelet-derived growth factor receptor expression in his tumor. We were unable to observe clinical benefits of imatinib in this patient. Therefore, cellular reactions of imatinib were investigated in vitro using 3 MPNST cell lines. Imatinib induced cytotoxicity in vitro with variable IC50 values (11.7 to > 30 mM). Induction of apoptosis was not a pivotal mechanism in the inhibitory effects. We found that the treatment of MPNST cell lines with imatinib induced autophagy. Suppression of the initiation of autophagy by 3-methyladenine or small interfering RNA (siRNA) against beclin-1 attenuated the imatinib-mediated cytotoxicity. In contrast, blocking the formation of autophagosomes or the development of autolysosomes using siRNA against microtubule-associated protein light chain 3B, bafilomycin A1, chloroquine, or an MEK1/2 inhibitor (U0126) enhanced the imatinib-induced cytotoxicity in MPNST cells. Our data showed that the imatinibmediated autophagy can function as a cytotoxic mechanism and that appropriate modulation of autophagy may sensitize MPNST cells to imatinib, which in turn may be a novel therapeutic strategy for MPNST. Key Words: malignant peripheral nerve sheath tumor, imatinib mesylate, platelet-derived growth factor receptor, autophagy
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alignant peripheral nerve sheath tumor (MPNST) is very rare in the pediatric age group and involves a highly aggressive soft-tissue tumor arising either from neurofibromatosis type-1 (NF1) or de novo from peripheral nerves. The overall 5-year survival rate has been reported to be approximately 30% because of resistance to conventional therapy.1–4 We experienced a case of a 7-year-old NF1 patient with MPNST and a tumor refractory to conventional therapy. Therefore, we sought to develop a novel therapeutic strategy based on investigation of the tumor biology. Recently, platelet-derived growth factor receptor (PDGFR) and its ligands have been suggested to be involved in malignant transformation and progression of MPNST.5–8 PDGFR is a kinase targeted by imatinib Received for publication August 5, 2012; accepted August 26, 2013. From the Department of Pediatrics, Kinki University Faculty of Medicine, Osaka, Japan. The authors declare no conflict of interest. Reprints: Naoki Sakata, MD, Department of Pediatrics, Kinki University Faculty of Medicine, 377-2 Onohigashi Osakasayama, Osaka 589-8511, Japan (e-mail: [email protected]). Supplemental Digital Content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s Website, www.jpho-online.com. Copyright r 2013 by Lippincott Williams & Wilkins
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mesylate (imatinib), which provides great benefits to patients with gastrointestinal stromal tumors and chronic myelogenous leukemia.9,10 Evidence that imatinib inhibits the cellular activity of several receptor kinases has led to the investigation of its use in various human cancers including MPNST.11–13 Therefore, we treated our patient with imatinib on the basis of the pathologic findings of PDGFR-a and PDGFR-b expression in the tumor. However, imatinib produced limited clinical effects. As cellular reactions after the treatment of MPNST cells with imatinib are not completely understood, it may be difficult to use new approaches for enhancement of the cytotoxicity of imatinib. Macroautophagy (referred to hereafter as autophagy) is a process by which proteins and organelles are degraded and materials are recycled in response to stress. Membranes called phagophores, also known as isolation membranes, elongate and form autophagosomes, which are double membrane– bound structures that sequester cytoplasmic organelles. The outer membrane of an autophagosome fuses with an endosome and a lysosome, resulting in formation of an autolysosome, and the internal materials are degraded. This process has been shown to be mediated by a highly organized and hierarchical team of autophagy-related (Atg) proteins.14 As there is emerging evidence that autophagy plays a crucial role in the regulation of malignant cell survival,15 it is recognized as a target for novel therapeutic strategies in cancer.16,17 In this study, we investigated the cellular reactions of imatinib in vitro using MPNST cell lines. The present report is the first to indicate that autophagy is associated with imatinib-induced cytotoxicity and that appropriate modulation of autophagy is important for sensitizing MPNST to imatinib.
MATERIALS AND METHODS Cell Lines and Culture YST-1, an MPNST cell line established by Nagashima et al,18 was kindly provided by the Cell Resource Center for Biomedical Research, Tohoku University, and cultured in RPMI 1640 with 10% heat-inactivated fetal calf serum (FCS) from Invitrogen (Karlsruhe, Germany). HS-PSS and TABLE 1. Summary of Expression of Imatinib-sensitive Tyrosine Kinase Receptors PDGF-a PDGF-b c-Kit c-Abl
YST-1
HS-PSS
HS-Sch-2
+ + + +
+ + – +
– + + –
PDGF indicates platelet-derived growth factor.
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A
B
STI 48 h
C 0
116 kDa
5
10
STI 72 h 20
0
5
10
20 μM PARP
89 kDa
cleaved PARP
17.4 kDa
cytochrome c cleaved caspase-3
17.4 kDa
β-actin
Cell viability
D
FIGURE 1. Imatinib induced cytotoxicity in MPNST cell lines but apoptosis was partially involved. A, YST-1, HS-PSS, or HS-Sch-2 cells were incubated with various concentrations of imatinib (STI) for 72 hours, and cell viability was analyzed by MTT assay. The results are presented as mean ± SE. Similar results were obtained from 3 independent experiments. Paired t test analysis comparing untreated cells versus imatinibtreated cells demonstrated a paired value of P < 0.05 (*); P < 0.05 (**). B, YST-1 cells were incubated with imatinib for 48 hours, and cell cycle analysis was carried out according to the Materials and methods section. *Significant differences from untreated controls (P < 0.05); NS, a difference that was not significant using paired t test analysis. C, YST-1 cells were incubated with various concentrations of imatinib for the indicated time, and cytochrome c, cleaved PARP, and cleaved caspase-3 were examined by Western blotting. D, YST-1 cells were incubated with zVAD-fmk (20 mM) and imatinib with or without zVAD-fmk for 72 hours, and cell viability was then examined by MTT assay. The control sample contained DMSO as a vehicle. The results are presented as mean ± SE. Similar results were obtained from 3 independent experiments.
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HS-Sch-2, provided by the RIKEN BioResource Center, were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% heat-inactivated FCS. The cells were cultured in RPMI 1640 or DMEM with 1% FCS when evaluating cell viability by MTT assay.
Reagents Antimicrotubule-associated protein light chain 3B (LC3B), b-actin, p62SQSTM1, poly(ADP-ribose) polymerase (PARP), cytochrome c, ERK1/2, phospho-ERK1/2, and beclin-1 antibodies were from Cell Signaling Technologies (Beverly, MA). Anticleaved caspase-3 antibody was purchased from Trevigen (Gaithersburg, MD). 3methyladenine (3-MA), pepstatin A, E-64d, chloroquine diphosphate (CQ), and bafilomycin A1 were purchased from Sigma Aldrich (St Louis, MO). SP600125, SB203580, and U0126 were purchased from Merck Chemicals (Darmstadt, Germany). Imatinib mesylate was kindly provided by Novartis Pharma AG (Basel, Switzerland). Imatinib was dissolved in distilled water at a concentration of 10 mM. Small interfering RNA (siRNA) against LC3B was purchased from Qiagen (Hilden, Germany) and siRNA against beclin-1 and a negative control were purchased from Cell Signaling Technology. The caspase inhibitor, z-Val-Ala-Asp (Ome)-CH2F (zVAD-fmk), was purchased from the Peptide Institute (Osaka, Japan). 3-MA was dissolved in phosphate-buffered saline (PBS) and CQ in distilled water. Other reagents were dissolved in dimethyl sulfoxide (DMSO).
MTT Assay Cells were cultured in 96-well culture plates and incubated with various reagents for 72 hours. Cell viability was analyzed using an MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide) cell proliferation assay kit (Cayman Chemical Company, MI) following the manufacturer’s instructions. All samples were assayed in triplicate, and the mean value for each experiment was calculated.
Flow Cytometry For cell cycle analysis, incubated cells were fixed in 70% ethanol and then incubated in ribonuclease A solution
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at 371C to remove RNA. After staining with propidium iodide, the cells were subjected to fluorescence-activated cell sorting. Formation of acidic vesicular organelles (AVOs), a morphologic characteristic of autophagy, was quantitated by acridine orange staining.19 Cells were stained with acridine orange (0.5 mg/mL; Dojindo, Kumamoto, Japan) for 15 minutes, removed from trypsin, and collected in PBS. Green (510 to 530 nm, FL1) and red (650 nm, FL3) fluorescence emission was measured by flow cytometry. AVOs were detected using a fluorescence microscope (Nikon Eclipse E600; Nikon Corp., Tokyo, Japan).
Western Blot Cells were incubated for the indicated times with the indicated concentration of reagents and subsequently lysed in lysis buffer. Immunoblotting was performed as in our previous study.20 The lysates were adjusted to the expression of b-actin.
Electron Microscopy HS-Sch-2 cells were treated with or without 20 mM imatinib for 48 hours at 371C. Cells were fixed in ice-cold 2.5% electron microscopy–grade glutaraldehyde in PBS overnight at 41C. Fixed samples were washed 3 times in PBS, and then postfixed in 1% osmium tetroxide containing 0.1% potassium ferricyanide for 1 hour. After 3 PBS washes, samples were dehydrated through a graded series of 30% to 100% ethanol, and then embedded in epoxy resin according to the standard procedure. Ultrathin (70 nm) sections were collected and stained with 2% uranyl acetate in 50% methanol for 10 minutes, followed by staining with 1% lead citrate for 7 minutes. Sections were imaged using a JH-7100 transmission electron microscope (Hitachi, Japan).
siRNA-mediated Knockdown Knockdown of siRNA was performed according to the manufacturer’s instructions. Before the experiment, we tested the efficacy of transfection with various conditions using 3 MPNST cell lines which were used in the study. To assess the effects of the siRNA-mediated knockdown of beclin-1 or LC3B, transfected cells were subjected to Western blot probed with anti-beclin-1 or LC3B antibodies.
FIGURE 2. Imatinib induced autophagy in MPNST cell lines. A, HS-Sch-2 or YST-1 cells were incubated with imatinib (STI) at the indicated concentrations from 24 to 72 hours, as indicated. Total cell lysates were resolved by SDS-PAGE and immunoblotted with antiLC3 or anti-b-actin antibodies. B, Electron microscopic assessment of autophagy formation. Untreated HS-Sch-2 cells (a); HS-Sch-2 cells were treated with imatinib (20 mM) for 60 hours. Autophagosomes (black arrowheads), autolysosomes (white arrowheads) (b); high magnification. Autophagosomes (white arrowheads), autolysosomes (black arrowheads) (c); high magnification. Double-membrane autophagosome sequestering a damaged mitochondrion (d). M indicates mitochondrion; N, nucleus. C, HS-Sch-2 cells were pretreated for 60 minutes with pepstatin A (100 mM) and E-64d (10 mM) and subsequently treated for 72 hours with imatinib (30 mM) in the continuous presence or absence of inhibitors, as indicated. DMSO as a vehicle was added in the indicated samples because pepstatin A and E-64d were dissolved in DMSO. Total cell lysates were resolved by SDS-PAGE and immunoblotted with anti-LC3 or anti-b-actin antibodies. The signals for the bands corresponding to LC3B-II and b-actin were quantitated by ImageJ software (NIH) and data are expressed as ratios of LC3B-II to b-actin. Similar results were obtained in two other independent experiments as well as the one shown. D, HS-Sch-2 cells were pretreated for 60 minutes with 3-MA (1 mM) or bafilomycin A1 (BFA, 3 nM), and were subsequently treated for 72 hours with imatinib (30 mM) in the continuous presence or absence of inhibitors, as indicated. DMSO as a vehicle was added in the indicated samples. Total cell lysates were resolved by SDS-PAGE and immunoblotted with anti-LC3 or anti-b-actin antibodies. The signals for the bands corresponding to LC3B-II and b-actin were quantitated using ImageJ software and data are expressed as ratios of LC3B-II to b-actin. Similar results were obtained in 2 other independent experiments and in the one shown. E, HS-Sch-2 cells were treated for 48 hours with various concentrations of imatinib, as indicated. Total cell lysates were resolved by SDS-PAGE and immunoblotted with anti-p62SQSTM1 or anti-b-actin antibodies. Similar results were obtained in 2 other independent experiments and in the one shown. F, YST-1 or HS-Sch-2 cells were treated with various concentrations of imatinib for 48 hours and then stained with acridine orange for quantitation of the formation of acidic vesicular organelles (AVOs). An increase in AVO formation was accompanied by an increase in FL3 fluorescence, reflecting induction of autophagy (right panel). AVOs were observed by fluorescence microscopy (left panel, a magnification of 200).
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Statistical Analysis
RESULTS
Data are presented as mean ± SE. Paired Student t test (2-tailed) was used for comparison between 2 groups. ANOVA and Turkey-Kramer test were used for multiple comparisons. Differences were considered statistically significant when P was 10 mM imatinib to induce an inhibitory effect in MPNST cells,7,8 glioma cells,36 and r
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neuroectodermal tumor cells,37 which implies that the inhibitory effect of imatinib does not depend on the inhibition of tyrosine kinases, such as PDGFR or c-kit, because lower concentrations of imatinib (0.1 to 1.0 mM) are sufficient to inhibit the phosphorylation of PDGFR or c-kit.37 However, in vitro experiments with uncertain factors in the FCS medium may influence the effects of imatinib; for example, a-1 acid glycoprotein, one of the plasma proteins, is known to bind to imatinib with high affinity and inhibit imatinib activity in vitro and in vivo.38 Therefore, we reduced the concentration of FCS to 1% when measuring cell viability after exposure to imatinib. The sensitivity to imatinib varied among the 3 MPNST cell lines used in this study, which might be dependent on the expression of imatinib-sensitive receptors. YST-1, the most sensitive cell line, expressed PDGFR-a, PDGFR-b, c-abl, and c-kit, and the next most sensitive cell line, HS-PSS, expressed PDGFR-a, PDGFR-b, and c-abl but not c-kit. HS-Sch-2 cell line that expressed PDGFR-b and c-kit was less sensitive (Table 1). Bond et al12 performed a pharmacokinetic study in pediatric patients and showed that the trough plasma imatinib concentration obtained on day 8 with administration of 440 mg/m2/d was 6.1 mM (median; range, 0.5 to 21.4 mM), suggesting that the inhibitory effect of imatinib may be clinically demonstrated in patients with MPNST if some type of enhancement is present. Little is known about the signaling pathways that regulate imatinib-mediated autophagy. We demonstrated here that ERK was phosphorylated after treatment with imatinib, and U0126, an MEK inhibitor, inhibited the autophagic machinery. Previous works reported evidence that the ERK signaling pathway is involved in the induction of autophagy.24,33,39–42 In particular, the ERKMEK signaling pathway played a crucial role in the
Modulation of Autophagy for Sensitizing MPNST Cells
development of autophagosomes to functional autolysosomes, and MEK inhibitor did not impede the initiation of autophagy,24,39 which is consistent with our data that the presence of U0126 abolished the upregulation of LC3B-II or the formation of AVOs, resulting in the augmentation of imatinib-mediated cytotoxicity. Interestingly, treatment with imatinib still induced the conversion of LC3B-I to LCB-II in beclin-1 siRNA-transfected YST-1 cells (Fig. 4B, right middle panel), suggesting that the accumulation of autophagosome after treatment with imatinib is not dependent on the expression of beclin-1 in YST-1. Furthermore, we detected the phosphorylation of ERK1/2 after treatment with imatinib in the beclin-1 siRNA-transfected YST-1 cells (see Figure, Supplemental Digital Content 1, http://links.lww.com/JPHO/ A54), indicating that the MEK/ERK activation may be independent of the pathway involved in beclin-1 (Fig. 6). Pharmacological inhibition of ERK1/2 resulted in the dysregulation of mitochondrial membrane potential followed by the release of cytochrome c in MPNST cells. In YST-1 cells, it subsequently activated the apoptosis pathway with the enhancement of imatinib-mediated cytotoxicity because there is crosstalk between the induction of apoptosis and autophagy.42 In contrast, in HS-Sch-2 cells, in which the late stage of autophagy is impeded, nonapoptotic cell death enhanced without the activation of apoptosis because HS-Sch-2 cells have a p53 mutation.29 Treatment with bafilomycin A1 or chloroquine resulted in the accumulation of LC3B-II and augmentation of imatinib-induced cytotoxicity. It has been reported that inhibition of imatinib-induced autophagy by bafilomycin A1 enhanced apoptosis associated with mitochondrial permeabilization and release of cathepsin D from mitochondria,43 which is consistent with the data in this study from YST-1 cells. Recently, Ghadimi et al44 reported that
Imatinib mesylate
(YST-1) PI3 kinase Class III
MEK/ERK activation
Beclin-1 lysosome X bafilomycin A1 X chloroquine
Protective effects autophagosome
X LC3B X MEK inhibitor
Cell death
autolysosome
Degradation of damaged organelles
X
Cell death
Activation of apoptosis or Enhancememt of non-apoptotic cell death FIGURE 6. Appropriate inhibition of autophagy mediated by imatinib-enhanced cytotoxicity in MPNST. r
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treatment with dual PI3K/mTOR inhibitors induced autophagy and suppressed MPNST xenograft growth, and the additional administration of chloroquine, as an autophagy inhibitor, enhanced these inhibitor-induced suppression of tumor growth by inducing apoptosis. Chloroquine represents a promising drug for cotreatment with chemotherapeutic agents, and several clinical trials of it are underway.35 In conclusion, imatinib may be an appropriate candidate for novel therapeutic strategies because MPNST expresses imatinib-sensitive tyrosine kinase receptors. We demonstrated that imatinib mediated autophagy in MPNST cells, which was associated with the cytotoxicity of imatinib. However, the subsequent autophagic machinery, formation of autophagosome and autolysosome in which degradation process, may play an important role of the imatinib resistance. We have provided evidence that impeding this process by genetic and pharmacological approaches sensitized MPNST cells to imatinib, resulting in the activation of apoptosis or nonapoptotic cell death pathways (Fig. 6). Thus, appropriate autophagy modulation is very important for cancer treatment because the induction of autophagy has a double-edged sword function. In order for us to develop successful autophagy-modulating strategies against cancer, we need to understand better how the roles of autophagy differ depending on the tumor cell type and genetic factors, and we need to determine how specific pathways of autophagy are activated or inhibited by the various anticancer therapies. Further research will be required for the development of new combinatorial therapeutic strategies that will hopefully sensitize tumor cells to anticancer therapy.
nerve sheath tumors, and its implications for imatinib sensitivity. Carcinogenesis. 2006;27:664–671. Aoki M, Nabeshima K, Koga K, et al. Imatinib mesylate inhibits cell invasion of malignant peripheral nerve sheath tumor induced by platelet-derived growth factor-BB. Lab Invest. 2007;87:767–779. Demetri GD, von Mehren M, Blanke CD, et al. Efficacy and safety of imatinib mesylate in advanced gastrointestinal stromal tumors. N Engl J Med. 2002;347:472–480. Druker BJ, Talpaz M, Resta DJ, et al. Efficacy and safety of a specific inhibitor of the BCR-ABL tyrosine kinase in chronic myeloid leukemia. N Engl J Med. 2001;344:1031–1037. Heinrich MC, Joensuu H, Demetri GD, et al. Phase II, open-label study evaluating the activity of imatinib in treating life-threatening malignancies known to be associated with imatinib-sensitive tyrosine kinases. Clin Cancer Res. 2008;14: 2717–2725. Bond M, Bernstein ML, Pappo A, et al. A phase II study of imatinib mesylate in children with refractory or relapsed solid tumors: a Children’s Oncology Group study. Pediatr Blood Cancer. 2008;50:254–258. Chugh R, Wathen JK, Maki RG, et al. Phase II multicenter trial of imatinib in 10 histologic subtypes of sarcoma using a Bayesian hierarchical statistical model. J Clin Oncol. 2009; 27:3148–3153. Klionsky DJ, Emr SD. Autophagy as a regulated pathway of cellular degradation. Science. 2000;290:1717–1721. Kondo Y, Kanzawa T, Sawaya R, et al. The role of autophagy in cancer development and response to therapy. Nat Rev Cancer. 2005;5:726–734. Dalby KN, Tekedereli I, Lopez Berestein G, et al. Targeting the prodeath and prosurvival functions of autophagy as novel therapeutic strategies in cancer. Autophagy. 2010;6:322–329. Kimmelman AC. The dynamic nature of autophagy in cancer. Genes Dev. 2011;25:1999–2010. Nagashima Y, Ohaki Y, Tanaka Y, et al. Establishment of an epithelioid malignant schwannoma cell line (YST-1). Virchows Arch B Cell Pathol Incl Mol Pathol. 1990;59:321–327. Paglin S, Hollister T, Delohery T, et al. A novel response of cancer cells to radiation involves autophagy and formation of acidic vesicles. Cancer Res. 2001;61:439–444. Sakata N, Patel HR, Terada N, et al. Selective activation of c-Jun kinase mitogen-activated protein kinase by CD40 on human B cells. J Biol Chem. 1995;270:30823–30828. Kabeya Y, Mizushima N, Ueno T, et al. LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J. 2000;19:5720–5728. Bjorkoy G, Lamark T, Johansen T. p62/SQSTM1: a missing link between protein aggregates and the autophagy machinery. Autophagy. 2006;2:138–139. Corcelle EA, Puustinen P, Jaattela M. Apoptosis and autophagy: targeting autophagy signalling in cancer cells— “trick or treats”? FEBS J. 2009;276:6084–6096. Corcelle E, Djerbi N, Mari M, et al. Control of the autophagy maturation step by the MAPK ERK and p38: lessons from environmental carcinogens. Autophagy. 2007;3:57–59. Sun T, Li D, Wang L, et al. c-Jun NH2-terminal kinase activation is essential for up-regulation of LC3 during ceramide-induced autophagy in human nasopharyngeal carcinoma cells. J Transl Med. 2011;9:161–170. Vazquez Martin A, Oliveras Ferraros C, Menendez JA. Autophagy facilitates the development of breast cancer resistance to the anti-HER2 monoclonal antibody trastuzumab. PLoS One. 2009;4:e6251. Chen N, Karantza V. Autophagy as a therapeutic target in cancer. Cancer Biol Ther. 2011;11:157–168. Solomon VR, Lee H. Chloroquine and its analogs: a new promise of an old drug for effective and safe cancer therapies. Eur J Pharmacol. 2009;625:220–233. Sonobe H, Takeuchi T, Furihata M, et al. A new human malignant peripheral nerve sheath tumour-cell line, HS-sch-2, harbouring p53 point mutation. Int J Oncol. 2000;17:347–352.
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ACKNOWLEDGMENTS The authors thank Shinji Kurashimo (Kinki University Life Science Research Institution) for technical support during flow cytometry analysis, Yoshitaka Horiuchi (Kinki University Life Science Research Institution) for technical support for imaging by transmission electron microscopy, and Masatomo Kimura (Department of Pathology, Kinki University Faculty of Medicine) and Mikiko Aoki (Department of Pathology, Fukuoka University School of Medicine) for analyzing the expression of PDGFR in the tumor samples.
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