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

Author Manuscript Published OnlineFirst on January 25, 2019; DOI: 10.1158/0008-5472.CAN-18-2558 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Interplay between TRAP1 and sirtuin-3 modulates mitochondrial respiration and oxidative stress to maintain stemness of glioma stem cells

Hye-Kyung Park1,6, Jun-Hee Hong2,6, 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,*

1Department of Biological Sciences, Ulsan National Institutes of Science and Technology (UNIST), Ulsan, 44919, Republic of Korea

2Division of Clinical Research, Research Institute and Hospital, National Cancer Center, Goyang, 10408, Republic of Korea

3Department of Cancer Biomedical Science, Graduate School of Cancer Science and Policy, National Cancer Center, Goyang, 10408, Republic of Korea 4Department of Pathology, Seoul National University Hospital, Seoul National University College of Medicine, Seoul, 03080, Republic of Korea

5Division of Radiation Cancer Research, Research Center for Radio-Senescence, Korea Institute of Radiological and Medical Sciences, Seoul, 01812, Republic of Korea

6These authors made an equal contribution.

*Correspondence: Jong Bae Park, Ph.D. (email: [email protected]; phone: +82-31-920-2450); Department of Cancer Biomedical Science, Graduate School of Cancer Science and Policy, National Cancer Center, Goyang, 10408, Republic of Korea Byoung Heon Kang, Ph.D. (email: [email protected]; Phone: +82-52-217-2521); Department of Biological Sciences, Ulsan National Institutes of Science and Technology (UNIST), Ulsan, 44919, Republic of Korea

Running title: TRAP1 and SIRT3 interplay in glioma stem cells

Disclosure of Potential Conflict of Interest: no potential conflicts of interest were disclosed

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on January 25, 2019; DOI: 10.1158/0008-5472.CAN-18-2558 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Abstract

Glioblastoma (GBM) cancer stem cells (CSC) are primarily responsible for metastatic dissemination, resistance to therapy, and relapse of GBM, the most common and aggressive brain tumor.

Development and maintenance of CSC require orchestrated metabolic rewiring and metabolic adaptation to a changing microenvironment. Here we show that cooperative interplay between the mitochondrial chaperone TRAP1 and the major mitochondria deacetylase sirtuin-3 (SIRT3) in glioma stem cells (GSC) increases mitochondrial respiratory capacity and reduces production of reactive oxygen species. This metabolic regulation endowed GSC with metabolic plasticity, facilitated adaptation to stress (particularly reduced nutrient supply), and maintained "stemness." Inactivation of

TRAP1 or SIRT3 compromised their interdependent regulatory mechanisms, leading to metabolic alterations, loss of stemness, and suppression of tumor formation by GSC in vivo. Thus, targeting the metabolic mechanisms regulating interplay between TRAP1 and SIRT3 may provide a novel therapeutic option for intractable GBM patients.

Significance: Discovery and functional analysis of a TRAP1-SIRT3 complex in glioma stem cells identify potential target proteins for glioblastoma treatment.

Introduction

Glioblastoma multiforme (GBM), the most common malignant brain tumor, quickly develops resistance to both radiation and cytotoxic chemotherapy, which inevitably leads to poor clinical outcomes and tumor recurrence despite multimodal therapy (1). A small subpopulation of self- renewing tumor cells, i.e., cancer stem cells (CSCs) or tumor-initiating cells, isolated from GBM patients is responsible for therapeutic resistance and tumor relapse (2,3). Thus, targeting glioma stem cells (GSCs) is a promising therapeutic approach for aggressive and relapsing tumors (3).

Some cancer cells are more dependent on aerobic glycolysis than on mitochondrial respiration; this phenomenon, called the Warburg effect, redirects metabolism toward biosynthetic pathways, reduces cellular oxidative stress, and supports proliferation and survival of cancer cells

(4,5). However, one caveat with respect to a metabolic shift toward glycolysis is a marked reduction 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 suggests that many cancer cells retain mitochondrial function for energy production (7,8), and that certain cancer cells and CSCs have a metabolic preference for mitochondrial respiration rather than glycolysis (9,10).

GSCs are extremely dependent on mitochondrial respiration for energy production and survival (9,11); however, little is known about the molecular mechanisms underlying metabolic reprogramming.

Tumor necrosis factor receptor-associated protein 1 (TRAP1) is a mitochondrial paralog of the 90 kDa heat shock protein (Hsp90) and is highly expressed by a variety of cancer cells (12,13).

TRAP1 endows cancer cells with a high cell death threshold and confers drug resistance by maintaining mitochondrial integrity (14-17). Thus, small molecule inhibitors targeting mitochondrial

TRAP1 have been developed to disrupt mitochondrial function and thereby induce cell death

(14,16,18,19). Recent reports suggest that metabolic regulation by TRAP1 alters mitochondrial respiration and subsequently reprograms cellular metabolism in some tumors (20-22). However, the underlying molecular mechanisms are unclear (23) and have not yet been examined in highly

tumorigenic CSCs.

Sirtuins (SIRTs) are NAD+-dependent protein deacetylases; among the seven mammalian

SIRTs (SIRT1–7) identified to date, SIRT3, SIRT4, and SIRT5 are localized to the mitochondria (24).

SIRT3, a major mitochondrial protein deacetylase, senses cellular metabolic stress and modulates energy and ROS metabolism (25-28), both of which are closely related to the pathophysiology of tumorigenesis. However, the contribution of SIRT3 to tumorigenesis is controversial; previous studies have identified both tumor-promoting and -suppressive roles (29,30). This suggests that the precise role of SIRT3 depends on the cellular context; therefore, the specific type of cancer must be taken into account (30,31). Studies suggest that SIRT3 has a tumor-promoting effect in the brain (32,33), but no report to date has described SIRT3-dependent metabolic regulation of GSCs.

Here, we found that interplay between TRAP1 and SIRT3 in GSCs rewired cellular metabolism to generate energy-efficient mitochondrial respiration without overproduction of ROS.

These metabolic reprogramming functions were crucial for maintenance of self-renewal capacity, lack of differentiation, and survival of GSCs, particularly under limited nutrient conditions. Inactivating either TRAP1 or SIRT3 compromised the metabolic pathways regulated by them and, subsequently, caused GSCs to lose stemness, leading to suppression of sphere formation in vitro and tumor growth in vivo.

Materials and methods

Chemicals and antibodies

All chemicals were purchased from Sigma. Anti-nestin and anti-Sox2 antibodies were purchased from

R&D Systems; anti-TRAP1, anti-SOD2, anti-NDUFA9, anti-UQCRC2, anti-ATPB, anti-GFAP, and anti-SIRT3 antibodies were purchased from Abcam; the anti-Cox IV antibody was purchased from

Cell Signaling Technology; anti-acetylated lysine, anti-GDH, anti-Ki-67, and anti-SDHB antibodies were purchased from Santa Cruz Biotechnology; the anti-β-actin antibody was obtained from MP

Biomedicals; and the anti-Hsp60 antibody was purchased from BD Biosciences.

Patients and specimens

A total of 150 patients with glioma were enrolled in this cohort study conducted at SNUH, Republic of Korea. This included 108 randomly selected consecutive patients with glioma who underwent curative resection at SNUH (performed by the same surgical team) in 2014–2015. Patients in this cohort were followed until June 2017, with a median observation time of 22.5 months (range, 2–47 months). Overall survival was defined as the interval between the date of surgery and date of death or last observation. Data for patients not experiencing relapse or death were censored at the last follow- up. All experiments involving human subjects were approved by the Institutional Review Board of

SNUH (no. 2014-08-004).

Tissue microarray analysis and immunohistochemistry

A tissue microarray (TMA) was constructed as described previously (34). All glioma cases were histologically reviewed by H&E staining. Briefly, two cores were taken from each representative

tumor tissue and from brain tissue adjacent to the tumor within a distance of 10 mm. These were used to construct TMA slides. Duplicate 1.0 cm diameter cylinders taken from intratumoral and peritumoral areas (a total of four punches per patient) were obtained and transferred to the recipient paraffin block at defined array positions (Biochip Company Ltd, Seoul, Korea). TMA blocks were cut into 4 μm sections and mounted onto slides coated with 3-aminopropyltrioxysilane. After deparaffinization and antigen retrieval in citrate buffer (pH 6.0) for 20 min in a pressure cooker, slides were treated for 10 min with 3% hydrogen peroxide to block endogenous peroxidase. Anti-TRAP1

(1:200, monoclonal, Abcam, #ab109323) and anti-Sox2 (1:200, polyclonal, R&D Systems, #AF2018) were the primary antibodies. The TMAs were subsequently incubated with the Polink-2 Plus HRP detection system (GBI Labs, USA) and visualized with 3,3’-diaminobenzidine (DAB). Negative controls were treated the same way, but without the primary antibodies. Immunohistochemical staining was assessed by two independent pathologists with no prior knowledge of patient characteristics. Discrepancies were resolved by consensus. Extent of staining was scored on a scale from 0 to 4, corresponding to the percentage of immune-reactive tumor cells (0%, 1–5%, 6–25%, 26–

75%, and 76–100%, respectively). Staining intensity was scored as negative (score = 0), weak (score

= 1), or strong (score = 2). A score ranging from 0 to 8 was calculated by multiplying the score for staining extent with that for intensity. Final grades (negative, 1+, 2+, and 3+) were assigned to each specimen with scores of 0–1, 2–3, 4–5, and 6–8, respectively.

Immunofluorescence staining

Formalin-fixed paraffin-embedded tissue samples were sectioned (7 μm thick) using a microtome and transferred to glass slides. After paraffin removal and antigen retrieval, the slides were blocked with 5%

FBS, 5% BSA, and 0.3% Triton X-100 in PBS, and then incubated overnight at 4°C with antibodies specific for Sox2 and TRAP1. Next, the slides were incubated for 2 h with Alexa Fluor 488 (green)-

or Alexa Fluor 633 (red)-conjugated secondary antibodies (Invitrogen) and mounted in a mounting medium containing DAPI (Vectashield). Stained slides were analyzed under a FV1000 confocal microscope (Olympus).

Brain tumor tissue samples, cells, and culture conditions

Resected brain tumor tissues were collected at SNUH with written informed consent in accordance with institutional guidelines, and graded pathologically according to WHO grades (35). GBM tissues were formalin-fixed and paraffin-embedded for immunohistochemistry or immunofluorescence analysis. All human GSC lines were cultured in DMEM/F-12 supplemented with B27 (Invitrogen),

EGF (10 ng/mL, R&D Systems), and bFGF (5 ng/mL, R&D Systems). Differentiated GSC cells were maintained in DMEM/F-12 supplemented with 10% fetal bovine serum. Brain cancer cell lines

(LN229, T98G, U87, and U251) and human astrocytes were purchased from the American Type

Culture Collection and maintained as recommended by the supplier. Cells were cultured in DMEM or

RPMI medium (GIBCO) containing 5% or 10% fetal bovine serum (FBS; GIBCO) and 1% penicillin/streptomycin (GIBCO) at 37°C in a humidified atmosphere of 5% CO2. To enrich stem cells,

LN229 and T98G cells were cultured for 3 weeks in DMEM/F12 (GIBCO) medium supplemented with 10 ng/mL EGF, 5 ng/mL bFGF, 0.04% B27, and 1% penicillin/streptomycin.

Measurement of mitochondrial respiration

OCR was measured using a Mito Stress Test Kit and a XF24 Extracellular Flux Analyzer (Seahorse

Bioscience, Billerica, MD, USA), according to the manufacturer’s protocol. Briefly, X01 or LN229 cells (2×104) were attached to laminin-coated Seahorse XF24 cell culture microplates (Seahorse

Bioscience) for 6 h and then incubated with drugs for 48 h at 37°C/5% CO2. At the time of OCR

measurement, the medium was changed to DMEM XF assay medium (Seahorse Bioscience) supplemented with 2.5 mM glutamine, 0.5 mM sodium pyruvate, and 17.5 mM glucose (pH 7.4 at

37°C). After equilibration for 30 min at 37°C in the absence of CO2, OCR was recorded using the

Seahorse XF Analyzer (Seahorse Bioscience). Oligomycin (0.4 μM), carbonyl cyanide p-

(trifluoromethoxy) phenylhydrazone (FCCP) (0.5 μM), and rotenone/antimycin A (0.5 μM) mixtures were injected sequentially to determine mitochondrial function. Readings were collected from each well every 8 min; each 8-min cycle comprised mixing (3 min), waiting (2 min), and recording (3 min).

After completion of OCR measurement, the amount of protein in each well was quantified to normalize the results.

Measurement of glucose uptake and lactate production

Cellular glucose uptake and lactate concentration in the culture medium were measured using the

Glucose Uptake Colorimetric Assay Kit (BioVision, Milpitas, CA, USA) and the Lactate Colorimetric

Assay Kit (BioVision), respectively, according to the manufacturer’s instructions. Briefly, to measure cellular uptake of glucose, X01 or LN229 cells (1×104) were plated in 96-well plates. Each well was washed three times with PBS and then starvation was induced by incubating the cells for 40 min with

100 μL of Krebs-Ringer-Phosphate-HEPES (KRPH) buffer. Cells were treated for 20 min with 1 mM

2-deoxyglucose and lysed with 90 μL of Extraction Buffer. Cell lysates were neutralized by addition of 10 μL of Neutralization Buffer. Fluorescence (Ex/Em = 535/587 nm) was measured in a microplate reader (Synergy™ NEO, BioTek, Winooski, VT, USA). To measure lactate production, culture medium was collected at the indicated times and mixed with Lactate Assay Buffer (50 μL/well) in a

96-well plate. Then, 50 μL of Reaction Buffer was added to each well for 30 min at room temperature.

Absorbance was measured at 570 nm in a microplate reader (Synergy™ NEO, BioTek).

Measurement of the NAD+/NADH ratio

To measure the NAD+/NADH ratio in mitochondria, X01 or 528NS cells (3×106) were seeded in 75T flasks and cultured in 5 mM or 25 mM glucose medium for 24 h. Mitochondria were isolated using a

Mitochondria Isolation Kit (Thermo Fisher Scientific), and the NAD+/NADH ratio was measured using the NAD+/NADH Quantification Colorimetric kit (BioVision), according to the manufacturer’s instructions. Briefly, isolated mitochondria were lysed by two freeze/thaw cycles in NAD/NADH

Extraction Buffer and vortexed for 10 sec. After centrifugation, half of the supernatant was used for measurement of NADt (total amount of NAD+ and NADH), and the other half was used for measurement of NADH (after decomposition of NAD+ at 60°C for 30 min). Samples were incubated for 5 min with NAD Cycling Mix, followed by NADH Developer solution for 2 h. Absorbance at 450 nm was then measured. The NAD+/NADH ratio was calculated as follows: (NADt−NADH)/NADH.

Treatment with siRNA

SiRNAs specific for TRAP1 and SIRT3 were synthesized by Genolution (Korea) using the following sequences: TRAP1-#1 5′-AAACATGAGTTCCAGGCCGAG-3′ and TRAP1-#2 5′-

CCCGGTCCCTGTACTCAGAAA-3′; SIRT3-#1 5′-GTGGGTGCTTCAAGTGTTGTT-3′ and SIRT3-

#2 5′-GCCCAACGTCACTCACTAC-3′; control 5′-ACUCUAUCUGCACGCUGAC-3′. Cells grown to 50–75% confluence on 6-well plates were transfected with 50–100 nM siRNA for 48 h using G-

Fectin (Genolution) and then analyzed by western blotting.

Flow cytometry analysis

To examine mitochondrial superoxide, cells were incubated for 20 min with 200 nM Mito-SOX red

(Invitrogen), washed with PBS, and then analyzed immediately using a FACS Calibur™ system (BD 9

Biosciences). Data were processed using FlowJo software (TreeStar). To measure induction of apoptosis, the DNA content (assessed using propidium iodide) and phosphatidylserine (PS) externalization were analyzed using the Annexin V/Dead Cell Apoptosis Kit (Invitrogen). Labeled cells were quantified using the FACS Calibur™ system (BD Biosciences), and data were analyzed using FlowJo software (TreeStar).

RNA extraction and reverse transcription-polymerase chain reaction (RT-PCR)

Total RNA was prepared from cultured cells using RNeasy mini kits (QIAGEN) according to the manufacturer’s instructions, and cDNA was synthesized using the ProtoScript® First Strand cDNA

Synthesis Kit (New England Biolabs) with an oligo(dT) primer. PCR reactions were performed in a

Mastercycler PCR machine (Eppendorf) with the following sets of forward and reverse oligonucleotide primers: GAPDH, 5′-CGGGAAGCTTGTCATCAATGG-3′ and 5′-

GGCAGTGATGGCATGGACTG-3′; TRAP1, 5′- AGCGCACTCATCAGGAAACT-3′ and 5′-

TCAAACTCACGAAGGTGCAG-3′; Nestin, 5′- AACAGCGACGGAGGTCTCTA-3′ and 5′-

CCTTTCCCAGGTTCTCTTCC-3′; Sox2, 5′-AACCAAGACGCTCATGAAGAAG-3′ and 5′-

GCGAGTAGGACATGCTGTAGGT-3′; GFAP, 5′-GCTTCCTGGAACAGCAAAAC-3′ and 5′-

CCTCCAGCGACTCAATCTTC-3′; SIRT3, 5′-CATGAGCTGCAGTGACTGGT-3′ and 5′-

GAGCTTGCCGTTCAACTAGG-3′; NDUFA9, 5′-CAATTGCTCAACTGTCCAAG-3′ and 5′-

AAGACTCTTGCTACCCATCG-3′; SDHB, 5′- TAAATGTGGCCCCATGGTAT-3′ and 5′-

GATACTGCTGCTTGCCTTCC-3′; UQCRC2, 5′-CAAAGTTGCCCCCAAAGTTA-3′ and 5′-

CATGAGTCTGCGGATTCTGA-3′; COX IV, 5′-CGAGCAATTTCCACCTCTGT-3′ and 5′-

GGGCCGTACACATAGTGCTT-3′; ATPB, 5′-AGCCCATGGTGGTTACTCTG-3′ and 5′-

GGCAGGGTCAGTCAAGTCAT-3′.

Immunoprecipitation and pull-down assay

For immunoprecipitation, 100–200 μg of protein lysate derived from GSCs incubated in IP lysis buffer (135 mM NaCl, 1% glycerol, 1% NP-40, 20 mM Tris-HCl, pH 8.0, and protease/phosphatase inhibitors) was precleared using 1 μg of rabbit or mouse IgG and 20 μL of Protein A/G PLUS agarose

(Santa Cruz) to remove non-specific proteins. The precleared lysates were then incubated at 4°C for 1 h with 1 μg of anti-TRAP1 antibody and then mixed with 20 μL of Protein A/G PLUS agarose at 4°C overnight. The immunocomplexes were then precipitated by centrifugation at 1000 g for 1 min at 4°C and washed three times with IP lysis buffer. Bound proteins were analyzed by western blotting. To perform the pull-down experiments, GST and GST-TRAP1 proteins were induced in E. coli BL21 by overnight incubation with 0.2 mM IPTG at 18°C. Cells were harvested by centrifugation and lysed by sonication in lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM MgCl2, and 1 mM dithiothreitol). After centrifugation at 15,000 g for 30 min, the soluble fractions were mixed with glutathione-Sepharose beads (GE Healthcare). Protein-bound beads were washed three times with wash buffer (50 mM Tris-HCl, pH 7.4, 500 mM NaCl, 5 mM MgCl2, and 1 mM dithiothreitol).

Mitochondria isolated from X01 cells were lysed for 1 h at 4°C in RIPA buffer containing 50 mM

Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 0.25% Na-deoxycholate, and proteinase inhibitor

(Invitrogen) under constant agitation. After centrifugation at 13,000 g for 5 min at 4°C, the supernatant was precleared for 1 h at 4°C with GSH beads (GE Healthcare). Then, 100–200 µg of precleared protein extract was incubated overnight at 4°C with GST and GST-TRAP1 beads. Bead- bound proteins were collected by centrifugation at 1000 g for 1 min at 4°C, washed three times with lysis buffer, and analyzed by western blotting.

Limiting dilution assay

For the in vitro limiting dilution assays, cells were plated in decreasing numbers (200, 100, 50, 25,

12.5, and 6.25) in 96-well plates containing DMEM/F-12 supplemented with B27, 10 ng/mL EGF, and 5 ng/mL bFGF. Extreme limiting dilution analysis was performed using software available at http://bioinf.wehi.edu.au/software/elda/. Images of spheres formed by each group were taken on Day

5 after plating.

Enzyme activity of SIRT3, SOD, Complex I, Complex II, and Complex V

The enzyme activity of SIRT3 in vitro or in vivo was determined using the SIRT3 Activity Assay Kit, according to the manufacturer’s instructions (Abcam). SOD activity was determined in vitro using the

SOD Activity Assay Kit, according to the manufacturer’s instructions (BioVision). The activity of

Complexes I (ab109721, Abcam), II (K660-100, BioVision), and V (ab109907, Abcam) was measured using respective activity assay kits according to the manufacturer’s instructions. Mitochondria were lysed in the provided buffers to obtain a protein solution (5 μg/μL) and loaded into the provided plates prior to immunocapture. After washing away unbound proteins, the provided substrate and dye were added to each well, and absorbance was measured as described in the instructions.

In vivo xenograft experiments

All animal experiments were conducted in accordance with protocols approved by the Institutional

Animal Care and Use Committee at the National Cancer Center, Republic of Korea (NCC-15-268).

Orthotopic GBM xenografts were produced as described previously (36). Briefly, cells were transplanted following resuspension in DMEM/F-12 medium supplemented with B27, 10 ng/mL EGF, and 5 ng/mL bFGF. Cells were injected stereo-tactically into the left striatum of 5-week-old female

BALB/c nude mice. The injection coordinates were 2.2 mm to the left of the midline and 0.2 mm

posterior to bregma, at a depth of 3.5 mm. The brain of each mouse was harvested and ﬁxed in 4% paraformaldehyde. For the subcutaneous mouse model, cells were injected into the hip area on both sides. Tumor growth was measured twice per week using electronic caliper. Tumor volume was calculated using the following formula: V = 1/2 × (width)2 × length. The mean tumor volume at the start of drug injection was approximately 50 mm3. Gamitrinib (10 mg/kg) or vehicle (DMSO) dissolved in 20% Cremophor EL (Sigma) in PBS was administered intraperitoneally every day. At the end of the experiment, animals were euthanized and tumors were collected for histology and western blotting.

Histology and IHC staining

To examine histologic characteristics, brains were removed, ﬁxed for 24 h at 4℃ with 4% paraformaldehyde, and stained with hematoxylin (DAKO) and 0.25% eosin (Merck). For IHC staining of CSC markers and TRAP1-associated genes (Sox2 (R&D Systems), Nestin (Abcam),

SIRT3 (Cell Signaling Technology), Ki-67 (Abcam), and cleaved caspase-3 (Cell Signaling

Technology)), sections were subjected to antigen retrieval in citrate buffer (pH 6.0) and endogenous peroxidase was blocked with 3% hydrogen peroxide. Tissue sections were then incubated overnight at

4℃ in a humidified chamber with appropriate primary antibodies in antibody diluent buffer (IHC

World). Staining results were visualized by sequential incubation of TMAs with components of the

Polink-2 Plus HRP detection system (GBI); DAB (Vector Laboratories) was used as the chromogen.

The number of cells positive for Ki-67 and cleaved caspase-3 per hematoxylin counter-stained area was counted (200 high-power fields were scanned).

Quantification and statistical analysis

All statistical analyses were performed using GraphPad Prism 7. All experiments were repeated at least twice, and data are presented as the mean ± SEM. Statistical significance of data derived from the following experiments was determined using Student’s t test: OCR, glucose uptake, lactate production, ATP production, SOD activity, SIRT3 activity, mitochondrial respiratory complex activity, tumor weight measurement, and quantification of IHC staining. A value of p<0.05 was considered significant. Pearson’s correlation coefficient (r) was used to determine the correlation between

TRAP1 and Sox2 expression in GBM cells and GSCs (using Prism 7). Survival was assessed using the Kaplan-Meier method and the log-rank test (using Prism 7).

Results

TRAP1 expression by GSCs and its positive correlation with brain tumor malignancy

To understand the relationship between TRAP1 and brain tumor malignancy, we examined expression of TRAP1 in brain tumor tissues derived from human patients. TRAP1 expression correlated significantly with brain tumor malignancy; in particular, TRAP1 expression in grade IV tumors (i.e.,

GBM) was higher than that in lower grade tumors (Fig. 1A and B, and Supplementary Table S1).

Similarly, expression of Sox2, a marker of stemness (37), was elevated in higher grade tumors (Fig.

1C and Supplementary Table S2). Interestingly, we found that expression of both TRAP1 and Sox2 at the single cell level was elevated in samples from glioma patients (Fig. 1D and E). Consistent with this, flow cytometry analysis of single cells from a GBM cell line (LN229) revealed a significant positive correlation between expression of TRAP1 and Sox2 (Pearson’s correlation coefficient r=0.6964; Supplementary Fig. S1A). The correlation was stronger in CSC-enriched LN229 cells

(r=0.8568) and the GSC line X01 (r=0.9284) (Supplementary Fig. S1A and B). Therefore, to better understand the clinical implications of TRAP1 and stemness marker protein levels in glioma patients, we constructed Kaplan-Meier curves using clinical information collected at Seoul National University

Hospital (SNUH). Analysis of these data indicated that poor survival of glioma patients correlated significantly with increased expression of TRAP1 and Sox2 (Fig. 1F and G; Supplementary Fig. S1C and D). Survival of patients with high expression of both TRAP1 and Sox2 was much worse than that of patients with low expression of both proteins (Fig. 1H and Supplementary Fig. S1E). The positive correlation between TRAP1 and Sox2 expression was significant for datasets derived from all glioma and GBM patients (Supplementary Fig. S1F and G).

TRAP1 is highly expressed by GSCs and regulates cell metabolism

To understand the function of TRAP1 in GSCs, we examined its expression in GSC lines X01 and

528NS. We found that TRAP1 expression at both the protein and mRNA levels was higher in GSCs than in differentiated counterparts (Fig. 2A and B). Furthermore, all ten GSC lines derived from glioma patients showed consistently higher expression of TRAP1 than normal astrocytes and GBM cell lines (Fig. 2C). Since TRAP1 regulates both mitochondrial respiration and ROS production by several cancers (14,23,38), we examined whether TRAP1 regulates mitochondrial metabolism in

GSCs. To examine TRAP1 function in mitochondria, we used the mitochondria-accumulating drug gamitrinib, a conjugate of triphenylphosphonium (for mitochondrial delivery) and geldanamycin (for

TRAP1 inhibition) (14,39). Inactivation of TRAP1 by gamitrinib or siRNA (14,19) led to a marked reduction in mitochondrial respiration in X01 cells, but had no significant effect on the differentiated counterpart (Fig. 2D–G); the basal oxygen consumption rate (OCR) of X01 cells fell by 33–58%, whereas spare respiratory capacity (SRC) fell by 73–92% (Fig. 2E and G). Consistent with this, inhibiting TRAP1 in X01 cells reduced the mitochondrial membrane potential (Supplementary Fig.

S2A) and increased lactate production (Supplementary Fig. S2B). In addition to mitochondrial respiration, the mitochondrial ROS concentration in GSCs increased after inactivating TRAP1 with either gamitrinib or siRNA (Fig. 2H and I). Similarly, when CSCs were enriched from GBM cell lines

T98G and LN229, the enriched stem cells expressed higher levels of TRAP1 than the original cells

(Supplementary Fig. S2C). Consequently, inhibiting TRAP1 expression by enriched stem cells reduced respiration, increased lactate production, and increased ROS production (Supplementary Fig.

S2D–F). Collectively, these data suggest that TRAP1 plays a critical role in maintaining mitochondrial respiration and reducing ROS concentrations in GSCs.

High mitochondrial respiration and low ROS production are required to maintain stemness and self-renewal properties of GSCs

The pro-respiratory function of TRAP1 appears to be the antithesis of the Warburg effect, an aerobic glycolytic metabolic characteristic of cancer cells (5). However, growing evidence suggests that tumor

metabolism is heterogeneous, and that certain tumors (or subpopulations of cells within tumors, including CSCs within GBM) depend primarily on mitochondrial respiration rather than on glycolysis

(9,40). Consistent with this, we found that X01 consumed less glucose and produced less lactate, and displayed slightly higher cellular ATP concentrations, than differentiated X01 cells (Supplementary

Fig. S3A–D). Furthermore, X01 cells had higher basal OCR and SRC than their differentiated counterpart (Supplementary Fig. S3E). Collectively, these data indicate that GSCs have a preference for using mitochondrial respiration for efficient ATP production, whereas the differentiated counterparts derive energy primarily from glycolysis. However, although respiration in GSCs was elevated, mitochondrial ROS production was significantly lower than that in the differentiated counterparts (Supplementary Fig. S3F). To examine the functional implications of mitochondrial respiration and ROS production with respect to maintaining GSC stemness, we exposed GSCs to the mitochondrial respiratory chain inhibitors antimycin A (Complex III) and oligomycin (Complex V), and to an exogenous ROS (hydrogen peroxide). The respiratory chain inhibitors and hydrogen peroxide not only reduced expression of stemness markers (nestin and Sox2) (Supplementary Fig.

S3G), but also inhibited sphere-forming activity by X01 cells (Supplementary Fig. S3H–J).

Collectively, these data suggest that mitochondrial respiration and antioxidant systems within the mitochondria are active in GSCs, and that they are necessary to maintain stemness.

TRAP1 supports stemness and the self-renewal properties of GSCs

To investigate whether the metabolic regulatory mechanisms associated with TRAP1 affect the stem cell properties of GSCs, we examined expression of stem cell markers after TRAP1 inactivation.

Treatment of X01 and 528NS cells with gamitrinib or TRAP1 siRNA reduced expression of Sox2 and nestin (Fig. 3A and B). Consistent with this, inactivation of TRAP1 by gamitrinib or TRAP1 knockdown reduced the sphere- and colony-forming activity of X01 cells (Fig. 3C and D;

Supplementary Fig. S4A–C). In addition to maintaining stemness, gamitrinib reduced the viability of

GSCs (X01 and 528NS) by inducing apoptotic cell death (Fig. 3E and F), but did not affect the viability of normal astrocytes and neural stem cells (Fig. 3E).

TRAP1 stabilizes and activates SIRT3 in GSCs

Similar to the functions of TRAP1 shown in this study, the major mitochondrial deacetylase SIRT3 modulates mitochondrial energy and ROS metabolism (25-27); therefore, we examined the mechanisms between these two proteins. To identify the relationship between TRAP1 and SIRT3, we first examined their expression in human glioma patients. We found a positive correlation between expression of TRAP1 and SIRT3 proteins in individual cancer cells (r=0.7392, p<0.0001; Fig. 4A and

B). A pull-down experiment revealed that TRAP1 interacts directly with SIRT3 (Fig. 4C). Consistent with this, endogenous TRAP1 co-immunoprecipitated with SIRT3 (Fig. 4D). Differentiation of GSCs reduced expression of both SIRT3 and TRAP1 at the protein level, but did not alter expression of

SIRT3 mRNA (Fig. 4E), suggesting that SIRT3 protein is destabilized during differentiation of GSCs.

Likewise, loss of TRAP1 function after siRNA or gamitrinib treatment led to reduced expression of

SIRT3 (Fig. 4F), most likely due to the reduced half-life of the SIRT3 protein (Supplementary Fig.

S5A); this suggests that TRAP1 acts as a chaperone that stabilizes SIRT3 protein. This is supported by the finding that SIRT3 enzyme activity in the mitochondrial extract fell markedly after TRAP1 inactivation (Fig. 4G). In addition to stabilizing SIRT3 in vivo, recombinant TRAP1 increased the deacetylase activity of purified SIRT3 in vitro (by more than 2-fold), a phenomenon reversed by exposure to the TRAP1 inhibitor gamitrinib (Fig. 4H). After normalizing enzyme activity to the

SIRT3 protein level, we found that gamitrinib still reduced SIRT3 activity (Supplementary Fig. S5B).

In addition, short-term (0.5 h) exposure of X01 cells to gamitrinib (which led to no detectable degradation of SIRT3), reduced the enzymatic activity of SIRT3 (Supplementary Fig. S5C).

Collectively, these data suggest that TRAP1 augments the deacetylase activity of SIRT3 by inhibiting degradation of the protein and by increasing its enzyme activity in GSCs.

The TRAP1-SIRT3 interaction inhibits production of ROS

Knocking down SIRT3 increased mitochondrial ROS production (Supplementary Fig. S5D), supporting previous studies showing that SIRT3 inhibits ROS (41) in GSCs. Here, we found that overexpression of SIRT3 fully inhibited the gamitrinib-triggered increase in cellular ROS production

(Fig. 4I), suggesting that TRAP1 acts as an upstream regulator of SIRT3 during ROS metabolism.

Likewise, overexpression of SIRT3 led to significant reversal of the cytotoxic activity of gamitrinib

(Supplementary Fig. S5E). To better understand the effectors downstream of SIRT3, we focused on superoxide dismutase 2 (SOD2), which is a SIRT3 substrate in mitochondria that regulates ROS metabolism (27,28). Inactivation of TRAP1 by gamitrinib increased the amount of acetylated mitochondrial proteins in X01 cells (Supplementary Fig. S5F). As a downstream effector of SIRT3,

SOD2 was highly acetylated after inhibition of TRAP1 (Fig. 4J and Supplementary Fig. S5G).

Treatment with gamitrinib led to a significant reduction in SOD enzyme activity in X01 mitochondria

(Fig. 4K) without altering expression of the protein (Supplementary Fig. S5H). Collectively, the data indicate that TRAP1 increases SIRT3 enzyme activity, resulting in reduced ROS production via deacetylation and subsequent activation of SOD2.

SIRT3 deacetylates TRAP1 Proteomics analyses have identified a number of acetylation sites within TRAP1 (PhosphoSitePlus database, http://www.phosphosite.org). Therefore, we assumed that these sites could be affected by the major mitochondrial deacetylase SIRT3 (25). As expected, nicotinamide (NAM), a SIRT inhibitor, increased acetylation of TRAP1 (Fig. 5A). Consistent with this, overexpression of SIRT3 reduced acetylation of TRAP1 (Fig. 5B), whereas knocking down SIRT3 increased acetylation of TRAP1 (Fig.

5C). Taken together, these data suggest that SIRT3 regulates acetylation of TRAP1.

Positive feedback between TRAP1 and SIRT3 augments mitochondrial respiration

Since TRAP1 (20-23) and SIRT3 (25,26) interact with and regulate electron transport chain complexes (ETCs), we next asked whether interplay between TRAP1 and SIRT3 regulates mitochondrial respiration. Pull-down (Fig. 5D) and co-immunoprecipitation experiments (Fig. 5E) showed that TRAP1 and SIRT3 were associated with Complexes I, II, IV, and V. Pharmacological and genetic inhibition of TRAP1 led to a marked reduction in expression of NDUFA9 (a Complex I subunit) and SDHB (a Complex II subunit) proteins without altering expression of the respective mRNAs (Fig. 5F–H), indicating increased degradation of ETC subunits upon TRAP1 inhibition.

SIRT3 knockdown also reduced expression of NDUFA9 and SDHB, but did not reduce expression of the SIRT3 substrate proteins SOD2 and glutamate dehydrogenase (GDH) (42) (Fig. 5I), suggesting that SIRT3-mediated increases in TRAP1 chaperone activity stabilize Complexes I and II. Gamitrinib increased acetylation of the SIRT3 substrate NDUFA9 (Fig. 5J); overexpression of SIRT3 increased basal OCR and SRC (Supplementary Fig. S6A) as previously reported (25,26). Overexpression of

SIRT3, however, did not reverse gamitrinib-induced dysfunction of ETCs (Supplementary Fig. S6A and S6B) due to disruption of Complex I and II after TRAP1 inactivation (Supplementary Fig. S6C).

Consistent with this, biochemical analyses showed that gamitrinib reduced the enzymatic activity of

Complexes I and II in the mitochondrial fraction of X01 cells without affecting activity of Complex V

(Fig. 5K–M). Consequently, SIRT3 knockdown reduced expression of nestin and Sox2 (Fig. 5I) and reduced the sphere- and colony-forming activity of X01 (Fig. 5N; Supplementary Fig. S7A and B).

Taken together, the data suggest that chaperone activity is required for formation of functional ETC complexes, and that SIRT3 activates ETC functions via deacetylation. Therefore, the positive interplay between TRAP1 and SIRT3 efficiently increases mitochondrial respiration by both quantitatively and qualitatively regulating ETCs in GSCs.

Interplay between TRAP1 and SIRT3 maintains the stemness properties of GSCs under reduced glucose conditions

To examine the role of SIRT3 during metabolic adaptation of GSCs, we incubated X01 and 528NS cells in low (5 mM) and high (25 mM) glucose DMEM medium. Low glucose conditions led to increased expression of SIRT3 and Complex I/II subunit proteins, resulting in increased expression of stemness markers (Fig. 6A and B). Accordingly, basal OCR and SRC in low glucose medium increased by 1.7-fold and 2.3-fold, respectively, compared with that in high glucose medium (Fig. 6C).

The increase of both ETC expression and OCR was suppressed by treatment with SIRT3 siRNA (Fig.

6D and E), suggesting TRAP1/SIRT3-dependent stabilization of respiratory complexes. Consistent with this, inhibition of TRAP1 by gamitrinib or siRNA reduced expression of Complex I/II subunits and stemness markers (Fig. 6F and G), and fully suppressed the basal OCR and SRC (Fig 6H).

Increased interplay between TRAP1 and SIRT3 could be triggered by increased levels of mitochondrial NAD+ upon exposure to low glucose medium (Fig. 6I and J). Consequently, GSCs were much more sensitive to TRAP1 inhibition under low glucose conditions than under high glucose conditions (Fig. 6K). These data suggest that interplay between TRAP1 and SIRT3 has a crucial role in metabolic adaptation under reduced nutrient conditions.

Inactivating TRAP1 inhibits the tumorigenic activity of GSCs in vivo

The interaction between TRAP1 and SIRT3 means that inactivating either of them effectively impairs crucial metabolic characteristics of GSCs coordinated by these proteins. To investigate the antitumor efficacy of targeting the interdependency between the proteins in vivo, X01 cells infected with a shTRAP1 lentivirus were transplanted orthotopically into the brains of nude mice. Lentiviral- mediated inhibition of TRAP1 led to a significant reduction in tumor growth (Supplementary Fig.

S8A) and subsequently prolonged the life span of the mice (Fig. 7A). Consistent with this, pharmacological inhibition of TRAP1 by gamitrinib reduced the growth of subcutaneously 21

transplanted 528NS and X01 cells (Fig. 7B and C, and Supplementary Fig. S8B).

Immunohistochemical and western blot analyses revealed that inhibition of TRAP1 reduced expression of Sox2, nestin, and SIRT3 in vivo (Fig. 7D, and Supplementary Fig. S8C and D).

Furthermore, gamitrinib reduced the number of Ki-67-positive cells, but increased the number of cells positive for cleaved caspase-3 (Fig. 7E and F), suggesting reduced cell proliferation and increased cell death in vivo.

Discussion

Here, we show that SIRT3 and TRAP1 are overexpressed in glioma stem cells (GSCs), and that interplay between these two molecules increases activity of ETCs without increasing the ROS concentration in GSC mitochondria. Nutrient conditions regulated interplay between these proteins and subsequently triggered reprograming of cellular metabolism. Inhibiting either TRAP1 or SIRT3 in

GSCs compromised this interplay and disrupted the function of both proteins, leading to metabolic dysregulation, overproduction of ROS, loss of stemness properties, and cell death 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.

A large proportion of cellular ROS is produced within mitochondrial respiratory complexes

(43). TRAP1 counters excess ROS production in many types of cancer cell (38). The molecular mechanisms underlying redox regulation have been elusive; however, we show here that activation of

SIRT3 by TRAP1 leads to activation of SOD2. ROS are not only toxic to cells, but also act as a signaling molecule that triggers differentiation of both normal and cancer stem cells (44,45). Similarly, we found that excess ROS in GSCs led to loss of stemness, indicating the importance of ROS scavenging systems for maintaining GSCs. Furthermore, since active scavenging of ROS is associated with GSC resistance to radiation and chemotherapy (45,46), the interplay between TRAP1 and SIRT3 may contribute to the therapeutic resistant of CSCs in vivo.

Fast and continuously growing tumor cells suffer local nutritional deprivation and need to generate adaptive responses to thrive under such stressful conditions (5,47). To meet nutrient demand under conditions of metabolic stresses, some cancer cells such as GSCs secrete angiogenic factors to induce angiogenesis in the tumor microenvironment, thereby allowing greater uptake of nutrients and oxygen (48,49). Furthermore, GSCs develop an internal strategy to adapt and survive under nutrient- poor conditions; they switch their metabolism to favor energy-efficient mitochondrial respiration

(9,11). Here, we showed that, under limiting glucose conditions, increased mitochondrial NAD+ concentrations in GSCs activate SIRT3. This subsequently activates TRAP1 via a positive feedback loop between TRAP1 and SIRT3, which consequently switches cellular metabolism toward energy- efficient respiration to allow adaptation to the harsh microenvironment. In addition, the reprogrammed mitochondrial metabolism increases the SRC, which avoids an ATP crisis under increased workload and stress conditions (50,51). Thus, this metabolic characteristic may contribute not only to cellular adaptation of GSCs to stress conditions, but also to development of therapeutic resistance. Since many drug-resistant CSCs depend primarily on mitochondrial respiration (40,52,53), other cancer cells could also exploit the TRAP1/SIRT3 interaction to improve metabolic adaptation and flexibility.

Although there is consensus regarding the metabolic rewiring function of TRAP1, the underlying molecular mechanism remains controversial (23). TRAP1 in some cancer cells (such as human colon carcinoma, osteosarcoma, and cervical carcinoma) suppresses mitochondrial respiration by inhibiting Complex II or IV (20,21), whereas TRAP1 in human prostate cancer, breast cancer, and glioblastoma increases mitochondrial respiration by increasing Complex II activity (22,54,55).

Considering that the TRAP1 chaperone functions differently in disparate cancer cells, the cellular context seems to define the metabolic functions of TRAP1 (23). This suggests that unique compositions of TRAP1 clients and regulators (such as the SIRT3-TRAP1 interaction examined in this study) may be the factors that determine TRAP1-driven metabolic regulation. With respect to the role of unique cellular composition, we suggest that TRAP1 interactors such as SIRT3 and the mitochondrial c-Src, which increase respiratory activity of ETC (21,26), may play a role. Interestingly,

TRAP1 regulates activity of these two enzymes in an opposing manner: it supports the enzyme activity of SIRT3 but inhibits that of c-Src. Therefore, we can speculate that TRAP1 inhibits or activates mitochondrial respiration in different cancer cell types depending on its preferential interaction with c-Src or SIRT3. TRAP1-dependent metabolic regulation may be better explained after comprehensive analyses of the TRAP1 networks in individual cancer cells

Collectively, the results presented herein reveal an important metabolic characteristic of

GSCs: high mitochondrial respiration without overproduction of ROS. This is closely associated with positive feedback interplay between TRAP1 and SIRT3. Since GSCs are responsible for failure of anticancer therapy and high recurrence rates , inhibiting TRAP1 and/or SIRT3 may be the Achilles heel of GSCs, leading to development of effective and novel strategies for treating GBM patients.

Acknowledgements

This work was supported by the National Cancer Center (NCC-1810121-1, Republic of Korea), by the

UNIST research fund (1.180018.01, Republic of Korea), by National Research Foundation of Korea

(NRF) grants funded by MSIT (NRF-2016R1A2B2012624, NRF-2016R1A6A3A11934753, NRF-

2017R1A2B4011741, NRF-2017R1D1A1B03033303, NRF-22A20130012280, NRF-

2018R1A5A1024340, NRF-2018R1A4A1025860), and by the Korea Drug Development Fund

(KDDF), funded by MSIT, MOTIE, and MOHW (KDDF-201512-02, Republic of Korea).

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Figure legends

Figure 1. Expression of TRAP1 and Sox2 in glioma patients.

A, Tumor grade and TRAP1 expression. Representative images showing immunohistochemical staining for TRAP1, according to World Health Organization glioma grading (35). The scores

(negative, 1+, 2+, and 3+) were calculated by quantifying both the extent and intensity of staining.

Scores of 2+ and 3+ are considered to indicate high TRAP1 expression, while negative scores and a score of 1+ are considered to indicate low TRAP1 expression. Scale bar, 20 μm.

B, C, Expression of TRAP1 and Sox2 in glioma patients. The percentage of patients showing high expression (2+ or higher) of TRAP1 or Sox2 is shown. Expression of both TRAP1 and Sox2 correlated significantly with tumor grade (TRAP1, p=0.0001; Sox2, p=0.0337; Chi-square test).

D, Co-expression of TRAP1 (green) and Sox2 (red). Tumor specimens from human GBM patients were analyzed by immunofluorescence staining. Scale bar, 20 μm.

E, Co-expression of TRAP1 and Sox2. The fluorescence intensity of (D) TRAP1 and SOX2 in individual cancer cells was averaged and presented as a scatter plot. Pearson’s correlation coefficient

(r) was calculated. A.U., arbitrary unit.

F, G, Overall survival of glioblastoma (GBM) patients. Kaplan-Meier survival curves were generated based on expression of TRAP1 or Sox2 in all glioblastoma patient specimens investigated.

H, Survival of GBM patients 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), (G), and (H) is denoted by staining scores of ≥2+ and ≤+1, respectively. The total number of patients with high and low expression is indicated. P-values were calculated using the log- rank test.

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 ten GSC cell lines (X01–0502) were analyzed by western blotting.

D, Oxygen consumption rate (OCR). X01 and its differentiated counterpart (X01(D)) were incubated for 24 h with 0.5 μM 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 spare respiratory capacity (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=0.04; **p=0.0012.

H, ROS production. Mito-SOX-labeled X01 cells were incubated for 6 h with gamitrinib (5 μM) 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.

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 h and then analyzed by western blotting.

B, TRAP1 knockdown by siRNA. X01 and 528NS cells were treated for 48 h 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 (+) or without serum (-), normal human neuronal stem cells (NSCs), 528NS cells, and X01 cells were incubated with gamitrinib for 48 h and analyzed in an MTT assay.

F, Apoptotic cell death. X01 cells were incubated for 24 h with gamitrinib (5 μM), labeled with

Annexin V and PI, and then analyzed by flow cytometry.

Figure 4. TRAP1 chaperones SIRT3 to reduce mitochondrial ROS production.

A, Co-expression of TRAP1 and SIRT3 in human GBM patients. Tumor specimens were analyzed by immunofluorescence staining. Blue, DAPI; red, TRAP1; green, SIRT3.

B, Scatter plot showing TRAP1 and SIRT3 expression in human GBM patients. The fluorescence intensity of TRAP1 and SIRT3 in each cancer cell was averaged and presented as a scatter plot. Person’s 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 h with TRAP1 siRNAs or gamitrinib as indicated and then analyzed by western blotting.

G, SIRT3 enzyme activity. X01 cells were incubated for 48 h with 0.5 μM gamitrinib, and

SIRT3 enzyme activity in mitochondrial extracts was measured. Relative SIRT3 activity was calculated after subtracting background signals generated by 1 mM nicotinamide adenine dinucleotide

(NAM)-treated samples. ***p<0.0001.

H, Activation of SIRT3 by TRAP1. SIRT3 (1 μM) was incubated with TRAP1 (2 μM), NAD

(200 μM), NAM (40 μM), gamitrinib (40 μM) 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 h with 1 μM 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 h with 0.5 μM gamitrinib, and SOD enzyme activity in mitochondrial extracts was analyzed. **p=0.0021. Data are expressed as the mean

± SEM from two independent experiments each performed using duplicate samples (in G, H, and K).

Figure 5. TRAP1/SIRT3 bind to and stabilize electron transport chain complexes (ETCs).

A, NAM treatment. X01 cells were treated for 24 h 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 co-immunoprecipitated with an anti-TRAP1 antibody and then analyzed by western blotting.

F, Mitochondrial fractionation. X01 cells were incubated with gamitrinib (0.5 μM) for 48 h.

The mitochondrial fraction was isolated from cells and analyzed by western blotting.

G, Expression of mRNAs. X01 cells were incubated with gamitrinib (0.5 μM) for 48 h.

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 panel) from DMSO or 0.5 μM gamitrinib-treated X01 cells were immunoprecipitated with an anti-NDUFA9 antibody and analyzed by western blotting (upper panel).

K, L, M, ETC activity. X01 cells were incubated with gamitrinib (0.5 μM) for 48 h. 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=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 (upper panel). Representative microscopic images of spheres (lower panel)

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 h in high (25 mM) or low (5 mM) 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 treatment. X01 and 528NS cells incubated in low glucose medium were treated with gamitrinib (0.5 μM) for 24 h 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 h with 0.5 μM gamitrinib and then analyzed using an XF analyzer. ***p<0.0002.

I, Increase in the mitochondrial NAD+ concentration under low glucose conditions.

Mitochondria were isolated from X01 and 528NS cells cultured in low or high glucose medium and examined using a NAD+/NADH quantitation kit. **p<0.003.

J, Schematic model showing mutual activation of SIRT3 and TRAP1. Increased NAD+ 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 h with gamitrinib as indicated and then analyzed in an MTT assay. ***p<0.0001.

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=7 per group). Survival was assessed using the Kaplan-Meier method and the log-rank test (p=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=5 per group). After tumor establishment, mice received a daily

intraperitoneal injection of vehicle or 10 mg/kg gamitrinib. Tumor volume was measured every other day.

C, Tumor weight. Tumors (n=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, Immunohistochemical analyses. X01 tumors collected in (B) were analyzed by hematoxylin and eosin (H&E) and immunohistochemical staining. Scale bar, 25 μm.

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 counter-stained area in 200 high-power fields was counted, and the results were compared. **p=0.003; *p=0.029.

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

Supplementary Access the most recent supplemental material at: Material http://cancerres.aacrjournals.org/content/suppl/2019/01/25/0008-5472.CAN-18-2558.DC1

Author Author manuscripts have been peer reviewed and accepted for publication but have not yet Manuscript been edited.

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