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GSCs- ROS-differentiation

Metabolic activation of mitochondria in glioma stem cells promotes cancer

development through a reactive oxygen species-mediated mechanism

Shuqiang Yuan 1,Yunxin Lu 1,Jing Yang 1,Gang Chen 2,Sangbae Kim 3,Li Feng 2,Marcia Ogasawara 2,Naima Hammoudi 2,Weiqin Lu 2,Hui Zhang 2,Jinyun Liu 2,Howard Colman 4,Ju-Seog Lee 3,Xiao-Nan Li 5,Rui-hua Xu 1*,Peng Huang 1,2*and Feng Wang 1*

Introduction

Recent studies indicate the existence of cancer stem cells (CSCs)in various types of cancers,including leukemia and solid tumors [1,2].Similar to normal stem cells,CSCs are able to self-renew and to generate the down-stream progeny.Although CSCs constitute a very small fraction of the total cancer cells in the tumor bulk,this

special subpopulation of malignant cells is thought to play a major role in cancer initiation and development and may be a key cause of resistance to chemotherapy and radiotherapy,leading to persistence of residual dis-ease and cancer recurrence [3].This phenomenon is due in part to the unique biological properties of CSCs,in-cluding high capacity of DNA repair,high expression of certain ATP-dependent drug exporting pumps,high levels of glutathione synthesis,and high expression of cell survival factors [4–6].A detailed understanding of factors that affect the ability of CSCs to maintain their self-renewal and promote disease progression is import-ant for developing new strategies to effectively kill CSCs.

*Correspondence:xurh@https://www.wendangku.net/doc/503573042.html, ;phuang@https://www.wendangku.net/doc/503573042.html, ;wangfeng@https://www.wendangku.net/doc/503573042.html, SY and YL are co-first authors.1

Sun Yat-sen University Cancer Center;State Key Laboratory of Oncology in South China;Collaborative Innovation Center for Cancer Medicine,651E Dongfeng Road,Guangzhou,Guangdong 510060,China

Full list of author information is available at the end of the

article

?2015Yuan et al.Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License (https://www.wendangku.net/doc/503573042.html,/licenses/by/4.0/),which permits unrestricted use,distribution,and

reproduction in any medium,provided you give appropriate credit to the original author(s)and the source,provide a link to the Creative Commons license,and indicate if changes were made.The Creative Commons Public Domain Dedication waiver (https://www.wendangku.net/doc/503573042.html,/publicdomain/zero/1.0/)applies to the data made available in this article,unless otherwise stated.

Yuan et al.Stem Cell Research &Therapy (2015) 6:198 DOI 10.1186/s13287-015-0174-2

Mounting evidence suggests that the tissue microenvir-onment may profoundly affect the biological properties and the fates of stem cells and CSCs[7].In vivo,normal stem cells or CSCs reside in special tissue locations known as stem cell niches,which are thought to provide the micro-environment important for the maintenance of their stem-ness[8].Although the exact nature of the stem cell niches remains to be defined,it is known that low oxygen and proper levels of certain growth factors such as epidermal growth factor(EGF)and basic fibroblast growth factor (bFGF)are important to maintain the stemness of the cells [9].Brain CSCs have been found in perivascular niches [8,10].Increasing the endothelial cells or blood vessels in orthotopic brain tumor xenografts enhances self-renewal of CSCs and accelerates the initiation and growth of tumors [10].However,exposure of CSCs to serum in vitro usually induces differentiation and presumably may compromise their self-renewal ability[11,12].CSCs cultured in serum-free media seem to closely mimic the genotype and gene expression profiles of their primary tumors in vivo than do CSCs cultured in standard serum-containing medium[9]. Although the ability of serum to induce apparent differenti-ation of CSCs has been known for a long time,the under-lying mechanisms remain largely unknown.It is also unclear whether exposure of CSCs to serum negatively or positively affects their ability to form tumor in vivo. Reactive oxygen species(ROS)are known to play a role in affecting the fates of normal stem cells[13,14]. Elevated ROS has been observed to induce differenti-ation of embryonic stem cells into cardiovascular and mesendodermal cells[7,15].The neural stem cells and hematopoietic stem cells contain lower levels of ROS than their mature progeny,whereas increased ROS levels are associated with lowered self-renewal capacity,in-creased cell cycling,and reduced viability[16–18].Previ-ous study showed that breast CSCs might have high ROS-scavenging capacity and contain lower cellular ROS compared with the corresponding non-tumorigenic cells[6].A recent study suggests that ROS might affect the differentiation state of CSC by activation of p38 MAPK[19].However,the role of ROS in serum-induce differentiation of CSCs and their physiological relevance in tumor development in vivo remain largely unclear. The present study was designed to investigate these im-portant questions.We showed that serum could activate mitochondrial respiration and promote generation of mitochondrial ROS,leading to apparent loss of certain stem cell markers and lower ability to form neuro-spheres.However,despite these seemingly differentiation phenotypes in vitro,the serum-induced glioma stem cells exhibited greater capacity to form tumor in vivo.Our study revealed a novel role of mitochondrial ROS in serum activation of CSCs to produce the downstream pro-geny and promote tumor development in vivo.The regulation of this redox signaling mechanism has potential implications in developing new strategies to target CSCs. Methods

Cell lines and cell culture

GSC11,GSC23,and GBM3752cell lines were originally established from fresh surgical specimens of glioblastoma multiforme at the University of Texas MD Anderson Cancer Center[20,21].GSC11and GSC23were maintained in Dulbecco’s modified Eagle’s medium with nutrient mixture F-12(DMEM/F12)(Mediatech Inc.,Manassas,VA,USA) supplemented with B27(Invitrogen,Carlsbad,CA,USA), 20ng/ml epidermal growth factor(Miltenyi Biotec,Auburn, CA,USA),20ng/ml of basic fibroblast growth factor (Miltenyi Biotec),and2mM L-glutamine(Mediatech Inc.) without serum(designated as“stem cell medium”).Cells were cultured in a humidified incubator maintained at37°C with5%CO2.GBM3752cells were obtained from GBM pa-tients undergoing surgery at Texas Children’s Hospital and maintained in severe combined immunodeficiency(SCID) mice orthotopically[22].The cells were freshly isolated from the tumors and cultured in stem cell medium for in vitro study within the first five passages.For serum treatment,cells were cultured in the stem cell medium with5%fetal bovine serum(FBS)with or without various concentrations of N-acetyl-cysteine(NAC)(Sigma-Aldrich,St.Louis,MO,USA). RNA isolation,RNA microarray analyses,and reverse transcription-polymerase chain reaction

GSC11and GSC23cells were cultured in stem cell medium with or without serum for1,3,or7days in triplicate.Total RNA was isolated from the cells by using an RNeasy Mini kit(Qiagen Inc.,Valencia,CA,USA).Sample labeling was performed with an RNA amplification kit in accordance with the conditions recommended by the manufacturer (Applied Biosystems,Foster City,CA,USA).Total RNA was reverse-transcribed by using a complementary DNA (cDNA)synthesis kit(Fermentas Inc.,Glen Burnie,MD, USA).The quantitative polymerase chain reaction analyses were carried out in a25-μl reaction mixture that contained 1μl cDNA,0.1μg oligonucleotide primer pairs,12.5μl SYBR Green Mix(Invitrogen),and diethylpyrocarbonate-treated water.Human HT-12v3expression beadchips containing48,000probes of25,000annotated genes were obtained from Illumina Inc.(San Diego,CA,USA).The gene expression microarray analysis was performed at the System Biology Department of the UT MD Anderson Cancer Center.Total RNA was extracted from GSC11cells and used for labeling and hybridization to human expres-sion beadchips in accordance with the protocols of the manufacturer.All experiments were performed in triplicate. Primary microarray data in this study are available in the National Cancer for Biotechnology Information Gene Expression Omnibus(GEO)database(GSE28220).The

following primer sets were used for quantitative reverse transcription-polymerase chain reaction(RT-PCR)analysis: SOX2-sense,5′-GCCTGGGCGCCGAGTGGA-3′;SOX2-antisense,5′-GGGCGAGCCGTTCATGTAGGTCTG-3′); Olig2-sense,5′-TGCGCAAGCTTTCCAAGA-3′;Olig2-antisense,5′-CAGCGAGTTGGTGAGCATGA-3′.

Flow cytometric analyses

Cells were dissociated into single-cell suspension by using accutase reagents(Sigma-Aldrich),stained with allophycocyanin(APC)-conjugated CD133antibody (clone AC133from MACS)or the control APC-IgG2b antibody(MACS)by using the conditions recom-mended by the manufacturer.APC fluorescence was quantitated by flow cytometry analysis.To measure intracellular ROS,cells were collected and dissociated into single-cell suspension by accutase,washed with phosphate-buffered saline(PBS)once,and resuspended in pre-warmed PBS containing freshly prepared CM-H2DCFDA(1μM)or MitoSOX-Red(5μM;Molecular Probes,Eugene,OR,USA).After incubation at37°C for30min(H2DCFDA)or15min(MitoSOX-Red),the cells were washed with PBS twice and then subjected to flow cytometric analyses.

Immunoblots

Cultured cells were washed with cold PBS before homogenization in lysate buffer.Whole cell lysate(20μg protein/sample)was used in Western blot analysis.Cell ly-sates were separated by electrophoresis on10–12%so-dium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes.After block-ing with5%non-fat milk/PBS with Tween20for1h,the membranes were incubated at4°C overnight with primary antibodies,including mouse anti-human CD133(Miltenyi Biotec),rabbit anti-human SOX2(Cell Signaling Technol-ogy Inc.,Danvers,MA,USA),rabbit anti-human Olig2 (Abcam,Cambridge,MA,USA),rabbit anti-human Catalase(EMD Chemicals,Gibbstown,NJ,USA),sheep anti-human SOD1(EMD Chemicals),rabbit anti-human SOD2(Santa Cruz Biotechnology Inc.,Santa Cruz,CA, USA),and anti-mouse total OXPHOS(Abcam).The Western blot signals were detected with horseradish peroxidase-conjugated secondary antibodies.The mem-branes were developed by using a Pierce Supersignal West Pico Chemiluminescent Substrate(Fisher Scientific Inc., Pittsburgh,PA,USA).

Immunofluorescence staining

Cells were fixed in4%formaldehyde,washed in PBS, and permeabilized for the analysis of intracellular markers(20min,0.25%Triton X-100;Sigma-Aldrich). The monolayers were then incubated with a blocking so-lution(PBS with5%FBS)(45min,room temperature),followed by incubation(overnight at4°C)with the primary antibodies:anti-glial fibrillary acidic protein(anti-GFAP) (Miltenyi Biotec),anti-β-III tubulin(Abcam),anti-Nestin (Abcam),and anti-O4(Miltenyi Biotec).After extensive washing in PBS,a second incubation(1h;37°C)with Alexa Fluor-488-or Alexa Fluor-547-specific anti-mouse or anti-rabbit secondary antibodies(all from Invitrogen) was performed.Cell nuclei were stained with4′,6-diami-dino-2-phenylindole(DAPI)(Sigma-Aldrich).Florescence labeling was observed by using a fluorescent microscope (Olympus,Tokyo,Japan).

Oxygen consumption assay

Samples were dissociated into singles cells,washed with PBS once,and suspended at4to approximately 10million cells per milliliter in stem cell medium. Oxygen consumption was measured in1-ml medium by using Oxytherm equipped with a Clark-type elec-trode(Hansatech Instruments Ltd,Norfolk,UK)as de-scribed previously[23].

Mouse xenografts

Subcutaneous xenografts:GSC11cells cultured under various conditions(stem cell medium without FBS,with 5%FBS,or with FBS and20mM NAC for7days)were collected,treated with accutase to make single-cell sus-pension,and inoculated into the right flank of nude mice (2×106cells per mouse).The mice were euthanized when the tumor diameter was greater than1.5cm.For orthotopic xenograft inoculation,GBM3752cells were first cultured in stem cell medium with or without serum(5%FBS).The cells were maintained in vitro under these two conditions for60passages.Cells were collected and inoculated intracranially into the brains of SCID mice(1×104cells per mouse).The mice were eu-thanized when they developed signs of neurological def-icit and became moribund.All experiments of the present study were performed in accordance with hu-man protocols approved by the Institutional Review Board at UT MD Anderson Cancer Center and Baylor College of Medicine as well as animal protocols(ACUF 11-98-08136,AN-4548)approved by the Institutional Animal Care and Use Committee of Baylor College of Medicine.Signed informed consent was obtained from all patients or their legal guardians prior to sample acquisition.

Results and Discussion

Induction of apparent differentiation of GSCs by serum and association with ROS stress responses

Both established glioma stem cell lines and primary gli-oma cells isolated from fresh tumor tissues were used in our study.GSC11and GSC23are two glioblastoma stem cell lines originally derived from glioblastoma multiforme

(GBM)surgical specimens and exhibit the in vitro stem cell characteristics of extensive self-renewal,the ability to differentiate to neurons and astrocytes,and the ability to initiate tumor in vivo .GSC11and GSC23cells were main-tained in serum-free stem cell culture medium as de-scribed previously (He,2010#274)[24].An orthotopic xenograft model (GBM3752)that preserves glioblastoma stem cells was originally established by directly inoculating primary tumor cells from fresh GBM specimen into the right cerebellum of SCID mice brain [22].The xenograft tumor cells preserve tumorigenicity,multi-lineage differen-tiation,and CD133+expression after being subtransplanted in mice brain.GBM3752cells were prepared freshly from tumors (maintained in SCID)mice for in vitro study.As shown in Fig.1,GSC11and GBM3752cells grew well in stem cell medium containing EGF and bFGF without serum and exhibited the morphology of stem-like neurospheres (Fig.1a)with high expression of CD133(Fig.1b,c).The addition of serum (%FBS)to the culture medium caused a significant change in cell morphology,manifested by a loss of neurosphere forma-tion and the appearance of differentiated cells attaching to the culture dish (Fig.1a).This was accompanied by a substantial decrease of CD133expression in a time-

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neurosphere formation and the expression of stem cell markers in glioblastoma stem cells.neurospheres in serum-free medium supplemented with epidermal growth factor and serum (5%FBS)for 3days led to a loss of neurosphere formation in both clones.b Western after exposure to serum for 1,3,and 7days.c Flow cytometry analysis of CD133expression serum for 7days.The right panel shows quantitation of the percentage of CD133+cells and 7days;*P <0.05.d Expression of SOX2,Olig2,and Notch1mRNA in GSC11cells mRNA was measured by quantitative reverse transcription-polymerase chain reaction.markers SOX2and Olig2and differentiation marker ANXA1.GSC11cells were exposed were detected by Western blot analysis.Cont control,D day,FBS fetal bovine serum,GBM

dependent manner(Fig.1b,c)and a decrease of Nestin (Additional file1:Figure S1).Quantitative RT-PCR and Western blot analyses revealed a significant decrease in expression of Sox2and Olig2,two transcription factors known to regulate neural stem cells and neural progenitor cells(Fig.1d,e).The expression of Notch1,a molecule important for promoting neural stem cell function[25], was also downregulated(Fig.1d).In contrast,the expres-sion of differentiation markers,including GFAP,β-III tubulin,O4(Additional file1:Figure S1),and ANXA1, were increased after serum exposure(Additional file1: Figure S1and Fig.1e).Similar results were observed in the third cell line GSC23(Additional file1:Figure S2). Surprisingly,this apparent differentiation induced by serum did not result in a decrease in tumorigenesis,and as will be described below,the glioma stem cells were activated by serum exposure(see in vivo study below). To investigate the molecular events and potential al-terations in the signaling pathways of GSCs in response to serum induction,we treated GSC11cells with5% FBS for1,3,and7days in triplicate cultures,and RNA was isolated from each sample for determination of gene expression profiles using microarray analyses.As shown in Additional file1:Figure S3A,clustering analysis of gene expression profiles revealed that the serum-treated GSC11cells exhibited gene expression profiles clearly distinct from that of the GSC11cells cultured in serum-free medium.There was a further shift of gene expres-sion profiles as the time of serum exposure prolonged. The fact that the three separated samples of the same time point(biological triplicate)displayed similar gene expression patterns and clustered in the same group demonstrated the high reproducibility of this experimen-tal https://www.wendangku.net/doc/503573042.html,ing the Ingenuity pathway analysis,we found that the oxidative stress response pathway was in-duced by serum most significantly(P=0.0005)at all time points tested.Additional file1:Figure S3B shows the genes involved in oxidative stress response identified by this analysis in GSC11cells.Among these genes, SOD2,catalase,NQO1,peroxiredoxin1,thioredoxin re-ductase1,and glutamate-cysteine ligase are involved in ROS scavenging.These results suggested that the homeo-stasis of reduction/oxidation(redox)balance might have been disrupted in the serum-induced GSCs.

Induction of mitochondrial ROS generation in glioma stem cells by serum through activation of electron transport chain

The observations that exposure of GSCs to serum caused consistent oxidative stress response in all tested time points prompted us to explore possible changes in cellular redox status.Since mitochondria are major sites of ROS production,we used MitoSOX-Red to de-tect mitochondrial superoxide(O2?)and5-(and-6)-chloromethyl-2,7-dichlorodihydrofluorescein diacetate acetyl ester(DCF-DA)to measure total cellular hydrogen peroxide(H2O2)and other ROS.The results showed that that serum induced a substantial increase of mitochon-drial O2?in a time-dependent manner,with an increase of the median value from46units in control cells(serum-free)to67,188,and268units on days1,3,and7after serum exposure,respectively(Fig.2a).Interestingly, total cellular ROS(as measured by DCF-DA)also showed a moderate increase,from84units in the con-trol to112,126,and159units on days1,3,and7,re-spectively(Fig.2a).Similar results were observed in another glioblastoma stem cell line GSC23(Additional file1:Figure S4).Exposure of GSC23cells to serum led to a5-and11-fold increase of mitochondrial O2?on days 3and7,respectively,and the total cellular ROS detected by DCF-DA also moderately increased(Additional file1: Figure S4A).

We then used two types of fresh glioma cells to further confirm the above observations.First,the stem-like GBM3752cells[22]were obtained freshly from tumor xenografts and divided into two portions;one portion was cultured in serum-free stem cell medium and the other portion was cultured in serum-containing medium. After7days,the mitochondrial ROS and total cellular ROS in each culture condition were measured by Mito-SOX and DC-FDA.As shown in Fig.2b,a3-fold in-crease in mitochondrial O2?and a moderate increase (26%)in total cellular ROS were observed,consistent with that seen in GSC11and GSC23cells.Furthermore, this pattern of redox alterations was consistently ob-served in primary glioma cells isolated from fresh GBM tumor tissues(Fig.2c and Additional file1:Figure S4B), suggesting that the induction of mitochondrial O2?gen-eration might be a highly consistent event in serum-induced changes in GSCs.

To test whether the increase in mitochondrial O2?and cellular ROS induced by serum in GSCs might cause stress response,we used Western blotting to analyze the expression of antioxidant molecules before and after serum exposure.As shown in Fig.2d,there was a time-dependent increase in expression of SOD2,a mitochon-drial superoxide dismutase that converts O2?to H2O2. Interestingly,the cytosolic superoxide dismutase(SOD1) did not exhibit significant change after GSC11and GSC23were exposed to serum(Fig.2d,Additional file1: Figure S4C),suggesting that the main source of ROS stress might be mainly from mitochondria and was con-sistent with the increase in mitochondrial O2?shown in Fig.2a-c.The expression of catalase,an enzyme that converts cellular H2O2to water and oxygen,increased after serum incubation(Fig.2d,Additional file1:Figure S4C).Cellular glutathione(GSH),a major endogenous antioxidant,decreased after serum exposure in GSC11

Cont D1 D3 D7

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cells (Fig.2e)and G23cells (Additional file 1:Figure S4D),reflecting a consumption of this antioxidant.These data together suggest that the increase in SOD2expression

might be a stress response to elevated mitochondrial O 2

?generation induced by serum.SOD2converted O 2?

to H 2O 2,which was then able to pass the mitochondrial membranes to cytosol,where it was converted to O 2and H 2O by catalase or neutralized by GSH,resulting in only a moderate increase of overall cellular ROS and a decrease in GSH.

Since mitochondrial O 2?is generated mainly during respiration because of the release of electrons from com-plexes I and III of the electron transport chain,we spec-ulated that the increased mitochondrial O 2?might be a result of active mitochondrial respiration induced by serum and not a consequence of a slower O 2?elimination since SOD2expression was increased.To test this possi-bility,we measured oxygen consumption in GSCs as an indicator of mitochondrial respiration.As shown in Fig.3a and b,exposure of GSC11cells to serum led to a time-dependent increase of oxygen consumption,with approximately a 100%increase by day 3.Interestingly,measurement of mitochondrial mass by using Mito-Tracker Green as well as mitochondria electron trans-port chain (ETC.)and the ATP synthase complex antibodies showed that serum did not cause any signifi-cant change in mitochondrial mass and complexes (Fig.3c and Additional file 1:Figure S5),suggesting that the increase in respiration was mainly a functional activa-tion of the pre-existing mitochondria.GBM3752cells from freshly dissected orthotopic tumor xenografts were cul-tured in either serum-free medium or serum-containing medium for 7days.A significantly higher oxygen con-sumption was observed in GBM3752cells cultured with serum (Additional file 1:Figure S6A)without any sig-nificant changes in mitochondrial mass (Additional file 1:Figure S6B).The increase in respiration without increase of mitochondrial mass was also consistently observed in GSC23cells (Additional file 1:Figure S6C).Despite the in-crease of mitochondrial respiration,cells at G 0/G 1phase were decreased only on day 1compared with cells cultured

in serum-free medium (Additional file 1:Figure S7).Cells at G 2/M phase were not changed.

Important role of mitochondrial ROS in mediating the serum effect on glioma stem cells

To evaluate the role of activation of mitochondrial res-piration and ROS generation in serum-induced apparent differentiation of GSCs,we first used https://www.wendangku.net/doc/503573042.html,plex I in-hibitor rotenone and complex III inhibitor antimycin to inhibit mitochondrial respiration in glioma stem cells and then tested whether this affected the ability of serum to induce apparent differentiation in GSCs.The results showed that both ETC.inhibitors disrupted mitochondrial respiration and caused a further increase of mitochondrial O 2?in the presence of serum (Fig.3d),but neither of them prevented the serum-induced GSCs from attaching to the flasks and exhibiting apparent differentiation morphology (data not shown).In fact,adding rotenone or antimycin even caused a further decrease of CD133at both mRNA (Fig.3e)and protein levels (Fig.3f).

Considering the observations that exposure of GSCs to serum caused an increase in mitochondrial respiration and O 2?generation but inhibition of mitochondria respir-ation by ETC.inhibitors (rotenone and antimycin)did not prevent serum to induce changes in GSCs,we speculate it was the increase in mitochondrial ROS gen-eration,not the respiration per se ,that plays a key role in mediating the serum effect on GSCs.To test this pos-sibility,we used exogenous H 2O 2to cause a level of in-crease in mitochondrial ROS comparable to that caused by serum in GSC11and GSC23cells (Fig.4a,b).Inter-estingly,a short-term treatment of GSCs with such ex-ogenous H 2O 2for 6h led to a significant decrease of SOX2,Olig2,and CD133mRNA expression in GSC11cells (Fig.4c)and GSC23cells (Fig.4d),similar to those observed in serum-induced cells.

To further validate this novel role of ROS,we used NAC,a precursor for glutathione synthesis with potent antioxidant property to reduce ROS stress,to test whether it could prevent the effect of serum on GSCs.As shown in Fig.5a,incubation of GSC11cells with serum for 7days

caused a significant increase of mitochondrial O 2?,and the presence of NAC effectively suppressed such ROS in-crease.Importantly,NAC also prevented serum-induced loss of ability to form stem-like neurospheres (Fig.5b)and partially preserved the expression of CD133in serum-in-duced cells (Fig.5c).These results suggest that the in-crease in mitochondrial ROS generation might play an important role in mediating the serum effect on GSCs.Since there was a significant decrease in expression of SOX2,Olig2,and Notch1in serum-induced GSCs,we tested whether NAC might also suppress this serum

effect.As shown in Fig.5d,quantitative RT-PCR re-vealed that the addition of NAC to the serum-treated GSC11cells largely blocked the decrease of SOX2and Olig2expression,suggesting that the expression of these two molecules might be redox-sensitive.Similar results were observed in GSC23(Additional file 1:Figure S8).Furthermore,gene expression analysis of molecules in-volved in the Notch pathway revealed that serum caused a significant decrease in the expression of Notch1,MFNG,LFNG,HESs,DTX3,and DLL1(Fig.5e).Consistently,the presence of antioxidant NAC largely prevented the

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mitochondrial activation and ROS generation in activation of glioblastoma stem cells.a Comparison exposure to serum for 1,3,and 7days.Oxygen consumption was measured by using Quantitative analysis of oxygen consumption in GSC11cells exposed to serum for 1,3,and mitochondrial mass in GSC11cells,measured by MitoTracker-Green.d GSC23cells were cultured FBS)in the presence or absence of 1μM rotenone (https://www.wendangku.net/doc/503573042.html,plex I inhibitor)or 2μM antimycin measured by flow cytometric analysis after cells were stained with MitoSOX-Red.e Effect cells were cultured in stem cell medium in the presence or absence of serum (5%FBS),expression levels were measured by quantitative reverse transcription-polymerase chain the presence or absence of serum (5%FBS),rotenone (1μM),or antimycin (2μM).CD133control,ETC .electron transport chain,FBS fetal bovine serum,GSC glioma stem cell,O 2?superoxide

downregulation of the Notch-related genes (Fig.5e),again suggesting the important role of ROS and redox signaling in regulation of GSCs.

Serum induction of GSCs promotes tumorigenesis in vivo

Because exposure of GSCs to serum caused the cells to exhibit apparent differentiation morphology and a de-crease in neurosphere formation,we used two in vivo models to test whether serum induction of such changes

might alter the ability of GSCs to form tumors in vivo .First,GSC11cells were incubated with or without serum for 7days,and the same numbers (2×106)of the con-trol cells or serum-induced cells were inoculated sub-cutaneously into the right flank of nude mice.Under these subcutaneous inoculation conditions,only two out of seven of the mice inoculated with the control GSC11cells (serum-free)form tumors,and surprisingly seven out of seven mice inoculated with the serum-induced

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GSC11cells developed tumor (Fig.6a),suggesting that exposure of GSC11cells to serum promotes their tumorigenesis.Interestingly,five out of seven mice inoc-ulated with GSC11cells treated with serum in the pres-ence of the antioxidant NAC formed tumors (Fig.6a).The overall survival of the mice inoculated with serum-induced GSC11cells was significantly shorter than that of the control mice inoculated serum-free GSC11cells (P =0.0019,Fig.6b).No significant difference (P =0.064)in overall survival was found between the control group

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R e l a t i v e m R N A l e v e l

Olig2

antioxidant NAC on serum-induced ROS generation and the expression of stem cell markers in GSCs.a Effect of NAC exposed to serum.Cells were cultured in stem cell medium with or without serum (5%FBS)in the presence Mitochondrial O 2?was measured by using MitoSOX-Red.b Comparison of neurosphere formation in GSC11serum-containing medium in the presence and absence of 20mM NAC for 7days.c NAC suppressed serum-induced were exposed to serum for 1,3,or 7days in the presence or absence of 20mM NAC as indicated,and CD133-positive flow cytometry analysis.*P <0.05.d GSC11cells were exposed to serum (5%FBS)for 3days in the presence and expression of SOX2and Olig2mRNA was measured by quantitative RT-PCR.**P <0.001.e NAC suppressed Notch pathway.GSC11cells were incubated without or with serum (5%FBS)for 3days in the presence isolated from each sample,and the expression of molecules involved in Notch signaling was measure bovine serum,GSC glioma stem cell,NAC N-acetyl-cysteine,O 2?superoxide,ROS reactive oxygen species,RT-PCR transcription-polymerase chain reaction

(serum-free)and the group of mice inoculated with GSC11cells exposed to serum in the presence of NAC (20mM).

We also used another mouse model,orthotopic inocu-lation of GBM3752cells into the SCID mice(Shu et al.

[22]),to further evaluate the role of serum exposure on tumorigenesis.The same numbers(1×104)of GBM3752 cells with or without serum exposure were inoculated into SCID mice intracranially,and the mice were observed for tumor development and survival.As shown in Fig.6c, the overall survival of mice inoculated with serum-induced GBM3752cells was significantly shorter than that of the mice bearing the control GBM3752cells cultured in stem cell medium without serum(P=0.0067).These results were consistent with those of the subcutaneous tumor model and suggest that serum exposure activated glioma stem cells and promoted tumor formation. Activation of the NF?B survival pathway in serum-induced glioma stem cells

To explore the possible mechanisms by which serum in-duction of mitochondrial ROS generation could lead to ac-tivation of GSCs and promote tumorigenesis,we further analyzed the gene expression microarray data from GSC11 cells exposed to serum for various times(Additional file1: Figure S2)and found that the expression of multiple genes downstream of nuclear factor-kappa-B(NFκB)(including CD44,IL-8,IL-11,CCND1,TFPI2,and PLAUR)consist-ently increased after incubation with serum(Fig.7a–g). Since oxidative stress is known to activate NFκB[26,27], it is possible that in vivo.Indeed,incubation of GSC23 cells with serum caused a substantial increase in IκBαphosphorylation and p65phosphorylation(Fig.7h),two molecular events indicative of NFκB activation.Import-antly,the addition of the antioxidant NAC partially sup-pressed the serum-induced phosphorylation of IκBαand p65,suggesting the role of ROS in mediating serum-induced NFκB activation.Similar results were observed in GSC11cells(Fig.7i).Serum exposure caused a significant increase in phosphorylated IκBαand p65(Fig.7i,lanes1 and2).The addition of NAC suppressed these phosphory-lations(Fig.7i,lane7).These data together with the known function of IKK in phosphorylating IκBαand p65 suggest a possibility that serum might activate NFκB in GSCs through ROS-induced activation of IKK,a redox-sensitive molecule known to be activated by ROS[28].To exam this possibility,we used a specific IKK inhibitor, BMS-345541,to test whether inhibition of IKK would pre-vent serum-induced phosphorylation of IκBαand p65.As shown in Fig.7i,BMS-345541at concentrations of10–20μM effectively suppressed serum-induced phosphoryl-ation of IκBαand p65,associated with a preservation of CD133expression of suppression of ANXA1.These data suggest that IKK might play an important role in mediat-ing serum-induced activation of NFκB through a redox-sensitive mechanism.

It has been known for some time that CSCs,similar to normal stem cells,require a certain tissue microenviron-ment to maintain their stemness[29,30]and that exposure of stem cells to serum in vitro usually induces differenti-ation phenotype[9].However,the underlying mecha-nisms remain unclear.Although apparent differentiation

phenotypes are also observed when CSCs are exposed to serum,it is unclear whether this would cause a decrease in tumorigenesis.Our study revealed a ROS-mediated mechanism by which serum induces apparent differenti-ation in glioma stem cells.We found that exposure of GSCs to serum resulted in activation of mitochondrial res-piration,leading to an increase of oxygen consumption

and high generation of mitochondrial O 2?,which induced

the expression of SOD2to convert O 2?

to H 2O 2.Owing to its relatively long half-life and ability to cross biological membranes,H 2O 2has been considered a second messen-ger that mediates redox-sensitive signaling in cellular re-sponse to growth factors [31,32].The ability of H 2O 2to cause oxidation of protein thiol via catalytic cysteine can

P-I B P-p65 CD133 ANXA1 -actin

FBS: - + + - NAC: - - + + D

a pathway by serum in glioblastoma stem cells is associated with ROS stress.a –g Induction of expression by serum exposure.GSC11cells were cultured in serum-free medium or serum-containing medium expression levels of the target genes were analyzed by microarray assays in triplicate for each time NF ?B pathway activation in GSC23cells.Protein samples were obtained from GSC23cells cultured the presence or absence of 20mM NAC for 3days.The expression of phospho-IKB α,phosphor-p65,Western blot.i IKK inhibitor BMS-345541and antioxidant NAC suppressed serum-induced NF ?B activation in serum-free or serum-containing medium with the indicated concentrations of BMS345541or phospho-IKB α,phosphor-p65,CD133,ANXA1,and β-actin was measured by Western blot analysis.CD44-5CD44transcript variant 5,FBS fetal bovine serum,GSC glioma stem cell,IL-8interleukin nuclear factor-kappa-B,PLAUR plasminogen activator urokinase receptor,ROS reactive oxygen species,

alter the function of the target proteins and thus provides a mechanism for redox signaling[32].Since O2?has an ex-tremely short half-life and cannot pass the mitochondrial membranes,the increased O2?within the mitochondria could not directly function as a second messenger to affect the nuclear gene expression.As such,conversion of the mitochondrial O2?to H2O2by SOD2seems important to relay the redox signal from mitochondria to cytosol and nucleus during serum-induced differentiation of GSCs. The upregulation of SOD2in serum-treated GSCs would facilitate the conversion of O2?to H2O2.

Although the ability of ROS to induce normal stem cell differentiation has been noticed in some experimen-tal systems[33,34],the exact mechanisms remain un-clear.Our study found that downregulation of SOX2, Olig2,and the Notch-related molecules by ROS might be a potential mechanism.The ability of exogenous anti-oxidant NAC to prevent the decreased expression of these genes and to block the serum-induced differenti-ation phenotype supports this notion.It is possible that the expression of SOX2,Olig2,and the Notch-related molecules is regulated via a redox-sensitive mechanism, with ROS being a negative regulator.Since SOX2,Olig2, and the Notch pathway are involved in regulation of neural stem cells[35,36],downregulation of these genes could promote apparent differentiation of GSCs and drive them to enter a process in which GSCs progress to become the downstream progeny cancer cells. Surprisingly,we found that although incubation of GSCs with serum induced apparent differentiation morphology and caused a downregulation of certain stem cell markers,including CD133,SOX2,and Olig2, this led to an increase of tumorigenicity and a reduction of survival in mice in two different mouse models(sub-cutaneous and orthotopic).These new findings seem to challenge the traditional view that CSCs are responsible for cancer development and their ability to form tumor would decrease once they are induced to undergo differ-entiation.Interestingly,a recent study showed that tumor cell self-renewal capacity did not predict tumor growth potential in vivo[37].The study by Barrett et al. showed that glioma cells with low self-renewal capacity were more tumorigenic and generate tumor more rap-idly than cells with high self-renewal capacity.These ob-servations are consistent with our findings.Furthermore, our study revealed that activation of the nuclear factor-kappa-B(NF?B)survival pathway by ROS might be a mechanism that promotes GSC survival and enhances tumorigenesis.The role of the NF?B pathway in normal stem cell proliferation has been implicated previously. For instance,NF?B is activated during human embryonic stem cell(hESC)differentiation,and inhibition of NF?B leads to a reduction of hESC proliferation and suppression of their progression toward primitive extraembryonic and embryonic lineages[38].As such,inhibition of NF?B may potentially prevent activation of CSCs and thus suppress tumor development.Interestingly,recent studies have shown that targeting the NF?B pathway may be effective against CSCs[39–41].Their studies showed that inhib-ition of NF?B by using compounds such as niclosamide and disulfiram suppressed acute myeloid leukemia stem cells and breast CSCs.

The NF?B signaling pathway plays a crucial role in cancer development and progression[42].It can either promote or inhibit carcinogenesis,depending on the cell types and experimental conditions.The NF?B signaling pathway is often activated by ROS through IKK phos-phorylation of I?B[43]and p65[44].Our study found that serum could cause the activation of NFκB,which could be blocked by the antioxidant NAC.These results suggest that the activation of NF?B in the serum-induced cells is probably mediated by a redox-regulatory mechanism due to ROS generation in the serum-activated mitochondria.It is worth noting that deletion of NF?BIA which encodes the NF?B inhibitor I?Bαseems to be an oncogenic event in GBM[45].Interest-ingly,a recent study showed that the NF?B pathway was activated in glioblastoma-initiating cells(GICs)after in-duction of differentiation and that blockade of the NF?B pathway could drive differentiating GSCs into senes-cence[40],again suggesting that activation of NF?B is important in maintaining the proliferation of differenti-ating GSCs.This effect was partly mediated by reduced levels of the NFκB target gene cyclin D1.Furthermore,a novel small-molecule inhibitor of the NFκB pathway in-duced senescence of tumor cells in a mouse model bear-ing human GIC-derived tumors.These findings reveal that activation of NFκB may keep differentiating GICs from acquiring a mature postmitotic phenotype,thus allowing cell proliferation.Our study showed that,in the case of GSC exposure to serum,activation of NF?B is likely through ROS stimulation of IKK,which promotes phosphorylation of I?Bαand p65.We found that anti-oxidant NAC or inhibition of IKK by BMS-345541could effectively prevent the serum-induced NF?B activation and loss of CD133,suggesting a novel role of ROS in driving the progression of GSCs toward downstream progeny cells to promote tumor development. Conclusions

In summary,our study showed that exposure of glioma stem cells to serum could stimulate mitochondrial res-piration leading to increased generation of mitochon-drial ROS,which activated NFκB to promote cancer cell survival and tumorigenesis.A key underlying mechan-ism is likely through a redox-mediated activation of IKK to phosphorylate IκBαand p65.Although the serum-induced elevation of ROS in GSCs also caused a

decrease in neurosphere formation in vitro and a re-duced expression of stem cell markers such as CD133,this apparent differentiation did not reduce the ability of the glioma stem cells to form tumor in vivo .Instead,the serum-induced GSCs exhibited greater tumorigenesis in both subcutaneous and orthotopic xenograft models.These new findings suggest that serum may activate gli-oma stem cells to progress toward the downstream can-cer progenitor cells and promote tumor formation and that activation of mitochondrial respiration and ROS generation may play a key role in redox signaling during this tumorigenesis process.It is also important to note that the apparent differentiation phenotype such as neu-rosphere formation and CD133expression in vitro observed might not necessarily predict tumorigenesis in vivo .

Accession numbers

The Gene Expression Omnibus (GEO)accession number for the RNA microarray data in this study is GSE28220.

Additional file

Abbreviations

APC:Allophycocyanin;bFGF:Basic fibroblast growth factor;CSC:Cancer stem cell;DCF-DA:5-(and-6)-chloromethyl-2,7-dichlorodihydrofluorescein diacetate acetyl ester;EGF:Epidermal growth factor;ETC.:Electron transport chain;FBS:Fetal bovine serum;GBM:Glioblastoma multiforme;GIC:Glioblastoma-initiating cell;GSC:Glioma stem cell;GSH:Glutathione;H 2O 2:Hydrogen peroxide;hESC:Human embryonic stem cell;NAC:N-acetyl-cysteine;NF κB:Nuclear factor-kappa-B;O 2?:Superoxide;PBS:Phosphate-buffered saline;ROS:Reactive oxygen species;RT-PCR:Reverse transcription-polymerase chain reaction;SCID:Severe combined immunodeficiency;SOD1:Cytosolic superoxide dismutase;UT:University of Texas.

Competing interests

The authors declare that they have no competing interests.

Authors ’contributions

SY and YL contributed to conception and design,collection/assembly of data,data analysis/interpretation,manuscript writing,and final approval and contributed equally to this article.JY,MO,HZ,JL,HC,and JL contributed to provision of study materials,manuscript writing,and final approval.GC and SK contributed to collection/assembly of data,manuscript writing,and final approval.LF,NH,WL,and XL contributed to design of animal study,data analysis/interpretation,manuscript writing,and final approval.RX,PH,and FW contributed to conception and design,provision of study materials,data analysis/interpretation,manuscript writing,and final approval.All authors read and approved the manuscript.

Acknowledgments

The authors thank Zahid H.Siddik,Anthony Lucci,and Hector Martinez-Valdez for helpful discussion.This work was supported in part by a grant from the major science and technology project of the National Basic Re-search (973)Program of China (2012CB967004),grants from National Natural Science Foundation of China (81302194and 81302144),a Specialized Re-search Fund for the Doctoral Program of Higher Education

(20130171120047),a grant from the National Institutes of Health (CA100428),and the Fundamental

Research Funds for the Central Universities (14ykpy40).FW is a recipient of

the Rosalie B.Hite Fellowship from UT MD Anderson Cancer Center and the Outstanding Young Talents of Sun Yat-sen University Cancer Center.Author details 1

Sun Yat-sen University Cancer Center;State Key Laboratory of Oncology in South China;Collaborative Innovation Center for Cancer Medicine,651E Dongfeng Road,Guangzhou,Guangdong 510060,China.2Department of Translational Molecular Pathology,The University of Texas MD Anderson Cancer Center,Houston,TX 77054,USA.3Department of Systems Biology,The University of Texas MD Anderson Cancer Center,Houston,TX 77054,USA.4Department of Neuro-Oncology,University of Utah,Salt Lake City,UT,USA.5Laboratory of Molecular Neuro-oncology,Texas Children ’s Cancer Center,Baylor College of Medicine,Houston,TX 77030,USA.Received:6March 2015Revised:15March 2015Accepted:1September

2015

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