High aldehyde dehydrogenase activity enhances stem cell features
High aldehyde dehydrogenase activity enhances stem cell features in breast cancer cells by activating hypoxia-inducible factor-2a
Ran-Ju Kim a ,b ,1,Jeong-Ran Park a ,1,Kyung-Jin Roh a ,A-Ram Choi a ,Soo-Rim Kim a ,Pyeung-Hyeun Kim b ,Jong Han Yu c ,Jong Won Lee c ,Sei-Hyun Ahn c ,Gyungyub Gong d ,Jae-Woong Hwang e ,Kyung-Sun Kang e ,Gu Kong f ,Yhun Yhong Sheen g ,Jeong-Seok Nam a ,?
a
Laboratory of Tumor Suppressor,Lee Gil Ya Cancer and Diabetes Institute,Gachon University,Incheon,Republic of Korea
b
Department of Molecular Bioscience,College of Biomedical Science,Kangwon National University,Chuncheon,Republic of Korea c
Department of Surgery,College of Medicine,University of Ulsan,Asan Medical Center,Seoul,Republic of Korea d
Department of Pathology,College of Medicine,University of Ulsan,Asan Medical Center,Seoul,Republic of Korea e
Laboratory of Stem Cell &Tumor Biology,Department of Veterinary Public Health,College of Veterinary Medicine,Seoul National University,Seoul,Republic of Korea f
Department of Pathology,College of Medicine,Hanyang University,Seoul,Republic of Korea g
College of Pharmacy,Ewha Womans University,Seoul,Republic of Korea
a r t i c l e i n f o Article history:
Received 13July 2012
Received in revised form 9October 2012Accepted 13November 2012
Keywords:Breast cancer Cancer stem cell
Aldehyde dehydrogenase Hypoxia-inducible factor-2a
a b s t r a c t
High aldehyde dehydrogenase (ALDH)activity has been recognized as a marker of cancer stem cells (CSCs)in breast cancer.In this study,we examined whether inhibition of ALDH activity suppresses stem-like cell properties in a 4T1syngeneic mouse model of breast cancer.We found that ALDH-positive 4T1cells showed stem cell-like properties in vitro and in vivo .Blockade of ALDH activity reduced the growth of CSCs in breast cancer cell lines.Treatment of mice with the ALDH inhibitor diethylaminobenz-aldehyde (DEAB)signi?cantly suppressed 4T1cell metastasis to the lung.Recent evidence suggests that ALDH affects the response of stem cells to hypoxia;therefore,we examined a possible link between ALDH and hypoxia signaling in breast cancer.Hypoxia-inducible factor-2a (HIF-2a )was highly dysregulated in ALDH-positive 4T1cells.We observed that ALDH was highly correlated with the HIF-2a expression in breast cancer cell lines and tissues.DEAB treatment of breast cancer cells reduced the expression of HIF-2a in vitro .In addition,reduction of HIF-2a expression suppressed in vitro self-renewal ability and in vivo tumor initiation in ALDH-positive 4T1cells.Therefore,our ?ndings may provide the evidence nec-essary for exploring a new strategy in the treatment of breast cancer.
ó2012Elsevier Ireland Ltd.All rights reserved.
1.Introduction
Breast cancer occurs at a high frequency in women worldwide [1].Early detection and anti-cancer treatment increase patient sur-vival.However,cancer relapse and metastasis are the most com-mon cause of mortality for breast cancer patients [2,3].Recent reports have demonstrated that cancer stem cells (CSCs)may be responsible for resistance to conventional therapies,which in many instances leads to cancer recurrence and metastasis [4–6].Therefore,CSC targeted therapies may prevent cancer relapse and provide more effective treatment.
The expression of aldehyde dehydrogenase (ALDH),as assessed by the Alde?uor assay,has been recognized as a marker for malig-nant stem cells in breast cancer [7].ALDH is a detoxifying enzyme responsible for oxidizing intracellular aldehydes and plays an important role in multiple biological activities,including drug resistance,cell differentiation and oxidative metabolism [8].Gines-tier and colleagues showed that breast CSCs with elevated ALDH activity are highly tumorigenic in a NOD/SCID xenograft model [7].Clinical data suggest that high ALDH expression is correlated with poor clinical outcome in breast cancer patients [7,9,10].Fur-thermore,Tanei and colleagues suggest that ALDH expression is a more signi?cant predictive marker than CD44+/CD24-for the iden-ti?cation of breast CSCs that are resistant to chemotherapy,although the CD44+/CD24-phenotype is an established marker of breast CSCs [11].Croker and colleagues reported that experimental inhibition of ALDH reduced chemotherapy and radiation resistance of breast CSCs in vitro [12].Studies have shown that a reduction in ALDH expression in lung cancer cell lines inhibits cell growth and motility by affecting the expression of a wide spectrum of genes [13].However,other reports showed that ALDH1de?ciency did not affect the function of stem cells in mice,and that ALDH inhibi-tion increased CSC population in some cancer cell lines [14–16].Collectively,these studies support that ALDH may be a stem cell
0304-3835/$-see front matter ó2012Elsevier Ireland Ltd.All rights reserved.https://www.sodocs.net/doc/9817347305.html,/10.1016/j.canlet.2012.11.026
?Corresponding authors.Tel.:+82328996072.
E-mail address:namjs@gachon.ac.kr (J.-S.Nam).1
These authors contribute equally to this work.
marker,but the molecular roles of ALDH in the stemness remain unclear.Therefore,the aim of this study was to investigate the function of ALDH in the maintenance and expansion of CSCs using a4T1syngeneic mouse model of breast cancer.In addition,we investigated whether selective inhibition of ALDH activity is a fea-sible strategy for CSC therapy in breast cancer.
Hypoxia is an important factor in the malignant progression of cancer[17].The hypoxic microenvironment drives cancer progres-sion by triggering a set of adaptive transcriptional responses that regulate tumor angiogenesis,tumor cell metabolism,motility and survival[18].Furthermore,the cellular response to hypoxia is important in the differentiation of embryonic,hematopoietic,mes-enchymal and neural stem cells[19].A growing body of evidence highlights the role of hypoxia in stem cell dynamics in cancer pro-gression.For example,cells in hypoxic tumor regions stabilized hy-poxia-inducible factors(HIFs)and activated the expression of adaptive genes,such as MDR1,ABCG2,telomerase,Notch and c-Myc,that could promote further dedifferentiation and enhance stem cell-like properties,such as self-renewal[20].Li and col-leagues showed that targeting HIFs in glioma stem cells inhibits self-renewal,proliferation,survival in vitro and attenuate tumor initiation potential of glioma stem cells in vivo[21].Recently,Nag-ano and colleagues showed that elevated ALDH activity may affect the proliferation and differentiation of mesenchymal stem cells during hypoxia[22].Genomic analysis showed that numerous genes in the ALDH superfamily are up-regulated due to oxidative stress in various tumors,which may disrupt cancer therapy[23]. Together,these data suggest a possible link between ALDH and hy-poxia signaling in CSCs.The purpose of this study was to examine the hypoxia-related transcription factors necessary for ALDH-dependent regulation of stem properties in breast cancer.We found that elevated ALDH activity promotes cancer stemness through a novel mechanism that involves the up-regulation of hy-poxia-inducible factor-2a(HIF-2a).
2.Materials and Methods
2.1.Cell culture
Mouse breast cancer cell lines(67NR,4TO7,4T1,TS/A and EMT6)and human breast cancer cell lines(BT-474,MCF-7,ZR-75B,T-47D,SK-BR3,MDA-MB-435, MDA-MB-231and Hs578T)were cultured in DMEM(Invitrogen,Grand Island, NY)containing10%FBS and1%penicillin/streptomycin(Invitrogen),as previously described[24,25].The ALDH inhibitor diethylaminobenzaldehyde(DEAB,Refs. [7,12,13])and paclitaxel were purchased from Sigma–Aldrich(St.Louis,MO).
2.2.Preparation of a single cell suspension of primary breast tumors
The use of fresh breast tumor specimens was approved by the research ethic committees at the Asan Medical https://www.sodocs.net/doc/9817347305.html,rmed consent was obtained from all pa-tients.All tumor tissues were digested into single cell suspension and used for FACS analysis as previously described[26].The clinicopathological characteristics of the breast tissue samples are described in Supplementary Table1.
2.3.Flow cytometry
FACS analysis and cell sorting were performed using FACS Calibur and FACS Aria machines(Becton Dickinson,Palo Alto,CA),respectively.FACS data were analyzed using Flowjo software(Tree Star,Ashland,OR).Antibodies to the following proteins were used:PE-conjugated Sca-1(dilution1/20),CD44(dilution1/40),CD61(dilu-tion1/40),CD117/c-Kit(dilution1/40,BD Pharmingen,San Diego,CA),APC-conju-gated CD133(dilution1/20,eBioscience,San Diego,CA)and HIF-2a(dilution1/200, Abcam).APC-conjugated rabbit IgG antibodies(dilution,1/500,Invitrogen,Carls-bad,CA)was used as the secondary antibody to visualize HIF-2a protein expression. The FACS gates were established by staining with isotype antibody or secondary antibody.The Alde?uor kit(Stem Cell Technologies,Vancouver,Canada)was used to isolate the population with a high ALDH enzymatic activity.Cells were stained for ALDH using the Alde?uor reagent according to the manufacturer’s instructions and analyzed on FACS Calibur.As negative control,for each sample of cell aliquot was treated with50l M DEAB,a speci?c ALDH inhibitor.Alde?uor pos cells were quanti?ed by calculating the percentage of total?uorescent cells compared with a control staining reaction.FACS Aria was used to sort Alde?uor-stained cells into Alde?uor neg and Alde?uor pos cell population.
2.4.Immunoblot analysis and immuno?uorescent staining
Immunoblot analysis and immuno?uorescent staining were performed as pre-viously described[27].Immunoblot bands was detected and scanned by the lumi-nescent image analyzer Image Quant LAS-4000(GE Healthcare,Tokyo,Japan).The degree of changes in immunoblot was determined using Image J software(NIH, Bethesda,MD)and normalized to b-actin.ALDH1expression was measured as the?uorescent intensity divided by area of the nucleus,as determined by DAPI staining,in three randomly selected?elds for each specimen using the Image-Pro Plus program(Media Cybernetics,Silver Spring,MD).Human breast cancer tissue arrays(AccuMax Array,Cat No.A312)were purchased from Isu Abxis(Seoul,Korea). Antibodies to the following proteins were used:ALDH1,HIF-1a,HIF-2a,OCT4(Ab-cam)and b-actin(Sigma–Aldrich).The clinicopathological characteristics of the breast tissue samples are described in Supplementary Table2.
2.5.Tumorsphere culture
Single cells were resuspended in serum-free DMEM(Invitrogen)containing B27 (Invitrogen),20ng/ml EGF,20ng/ml bFGF(Peprotech,Rocky Hill,NJ)and4l g/ml heparin(Sigma–Aldrich),as previously described[27].Primary tumorspheres were derived by plating50,000–200,000single cells/well into six-well ultra-low attach-ment dishes(Corning,Lowell,MA).Secondary and tertiary tumorspheres were pla-ted at50,000cells/well and50,000cells/well,respectively.Dishes were cultivated for7days to enumeration of spheres.Individual spheres P100l m from each rep-licate well(n P9wells)were counted under an inverted microscope at50x magni-?cation using the Image-Pro Plus program(Media Cybernetics).The percentage of cells capable of forming spheres,termed the‘tumorsphere formation ef?ciency (TSFE)’,was calculated as follows:[(number of sphere formed/number of single cells plated)?100],as previously described[28].
2.6.In vivo limiting dilution and metastasis assay
All animals were maintained according to IACUC-approved protocols of the Lee Gil Ya Cancer and Diabetes Institute.For titration experiments,the left thoracic(#2) mammary fat pad(m.f.p.)of anesthetized7-week-old female Balb/c mice(Orient Charles River Technology,Seoul,Korea)was surgically exposed,and sorted4T1cells were inoculated into the m.f.p.in a50-l L volume(n=6–8for each group).In order to evaluate the tumor incidence,we con?rmed the presence of tumor in inoculated sites at necropsy.Mice were euthanized,and primary tumors were collected on day 21.The volume of the primary tumor was measured as previously described[27]. For the metastasis experiment,the inguinal(#4)m.f.p.was chosen for the cell inoc-ulation,because it was dif?cult to execute the surgery of the tumor formed in the thoracic(#2)m.f.p.for the anatomical complexity of blood vessel formation.The left inguinal(#4)m.f.p.of anesthetized7-week-old female Balb/c mice was surgi-cally exposed,and5?1044T1cells were inoculated into the m.f.p.in a50-l L vol-ume.After inoculation,the mice were randomly assigned to two treatment groups and one control group with9–10animals per group.Primary tumors were surgi-cally excised on day7.DEAB(1and10mg/kg B.W.)was administered three times per week i.p.,starting1day after removal of primary tumor.The control group re-ceived the same dosage/volume of the0.2%DMSO vehicle.Mice were euthanized on day28,and autopsies were performed immediately.The lungs were removed,in-?ated,and?xed in10%buffered formalin.Macroscopic quantitation of metastases was performed by counting the number of nodules on the surface of the lung as pre-viously described[25].
2.7.Apoptosis analysis
Apoptosis was measured using the Annexin V-FITC apoptosis kit(BD Biosci-ences).Brie?y,cells at$70%con?uency were exposed to UV irradiation(UV Cross-linker,Spectroline,Westbury,NY)or paclitaxel treatment and harvested.
2.8.Quantitative reverse-transcription-PCR(qRT-PCR)
qRT-PCR was performed to quantitate mRNA expression using the SYBR green PCR master mix(Applied Biosystems,Foster City,CA)and the ABI7300Real-Time PCR system according to the manufacturer’s instructions.Mouse and human mRNA levels were normalized to mouse hypoxanthine–guanine phosphoribosyltransfer-ase(HPRT)and human cyclophilin A(PPIA)mRNA,respectively.Primer sequences are provided in Supplementary Tables3and4.
2.9.Small interference RNA and short hairpin RNA
Small interfering RNA(siRNA)targeting mouse and human ALDH1and HIF-2a and non-targeting siRNA were purchased from Bioneer,Inc.(Daejeon,Korea). Transfection was performed using Lipofectamine2000(Invitrogen)according to
R.-J.Kim et al./Cancer Letters333(2013)18–3119
the manufacturer’s instructions.For the siRNA study,we have tested each three siR-NA duplexes designed from the target gene sequence.We have chosen the siRNA that resulted in the greatest inhibition of ALDH1and HIF-2a mRNA,respectively (Supplementary Table5).The siRNA-mediated reduction of target gene expression persisted for at least6days in culture(data not shown).Lentiviral-based plasmids containing short hairpin RNA(shRNA)targeting mouse HIF-2a were constructed using the chosen HIF-2a-coding siRNA oligos.ShRNA infection was performed as previously described[24].Stable transfectants were selected by incubation with puromycin(2l g/ml;Sigma–Aldrich),and knockdown of HIF-2a expression was determined by qRT-PCR and immunoblot analysis.
2.10.Transient plasmid transfection
The pcDNA3-HIF-2a and pcDNA3control expression vectors were kindly pro-vided by Dr.Futoshi Shibasaki(Tokyo Metropolitan Institute of Medical Science,To-kyo,Japan)[29].Transfection was done with Lipofectamine2000transfection reagent(Invitrogen)according to the manufacturer’s instructions.
2.11.Promoter reporter assay
Luciferase activity assay was performed using the Luciferase Assay(Promemga, Madison,WI),as previously described[24].Brie?y,cells in12-well?at-bottomed plates were transfected with a total of500ng/well promoter reporter plasmid with 250ng/well b-gal plasmid DNA(for normalization)using Lipofectamine2000 according to the manufacturer’s instructions.After transfection,cells were incu-bated for24h,and cell lysates were harvested.Luminescence was measured using VICTOR3software(Perkin-Elmer Life and Analytical Science,Boston,MA).The pGL3-HIF-2a-LUC promoter reporter was kindly provided by Dr.Futoshi Shibasaki[29]. The pOCT4-LUC promoter reporter plasmid was purchased from Addgene,Inc. (Addgene plasmid,cat no.17221).
2.12.Statistical analysis
All experiments were conducted with a minimum of three samples,and the re-sults were expressed as the mean±SD.Statistical analysis was performed using an unpaired parametric Student’s t-test or nonparametric Mann–Whitney U test,un-less otherwise indicated in the text.
3.Results
3.1.ALDH expression in breast cancer tissues and cell lines
Mouse and human stem cells have high ALDH activity[7].Class 1of the ALDH family(known as ALDH1or ALDH1A1)is the isoform that predominates in mammals[30,31].The recent report suggests that different isoforms besides ALDH1may be responsible for ALDH activity in cancer cells,such as ALDH1B1,ALDH1A3,ALDH2 and ALDH3A1[10,32,33].However,both clinical and experimental data suggest that the ALDH1marker could be primarily responsible for the ALDH activity of stem cells in breast cancer[7,34,35].In addition,we analyzed the particular ALDH isoforms expressed by Alde?uor pos4T1cells.qPT-PCR showed that the most upregulated gene was ALDH1in Alde?uor pos cells compared to Alde?uor neg cells.(Supplementary Fig.S1).Therefore,these?ndings supported that ALDH1might be a major ALDH isoform that contributes to Alde?uor activity in breast cancer cells.
To determine whether ALDH has any relevance in breast carci-noma,we analyzed the expression of ALDH1,major member of the ALDH family[36],in the matched normal human breast and breast cancer samples.Immuno?uorescent staining showed that ALDH1 expression was elevated$2–14-fold in cancerous tissues com-pared with non-cancerous tissues from the same breast cancer pa-tients(Fig.1A and Supplementary Fig.S2).We also examined the Alde?uor pos cell population in various breast cancer cell lines using the Alde?uor assay.FACS analysis showed that Alde?uor pos cells accounted for less than3%of all cells in human breast cancer.In contrast,mouse breast cancer cells contained a$6–30%Alde-lfuor pos cells(Fig.1B and Supplementary Fig.S3).We then focused on the role of ALDH in CSC dynamics in the4T1cell line,which is widely considered to be one of the best mouse models of breast cancer[37,38].3.2.The Alde?uor pos4T1cell population has CSC properties in vitro and in vivo
It has been reported that cancer stem-like cells can be cultured in vitro to determine stem cell properties,such as self-renewal in serum-free medium with bFGF and EGF[39].To determine the sphere-forming ability of4T1Alde?uor pos cells,we plated freshly-isolated Alde?uor pos and Alde?uor neg4T1cells in sphere-forming culture conditions.Only the Alde?uor pos4T1cell popula-tion grew into typical tumorspheres(Fig.1C).These cells also showed much larger and greater number of tumorspheres than the Alde?uor neg4T1cells(Fig.1C).In vitro culture of Hs578T hu-man breast cancer cells con?rmed that Alde?uor pos cells have high tumorsphere formation ability(Fig.1C).Ginestier and colleagues showed that small number of Alde?uor pos breast carcinoma cells gave rise to a differentiated mammary carcinoma in NOD/SCID mice[7].However,xenografts in immunode?cient mice may not fully recapitulate tumor development observed in human patients [40].Therefore,to investigate in vivo tumorigenicity ability of the Alde?uor pos cells in immune competent mice,we sorted4T1cells into Alde?uor neg and Alde?uor pos cell population,and injected into the mammary glands of female balb/c mice.The frequency of CSCs in a particular cell type was determined using ELDA webtool at https://www.sodocs.net/doc/9817347305.html,.au/software/elda.ELDA analysis revealed a $3311-fold increase in CSC frequency from Alde?uor pos4T1cells to Alde?uor neg4T1cells(Table1).Data were also statistically sig-ni?cant on the basis of the Fisher’s exact test(Table1).At the same injection dose(10,000-cells),the primary tumor generated by Alde?uor pos4T1cells showed a$27-fold greater volume than that of the Alde?uor neg4T1cells(Fig.1D).These results demonstrate that the Alde?uor pos cells have a greater tumorigenic potential in vivo in an immune competent microenvironment.
3.3.Inhibition of ALDH activity suppresses characteristics of CSCs
in vitro and in vivo
The above observations prompted us to ask whether inhibition of ALDH activity reduces cancer stem-like characteristics.To inves-tigate this possibility,after breast cancer cells were cultured under adherent conditions for48h with DEAB,a speci?c inhibitor to ALDH,cell were detached and suspension cultured under serum-free conditions without DEAB treatment for7days(Fig.2A).The recent report suggests that DEAB may be more speci?c inhibitor for ALDH3A1[33].However,DEAB has been also used as a speci?c inhibitor for ALDH1[41].In addition,many independent studies have used DEAB as a speci?c ALDH inhibitor,regardless of particu-lar ALDH isoforms[7,12,13].Therefore,we used DEAB so as to in-hibit the elevated ALDH activity in this study.Before investigating the effect of DEAB in CSC dynamics,we determined the cytoxic concentration of DEAB in breast cancer cells.MTT analysis showed that the IC50for67NR,4T1,MCF-7and Hs578T cells were138.5, 321.6,132.3and132.7l M,respectively(Supplementary Fig.S4A).DEAB did not reduce viability of breast cancer cells as concentration of up to15l M.The BrdU FACS analysis showed that the treatment with15l M DEAB did not affect cell proliferation in ALDH1neg and ALDH1pos cells compared to control treatment(Sup-plementary Fig.S4B).Therefore,the concentration of DEAB of 15l M was chosen to study the further effects of DEAB on cancer stemness.DEAB pretreatment signi?cantly reduced the ef?ciency of formation of tumorspheres more than100l m in diameter in both mouse and human breast cancer cell lines(Fig.2A).Next, we investigated whether DEAB treatment affects tumorsphere for-mation upon serial passage.Concentrations up to50l M DEAB were used to study the de?nite effect of DEAB treatment on tumor-sphere formation upon serial passage.4T1cells were cultured in low attachment dishes with0,15or50l M DEAB treatment for
20R.-J.Kim et al./Cancer Letters333(2013)18–31
7days,and then examined the ability of DEAB-treated 4T1primary tumorspheres to form secondary and tertiary tumorspheres with-out additional treatment.The number of secondary and tertiary
tumorspheres generated from the 15or 50l M DEAB-treated tumorspheres was signi?cantly lower than the control counter-parts (Fig.2B).We also investigated whether the inhibition ALDH1
A2
A4
B2
B4
C2
F5
F7
20
40
B
T 474M C
F -7Z R -75B T -47D S K -B R 3M D A -M B -468M D A -M B -435M D A -M B -231H s 578T 67
N R 4T
O
74T 1E M T 6T S /A cells show an increase in stem cell-like properties in vitro and in vivo.(A)Immuno?uorescent analysis of ALDH1in human breast cancer patient-matched normal breast tissues.Tissues were stained using antibodies to ALDH1(green).All nuclei were counterstained with DAPI (blue).cytometric pro?les of Alde?uor pos cells in mouse and human breast cancer cell lines.Alde?uor pos cells were quanti?ed by calculating the percentage compared with a DEAB staining reaction.(C)The tumorsphere formation ability of Alde?uor neg and Alde?uor pos cells isolated from breast cancer show the percentage of Aldel?uor neg (R1)and Alde?uor pos (R2)cells from 4T1and Hs578T cells,respectively.Values represent mean ±bar,100l m.(D)Primary tumor growth in the Alde?uor neg and Alde?uor pos 4T1cells after a 10,000-cells inoculation.Sorted cells were injected mice (n =6for each group).Results are presented as mean ±S.D.?P <0.05.(For interpretation of the references to colour in this ?gure legend,version of this article.)
R.-J.Kim et al./Cancer Letters 333(2013)18–3121
expression by siRNA affects tumorsphere formation in 4T1and Hs578T cells.Immunoblot analysis showed that ALDH1expression in 4T1and Hs578T cells was signi?cantly suppressed by ALDH1siRNA (siALDH1)but not by control siRNA (siCON)(Fig.2C).We found that the knockdown of ALDH1reduced the ef?ciency of for-mation of tumorspheres more than 100l m in diameter in 4T1and Hs578T cells (Fig.2D).In addition,we examined whether inhibi-tion of ALDH activity affects the CSC population.The previous re-ports described that Sca-1,CD133,CD117/c-Kit,CD44and CD61could be cell surface markers of breast CSCs [26,27,42–44].FACS analysis showed that DEAB treatment of 4T1cells caused a signif-icant decrease in the number of breast CSCs were characterized by Sca-1,CD133and CD117/c-Kit expression (Fig.2E).However,the 4T1CSC population characterized by CD44and CD61expression were unaffected by DEAB treatment.SiALDH1treatment of 4T1cells affected the CSC populations similar to DEAB treatment (Fig.2E).The CSC hypothesis proposes that CSCs may be responsi-ble for metastasis.Therefore,we evaluated whether DEAB treat-ment could regulate 4T1cell metastasis in syngeneic Balb/c mice following orthotopic implantation into the mammary gland.High-dose DEAB treatment (10mg/kg B.W.)signi?cantly reduced 4T1cell metastasis to the lungs as evidenced by the reduction ($2-fold)in number of macroscopically visible metastatic nodules in the mice as compared with the control group or low-dose DEAB
Fluorescent Aldefluor Activity
S S C
R2
R1
R2
R1
Fluorescent Aldefluor Activity
S S C
Aldefluor
Aldefluor
Aldefluor neg
Aldefluor pos
*
1500Table 1
Tumor-initiating ability of Alde?uor pos cell population in the 4T1syngeneic mouse model.Estimated cell frequency
Cell type a
Aldel?uor neg cells
Alde?uor pos cells P values b Cell no.of inoculation,%(n)10,000100%(6of 6)100%(6of 6)–
500084%(5of 6)100%(6of 6) 1.00e+00100017%(1of 6)100%(6of 6) 1.50e à02500
0%(0of 6)
100%(6of 6) 2.00e à03Estimated CSC frequency by ELDA (95%CI)
1/3311(1/6346-1728)
1/1(1/651-1/1)
6.38e à08
a
Cells injected into the left #2m.f.p.of Balb/c mice at varying concentrations.The estimated frequency of CSC was determined using available ELDA analysis.ELDA:Extreme limiting dilution analysis;CI:con?dence interval.b
P -values were calculated using Fisher’s exact test.ELDA:Extreme limiting dilution analysis;CI:con?dence interval.
22R.-J.Kim et al./Cancer Letters 333(2013)18–31
treatment (1mg/kg B.W.)group (Fig.2F).Taken together,these re-sults show that the blockade of ALDH activity may reduce CSC properties and suppress the metastatic potential of CSCs.3.4.The Alde?uor pos cell population has high HIF-2a expression To determine whether hypoxia affect expansion of Alde?uor pos breast cancer cells,cells were cultured under normoxic (21%O 2)or hypoxic conditions (1%O 2)for 18h.We did not observe signi?cant change of Alde?uor pos cell population in breast cancer cultures when cells were introduced to hypoxic conditions (Supplementary Fig.S5A ).To further investigate the link between ALDH activity and hypoxia signaling,we compared the mRNA expression pro?le of hypoxia-related transcription factors (HRTFs)in Alde?uor neg and Alde?uor pos 4T1cell population under normoxic or hypoxic condi-tions.qRT-PCR analysis showed that the most dysregulated gene
013
10(days)
Seeding
Treatment
Replating
Assay
Adherent culture
Sphere culture
A
7
1421(days)
Treatment #Passage
123
1st
2
nd
3
rd
B
CON
DEAB (50 μM)
None
siCON
siALDH1
None
siCON
siALDH1
N o n
e s
i C O
N s i
A L D
H 1N o n
e s i C
O N
s i A
L D H
1
D
NS
*
*
4T1
Hs578T
NS
*
*
(Passage)
DEAB (15 μM)0.00
0.050.100.15
E
F S T n a e M *
**
**
*
E
F S T n a e M 0.00
0.030.060.090.20
0.10
0.00
C
N o n
e
s i C
O N
s i A
L D H
1
N o n
e
s i C
O N
s i A
L D H
1
ALDH1β-Actin
ALDH1β-Actin
4T1
Hs578T
Fold Changes
9
1.04
9.00
0.19
0.03
1.10
0.1ALDH1
+
67NR 4T1MCF-7Hs578T
_
DEAB
+
_
+
_
+
_
+
_
****
DEAB
+
_
+
_
+
_
0.010.020.030
0.1
0.20.30.00
0.02
0.040.06E
F S T n a e M 0.0
0.1
0.20.3Blockade of ALDH activity reduces sphere-forming ability and metastasis of 4T1cells.(A)Effect of DEAB pretreatment on tumorsphere formation of breast h pretreatment with 0or 15l M DEAB in adherent condition,cells were detached and suspension cultured under serum free conditions without days .Left panel,experimental schema.(B)The sphere forming assay of subsequent passages that were derived from DEAB-treated 4T1primary tumorspheres.tumorsphere culture was performed with DEAB cotreatment (0,15or 50l M).The secondary and tertiary tumorsphere culture was performed without panel,experimental schema.(C)Immunoblot analysis of ALDH1expression in breast cancer cell lysates of control cells (None;non-transfectant),with non-speci?c control siRNA (siCON)or ALDH1-targeted siRNA (siALDH1).b -Actin was used as a normalization control.Immunoblot data was normalized J (NIH,Bethesda,MD)and results are reported as fold changes.(D)Effects of ALDH1knockdown on tumorsphere formation in breast cancer cell transfection under adherent conditions,cells were cultured as stated in Fig.2A.(E)Characterization of the CSC population in DEAB (left panel )-and ALDH14T1cells.Values (A,B and D,E)represent mean ±SD for three determinations.(F)Effects of DEAB treatment on 4T1metastatic ef?ciency.Lines ,median metastases for each group (n =9–10for each group).Left panel,experimental schema.?P <0.05.NS,no signi?cance.Scale bar,100l m.
R.-J.Kim et al./Cancer Letters 333(2013)18–3123
was HIF-2a,but not HIF-1a,under normoxic or hypoxic conditions (Figs.3A and B).Immunoblotting of Alde?uor neg and Aldel?uor pos 4T1cells grown under normoxia con?rmed a robust increase in HIF-2a,but not HIF-1a(Fig.3C).We further examined HIF-2a expression in Alde?uor neg and Alde?uor pos cells under hypoxia for0,18or48h.FACS analysis showed that HIF-2a expression in-creased in Alde?uor pos cells in each time point,compared to Alde-?uor neg cells(Supplementary Fig.S5B).To validate the correlation between high ALDH activity and HIF-2a expression,we examined HIF-2a expression in Alde?uor neg and Alde?uor pos cells from vari-ous breast cancer cell lines and tissues.FACS analysis showed that HIF-2a expression signi?cantly increased($3–18-fold)in Alde?u-or pos cells compared to Alde?uor neg cells in the breast cancer cell lines(Fig.3D).Furthermore,a signi?cant increase($3–137-fold) in HIF-2a expression was observed in Alde?uor pos cells compared to Alde?uor neg cells in breast cancer tissues(Figure3E).Immuno-?uorescent staining also showed that the revelation of HIF-2a expression increased when ALDH1expression was strongly re-vealed in breast cancer tissues(Fig.3F).Next,we evaluated HIF-2a expression in the CSC population characterized by different stem cell markers,including Sca-1and CD133[27,30].FACS analy-sis showed a signi?cant increase in HIF-2a expression in Sca-1pos and CD133pos4T1cells(17.3and1.8-fold,respectively)(Fig.3G). Taken together,these results show that HIF-2a expression is highly dysregulated in the Alde?uor pos CSC population in breast carcinoma.
3.5.Inhibition of ALDH activity suppresses HIF-2a expression
We next examined whether DEAB treatment of4T1cells in?u-enced the HRTF gene expression pro?le.qRT-PCR analysis showed a signi?cant decrease($1.5–3-fold)in BHLHB2,HTATIP,ID1, NOTCH1,PPAR a,HIF-2a,HIF-3a and FOXO4gene expression in DEAB-treated4T1cells as compared to control-treated cells (Fig.4A).Among these HRTF genes,HIF-2a mRNA expression de-creased most signi?cantly after DEAB treatment.Immunoblot anal-ysis showed that DEAB treatment of breast cancer cells signi?cantly reduced HIF-2a protein expression,but not HIF-1a protein expression(Fig.4B).Experimental knockdown of ALDH1 by siRNA also produced statistically signi?cant decrease($2.2-fold)of HIF-2a mRNA expression,but not HIF-1a in4T1cells(Sup-plementary Fig.S6).In addition,we found that DEAB treatment sig-ni?cantly down-regulated transcription from a HIF-2a promoter reporter construct in4T1and Hs578T cells(Fig.4C).These data suggest that inhibition of ALDH activity down-regulates HIF-2a at the transcriptional level in breast cancer cells.
3.6.Knockdown of HIF-2a expression reduces resistance to apoptosis
CSCs exhibit characteristics of high survival,invasiveness and resistance to apoptosis[45].To determine whether HIF-2a in?u-ences these characteristics,we examined proliferation,motility and apoptosis in breast cancer cells using siRNA targeting HIF-2a.Immunoblot analysis showed that HIF-2a protein expression was ef?ciently suppressed in breast cancer cells transfected with HIF-2a-targeted siRNA(siHIF2a),whereas the non-transfected cells(None)and cells transfected with control siRNA(siCON)have similar HIF-2a expression(Fig.5A).HIF-2a knockdown did not af-fect cell cycle or viability(Supplementary Figs.S7A and B).Simi-larly,siHIF2a treatment did not affect the motility of breast cancer cells(Supplementary Fig.S7C).However,we observed that the amount of apoptosis after UV exposure remarkably increased in siHIF2aàtransfected breast cancer cells(Fig.5B).In addition, the rate of paclitaxel-induced apoptosis in siHIF2a-transfected cells was higher than that in the control cells(Fig.5C).These re-sults suggest that HIF-2a may enhance the survival of breast can-cer cells.
3.7.Knockdown of HIF-2a expression suppresses CSC characteristics and reduces expression of the OCT4stem cell transcription factor Before determining the effect of HIF-2a on tumorsphere forma-tion,we compared the ALDH activity of breast cancer cell lines fol-lowing transfection with siHIF2a and siCON.FACS analysis showed that knockdown of HIF-2a did not affect the change of Alde?uor pos cell population in breast cancer cells compared to control counter-parts(Supplementary Fig.S8A).In addition,we examined whether knockdown of HIF-2a affects the CSC population by using other CSC markers.FACS analysis showed that siHIF2a treatment of 4T1cells did not affect CSC population characterized by cell surface markers of breast CSC,including Sca-1,CD133,CD117/c-kit,CD44 and CD61(Supplementary Fig.S8B).The tumorsphere assay showed that HIF-2a knockdown in breast cancer cells reduced the ef?ciency of formation of tumorspheres that were more than 100l m in diameter(Fig.6A).Interestingly,the knockdown of HIF-2a signi?cantly decreased the sphere-forming ability of Alde-
24R.-J.Kim et al./Cancer Letters333(2013)18–31
?uor pos 4T1cells but did not affect tumorsphere formation in Alde-?uor neg cells (Fig.6B).The above observations prompted us to ask whether HIF-2a in?uences the tumor initiation ability of Alde?u-or pos CSC in vivo .We stably knock-downed HIF-2a protein expres-sion in 4T1cells using lentiviral shRNA (Supplementary Fig.S8C )and then examined in vivo tumorigenic potential of Alde?uor pos 4T1cells.ELDA analysis showed a $11-fold reduction in CSC fre-quency from Alde?uor pos 4T1cells infected with HIF-2a shRNA to Alde?uor pos 4T1cell infected control shRNA.Data were also sta-tistically signi?cant on the basis of the Fisher’s exact test (Table 2).These results demonstrate that suppression of HIF-2a attenuated the tumor initiation ability of Alde?uor pos 4T1cells in vivo .Takah-ashi and colleagues demonstrated that pluripotent stem cells can be induced from mouse ?broblasts by the retroviral transduction of four transcription factors,OCT4,SOX2,c-MYC and KLF4[46].In addition,Covello and colleagues have demonstrated that HIF-2a is involved in embryonic stem cell pluripotency [47].The recent reports described that the identi?cation of the stem cell regulator OCT4as a HIF-2a target gene directly links HIF-2a to embryonic and glioma stem cell biology [21,47].Based on these reports,we
67NR 4T1
MCF-7Hs578T
HIF-2α
% o f M a x i m u m
Aldefluor
neg
Aldefluor
A A HIF-1HIF-2β1.000.871.00
16.13
cells have elevated HIF-2a expression Alde?uor pos 4T1cells under normoxic (1%O 2)conditions for 18h,after which Alde?uor pos 4T1cells under normoxic fold changes.(D and E)Flow cytometric tissues.(F)Immuno?uorescent analysis Tissues were stained with H&E.Scale ±SD for three determinations.?R.-J.Kim et al./Cancer Letters 333(2013)18–3125
used qRT-PCR,immunoblot and reporter assay to con?rm the OCT4as HIF-2a target gene in breast cancer cells.qRT-PCR and immuno-blot analysis showed a $2-fold reduction of OCT4expression in si-HIF2a -transfected cells compared to siCON-transfected cells;however,no signi?cant change was observed in the expression of other stem transcription factors (Fig.6C).In addition,DEAB treat-ment reduced OCT4expression in breast cancer cell lines,including 67NR,4T1,MCF-7and Hs578T cells (Supplementary Fig.S9).We also found that the transient transfection of HIF-2a expression vec-tor signi?cantly up-regulated the transcription from an OCT4pro-moter reporter construct in Hs578T cells (Fig.6D).Though our study did not fully address the role of HIF-2a in breast cancer stemness,our data suggest that HIF-2a at least in part contributes to cancer stemness though the activation of OCT4[21].
Tissue 3Tissue 1Tissue 2Tissue 4Tissue 5Tissue 6Tissue 7Tissue 8
HIF-2α
neg 5.45 4.74 2.690.380.742.81 3.148.59H&E
DAPI
ALDH1
α
Tissue 6
Tissue 4Tissue 3Fig.3.(continued )
26R.-J.Kim et al./Cancer Letters 333(2013)18–31
4.Discussion
High ALDH activity is a major biomarker for normal and malig-nant breast stem cells[7].However,the molecular role of ALDH in cancer stemness has not yet been fully elucidated.In this study,we enriched for putative CSCs according to ALDH enzymatic activity in the4T1mouse model.To our knowledge,this report is the?rst that elevated ALDH activity is a marker of CSCs in an immune-compe-tent syngeneic tumor microenvironment.We also found that ALDH controls the maintenance and expansion of putative CSCs at least in part by activation of HIF-2a.This is supported by the following ?ndings:(1)the size of the Alde?uor pos4T1cell population was correlated with tumorigenicity in immune-competent mice;(2)inhibition of ALDH activity down-regulated HIF-2a expression at the transcriptional level;and(3)knockdown of HIF-2a expression reduced in vitro tumorsphere formation and in vivo tumor initia-tion of Alde?uor pos4T1cells.Our results support that ALDH1could be a potentially good marker for CSC characterization in a synge-neic xenograft model.However,recent reports suggest that differ-ent isoforms beside ALDH1may be responsible for ALDH activity in various forms of cancer[10,32,33].Therefore,additional studies will surely lead to a classi?cation of relative contribution of ALDH isoforms in cancer stemness.
The CSC hypothesis proposes that CSCs are the root of cancer and contribute to cancer metastasis and recurrence.Accumulating evidence suggests that effectively targeting CSCs may signi?cantly improve the outcome for cancer patients.During the past decade, several signal pathways important for the maintenance of CSCs have been elucidated[48].Recent reports have shown that CSCs found in brain cancer are dependent on HIF activity for survival, self-renewal and tumor growth.In particular,HIF-2a expression is signi?cantly associated with the survival rate of brain cancer pa-tients.Therefore,HIF-2a is a promising therapeutic target for brain cancer treatment[21].Our data suggest that inhibition of HIF-2a may attenuate the self-renewal ability and tumor initiation poten-tial of breast CSCs.In silico analysis of clinical microarray data showed that HIF-2a mRNA expression was elevated in invasive breast cancer as compared to normal breast tissue as well as in recurrent breast cancer compared to non-recurrent breast cancer (Supplementary Fig.S10).Thus,our work supports the hypothesis that blockade of HIF-2a may reduce CSC survival in patients with early-stage or advanced-stage cancer[21,49,50].
HIF-2
β?
+++
HIF-1
1.00 1.12 1.000.091.00 1.03
1.000.33
1.000.85
1.000.27
1.000.78
1.000.11
+
HIF-2
Sca-1CD133
Sca-1CD133
neg pos neg pos
2.90 50.18 34.28 64.40
R.-J.Kim et al./Cancer Letters333(2013)18–3127
OCT4is a primary regulator of stem cell self-renewal and differentiation and has been recently validated as a CSC therapeutic target [51].In addition,recent studies suggest that HIF-2a -mediated up-regulation of OCT4expression may enhance CSC proliferation and survival [21,51].Our and other studies have provided evidence that the OCT4promoter is regulated by HIF-2a [47].Furthermore,we recently reported that 4T1breast CSCs with elevated OCT4expression are highly tumorigenic in a xenograft model [52].Collectively,these data support the hypothesis that HIF-2a regulates the tumorigenic capacity of stem cells through activation of OCT4[20,21].Recently,it has been reported that HIF-2a is involved in molecular signaling pathways,such as Notch and Wnt signaling,during development [50,53].In addition,SERPINB9up-regulation by HIF-2a may prevent immune detection of CSCs [54,55].Collectively,these data suggest that HIF-2a may modulate CSC dynamics through multiple pathways.Therefore,studies that determine the molecular signaling downstream of HIF-2a during the maintenance and expansion of CSCs are needed.
Our work is the ?rst report that elevated ALDH activity pro-motes cancer stemness through the up-regulation of HIF-2a .However,our present report does not fully explain the molecular mechanism of HIF-2a regulation by ALDH.Thus,we are still inves-tigating possible mechanisms.One possibility is a mechanism that that operates through the retinoid signaling,because ALDH induces the differentiation of human hematopoietic stem cells by oxidizing retinol to retinoic acid [14].In preliminary study using our model systems,we investigated the possible link among ALDH,HIF-2a ,and retinoid signaling.However,our data suggest that ALDH activation of retinol may not be involved mechanically in the regulation of HIF-2a expression (Supplementary Fig.S11).Other possible mechanism is that the elevated ALDH activity may enhance HIF-2a expression through reducing reactive oxygen species (ROS)level.ALDH enzymes have a critical role in scaveng-ing ROS and induced UV-induced oxidative stress [56,57].It has been reported that generation of ROS result in reduction of stem-like properties in CSCs derived from pancreatic cancer and leuke-mia [58,59].In preliminary studies,FACS analysis showed that Alde?uor pos 4T1cells have low level of ROS compared to Alde?u-or neg 4T1cells (Supplementary Fig.S12A ).DEAB treatment in-creased ROS level in 4T1cells (Supplementary Fig.S12B ).H 2O 2-induced ROS production down-regulated transcription from a HIF-2a promoter reporter construct in 4T1cells (Supplementary Fig.S13A ).N-acetylcysteine,ROS scavenger,reversed the DEAB treatment-mediated down-regulation of HIF-2a transcription in 4T1cells (Supplementary Fig.S13B ).These ?ndings support the possibility that ALDH activation might be involved in the regula-tion of HIF-2a expression through a mechanism involving changes in ROS production;therefore,these molecular details are under investigation.
s s HIF-2β4T1
s s Hs578T
1.00
0.81
0.07
1.00
1.40
0.08
28R.-J.Kim et al./Cancer Letters 333(2013)18–31
α-Actin
N o n e s i C O N
s i H I F 2
αsiCON
siHIF2α
siCON
siHIF2α
N o n e s i C O N
s i H I F 2αN o n e s i C O N
s i H I F 2αN o n e s i C O N
s i H I F 2
α0.00
0.0
0.00
0.00
1.00 1.050.15 1.00 1.260.08 1.000.890.10 1.000.910.10OCT4
βOCT4
β+*
HIF-2βα
1.00
0.95
0.21
1.00
1.18
0.15
1.00
1.13
3.10
R.-J.Kim et al./Cancer Letters 333(2013)18–3129
The other possible mechanism is that high ALDH activity may up-regulate HIF-2a expression through the control of proteasome activity.Disul?ram,a member of dithiocarbamate family capable of binding copper and an inhibitor of ALDH,is currently being used clinically for the treatment of alcoholism.Chen ad colleagues re-ported that disul?ram–copper complex may have antitumor activ-ity in breast cancer cultures and xenografts by inhibition of the proteasome activity[60].Yip and colleagues showed that combina-tion of disul?ram with copper inhibited CSCs in breast cancer cell lines via inhibition of the proteasome-NF-j B pathway[56].In pre-liminary promoter-reporter studies,inhibition of NF-j B activity suppressed the up-regulation of HIF-2a transcription(Supplemen-tary Fig.S14).Recent reports suggest that an ubiquitin–protea-some system may be involved in switching cells from HIF-1a to HIF-2a dependent signaling promoting stem cell characteristics, aggressive tumor growth and invasion[61,62].Therefore,addi-tional studies are necessary to determine whether high ALDH activity can control HIF-2a activation,but not HIF-1a,through reg-ulation of the proteasome pathway.
In summary,many independent studies suggest that high ALDH activity may be a biomarker for CSCs in a variety of cancers.Our present work suggests that the therapeutic targeting of ALDH activity may be useful for the treatment of cancer stem cells.We predict that the further characterization of the pinned speci?c role of ALDH in CSC signaling complexes will aid in the development of more effective cancer therapies.
Con?icts of interest
The authors indicate no potential con?icts of interest. Acknowledgements
We thank https://www.sodocs.net/doc/9817347305.html,lage M.Wake?eld(National Cancer Institute, USA)for critical reading of our manuscript.This research was sup-ported by Basic Science Research Program through the National Re-search Foundation of Korea(NRF)funded by the ministry of Education,Science and Technology(2012-0003630),by a grant from the National R&D Program for Cancer Control,Ministry for Health,and Welfare,Republic of Korea(0820180).
Appendix A.Supplementary material
Supplementary data associated with this article can be found,in the online version,at https://www.sodocs.net/doc/9817347305.html,/10.1016/j.canlet.2012.
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Table2
The effects of HIF-2a knockdown on tumor-initiating ability of Alde?uor pos cell population in the4T1syngeneic mouse model.
Estimated cell frequency Cell type a
b ShCON Alde?uor pos cells
c ShHIF2a Alde?uor pos cells P values d
Cell no.of inoculation,n(%)
1000–––
500100%(8of8)13%(1of8) 1.40eà03
10013%(1of8)0%(0of8) 1.00e+00
250%(0of8)0%(0of8)–
Estimated CSC frequency by ELDA(95%CI)1/368(1/696-1/195)1/3945(1/27959-1/557) 3.50eà03
The estimated frequency of CSC was determined using available ELDA analysis.P-values between cell type for each cell density
tested are listed.ELDA:Extreme limiting dilution analysis;CI:con?dence interval.
a Cells injected into the left#2m.f.p.of Balb/c mice at varying concentrations.
b Aldel?uor pos4T1cells infected with non-speci?
c control shRNA(shCON).
c Alde?uor pos4T1cells infecte
d with HIF-2a-targeted shRNA(shHIF2a).
d P-values wer
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