Canonical autophagy does not contribute to cellular radioresistance

Radiotherapy and Oncology 114 (2015) 406–412

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Cellular radiobiology

Canonical autophagy does not contribute to cellular radioresistance Marco B.E. Schaaf a, Barry Jutten a, Tom G. Keulers a, Kim G.M. Savelkouls a, Hanneke J.M. Peeters a, Twan van den Beucken b, Frederik-Jan van Schooten c, Roger W. Godschalk c, Marc Vooijs a, Kasper M.A. Rouschop a,⇑ a Department of Radiation Oncology (Maastro Lab), GROW School for Oncology & Developmental Biology, Maastricht University Medical Center+; b Department of Toxicogenomics; and c Department of Toxicology, NUTRIM School for Nutrition, Toxicology and Metabolism, Maastricht University, The Netherlands

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Article history: Received 17 October 2014 Received in revised form 11 February 2015 Accepted 19 February 2015 Available online 13 March 2015 Keywords: Autophagy Ionizing radiation ATG7 LC3b Chloroquine 3-Methyladenine

a b s t r a c t Background: (Pre)clinical studies indicate that autophagy inhibition increases response to anti-cancer therapies. Although promising, due to contradicting reports, it remains unclear if radiation therapy changes autophagy activity and if autophagy inhibition changes the cellular intrinsic radiosensitivity. Discrepancies may result from different assays and models through off-target effects and influencing other signaling routes. In this study, we directly compared the effects of genetic and pharmacological inhibition of autophagy after irradiation in human cancer cell lines. Materials and methods: Changes in autophagy activity after ionizing radiation (IR) were assessed by flux analysis in eight cell lines. Clonogenic survival, DNA damage (COMET-assay) and H2AX phosphorylation were assessed after chloroquine or 3-methyladenine pretreatment and after ATG7 or LC3b knockdown. Results: IR failed to induce autophagy and chloroquine failed to change intrinsic radiosensitivity of cells. Interestingly, 3-methyladenine and ATG7- or LC3b-deficiency sensitized cancer cells to irradiation. Surprisingly, the radiosensitizing effect of 3-methyladenine was also observed in ATG7 and LC3b deficient cells and was associated with attenuated c-H2AX formation and DNA damage repair. Conclusion: Our data demonstrate that the anti-tumor effects of chloroquine are independent of changes in intrinsic radioresistance. Furthermore, ATG7 and LC3b support radioresistance independent of canonical autophagy that involves lysosomal degradation. Ó 2015 Elsevier Ireland Ltd. All rights reserved. Radiotherapy and Oncology 114 (2015) 406–412

Autophagy is a therapeutic target for cancer treatment due to its ability to promote survival of cells exposed to stresses observed in the tumor microenvironment and exposed to anti-cancer therapies. Currently, multiple clinical trials have been initiated that combine conventional anti-cancer therapy with autophagy inhibition. Upon activation, a complex consisting of Beclin1 and Vps34, a class III PI3K, triggers the formation of double-membrane vesicles. These autophagosomes engulf cellular constituents and incorporate microtubule-associated protein 1 light chain b (MAP1LC3b, LC3b hereafter) into its membranes [1,2]. For membrane association, cytosolic LC3b (LC3b-I) requires phosphatidylethanolamine (PE) conjugation (LC3b-II). This ubiquitin-like process is mediated through a series of autophagy-related proteins (ATGs) including ATG7 [1,3]. After fusion of the mature autophagosome with a lysosome, acidic hydrolase enzymes digest the content, allowing cells to recycle parts of its constituents. Enzyme activity and fusion ⇑ Corresponding author at: Maastricht University Medical Center+, Universiteitssingel 50, Room 3.318, 6200MD Maastricht, The Netherlands. E-mail address: [email protected] (K.M.A. Rouschop). http://dx.doi.org/10.1016/j.radonc.2015.02.019 0167-8140/Ó 2015 Elsevier Ireland Ltd. All rights reserved.

require acidic lysosomal compartments [4] and are inhibited by lysosomotropic drugs such as chloroquine (CQ) [5] and Bafilomycin A1 [6]. Treatment with CQ or its derivative, hydroxyCQ, is therapeutically attractive with acceptable toxicity [7] and low costs. Previously we showed that hypoxic tumor cells, a general feature of solid tumors that contributes to therapy resistance, are dependent on autophagy for survival. CQ treatment decreases hypoxia tolerance, reduces tumor hypoxia and sensitizes tumors to irradiation [8]. In addition to the effect on tumor hypoxia and altering the tumor phenotype into a more radiosensitive tumor, reports indicate that the inhibition of autophagy may alter the intrinsic radiosensitivity of cells. These reports have shown contradicting results as both radiosensitizing [9–13] as well as radioresistance enhancing [14–17] effects are observed. These contradictions were also observed when using genetic models e.g., ATG5, Beclin1, ATG12 and ATG3 [14,18]. Discrepancies in results may have arisen from the lack of our understanding of the mechanism at play, use of non-gold standard techniques and use of autophagy inhibitors at toxic concentrations. Furthermore, as targeting autophagy is now increasingly combined with

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anti-cancer therapies in forthcoming and current clinical studies, investigating the precise mechanism by which autophagy inhibition results in increased tumor responsiveness is evidently relevant. Therefore, we studied the role of autophagy in the response of cancer cells to ionizing radiation (IR) using commonly used autophagy inhibitors and by silencing crucial autophagy gene expression. Materials and methods Cell models Colorectal adenocarcinoma (HT29, HCT116), mammary adenocarcinoma (MCF7, MDA-MB-231), mammary ductal carcinoma (T47D), lung carcinoma (A549), cervix adenocarcinoma (HeLa) and prostate carcinoma (DU145) cells were maintained as described by ATCC. Doxycycline-inducible shRNA targeting ATG7 [ATACAGTGTTCCAATAGCTGGG] or scrambled control [CGAGGGC GACTTAACCTTAGG] was achieved through lentiviral pTripZ (Thermo Scientific) expression. shRNA targeting LC3b [TTTCTCA CTCTCATACACCTCT] was cloned into inducible Tet-pLKO-puro [19]. Virus particle transduction was done as described previously [20]. Cells were incubated with 1 lg/ml doxycycline to induce shRNA expression. Exposure Cells were irradiated using a MCN 225 industrial X-ray tube (Phillips) operated at 225 kV and 10 mA. Cells were exposed to hypoxia (MACS VA500, Don Whitley Scientific), 3-MA (3-methyladenine; Selleckchem) and chloroquine (Sigma–Aldrich). Immunoblot Cells were lysed and processed as described previously [21]. After transfer, proteins were probed with antibodies against MAP1LC3b (Cell Signaling), phospho(Ser139)-H2AX (Millipore), ATG7 (Cell signaling), phospho(Thr26/Ser28)-Chk2 (Cell Signaling) and Actin (MP Biomedicals, 8961001). Bound antibodies were visualized using HRP-linked anti-rabbit or antimouse (Cell Signaling) antibodies. Clonogenic assay Cells were pretreated 16 h with 3-MA or chloroquine and/or 5–6 days with doxycycline to induce knockdown before irradiation. Twenty-four hours post-IR floating and attached cells were collected and single cells were seeded in medium without doxycycline and incubated for 10 days to form colonies. Colonies (>50 cells) were counted manually. Immunocytochemistry Cells were fixed with 100% methanol at 20 °C (c-H2AX) or 4% paraformaldehyde (LC3b), permeabilized with 0.05% Tween-PBS and blocked using normal goat serum. Cells were stained with anti-phospho(Ser139)-H2AX (Millipore) or anti-LC3b (Cell signaling) antibodies, anti-rabbit Alexa Fluor 488 (Invitrogen) and Hoechst33342 (Sigma–Aldrich). COMET assay After exposure, cells were stored ( 70 °C) in RPMI containing 10% heat inactivated fetal calf serum, 10% DMSO and 1% Pen/Strep. For single cell gel electrophoresis, cells were embedded in 0.7% low melting point agarose (in PBS). Cells were lysed overnight at 4 °C in lysis buffer (1% Triton X-100, 2.5 M NaCl, 0.1 M EDTA, 10 mM Tris, pH 10) and placed in an alkaline solution (0.3 M NaOH, 1 mM EDTA, pH > 13) for 40 min, followed by electrophoresis (30 min at 25 V (1.2 V/cm) and 300 mA). DNA was visualized with ethidium bromide (10 lg/ml) and the comets were analyzed by computerized image analysis (Perceptive Instruments, COMET III-software). Fifty randomly selected cells on each slide were scored and the median% DNA in the tail was used as endpoint. Cell cycle Cells were treated with 3-MA or chloroquine for 16 h and incubated with 10 lM BrdU for 30 min, trypsinized and fixed with methanol. After pepsin (0.4 mg/ml in 0.1 M HCl), HCl (2 M) and Sodiumtetraborate (0.1 M pH 8.5) digestion, cells were stained with rat-anti-BrdU (AbD Serotec) and Alexa Fluor 488 Goat Anti-Rat IgG (H + L) Antibody (Life Technologies). Cells were resuspended in cold PBS/1% BSA/RNase (100 lg/ml)/Propidium Iodide (20 lg/ml) solution and analyzed on a BD FACSCanto II flow cytometer.

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Statistics Data were analyzed using GraphPad Prism. Student’s t test was used for single comparisons. Multiple testing was done using a repeated measures ANOVA with a Bonferroni post hoc test. P-values <0.05 were considered statistically significant. Results Irradiation does not induce autophagy activity Several stimuli result in induction of autophagy, such as starvation, hypoxia and chemotherapy [22]. In this study we determined autophagy activity using a gold standard technique which includes activity over the complete process of autophagy from activation to lysosomal degradation; autophagic flux [23]. In short, during autophagy, LC3b-II is partly degraded by lysosomes and the magnitude is a measure for autophagy activity. Preventing lysosomal degradation by CQ addition accumulates LC3b-II. The difference in LC3b-II levels in the presence and absence of CQ is indicative of autophagy activity. Eight different cell lines were irradiated (1.4 Gy and 5.6 Gy) and autophagic flux was determined using LC3b immunoblotting. In 6 cell lines, irradiation did not alter flux (Fig. 1A) while in two cell lines, turnover of autophagosomes was inhibited as illustrated by elevated LC3b-II levels in the absence of CQ after irradiation (Fig. 1B). LC3b immunofluorescence in HT29 cells four hours post-IR showed a comparable number of LC3b foci (Fig. 1C). In contrast, in HCT116 cells the number of LC3b foci increased after IR (Fig. 1D). These results indicate that single-dose irradiation inhibits or does not change autophagic flux and depends on the cell line tested. In addition to single-dose irradiation we evaluated the effect of fractionated irradiation on autophagic flux. Irradiation on two consecutive days further reduced autophagic flux in HT29, MDA-MB-231 and A549 cells (Suppl. Fig. 1). Chloroquine does not increase the intrinsic radiosensitization of cancer cell lines Although flux is not enhanced after irradiation, basal autophagy could still contribute to radioresistance as it is implicated in response to oxidative stress [24]. Since CQ is tested in multiple clinical trials, we investigated its radiosensitizing effect by clonogenic survival. Cells were exposed to CQ for 16 h pre-irradiation at the highest concentration that inhibits autophagy without any toxicity [8,25] (Suppl. Fig. 2), followed by a single dose irradiation. In agreement with our previous work [8], CQ addition had no added effect on clonogenic survival in HT29, HCT116 and U373 cells after irradiation. Similar results were obtained in MDA-MB231, A549 and MCF7 cells exposed to 5.6 Gy (Fig. 2). These data indicate that CQ does not alter intrinsic cellular radiosensitivity. Knockdown of autophagy related proteins and 3-MA treatment sensitizes cells to irradiation We observed no radiosensitizing effect after blocking autophagy with CQ. Other autophagy inhibitors, such as the PI3K class III inhibitor, 3-MA, have been reported to increase radiation sensitivity [9,11,13]. Clonogenic survival analysis in HT29 cells confirmed that 3-MA has radiosensitizing effects at 1 mM (p < 0.0001), without affecting survival of unirradiated cells (Fig. 3A). Interestingly, this concentration had no effect on autophagy activity (Fig. 3B). In HCT116 and MDA-MB-231 cells, comparable effects of 3-MA on radiosensitivity were observed (Fig. 3C and D and Suppl. Fig. 3A; p < 0.05 and p = 0.05 respectively). In A549 cells, 3-MA had no radiosensitizing effect (Fig. 3E and Suppl. Fig. 3A). Consistent with HT29 cells, autophagy activity was not changed by 3-MA treatment after irradiation in all three

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Fig. 1. Ionizing radiation blocks or does not affect autophagy activity. (A) Immunoblots against LC3b of HT29, MDA-MB-231, A549, T47D, MCF7, HeLa, (B) HCT116 and DU145 cancer cells 24 h after exposure to 1.4 Gy (low bar) or 5.6 Gy (high bar) in the presence or absence of chloroquine (CQ) i.e. flux analysis. As indicated, long and short exposures are shown. (C) Representative images and quantifications of immunofluorescent analysis of LC3b (green) and nuclei (blue) of HT29 and (D) HCT116 cells. Insets show magnifications. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 2. Chloroquine does not sensitize cells to irradiation. Clonogenic survival was assessed in HT29, HCT116 and U373 cells after exposure to a range of single irradiation doses in presence or absence of CQ. Similar assay was performed in MDA-MB-231, A549 (n = 4) and MCF7 cells after 5.6 Gy (IR). n = 3, mean ± SEM.

cell lines (Fig. 3F). These data indicate that in the majority of cell lines tested, 3-MA increases radiosensitivity without affecting autophagy activity. To further assess if autophagy is crucial for the survival and response of cancer cells after irradiation, we used ATG7 and LC3b knockdown cell lines. As long-term impairment of autophagy often leads to toxicity and cell killing, we engineered inducible knockdown models. This is illustrated by comparable plating efficiencies

(Suppl. Fig. 3B). ATG7 knockdown inhibited autophagy as demonstrated by the diminished lysosomal LC3b-II turnover and accumulation of LC3b-I, even in absence of CQ (Fig. 3G; Suppl. Fig. 3C). LC3b knockdown resulted in markedly reduced protein levels of both LC3b-I and LC3b-II (Fig. 3H). We observed that ATG7 and LC3b knockdown radiosensitized HT29 cells (Fig. 3I and J; at 5.6 Gy both p < 0.05). This effect was most pronounced at the highest dose of IR and dependent on the expression of the shRNA

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Fig. 3. Radiosensitization by 3-MA and knockdown of autophagy-related proteins. (A) HT29 cells (n = 4) were pretreated with indicated 3-MA concentrations and irradiated at 5.6 Gy (squares). (B) Flux analysis of control and irradiated HT29 cells in the presence or absence of 3-MA. (C) Clonogenic survival of irradiated (5.6 Gy) HCT116 (n = 6), (D) MDA-MB-231 (n = 3) and (E) A549 (n = 4) cells in the presence or absence of 1 mM 3-MA. This concentration was non-toxic as indicated by plating efficiencies (Suppl. Fig. 3A). (F) Flux analysis of control and 3-MA treated HCT116, MDA-MB-231 and A549 cells. (G) LC3b immunoblot of doxycycline treated HT29 cells expressing shRNA against ATG7 or (H) LC3b. (I) Clonogenic survival after single-dose irradiations of HT29 cells depleted of ATG7 (n = 3) or (J) LC3b (n = 4), alone or co-treated with 3-MA. (K) Clonogenic survival of HT29 cells when 3-MA was removed from culture medium immediately after IR (5.6 Gy) (white bars)(n = 3). A,C,D,E,I,J,K, mean ± SEM.

(Suppl. Fig. 3D). Interestingly, both ATG7 and LC3B knockdown cells were further radiosensitized after 3-MA exposure (Fig. 3I and J at 5.6 Gy both p < 0.05). Interestingly, the removal of 3-MA immediately after IR (3-MA pre-incubation) abrogated the radiosensitizing effect of 3-MA (Fig. 3K). Together, these data indicate that targeting autophagyassociated genes and 3-MA, increases radiation sensitivity postirradiation and independent of autophagy. 3-MA changes the response of cells after irradiation As the extent of DNA damage inflicted and repaired after irradiation influences cell survival, we investigated whether 3MA affected H2AX phosphorylation. As expected, IR increased the number of c-H2AX foci per cell in HT29 and HCT116 cells, regardless of 3-MA pretreatment. Surprisingly, the number of foci in 3MA treated HT29 and HCT116 cells 2 h post-IR was significantly lower compared to controls (Fig. 4A; HT29 p < 0.05). Additionally, we observed that 3-MA reduced the number of c-H2AX foci in unirradiated HT29 cells (Suppl. Fig. 4A). We also observed an attenuated induction of c-H2AX by immunoblot analysis in irradiated 3-MA pretreated cells compared to controls (Fig. 4B; p = 0.05). Taken together these results show that radiation-induced phosphorylation of H2AX is less abundant after exposure to 3-MA.

Phosphorylation of H2AX is mediated by PI3K-like kinases; ATM, ATR and DNA-PK [26], and is required for the assembly of DNA repair proteins to the damaged site (reviewed in [27]). Yoon et al. demonstrated that 3-MA (10 mM) treatment inhibits ATM and DNA-PKcs phosphorylation in MCF7 cells after exposure to an inducer of oxidative DNA damage, capsaicin [28]. To determine if ATM activity is also reduced after exposure to 1 mM 3-MA, phosphorylation of Chk2 at 60 and 120 post IR was assessed (Suppl. Fig. 4B). Irradiation increased Chk2 phosphorylation post-IR, but was unaffected by 3-MA treatment, suggesting that the radiosensitizing effect of 3-MA is not a result of reduced ATM activity. Furthermore, we tested whether 3-MA was able to radiosensitize HCT116 DNA-PK catalytic subunit (DNA-PKcs) knock-out cells. Knock-out of DNA-PKcs severely decreased clonogenic survival after IR, but the sensitizing effect of 3-MA remained (Suppl. Fig. 4C and D). Together, these data indicate that the radiosensitizing effect of 3-MA is independent of ATM and DNA-PKcs. To determine whether the limited H2AX phosphorylation results from attenuated phosphorylation events or reduced DNA damage, we assessed DNA-damage directly by single cell gel electrophoresis (neutral COMET assay). The percentage DNA in the tail after migration is indicative of double strand DNA breaks. In irradiated HT29 cells, we observed that damage is induced and repaired, returning to basal level at eight hours post-IR (Fig. 4C). Unexpectedly,

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Fig. 4. Cellular DNA damage response after IR is altered by 3-MA. (A) Representative images and quantifications of immunofluorescent stainings of (un)irradiated HT29 and HCT116 cells showing c-H2AX (green) and nuclei (blue) directly and 120 min after IR. Insets show magnifications. (B) Representative immunoblot and quantifications of (un)irradiated HT29 cells in presence or absence of 3-MA. (C) Quantified COMET assay data of HT29 cells plotted for percentage DNA in tail corrected for background (unirradiated) at different time points post-IR (5.6 Gy) in presence or absence of 3-MA. (D) Cell cycle distribution of HT29, HCT116, MDA-MB-231 (n = 2) and A549 cells after 16 h of treatment with 3-MA, CQ or control. (E) Representative images and quantifications of c-H2AX immunofluorescent staining of (un)irradiated HT29 control or ATG7 depleted cells 60 min post-IR in presence or absence of 3-MA. (A–E) n = 3, mean ± SEM. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

irradiated HT29 cells treated with 3-MA showed a prolonged induction of DNA damage up to 1 h post-IR, which was rapidly resolved and returned to control levels 2 h post-IR (Fig. 4C; Suppl. Fig. 5; p < 0.05). The initial damage after IR is lower when treated with 3-MA which could be a result of changes in cell cycle distribution. Indeed, a reduced number of cells in S-phase were observed in HT29, but not in HCT116, MDA-MB-231 and A549 cells (Fig. 4D) indicating that the radiosensitizing effect is independent of cell cycle effects. DNA damage kinetics of both CQ treatment and ATG7 deficiency were comparable to control (Suppl. Fig. 6). In line with the observed autophagy independent effect of 3-MA, the number of c-H2AX foci at one hour post-IR was reduced in ATG7 knockdown cells after 3-MA treatment (Fig. 4E). These data further support an autophagy independent radiosensitizing effect by 3-MA, possibly due to changes in DNA repair.

Discussion Autophagy is a pro-survival mechanism of metabolically stressed cells. For example, cells exposed to hypoxia rapidly increase autophagic flux [8]. Targeting autophagy [29] genetically or pharmacologically, sensitizes cells to hypoxia and reduces the hypoxic tumor fraction which leads to sensitization of tumors to irradiation [1]. In contrast to metabolic stresses, the role of autophagy following irradiation remains elusive as contradicting reports describe enhanced radioresistance [13–16] as well as radiosensitization [8–12] after autophagy inhibition. Numerous clinical trials have been initiated that combine conventional anti-cancer therapy (including irradiation) with autophagy inhibition, it is therefore important to understand its effect.

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Flux analysis is a reliable assay as it enables segregation between induced and blocked autophagy activity. Immunofluorescent analysis lacks this advantage, for example we observed an increase in the number of LC3b foci after hypoxia exposure (autophagy-induction) and after irradiation (autophagyinhibition) in HCT116 cells (Suppl. Fig. 7 and Fig. 1D). This illustrates that autophagic activity should be monitored dynamically [23]. Interestingly, we observe two different effects of IR on autophagy, (1) it remains unchanged or (2) it is blocked. This indicates that variation in response to IR is cell line dependent and may contribute to the reported discrepancies. Contributing to the elusive role of autophagy in radiation response is the use of single assays. In this report we show that both CQ and knockdown of the essential autophagy genes, ATG7 and LC3b, effectively inhibit autophagy; however, only knockdown of LC3b or ATG7 reduced survival. Interestingly, this was not observed for CQ. This indicates a radioprotective role of these autophagy-associated genes, yet independent on lysosomal degradation, and thus unrelated to canonical autophagy. The mechanism of autophagy has been implicated in additional cellular responses. Most proteins that are secreted contain a Nterminal secretion signal that allows trafficking through the ER and golgi and transit through the secretory pathway. Secreted proteins that lack this signal are secreted through unconventional protein secretion (UPS). The involvement of autophagy machinery in UPS is illustrated by ATG5 dependent interleukin 1b and HMGB1 secretion by mammalian macrophages [30] and ATG5 and ATG16L dependent lysozyme secretion by granule exocytosis in Paneth cells [31]. Several proteins, associated with radioresistance are secreted through UPS [32]. For example, FGF-2 and galectin-1 contribute to radioresistance by increasing the cells’ capability to repair DNA double strand breaks [33,34] and IL-1a downregulates the apoptotic response after irradiation [35]. Further research will be required to elucidate the role of UPS in mediating radioresistance. Unexpectedly, the observed radiosensitization of 3-MA is autophagy independent, but effectively attenuates H2AX phosphorylation and changes DNA damage repair kinetics after IR (Fig. 4). Yoon et al. demonstrated that 10 mM 3-MA reduces ATM and DNA-PKcs activation in response to capsaicin [28]. At the concentration we used (1 mM), 3-MA did not inhibit autophagy or ATM activity and was still capable of radiosensitizing DNA-PKcs knockout cells. Here we demonstrate that already at a relatively low concentration 3-MA has effects unrelated to autophagy that induce radiosensitization. Though it may also inhibit autophagy at higher concentrations, this is an important observation as this exposes additional effects which enable misinterpretation. Yet, the exact mechanism by which 3-MA influences radiosensitivity remains unknown. The added value of CQ or hydroxyCQ addition to current anticancer therapies is momentarily evaluated in more than 55 clinical trials. Here, we observed no intrinsic changes in radiosensitivity after CQ addition; however, we have previously demonstrated that CQ treatment decreases hypoxia tolerance [8]. In addition, Maes et al. have shown that CQ induces vessel normalization and chemotherapy delivery in an autophagy independent manner [36]. Taken together, the anti-tumor effects of CQ are mediated through reducing the hypoxic radioresistant cell population within the tumor, rather than affecting intrinsic radiosensitivity of cancer cells. It is therefore an attractive therapeutic option to pursue clinically.

Conflict of interest statement None of the authors have a conflict of interest to declare.

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Acknowledgments The authors would like to thank Daniëlle Pachen for her excellent technical support. This work was financially supported by the Dutch Cancer Society (KWF Grants UM 2010-4714 and 2012-5506 to K.R.), STOPhersentumoren.nl and zeldzame ziektenfonds (to K.R.), and European Research Council under the European Union’s Seventh Framework Programme (FP/20072013)/ERC Grant Agreement n. 617060 (to M.V.). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.radonc.2015.02. 019. References [1] Ichimura Y, Kirisako T, Takao T, Satomi Y, Shimonishi Y, Ishihara N, et al. A ubiquitin-like system mediates protein lipidation. Nature 2000;408:488–92. [2] Kabeya Y, Mizushima N, Ueno T, Yamamoto A, Kirisako T, Noda T, et al. LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J 2000;19:5720–8. [3] Nath S, Dancourt J, Shteyn V, Puente G, Fong WM, Nag S, et al. Lipidation of the LC3/GABARAP family of autophagy proteins relies on a membrane-curvaturesensing domain in Atg3. Nature cell Biol 2014;16:415–24. [4] Kawai A, Uchiyama H, Takano S, Nakamura N, Ohkuma S. Autophagosomelysosome fusion depends on the pH in acidic compartments in CHO cells. Autophagy 2007;3:154–7. [5] Ding WX, Ni HM, Gao W, Yoshimori T, Stolz DB, Ron D, et al. Linking of autophagy to ubiquitin-proteasome system is important for the regulation of endoplasmic reticulum stress and cell viability. Am J Pathol 2007;171:513–24. [6] Yamamoto A, Tagawa Y, Yoshimori T, Moriyama Y, Masaki R, Tashiro Y. Bafilomycin A1 prevents maturation of autophagic vacuoles by inhibiting fusion between autophagosomes and lysosomes in rat hepatoma cell line, H-4II-E cells. Cell Struct Funct 1998;23:33–42. [7] Rojas-Puentes LL, Gonzalez-Pinedo M, Crismatt A, Ortega-Gomez A, GamboaVignolle C, Nunez-Gomez R, et al. Phase II randomized, double-blind, placebocontrolled study of whole-brain irradiation with concomitant chloroquine for brain metastases. Radiat Oncol 2013;8:209. [8] Rouschop KM, van den Beucken T, Dubois L, Niessen H, Bussink J, Savelkouls K, et al. The unfolded protein response protects human tumor cells during hypoxia through regulation of the autophagy genes MAP1LC3B and ATG5. J Clin Invest 2010;120:127–41. [9] Chaachouay H, Ohneseit P, Toulany M, Kehlbach R, Multhoff G, Rodemann HP. Autophagy contributes to resistance of tumor cells to ionizing radiation. Radiother Oncol 2011;99:287–92. [10] Chen YS, Song HX, Lu Y, Li X, Chen T, Zhang Y, et al. Autophagy inhibition contributes to radiation sensitization of esophageal squamous carcinoma cells. Dis Esophagus 2011;24:437–43. [11] Lomonaco SL, Finniss S, Xiang C, Decarvalho A, Umansky F, Kalkanis SN, et al. The induction of autophagy by gamma-radiation contributes to the radioresistance of glioma stem cells. Int J Cancer 2009;125:717–22. [12] Paglin S, Hollister T, Delohery T, Hackett N, McMahill M, Sphicas E, et al. A novel response of cancer cells to radiation involves autophagy and formation of acidic vesicles. Cancer Res 2001;61:439–44. [13] Tseng HC, Liu WS, Tyan YS, Chiang HC, Kuo WH, Chou FP. Sensitizing effect of 3-methyladenine on radiation-induced cytotoxicity in radio-resistant HepG2 cells in vitro and in tumor xenografts. Chem Biol Interact 2011;192:201–8. [14] Kim KW, Hwang M, Moretti L, Jaboin JJ, Cha YI, Lu B. Autophagy upregulation by inhibitors of caspase-3 and mTOR enhances radiotherapy in a mouse model of lung cancer. Autophagy 2008;4:659–68. [15] Kuwahara Y, Oikawa T, Ochiai Y, Roudkenar MH, Fukumoto M, Shimura T, et al. Enhancement of autophagy is a potential modality for tumors refractory to radiotherapy. Cell Death Dis 2011;2:e177. [16] Lin CI, Whang EE, Abramson MA, Jiang X, Price BD, Donner DB, et al. Autophagy: a new target for advanced papillary thyroid cancer therapy. Surgery 2009;146:1208–14. [17] Palumbo S, Pirtoli L, Tini P, Cevenini G, Calderaro F, Toscano M, et al. Different involvement of autophagy in human malignant glioma cell lines undergoing irradiation and temozolomide combined treatments. J Cell Biochem 2012;113:2308–18. [18] Apel A, Herr I, Schwarz H, Rodemann HP, Mayer A. Blocked autophagy sensitizes resistant carcinoma cells to radiation therapy. Cancer Res 2008;68:1485–94. [19] Wee S, Wiederschain D, Maira SM, Loo A, Miller C, deBeaumont R, et al. PTENdeficient cancers depend on PIK3CB. Proc Natl Acad Sci U S A 2008;105:13057–62. [20] Ramaekers CH, van den Beucken T, Meng A, Kassam S, Thoms J, Bristow RG, et al. Hypoxia disrupts the Fanconi anemia pathway and sensitizes cells to

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