Interplay Between TRAP1 and Sirtuin-3 Modulates Mitochondrial

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Published OnlineFirst January 25, 2019; DOI: 10.1158/0008-5472.CAN-18-2558 Cancer Metabolism and Chemical Biology Research

Interplay between TRAP1 and Sirtuin-3 Modulates Mitochondrial Respiration and Oxidative Stress to Maintain Stemness of Glioma Stem Cells Hye-Kyung Park1, Jun-Hee Hong2, Young Taek Oh2, Sung Soo Kim2, Jinlong Yin2, An-Jung Lee1, Young Chan Chae1, Jong Heon Kim3, Sung-Hye Park4, Chul-Kee Park4, Myung-Jin Park5, Jong Bae Park2,3, and Byoung Heon Kang1

Abstract

Glioblastoma (GBM) cancer stem cells (CSC) are primar- stress (particularly reduced nutrient supply), and main- ily responsible for metastatic dissemination, resistance to tained "stemness." Inactivation of TRAP1 or SIRT3 compro- therapy, and relapse of GBM, the most common and aggres- mised their interdependent regulatory mechanisms, leading sive brain tumor. Development and maintenance of CSCs to metabolic alterations, loss of stemness, and suppression require orchestrated metabolic rewiring and metabolic of tumor formation by GSC in vivo. Thus, targeting the adaptation to a changing microenvironment. Here, we show metabolic mechanisms regulating interplay between TRAP1 that cooperative interplay between the mitochondrial chap- and SIRT3 may provide a novel therapeutic option for erone TRAP1 and the major mitochondria deacetylase sir- intractable patients with GBM. tuin-3 (SIRT3) in glioma stem cells (GSC) increases mitochondrial respiratory capacity and reduces production of Signiﬁcance: Discovery and functional analysis of a reactive oxygen species. This metabolic regulation endowed TRAP1–SIRT3 complex in glioma stem cells identify potential GSCs with metabolic plasticity, facilitated adaptation to target proteins for glioblastoma treatment.

Introduction is a promising therapeutic approach for aggressive and relapsing Glioblastoma multiforme (GBM), the most common malig- tumors (3). nant brain tumor, quickly develops resistance to both radiation Some cancer cells are more dependent on aerobic glycolysis and cytotoxic chemotherapy, which inevitably leads to poor than on mitochondrial respiration; this phenomenon, called the clinical outcomes and tumor recurrence despite multimodal Warburg effect, redirects metabolism toward biosynthetic path- therapy (1). A small subpopulation of self-renewing tumor cells, ways, reduces cellular oxidative stress, and supports proliferation that is, cancer stem cells (CSC) or tumor-initiating cells, isolated and survival of cancer cells (4, 5). However, one caveat with from patients with GBM is responsible for therapeutic resistance respect to a metabolic shift toward glycolysis is a marked reduc- and tumor relapse (2, 3). Thus, targeting glioma stem cells (GSC) tion in the ATP yield, which ultimately requires cancer cells to increase uptake of glucose (5, 6). This may limit tumor progression under reduced nutrient conditions. Growing evidence sug- 1Department of Biological Sciences, Ulsan National Institutes of Science and gests that many cancer cells retain mitochondrial function for 2 Technology (UNIST), Ulsan, Republic of Korea. Division of Clinical Research, energy production (7, 8), and that certain cancer cells and CSCs Research Institute and Hospital, National Cancer Center, Goyang, Republic of have a metabolic preference for mitochondrial respiration rather Korea. 3Department of Cancer Biomedical Science, Graduate School of Cancer Science and Policy, National Cancer Center, Goyang, Republic of Korea. than glycolysis (9, 10). GSCs are extremely dependent on mito- 4Department of Pathology, Seoul National University Hospital, Seoul National chondrial respiration for energy production and survival (9, 11); University College of Medicine, Seoul, Republic of Korea. 5Division of Radiation however, little is known about the molecular mechanisms under- Cancer Research, Research Center for Radio-Senescence, Korea Institute of lying metabolic reprogramming. Radiological and Medical Sciences, Seoul, Republic of Korea. TNF receptor-associated protein 1 (TRAP1) is a mitochon- Note: Supplementary data for this article are available at Cancer Research drial paralog of the 90-kDa HSP (Hsp90) and is highly Online (http://cancerres.aacrjournals.org/). expressed by a variety of cancer cells (12, 13). TRAP1 endows H.-K. Park and J.-H. Hong contributed equally to this article. cancer cells with a high cell death threshold and confers drug – Corresponding Authors: Byoung Heon Kang, Department of Biological resistance by maintaining mitochondrial integrity (14 17). Sciences, Ulsan National Institutes of Science and Technology (UNIST), 50 Thus, small-molecule inhibitors targeting mitochondrial UNIST St., Ulsan 44919, Republic of Korea (South). Phone: 82-52-217-2521; TRAP1 have been developed to disrupt mitochondrial function Fax: 82-52-217-2639; E-mail: [email protected]; and Jong Bae Park, Depart- and thereby induce cell death (14, 16, 18, 19). Recent reports ment of Cancer Biomedical Science, Graduate School of Cancer Science suggest that metabolic regulation by TRAP1 alters mitochon- and Policy, National Cancer Center, Goyang 10408, Republic of Korea. Phone: 82- drial respiration and subsequently reprograms cellular metab- 31-920-2450; E-mail: [email protected] olism in some tumors (20–22). However, the underlying doi: 10.1158/0008-5472.CAN-18-2558 molecular mechanisms are unclear (23) and have not yet been 2019 American Association for Cancer Research. examined in highly tumorigenic CSCs.

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þ Sirtuins (SIRT) are NAD -dependent protein deacetylases; punches per patient) were obtained and transferred to the recip- among the seven mammalian SIRTs (SIRT1–7) identified to date, ient paraffin block at defined array positions (Biochip Company SIRT3, SIRT4, and SIRT5 are localized to the mitochondria (24). Ltd). TMA blocks were cut into 4-mm sections and mounted onto SIRT3, a major mitochondrial protein deacetylase, senses cellular slides coated with 3-aminopropyltrioxysilane. After deparaffini- metabolic stress and modulates energy and ROS metabo- zation and antigen retrieval in citrate buffer (pH 6.0) for 20 lism (25–28), both of which are closely related to the pathophys- minutes in a pressure cooker, slides were treated for 10 minutes iology of tumorigenesis. However, the contribution of SIRT3 to with 3% hydrogen peroxide to block endogenous peroxidase. tumorigenesis is controversial; previous studies have identified Anti-TRAP1 (1:200, monoclonal, Abcam, #ab109323) and anti- both tumor-promoting and -suppressive roles (29, 30). This Sox2 (1:200, polyclonal; R&D Systems, #AF2018) were the pri- suggests that the precise role of SIRT3 depends on the cellular mary antibodies. The TMAs were subsequently incubated with the context; therefore, the specific type of cancer must be taken Polink-2 Plus HRP detection system (GBI Labs) and visualized into account (30, 31). Studies suggest that SIRT3 has a tumor- with 3,30-diaminobenzidine (DAB). Negative controls were trea- promoting effect in the brain (32, 33), but no report to date has ted the same way, but without the primary antibodies. IHC described SIRT3-dependent metabolic regulation of GSCs. staining was assessed by two independent pathologists with no Here, we found that interplay between TRAP1 and SIRT3 in prior knowledge of patient characteristics. Discrepancies were GSCs rewired cellular metabolism to generate energy-efficient resolved by consensus. Extent of staining was scored on a scale mitochondrial respiration without overproduction of ROS. These from 0 to 4, corresponding to the percentage of immune-reactive metabolic reprogramming functions were crucial for maintenance tumor cells (0%, 1–5%, 6%–25%, 26%–75%, and 76%–100%, of self-renewal capacity, lack of differentiation, and survival of respectively). Staining intensity was scored as negative (score ¼ 0), GSCs, particularly under limited nutrient conditions. Inactivating weak (score ¼ 1), or strong (score ¼ 2). A score ranging from 0 either TRAP1 or SIRT3 compromised the metabolic pathways to 8 was calculated by multiplying the score for staining extent regulated by them and, subsequently, caused GSCs to lose stem- with that for intensity. Final grades (negative, 1þ,2þ, and 3þ) ness, leading to suppression of sphere formation in vitro and were assigned to each specimen with scores of 0–1, 2–3, 4–5, and tumor growth in vivo. 6–8, respectively.

Immunofluorescence staining Materials and Methods Formalin-fixed, paraffin-embedded tissue samples were sec- Chemicals and antibodies tioned (7-mm thick) using a microtome and transferred to glass All chemicals were purchased from Sigma. Anti-nestin and anti- slides. After paraffin removal and antigen retrieval, the slides were Sox2 antibodies were purchased from R&D Systems; anti-TRAP1, blocked with 5% FBS, 5% BSA, and 0.3% Triton X-100 in PBS, and anti-SOD2, anti-NDUFA9, anti-UQCRC2, anti-ATPB, anti-GFAP, then incubated overnight at 4C with antibodies specific for Sox2 and anti-SIRT3 antibodies were purchased from Abcam; the and TRAP1. Next, the slides were incubated for 2 hours with Alexa anti-Cox IV antibody was purchased from Cell Signaling Tech- Fluor 488 (green)- or Alexa Fluor 633 (red)-conjugated secondary nology; anti-acetylated lysine, anti-GDH, anti-Ki-67, and anti- antibodies (Invitrogen) and mounted in a mounting medium SDHB antibodies were purchased from Santa Cruz Biotechnolo- containing DAPI (Vectashield). Stained slides were analyzed gy; the anti-b-actin antibody was obtained from MP Biomedicals; under a FV1000 confocal microscope (Olympus). and the anti-Hsp60 antibody was purchased from BD Biosciences. Brain tumor tissue samples, cells, and culture conditions Patients and specimens Resected brain tumor tissues were collected at SNUH (Seoul, A total of 150 patients with glioma were enrolled in this cohort Republic of Korea) with written informed consent in accordance study conducted at Seoul National University Hospital (SNUH, with institutional guidelines, and graded pathologically accord- Seoul, Republic of Korea). This included 108 randomly selected ing to WHO grades (35). GBM tissues were formalin-fixed and consecutive patients with glioma who underwent curative resec- paraffin-embedded for IHC or immunofluorescence analysis. All tion at SNUH (performed by the same surgical team) in 2014– human GSC lines were cultured in DMEM/F-12 supplemented 2015. Patients in this cohort were followed until June 2017, with a with B27 (Invitrogen), EGF (10 ng/mL; R&D Systems), and basic median observation time of 22.5 months (range, 2–47 months). fibroblast growth factor (bFGF; 5 ng/mL; R&D Systems). Differ- Overall survival was defined as the interval between the date of entiated GSC cells were maintained in DMEM/F-12 supplemen- surgery and date of death or last observation. Data for patients not ted with 10% FBS. Brain cancer cell lines (LN229, T98G, U87, and experiencing relapse or death were censored at the last follow-up. U251) and human astrocytes were purchased from the ATCC All experiments involving human subjects were approved by and maintained as recommended by the manufacturer. Cells were the Institutional Review Board (IRB) of SNUH (IRB no. 2014- cultured in DMEM or RPMI medium (Gibco) containing 5% or 08-004). 10% FBS (Gibco) and 1% penicillin/streptomycin (Gibco) at 37 C in a humidified atmosphere of 5% CO2. To enrich stem Tissue microarray analysis and IHC cells, LN229 and T98G cells were cultured for 3 weeks in DMEM/ A tissue microarray (TMA) was constructed as described pre- F12 (Gibco) medium supplemented with 10 ng/mL EGF, 5 ng/mL viously (34). All glioma cases were histologically reviewed by bFGF, 0.04% B27, and 1% penicillin/streptomycin. hematoxylin and eosin staining. Briefly, two cores were taken from each representative tumor tissue and from brain tissue Measurement of mitochondrial respiration adjacent to the tumor within a distance of 10 mm. These were Oxygen consumption rate (OCR) was measured using a Mito used to construct TMA slides. Duplicate 1.0-cm diameter cylinders Stress Test Kit and a XF24 Extracellular Flux Analyzer (Seahorse taken from intratumoral and peritumoral areas (a total of four Bioscience), according to the manufacturer's protocol. Briefly, X01

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or LN229 cells (2 104) were attached to laminin-coated CATGAGTTCCAGGCCGAG-30 and TRAP1-#2 50-CCCGGTCCC- Seahorse XF24 cell culture microplates (Seahorse Bioscience) for TGTACTCAGAAA-30; SIRT3-#1 50-GTGGGTGCTTCAAGTGTT- 6 hours and then incubated with drugs for 48 hours at 37C/5% GTT-30 and SIRT3-#2 50-GCCCAACGTCACTCACTAC-30; control 0 0 CO2. At the time of OCR measurement, the medium was changed 5 -ACUCUAUCUGCACGCUGAC-3 . Cells grown to 50%–75% to DMEM XF assay medium (Seahorse Bioscience) supplemented confluence on 6-well plates were transfected with 50–100 nmol/L with 2.5 mmol/L glutamine, 0.5 mmol/L sodium pyruvate, and siRNA for 48 hours using G-Fectin (Genolution) and then ana- 17.5 mmol/L glucose (pH 7.4 at 37C). After equilibration for 30 lyzed by Western blotting. minutes at 37 C in the absence of CO2, OCR was recorded using the Seahorse XF Analyzer (Seahorse Bioscience). Oligomycin Flow cytometry analysis (0.4 mmol/L), carbonyl cyanide p-(trifluoromethoxy) phenylhy- To examine mitochondrial superoxide, cells were incubated for drazone (FCCP; 0.5 mmol/L), and rotenone/antimycin A 20 minutes with 200 nmol/L Mito-SOX red (Invitrogen), washed (0.5 mmol/L) mixtures were injected sequentially to determine with PBS, and then analyzed immediately using a FACSCalibur mitochondrial function. Readings were collected from each well System (BD Biosciences). Data were processed using FlowJo every 8 minutes; each 8-minute cycle comprised mixing (3 min- software (TreeStar). To measure induction of apoptosis, the DNA utes), waiting (2 minutes), and recording (3 minutes). After content (assessed using propidium iodide) and phosphatidylser- completion of OCR measurement, the amount of protein in each ine (PS) externalization were analyzed using the Annexin V/Dead well was quantified to normalize the results. Cell Apoptosis Kit (Invitrogen). Labeled cells were quantified using the FACSCalibur system (BD Biosciences) and data were Measurement of glucose uptake and lactate production analyzed using FlowJo software (TreeStar). Cellular glucose uptake and lactate concentration in the culture medium were measured using the Glucose Uptake Colorimetric RNA extraction and RT-PCR Assay Kit (BioVision) and the Lactate Colorimetric Assay Kit Total RNA was prepared from cultured cells using RNeasy Mini (BioVision), respectively, according to the manufacturer's instruc- Kits (QIAGEN) according to the manufacturer's instructions, and tions. Briefly, to measure cellular uptake of glucose, X01 or LN229 cDNA was synthesized using the ProtoScript First Strand cDNA cells (1 104) were plated in 96-well plates. Each well was washed Synthesis Kit (New England Biolabs) with an oligo(dT) primer. three times with PBS and then starvation was induced by incu- PCR reactions were performed in a Mastercycler PCR machine bating the cells for 40 minutes with 100 mL of Krebs-Ringer- (Eppendorf) with the following sets of forward and reverse Phosphate-HEPES (KRPH) buffer. Cells were treated for 20 min- oligonucleotide primers: GAPDH, 50-CGGGAAGCTTGTCAT- utes with 1 mmol/L 2-deoxyglucose and lysed with 90 mLof CAATGG-30 and 50-GGCAGTGATGGCATGGACTG-30; TRAP1, Extraction Buffer. Cell lysates were neutralized by addition of 50- AGCGCACTCATCAGGAAACT-30 and 50-TCAAACTCAC- 10 mL of Neutralization Buffer. Fluorescence (Ex/Em ¼ 535/587 GAAGGTGCAG-30; Nestin, 50- AACAGCGACGGAGGTCTCTA-30 nm) was measured in a microplate reader (Synergy NEO, BioTek). and 50-CCTTTCCCAGGTTCTCTTCC-30; Sox2, 50-AACCAA- To measure lactate production, culture medium was collected GACGCTCATGAAGAAG-30 and 50-GCGAGTAGGACATGCTG- at the indicated times and mixed with Lactate Assay Buffer TAGGT-30; GFAP, 50-GCTTCCTGGAACAGCAAAAC-30 and 50- (50 mL/well) in a 96-well plate. Then, 50 mL of Reaction Buffer CCTCCAGCGACTCAATCTTC-30; SIRT3, 50-CATGAGCTGCAGT- was added to each well for 30 minutes at room temperature. GACTGGT-30 and 50-GAGCTTGCCGTTCAACTAGG-30; NDUFA9, Absorbance was measured at 570 nm in a microplate reader 50-CAATTGCTCAACTGTCCAAG-30 and 50-AAGACTCTTGCTA- (Synergy NEO, BioTek). CCCATCG-30; SDHB, 50- TAAATGTGGCCCCATGGTAT-30 and 50-GATACTGCTGCTTGCCTTCC-30; UQCRC2, 50-CAAAGTTG- þ Measurement of the NAD /NADH ratio CCCCCAAAGTTA-30 and 50-CATGAGTCTGCGGATTCTGA-30; þ To measure the NAD /NADH ratio in mitochondria, X01 or COX IV, 50-CGAGCAATTTCCACCTCTGT-30 and 50-GGGCCGTA- 528NS cells (3 106) were seeded in 75T flasks and cultured in CACATAGTGCTT-30; and ATPB, 50-AGCCCATGGTGGTTACT- 5 mmol/L or 25 mmol/L glucose medium for 24 hours. Mito- CTG-30 and 50-GGCAGGGTCAGTCAAGTCAT-30. chondria were isolated using a Mitochondria Isolation Kit þ (Thermo Fisher Scientific), and the NAD /NADH ratio was mea- Immunoprecipitation and pull-down assay þ sured using the NAD /NADH Quantification Colorimetric kit For immunoprecipitation, 100–200 mg of protein lysate (BioVision), according to the manufacturer's instructions. Briefly, derived from GSCs incubated in immunoprecipitation (IP) lysis isolated mitochondria were lysed by two freeze/thaw cycles in buffer (135 mmol/L NaCl, 1% glycerol, 1% NP-40, 20 mmol/L NAD/NADH Extraction Buffer and vortexed for 10 seconds. After Tris-HCl, pH 8.0, and protease/phosphatase inhibitors) was pre- centrifugation, half of the supernatant was used for measurement cleared using 1 mg of rabbit or mouse IgG and 20 mL of Protein A/G þ of NADt (total amount of NAD and NADH), and the other PLUS agarose (Santa Cruz Biotechnology) to remove nonspecific half was used for measurement of NADH (after decomposition proteins. The precleared lysates were then incubated at 4C for 1 þ of NAD at 60C for 30 minutes). Samples were incubated for hour with 1 mg of anti-TRAP1 antibody and then mixed with 20 mL 5 minutes with NAD Cycling Mix, followed by NADH of Protein A/G PLUS agarose at 4C overnight. The immunocom- Developer solution for 2 hours. Absorbance at 450 nm was then plexes were then precipitated by centrifugation at 1,000 g for 1 þ measured. The NAD /NADH ratio was calculated as follows: minute at 4C and washed three times with IP lysis buffer. Bound (NADtNADH)/NADH. proteins were analyzed by Western blotting. To perform the pull- down experiments, GST and GST-TRAP1 proteins were induced in Treatment with siRNA E. coli BL21 by overnight incubation with 0.2 mmol/L IPTG at siRNAs specific for TRAP1 and SIRT3 were synthesized 18C. Cells were harvested by centrifugation and lysed by soni- by Genolution using the following sequences: TRAP1-#1 50-AAA- cation in lysis buffer (50 mmol/L Tris-HCl, pH 7.4, 150 mmol/L

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NaCl, 5 mmol/L MgCl2, and 1 mmol/L dithiothreitol). After (DMSO) dissolved in 20% Cremophor EL (Sigma) in PBS was centrifugation at 15,000 g for 30 minutes, the soluble fractions administered intraperitoneally every day. At the end of the exper- were mixed with glutathione-Sepharose beads (GE Healthcare). iment, animals were euthanized and tumors were collected for Protein-bound beads were washed three times with wash buffer histology and Western blotting. (50 mmol/L Tris-HCl, pH 7.4, 500 mmol/L NaCl, 5 mmol/L MgCl2, and 1 mmol/L dithiothreitol). Mitochondria isolated Histology and IHC staining from X01 cells were lysed for 1 hour at 4C in RIPA buffer To examine histologic characteristics, brains were removed, containing 50 mmol/L Tris-HCl, pH 7.4, 150 mmol/L NaCl, fixed for 24 hours at 4C with 4% paraformaldehyde, and stained 1% NP-40, 0.25% sodium deoxycholate, and proteinase inhibitor with hematoxylin (DAKO) and 0.25% eosin (Merck). For IHC (Invitrogen) under constant agitation. After centrifugation at staining of CSC markers and TRAP1-associated genes [Sox2 (R&D 13,000 g for 5 minutes at 4C, the supernatant was precleared Systems), Nestin (Abcam), SIRT3 (Cell Signaling Technology), for 1 hour at 4C with glutathione beads (GE Healthcare). Then, Ki-67 (Abcam), and cleaved caspase-3 (Cell Signaling Tech- 100–200 mg of precleared protein extract was incubated overnight nology)], sections were subjected to antigen retrieval in citrate at 4C with GST and GST-TRAP1 beads. Bead-bound proteins buffer (pH 6.0) and endogenous peroxidase was blocked with 3% were collected by centrifugation at 1,000 g for 1 minute at 4C, hydrogen peroxide. Tissue sections were then incubated overnight washed three times with lysis buffer, and analyzed by Western at 4C in a humidified chamber with appropriate primary blotting. antibodies in antibody diluent buffer (IHC World). Staining results were visualized by sequential incubation of TMAs with Limiting dilution assay components of the Polink-2 Plus HRP detection system (GBI); For the in vitro limiting dilution assays, cells were plated in DAB (Vector Laboratories) was used as the chromogen. The decreasing numbers (200, 100, 50, 25, 12.5, and 6.25) in 96-well number of cells positive for Ki-67 and cleaved caspase-3 per plates containing DMEM/F-12 supplemented with B27, 10 ng/mL hematoxylin counterstained area was counted (200 high-power EGF, and 5 ng/mL bFGF. Extreme limiting dilution analysis was fields were scanned). performed using software available at http://bioinf.wehi.edu.au/ software/elda/. Images of spheres formed by each group were Quantification and statistical analysis taken on day 5 after plating. All statistical analyses were performed using GraphPad Prism 7. All experiments were repeated at least twice, and data are pre- Enzyme activity of SIRT3, SOD, complex I, complex II, and sented as the mean SEM. Statistical significance of data derived complex V from the following experiments was determined using Student The enzyme activity of SIRT3 in vitro or in vivo was determined t test: OCR, glucose uptake, lactate production, ATP production, using the SIRT3 Activity Assay Kit, according to the manufacturer's SOD activity, SIRT3 activity, mitochondrial respiratory complex instructions (Abcam). SOD activity was determined in vitro using activity, tumor weight measurement, and quantification of IHC the SOD Activity Assay Kit, according to the manufacturer's staining. A value of P < 0.05 was considered significant. Pearson instructions (BioVision). The activity of Complexes I (ab109721, correlation coefficient (r) was used to determine the correlation Abcam), II (K660-100, BioVision), and V (ab109907, Abcam) was between TRAP1 and Sox2 expression in GBM cells and GSCs measured using respective activity assay kits according to the (using Prism 7). Survival was assessed using the Kaplan–Meier manufacturer's instructions. Mitochondria were lysed in the pro- method and the log-rank test (using GraphPad Prism 7). vided buffers to obtain a protein solution (5 mg/mL) and loaded into the provided plates prior to immunocapture. After washing away the unbound proteins, the provided substrate and dye were Results added to each well and absorbance was measured as described in TRAP1 expression by GSCs and its positive correlation with the instructions. brain tumor malignancy To understand the relationship between TRAP1 and brain In vivo xenograft experiments tumor malignancy, we examined expression of TRAP1 in brain All animal experiments were conducted in accordance with tumor tissues derived from human patients. TRAP1 expression protocols approved by the Institutional Animal Care and Use correlated significantly with brain tumor malignancy; in partic- Committee at the National Cancer Center, Republic of Korea ular, TRAP1 expression in grade IV tumors (i.e., GBM) was higher (NCC-15-268). Orthotopic GBM xenografts were produced as than that in lower grade tumors (Fig. 1A and B; Supplementary described previously (36). Briefly, cells were transplanted follow- Table S1). Similarly, expression of Sox2, a marker of stem- ing resuspension in DMEM/F-12 medium supplemented with ness (37), was elevated in higher grade tumors (Fig. 1C; Supple- B27, 10 ng/mL EGF, and 5 ng/mL bFGF. Cells were injected mentary Table S2). Interestingly, we found that expression of both stereotactically into the left striatum of 5-week-old female TRAP1 and Sox2 at the single-cell level was elevated in samples BALB/c nude mice. The injection coordinates were 2.2 mm to from patients with glioma (Fig. 1D and E). Consistent with this, the left of the midline and 0.2 mm posterior to bregma, at a depth flow cytometry analysis of single cells from a GBM cell line of 3.5 mm. The brain of each mouse was harvested and fixed in 4% (LN229) revealed a significant positive correlation between paraformaldehyde. For the subcutaneous mouse model, cells expression of TRAP1 and Sox2 (Pearson correlation coefficient were injected into the hip area on both sides. Tumor growth was r ¼ 0.6964; Supplementary Fig. S1A). The correlation was stronger measured twice per week using electronic caliper. Tumor volume in CSC-enriched LN229 cells (r ¼ 0.8568) and the GSC line X01 was calculated using the following formula: V ¼ 1/2 (width)2 (r ¼ 0.9284; Supplementary Fig. S1A and S1B). Therefore, to length. The mean tumor volume at the start of drug injection was better understand the clinical implications of TRAP1 and stem- approximately 50 mm3. Gamitrinib (10 mg/kg) or vehicle ness marker protein levels in glioma patients, we constructed

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Figure 1. Expression of TRAP1 and Sox2 in patients with glioma. A, Tumor grade and TRAP1 expression. Representative images showing IHC staining for TRAP1, according to World Health Organization glioma grading (35). The scores (negative, 1þ,2þ,and3þ) were calculated by quantifying both the extent and intensity of staining. Scores of 2þ and 3þ are considered to indicate high TRAP1 expression, while negative scores and a score of 1þ are considered to indicate low TRAP1 expression. Scale bar, 20 mm. B and C, Expression of TRAP1 and Sox2 in patients with glioma. The percentage of patients showing high expression (2þ or higher) of TRAP1 or Sox2 is shown. Expression of both TRAP1 and Sox2 correlated significantly with tumor grade (TRAP1, P ¼ 0.0001; Sox2, P ¼ 0.0337; c2 test). D, Coexpression of TRAP1 (red) and Sox2 (green). DAPI, blue. Tumor specimens from human patients with GBM were analyzed by immunofluorescence staining. Scale bar, 20 mm. E, Coexpression of TRAP1 and Sox2. The fluorescence intensity of TRAP1 and SOX2 in individual cancer cells (D) was averaged and is presented as a scatter plot. Pearson correlation coefficient (r) was calculated. A.U., arbitrary unit. F and G, Overall survival of patients with GBM. Kaplan–Meier survival curves were generated on the basis of expression of TRAP1 or Sox2 in all glioblastoma patient specimens investigated. H, Survival of patients with GBM with high expression of both TRAP1 and Sox2. Kaplan–Meier plots were generated with respect to high and low expression of both TRAP1 and Sox2. High and low expression in F–H is denoted by staining scores of 2þ and þ1, respectively. The total number of patients with high and low expression is indicated. P values were calculated using the log-rank test.

Kaplan–Meier curves using clinical information collected at Seoul expression at both the protein and mRNA levels was higher in National University Hospital (SNUH, Seoul, Republic of Korea). GSCs than in differentiated counterparts (Fig. 2A and B). Fur- Analysis of these data indicated that poor survival of patients with thermore, all ten GSC lines derived from patients with glioma glioma correlated significantly with increased expression of showed consistently higher expression of TRAP1 than normal TRAP1 and Sox2 (Fig. 1F and G; Supplementary Fig. S1C and astrocytes and GBM cell lines (Fig. 2C). Because TRAP1 regulates S1D). Survival of patients with high expression of both TRAP1 and both mitochondrial respiration and ROS production by several Sox2 was much worse than that of patients with low expression of cancers (14, 23, 38), we examined whether TRAP1 regulates both proteins (Fig. 1H; Supplementary Fig. S1E). The positive mitochondrial metabolism in GSCs. To examine TRAP1 function correlation between TRAP1 and Sox2 expression was significant in mitochondria, we used the mitochondria-accumulating drug for datasets derived from all patients with glioma and GBM gamitrinib, a conjugate of triphenylphosphonium (for mitochon- (Supplementary Fig. S1F and S1G). drial delivery) and geldanamycin (for TRAP1 inhibition; refs. 14, 39). Inactivation of TRAP1 by gamitrinib or siRNA (14, 19) led to TRAP1 is highly expressed by GSCs and regulates cell a marked reduction in mitochondrial respiration in X01 cells, but metabolism had no significant effect on the differentiated counterpart To understand the function of TRAP1 in GSCs, we examined its (Fig. 2D–G); the basal OCR of X01 cells fell by 33%–58%, expression in GSC lines X01 and 528NS. We found that TRAP1 whereas spare respiratory capacity (SRC) fell by 73%–92%

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Figure 2. TRAP1 is required to maintain the metabolic characteristics of GSCs. A, Expression of TRAP1 protein. GSCs (Con), X01, and 528NS cells were differentiated for 3 (D3), 5 (D5), or 7 days (D7) and then analyzed by Western blotting. B, TRAP1 transcription. mRNA was isolated from X01 and 528NS cells and then analyzed by RT-PCR. C, TRAP1 expression in GSCs. Astrocytes, GBM cell lines (U87 and U251), and 10 GSC cell lines (X01–0502) were analyzed by Western blotting. D, OCR. X01 and its differentiated counterpart (X01; D) were incubated for 24 hours with 0.5 mmol/L gamitrinib (Gami) and then examined using a Seahorse XF Analyzer. O, F, R, and A indicate treatment with oligomycin, FCCP, rotenone, and antimycin A, respectively. E, Basal OCR and SRC after gamitrinib treatment. The basal OCR and SRC were calculated and compared. The data are expressed as the mean SEM from two independent experiments, each performed using duplicate samples. , P < 0.0001; NS, not significant. F, OCR after TRAP1 knockdown. X01 cells were incubated with scrambled (siControl) or TRAP1 siRNA (siTRAP1) and examined using the XF analyzer. G, Basal OCR and SRC after TRAP1 knockdown. The basal OCR and SRC were calculated after TRAP1 siRNA treatment and compared. Data are expressed as the mean SEM from two independent experiments, each performed using duplicate samples. , P ¼ 0.04; , P ¼ 0.0012. H, ROS production. Mito-SOX-labeled X01 cells were incubated for 6 hours with gamitrinib (5 mmol/L) and analyzed by flow cytometry. I, ROS production after TRAP1 knockdown. Mito-SOX–labeled X01 cells were incubated with TRAP1 siRNA and analyzed by flow cytometry.

(Fig. 2E and G). Consistent with this, inhibiting TRAP1 in X01 High mitochondrial respiration and low ROS production are cells reduced the mitochondrial membrane potential (Supple- required to maintain stemness and self-renewal properties mentary Fig. S2A) and increased lactate production (Supplemen- of GSCs tary Fig. S2B). In addition to mitochondrial respiration, the The pro-respiratory function of TRAP1 appears to be the antith- mitochondrial ROS concentration in GSCs increased after inacti- esis of the Warburg effect, an aerobic glycolytic metabolic char- vating TRAP1 with either gamitrinib or siRNA (Fig. 2H and I). acteristic of cancer cells (5). However, growing evidence suggests Similarly, when CSCs were enriched from GBM cell lines T98G that tumor metabolism is heterogeneous, and that certain tumors and LN229, the enriched stem cells expressed higher levels of (or subpopulations of cells within tumors, including CSCs within TRAP1 than the original cells (Supplementary Fig. S2C). Conse- GBM) depend primarily on mitochondrial respiration rather than quently, inhibiting TRAP1 expression by enriched stem cells on glycolysis (9, 40). Consistent with this, we found that X01 reduced respiration, increased lactate production, and increased consumed less glucose and produced less lactate, and displayed ROS production (Supplementary Fig. S2D–S2F). Collectively, slightly higher cellular ATP concentrations, than differentiated these data suggest that TRAP1 plays a critical role in maintaining X01 cells (Supplementary Fig. S3A–S3D). Furthermore, X01 cells mitochondrial respiration and reducing ROS concentrations had higher basal OCR and SRC than their differentiated coun- in GSCs. terpart (Supplementary Fig. S3E). Collectively, these data indicate

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Figure 3. TRAP1 is required to maintain the stem cell properties of GSCs. A, Loss of stemness after gamitrinib treatment. X01 and 528NS cells were incubated with gamitrinib for 24 hours and then analyzed by Western blotting. B, TRAP1 knockdown by siRNA. X01 and 528NS cells were treated for 48 hours with siRNA and then analyzed by Western blotting. C, Limiting dilution assay after gamitrinib treatment. X01 cells were incubated with gamitrinib and analyzed in a limiting dilution assay. D, Limiting dilution assay after TRAP1 knockdown. X01 cells were treated with shTRAP1-containing lentiviruses and then analyzed in a limiting dilution assay. E, Cell viability assay. Normal human astrocytes cultured with (þ)or without serum (), normal human neuronal stem cells (NSC), 528NS cells, and X01 cells were incubated with gamitrinib for 48 hours and analyzed in an MTT assay. F, Apoptotic cell death. X01 cells were incubated for 24 hours with gamitrinib (5 mmol/L), labeled with Annexin V and PI, and then analyzed by ﬂow cytometry.

that GSCs have a preference for using mitochondrial respiration In addition to maintaining stemness, gamitrinib reduced the for efficient ATP production, whereas the differentiated counter- viability of GSCs (X01 and 528NS) by inducing apoptotic cell parts derive energy primarily from glycolysis. However, although death (Fig. 3E and F), but did not affect the viability of normal respiration in GSCs was elevated, mitochondrial ROS production astrocytes and neural stem cells (Fig. 3E). was significantly lower than that in the differentiated counterparts (Supplementary Fig. S3F). To examine the functional implica- TRAP1 stabilizes and activates SIRT3 in GSCs tions of mitochondrial respiration and ROS production with Similar to the functions of TRAP1 shown in this study, the respect to maintaining GSC stemness, we exposed GSCs to major mitochondrial deacetylase SIRT3 modulates mitochondri- the mitochondrial respiratory chain inhibitors antimycin A al energy and ROS metabolism (25–27); therefore, we examined (Complex III) and oligomycin (Complex V), and to an exogenous the mechanisms between these two proteins. To identify the ROS (hydrogen peroxide). The respiratory chain inhibitors and relationship between TRAP1 and SIRT3, we first examined their hydrogen peroxide not only reduced expression of stemness expression in human patients with glioma. We found a positive markers (nestin and Sox2; Supplementary Fig. S3G), but also correlation between expression of TRAP1 and SIRT3 proteins in inhibited sphere-forming activity by X01 cells (Supplementary individual cancer cells (r ¼ 0.7392, P < 0.0001; Fig. 4A and B). A Fig. S3H–S3J). Collectively, these data suggest that mitochondrial pull-down experiment revealed that TRAP1 interacts directly with respiration and antioxidant systems within the mitochondria are SIRT3 (Fig. 4C). Consistent with this, endogenous TRAP1 coim- active in GSCs, and that they are necessary to maintain stemness. munoprecipitated with SIRT3 (Fig. 4D). Differentiation of GSCs reduced expression of both SIRT3 and TRAP1 at the protein level, TRAP1 supports stemness and the self-renewal properties of but did not alter expression of SIRT3 mRNA (Fig. 4E), suggesting GSCs that SIRT3 protein is destabilized during differentiation of GSCs. To investigate whether the metabolic regulatory mechanisms Likewise, loss of TRAP1 function after siRNA or gamitrinib treat- associated with TRAP1 affect the stem cell properties of GSCs, ment led to reduced expression of SIRT3 (Fig. 4F), most likely due we examined expression of stem cell markers after TRAP1 inac- to the reduced half-life of the SIRT3 protein (Supplementary Fig. tivation. Treatment of X01 and 528NS cells with gamitrinib or S5A); this suggests that TRAP1 acts as a chaperone that stabilizes TRAP1 siRNA reduced expression of Sox2 and nestin (Fig. 3A SIRT3 protein. This is supported by the finding that SIRT3 enzyme and B). Consistent with this, inactivation of TRAP1 by gamitrinib activity in the mitochondrial extract fell markedly after TRAP1 or TRAP1 knockdown reduced the sphere- and colony-forming inactivation (Fig. 4G). In addition to stabilizing SIRT3 in vivo, activity of X01 cells (Fig. 3C and D; Supplementary Fig. S4A–S4C). recombinant TRAP1 increased the deacetylase activity of purified

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Figure 4. TRAP1 chaperones SIRT3 to reduce mitochondrial ROS production. A, Coexpression of TRAP1 and SIRT3 in human patients with GBM. Tumor specimens were analyzed by immunofluorescence staining. Blue, DAPI; red, TRAP1; green, SIRT3. B, Scatter plot showing TRAP1 and SIRT3 expression in human patients with GBM. The fluorescence intensity of TRAP1 and SIRT3 in each cancer cell was averaged and presented as a scatter plot. Pearson correlation coefficient (r) was calculated to determine the correlation between TRAP1 and SIRT3 expression. A.U., arbitrary unit. C, In vitro pull-down experiment. GST and GST-TRAP1 beads were incubated with 35S-labeled SIRT3. Bead-bound proteins were analyzed by autoradiography (top) and Coomassie staining (bottom). D, Immunoprecipitation of SIRT3 and TRAP1. Mitochondrial extracts prepared from X01 cells were immunoprecipitated with anti-SIRT3 or anti-TRAP1 antibodies. Bound proteins were analyzed by Western blotting. E, Expression of SIRT3. Whole protein extracts and isolated mRNA from X01 and 528NS cells and their differentiated counterparts were analyzed by Western blotting and RT-PCR, respectively. F, SIRT3 degradation upon TRAP1 inhibition. X01 and 528NS cells were incubated for 24 hours with TRAP1 siRNAs or gamitrinib (Gami) as indicated and then analyzed by Western blotting. G, SIRT3 enzyme activity. X01 cells were incubated for 48 hours with 0.5 mmol/L gamitrinib, and SIRT3 enzyme activity in mitochondrial extracts was measured. Relative SIRT3 activity was calculated after subtracting background signals generated by 1 mmol/L nicotinamide adenine dinucleotide (NAM)-treated samples. , P < 0.0001. H, Activation of SIRT3 by TRAP1. SIRT3 (1 mmol/L) was incubated with TRAP1 (2 mmol/L), NAD (200 mmol/L), NAM (40 mmol/L), gamitrinib (40 mmol/L) as indicated, and deacetylase enzyme activity was measured. , P < 0.004. I, Mitochondrial ROS production. Mito-SOX–labeled X01 cells overexpressing (or not) SIRT3 were incubated for 12 hours with 1 mmol/L gamitrinib and analyzed by flow cytometry. J, Acetylation of SOD2. Mitochondrial extracts from gamitrinib-treated X01 cells were immunoprecipitated with an anti-SOD2 antibody and analyzed by Western blotting. K, SOD enzyme activity. X01 cells were incubated for 48 hours with 0.5 mmol/L gamitrinib, and SOD enzyme activity in mitochondrial extracts was analyzed. , P ¼ 0.0021. Data are expressed as the mean SEM from two independent experiments, each performed using duplicate samples (G, H,andK).

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SIRT3 in vitro (by more than 2-fold), a phenomenon reversed by nase (GDH; Fig. 5I; ref. 42), suggesting that SIRT3-mediated exposure to the TRAP1 inhibitor gamitrinib (Fig. 4H). After increases in TRAP1 chaperone activity stabilize Complexes I and normalizing enzyme activity to the SIRT3 protein level, we found II. Gamitrinib increased acetylation of the SIRT3 substrate that gamitrinib still reduced SIRT3 activity (Supplementary NDUFA9 (Fig. 5J); overexpression of SIRT3 increased basal OCR Fig. S5B). In addition, short-term (0.5 hour) exposure of X01 andSRC(SupplementaryFig.S6A)aspreviously reported (25,26). cells to gamitrinib (which led to no detectable degradation of Overexpression of SIRT3, however, did not reverse gamitrinib- SIRT3), reduced the enzymatic activity of SIRT3 (Supplementary induced dysfunction of ETCs (Supplementary Fig. S6A and S6B) Fig. S5C). Collectively, these data suggest that TRAP1 augments due to disruption of Complex I and II after TRAP1 inactivation the deacetylase activity of SIRT3 by inhibiting degradation of the (Supplementary Fig. S6C). Consistent with this, biochemical protein and by increasing its enzyme activity in GSCs. analyses showed that gamitrinib reduced the enzymatic activity of Complexes I and II in the mitochondrial fraction of X01 cells The TRAP1–SIRT3 interaction inhibits production of ROS without affecting activity of Complex V (Fig. 5K–M). Consequent- Knocking down SIRT3 increased mitochondrial ROS produc- ly, SIRT3 knockdown reduced expression of nestin and Sox2 tion (Supplementary Fig. S5D), supporting previous studies (Fig. 5I) and reduced the sphere- and colony-forming activity of showing that SIRT3 inhibits ROS (41) in GSCs. Here, we found X01 (Fig. 5N; Supplementary Fig. S7A and S7B). Taken together, that overexpression of SIRT3 fully inhibited the gamitrinib-trig- the data suggest that chaperone activity is required for formation gered increase in cellular ROS production (Fig. 4I), suggesting that of functional ETC complexes, and that SIRT3 activates ETC func- TRAP1 acts as an upstream regulator of SIRT3 during ROS metab- tions via deacetylation. Therefore, the positive interplay between olism. Likewise, overexpression of SIRT3 led to significant reversal TRAP1 and SIRT3 efficiently increases mitochondrial respiration of the cytotoxic activity of gamitrinib (Supplementary Fig. S5E). by both quantitatively and qualitatively regulating ETCs in GSCs. To better understand the effectors downstream of SIRT3, we focused on superoxide dismutase 2 (SOD2), which is a SIRT3 Interplay between TRAP1 and SIRT3 maintains the stemness substrate in mitochondria that regulates ROS metabo- properties of GSCs under reduced glucose conditions lism (27, 28). Inactivation of TRAP1 by gamitrinib increased the To examine the role of SIRT3 during metabolic adaptation of amount of acetylated mitochondrial proteins in X01 cells (Sup- GSCs, we incubated X01 and 528NS cells in low (5 mmol/L) and plementary Fig. S5F). As a downstream effector of SIRT3, SOD2 high (25 mmol/L) glucose DMEM medium. Low glucose condi- was highly acetylated after inhibition of TRAP1 (Fig. 4J; Supple- tions led to increased expression of SIRT3 and Complex I/II mentary Fig. S5G). Treatment with gamitrinib led to a significant subunit proteins, resulting in increased expression of stemness reduction in SOD enzyme activity in X01 mitochondria (Fig. 4K) markers (Fig. 6A and B). Accordingly, basal OCR and SRC in low without altering expression of the protein (Supplementary Fig. glucose medium increased by 1.7-fold and 2.3-fold, respectively, S5H). Collectively, the data indicate that TRAP1 increases SIRT3 compared with that in high glucose medium (Fig. 6C). The enzyme activity, resulting in reduced ROS production via deace- increase of both ETC expression and OCR was suppressed tylation and subsequent activation of SOD2. by treatment with SIRT3 siRNA (Fig. 6D and E), suggesting TRAP1/SIRT3-dependent stabilization of respiratory complexes. SIRT3 deacetylates TRAP1 Consistent with this, inhibition of TRAP1 by gamitrinib or siRNA Proteomics analyses have identified a number of acetylation reduced expression of Complex I/II subunits and stemness mar- sites within TRAP1 (PhosphoSitePlus database, http://www. kers (Fig. 6F and G), and fully suppressed the basal OCR and SRC phosphosite.org). Therefore, we assumed that these sites could (Fig 6H). Increased interplay between TRAP1 and SIRT3 could be þ be affected by the major mitochondrial deacetylase SIRT3 (25). triggered by increased levels of mitochondrial NAD upon expo- As expected, nicotinamide, a SIRT inhibitor, increased acetyla- sure to low glucose medium (Fig. 6I and J). Consequently, GSCs tion of TRAP1 (Fig. 5A). Consistent with this, overexpression of were much more sensitive to TRAP1 inhibition under low glucose SIRT3 reduced acetylation of TRAP1 (Fig. 5B), whereas knock- conditions than under high glucose conditions (Fig. 6K). These ing down SIRT3 increased acetylation of TRAP1 (Fig. 5C). data suggest that interplay between TRAP1 and SIRT3 has a crucial Taken together, these data suggest that SIRT3 regulates acety- role in metabolic adaptation under reduced nutrient conditions. lation of TRAP1. Inactivating TRAP1 inhibits the tumorigenic activity of GSCs Positive feedback between TRAP1 and SIRT3 augments in vivo mitochondrial respiration The interaction between TRAP1 and SIRT3 means that inacti- Because TRAP1 (20–23) and SIRT3 (25, 26) interact with and vating either of them effectively impairs crucial metabolic char- regulate electron transport chain complexes (ETC), we next asked acteristics of GSCs coordinated by these proteins. To investigate whether interplay between TRAP1 and SIRT3 regulates mitochon- the antitumor efficacy of targeting the interdependency between drial respiration. Pull-down (Fig. 5D) and coimmunoprecipita- the proteins in vivo, X01 cells infected with a shTRAP1 lentivirus tion experiments (Fig. 5E) showed that TRAP1 and SIRT3 were were transplanted orthotopically into the brains of nude mice. associated with Complexes I, II, IV, and V. Pharmacologic and Lentiviral-mediated inhibition of TRAP1 led to a significant genetic inhibition of TRAP1 led to a marked reduction in expres- reduction in tumor growth (Supplementary Fig. S8A) and sub- sion of NDUFA9 (a Complex I subunit) and SDHB (a Complex II sequently prolonged the life span of the mice (Fig. 7A). Consistent subunit) proteins without altering expression of the respective with this, pharmacologic inhibition of TRAP1 by gamitrinib mRNAs (Fig. 5F–H), indicating increased degradation of ETC reduced the growth of subcutaneously transplanted 528NS and subunits upon TRAP1 inhibition. SIRT3 knockdown also reduced X01 cells (Fig. 7B and C; Supplementary Fig. S8B). Immunohis- expression of NDUFA9 and SDHB, but did not reduce expression tochemical and Western blot analyses revealed that inhibition of of the SIRT3 substrate proteins SOD2 and glutamate dehydroge- TRAP1 reduced expression of Sox2, nestin, and SIRT3 in vivo

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Figure 5. TRAP1/SIRT3 bind to and stabilize electron transport chain complexes. A, NAM treatment. X01 cells were treated for 24 hours with nicotinamide (NAM) as indicated and then analyzed by Western blotting. B, Reduced TRAP1 acetylation after SIRT3 overexpression. TRAP1 was immunoprecipitated from extracts of empty vector- or SIRT3-overexpressed X01 cells using an anti-TRAP1 antibody and then analyzed by Western blotting. C, SIRT3 knockdown increases acetylation of TRAP1. TRAP1 was immunoprecipitated from extracts of SIRT3 siRNA-treated cells and then analyzed by Western blotting. D, Pull-down assay. Mitochondrial extracts of X01 cells were incubated with GST or GST-TRAP1 beads. Bound proteins were analyzed by Western blotting. NDUFA9, SDHB, UQCRC2, Cox IV, and ATPB are subunits of Complexes I, II, III, IV, and V, respectively. E, Immunoprecipitation of TRAP1. TRAP1-bound proteins from mitochondrial extracts were coimmunoprecipitated with an anti-TRAP1 antibody and then analyzed by Western blotting. F, Mitochondrial fractionation. X01 cells were incubated with gamitrinib (Gami; 0.5 mmol/L) for 48 hours. The mitochondrial fraction was isolated from cells and analyzed by Western blotting. G, Expression of mRNAs. X01 cells were incubated with gamitrinib (0.5 mmol/L) for 48 hours. Expression of mRNA was analyzed by reverse transcription, followed by PCR. H, TRAP1 knockdown. X01 and 528NS cells were treated with TRAP1 siRNAs and then analyzed by Western blotting. I, SIRT3 knockdown. X01 and 528NS cells were treated with SIRT3 siRNAs and then analyzed by Western blotting. J, Acetylation of NDUFA9 after gamitrinib treatment. Mitochondrial extracts (bottom) from DMSO or 0.5 mmol/L gamitrinib-treated X01 cells were immunoprecipitated with an anti-NDUFA9 antibody and analyzed by Western blotting (top). K–M, ETC activity. X01 cells were incubated with gamitrinib (0.5 mmol/L) for 48 hours. After mitochondrial fractionation, complex activity was measured as described in Materials and Methods. Data are expressed as the mean SEM from two independent experiments, each performed using duplicate samples. , P < 0.05; , P ¼ 0.0036; NS, not signiﬁcant. N, Limiting dilution assay after SIRT3 knockdown. X01 cells were treated with SIRT3 siRNAs and analyzed in a limiting dilution assay (top). Representative microscopic images of spheres (bottom).

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Figure 6. Interplay between SIRT3 and TRAP1 under low glucose conditions. A, Glucose concentration and SIRT3 expression. X01 and 528NS cells were incubated for 24 hours in high (25 mmol/L) or low (5 mmol/L) glucose DMEM medium and then analyzed by Western blotting. B, Glucose concentration and expression of SIRT3 mRNA. Cells were cultured as in A and then analyzed by RT-PCR. C, Measurement of OCR. The OCR of X01 cells incubated in low and high glucose media was analyzed using an XF analyzer, and the results were compared. , P < 0.003. D, SIRT3 knockdown. X01 and 528NS cells incubated in low glucose medium were treated with SIRT3 siRNAs and then analyzed by Western blotting. E, OCR measurement after SIRT3 knockdown. X01 cells were incubated with SIRT3 siRNA and then analyzed using an XF analyzer. , P < 0.016. F, Gamitrinib (Gami) treatment. X01 and 528NS cells incubated in low glucose medium were treated with gamitrinib (0.5 mmol/L) for 24 hours and analyzed by Western blotting. G, TRAP1 knockdown. X01 cells were incubated with TRAP1 siRNA in low glucose medium and then analyzed by Western blotting. H, OCR measurement after gamitrinib treatment. X01 cells incubated in low glucose medium were treated for 24 hours with 0.5 mmol/L gamitrinib and then analyzed using an XF analyzer. , P < 0.0002. I, Increase in the mitochondrial NADþ concentration under low glucose conditions. Mitochondria were isolated from X01 and 528NS cells cultured in low or high glucose medium and examined using a NADþ/NADH quantitation kit. , P < 0.003. J, Schematic model showing mutual activation of SIRT3 and TRAP1. Increased NADþ concentrations in the mitochondria under reduced glucose conditions activate SIRT3. Subsequent activation of TRAP1 triggers a positive feedback loop that activates TRAP1 and SIRT3 via deacetylation and protein chaperoning, respectively. K, Cell viability. X01 and 528NS cells incubated in low and high glucose DMEM medium were treated for 24 hours with gamitrinib as indicated and then analyzed in an MTT assay (, P < 0.0001).

(Fig. 7D; Supplementary Fig. S8C and S8D). Furthermore, reprograming of cellular metabolism. Inhibiting either TRAP1 or gamitrinib reduced the number of Ki67-positive cells, but SIRT3 in GSCs compromised this interplay and disrupted the increased the number of cells positive for cleaved caspase-3 function of both proteins, leading to metabolic dysregulation, (Fig. 7E and F), suggesting reduced cell proliferation and increased overproduction of ROS, loss of stemness properties, and cell death cell death in vivo. both in vitro and in vivo. Thus, the results clearly indicate that TRAP1 and SIRT3 play an important role by maintaining stem cell populations during development of malignant glioma. Discussion A large proportion of cellular ROS is produced within mito- Here, we show that SIRT3 and TRAP1 are overexpressed in chondrial respiratory complexes (43). TRAP1 counters excess ROS glioma stem cells (GSC), and that interplay between these two production in many types of cancer cell (38). The molecular molecules increases activity of ETCs without increasing the ROS mechanisms underlying redox regulation have been elusive; concentration in GSC mitochondria. Nutrient conditions regu- however, we show here that activation of SIRT3 by TRAP1 leads lated interplay between these proteins and subsequently triggered to activation of SOD2. ROS are not only toxic to cells, but also act

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Figure 7. Suppression of tumor growth by TRAP1 inactivation in vivo. A, Survival curves. X01 cells (1 104) infected with lentivirus harboring shControl or shTRAP1 were implanted into nude mice (n ¼ 7 per group). Survival was assessed using the Kaplan–Meier method and the log-rank test (P ¼ 0.0001). Mean survival of shControl- and shTRAP1-treated cells was 49 and 59 days, respectively (t test, P < 0.0001). B, Tumor growth inhibition. 528NS (1 106; left) or X01 GSCs (1 106; right) were implanted subcutaneously into nude mice (n ¼ 5 per group). After tumor establishment, mice received a daily intraperitoneal injection of vehicle or 10 mg/kg gamitrinib (Gami). Tumor volume was measured every other day. C, Tumor weight. Tumors (n ¼ 5 per group) collected in B (528NS tumors after 24 days of drug treatment and X01 after 12 days of treatment) were weighed and the results compared. , P < 0.025. D, IHC analyses. X01 tumors collected in B were analyzed by hematoxylin and eosin (H&E) and IHC staining. Scale bar, 25 mm. E, Staining for Ki-67 and cleaved caspase-3. X01-xenograft tumors in B were analyzed immunohistochemically. F, Quantiﬁcation of Ki-67 and caspase-3 staining. The number of Ki-67- and active caspase-3–positive cells per hematoxylin counterstained area in 200 high-power ﬁelds was counted, and the results were compared (, P ¼ 0.029; , P ¼ 0.003).

as a signaling molecule that triggers differentiation of both conditions; they switch their metabolism to favor energy-efficient normal and cancer stem cells (44, 45). Similarly, we found that mitochondrial respiration (9, 11). Here, we showed that, under þ excess ROS in GSCs led to loss of stemness, indicating the limiting glucose conditions, increased mitochondrial NAD con- importance of ROS scavenging systems for maintaining GSCs. centrations in GSCs activate SIRT3. This subsequently activates Furthermore, because active scavenging of ROS is associated with TRAP1 via a positive feedback loop between TRAP1 and SIRT3, GSC resistance to radiation and chemotherapy (45, 46), the which consequently switches cellular metabolism toward energy- interplay between TRAP1 and SIRT3 may contribute to the ther- efficient respiration to allow adaptation to the harsh microenvi- apeutic resistance of CSCs in vivo. ronment. In addition, the reprogrammed mitochondrial metab- Fast and continuously growing tumor cells suffer local nutri- olism increases the SRC, which avoids an ATP crisis under tional deprivation and need to generate adaptive responses to increased workload and stress conditions (50, 51). Thus, this thrive under such stressful conditions (5, 47). To meet nutrient metabolic characteristic may contribute not only to cellular adap- demand under conditions of metabolic stresses, some cancer cells tation of GSCs to stress conditions, but also to development of such as GSCs secrete angiogenic factors to induce angiogenesis in therapeutic resistance. Because many drug-resistant CSCs depend the tumor microenvironment, thereby allowing greater uptake primarily on mitochondrial respiration (40, 52, 53), other cancer of nutrients and oxygen (48, 49). Furthermore, GSCs develop cells could also exploit the TRAP1/SIRT3 interaction to improve an internal strategy to adapt and survive under nutrient-poor metabolic adaptation and flexibility.

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Although there is a consensus regarding the metabolic rewiring Disclosure of Potential Conflicts of Interest function of TRAP1, the underlying molecular mechanism remains No potential conflicts of interest were disclosed. controversial (23). TRAP1 in some cancer cells (such as human colon carcinoma, osteosarcoma, and cervical carcinoma) sup- Authors' Contributions presses mitochondrial respiration by inhibiting Complex II or Conception and design: H.-K. Park, J.-H. Hong, J.H. Kim, J.B. Park, B.H. Kang IV (20, 21), whereas TRAP1 in human prostate cancer, breast Development of methodology: H.-K. Park, Y.T. Oh, S.-H. Park, B.H. Kang cancer, and glioblastoma increases mitochondrial respiration by Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): H.-K. Park, Y.T. Oh, A.-J. Lee, S.-H. Park, C.-K. Park, increasing Complex II activity (22, 54, 55). Considering that the J.B. Park, B.H. Kang TRAP1 chaperone functions differently in disparate cancer cells, the Analysis and interpretation of data (e.g., statistical analysis, biostatistics, cellular context seems to define the metabolic functions of computational analysis): H.-K. Park, J.-H. Hong, Y.T. Oh, S.S. Kim, J. Yin, TRAP1 (23). This suggests that unique compositions of TRAP1 J.H. Kim, B.H. Kang clients and regulators (such as the SIRT3–TRAP1 interaction exam- Writing, review, and/or revision of the manuscript: H.-K. Park, J.-H. Hong, ined in this study) may be the factors that determine TRAP1-driven Y.C. Chae, J.B. Park, B.H. Kang Administrative, technical, or material support (i.e., reporting or organizing metabolic regulation. With respect to the role of unique cellular data, constructing databases): Y.T. Oh, S.S. Kim, J. Yin, J.H. Kim, S.-H. Park composition, we suggest that TRAP1 interactors such as SIRT3 and Study supervision: M.-J. Park, J.B. Park, B.H. Kang the mitochondrial c-Src, which increase respiratory activity of ETC (21, 26), may play a role. Interestingly, TRAP1 regulates Acknowledgments activity of these two enzymes in an opposing manner: it supports This work was supported by the National Cancer Center (NCC-1810121-1, the enzyme activity of SIRT3 but inhibits that of c-Src. Therefore, we Republic of Korea), by the UNIST research fund (1.180018.01, Republic can speculate that TRAP1 inhibits or activates mitochondrial of Korea), by National Research Foundation of Korea (NRF) grants funded by MSIT (NRF-2016R1A2B2012624, NRF-2016R1A6A3A11934753, NRF- respiration in different cancer cell types depending on its prefer- 2017R1A2B4011741, NRF-2017R1D1A1B03033303, NRF-22A20130012280, ential interaction with c-Src or SIRT3. TRAP1-dependent metabolic NRF-2018R1A5A1024340, NRF-2018R1A4A1025860), and by the Korea Drug regulation may be better explained after comprehensive analyses of Development Fund (KDDF), funded by MSIT, MOTIE, and MOHW (KDDF- the TRAP1 networks in individual cancer cells. 201512-02, Republic of Korea). Collectively, the results presented herein reveal an important metabolic characteristic of GSCs: high mitochondrial respiration The costs of publication of this article were defrayed in part by the without overproduction of ROS. This is closely associated with payment of page charges. This article must therefore be hereby marked positive feedback interplay between TRAP1 and SIRT3. Because advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate GSCs are responsible for failure of anticancer therapy and high this fact. recurrence rates, inhibiting TRAP1 and/or SIRT3 may be the Achilles heel of GSCs, leading to development of effective and Received August 17, 2018; revised December 15, 2018; accepted January 22, novel strategies for treating patients with GBM. 2019; published first January 25, 2019.

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OF14 Cancer Res; 79(7) April 1, 2019 Cancer Research

Interplay between TRAP1 and Sirtuin-3 Modulates Mitochondrial Respiration and Oxidative Stress to Maintain Stemness of Glioma Stem Cells

Hye-Kyung Park, Jun-Hee Hong, Young Taek Oh, et al.

Cancer Res Published OnlineFirst January 25, 2019.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-18-2558

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