hypoxia and human genome stability: downregulation of ... · showed that hypoxia reduces cell...

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 746858 8 pageshttpdxdoiorg1011552013746858

Research ArticleHypoxia and Human Genome Stability Downregulation ofBRCA2 Expression in Breast Cancer Cell Lines

Daniele Fanale1 Viviana Bazan1 Stefano Caruso1 Marta Castiglia1 Giuseppe Bronte1

Christian Rolfo2 Giuseppe Cicero1 and Antonio Russo1

1 Section of Medical Oncology Department of Surgical Oncological and Stomatological SciencesUniversity of Palermo 90127 Palermo Italy

2 Phase I-Early Clinical Trials Unit Oncology Department and Multidisciplinary Oncology Center Antwerp (MOCA)Antwerp University Hospital 2650 Edegem Belgium

Correspondence should be addressed to Antonio Russo antoniorussousanet

Received 30 April 2013 Accepted 21 August 2013

Academic Editor Dominic Fan

Copyright copy 2013 Daniele Fanale et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Previously it has been reported that hypoxia causes increased mutagenesis and alteration in DNA repair mechanisms In 2005 aninteresting study showed that hypoxia-induced decreases in BRCA1 expression and the consequent suppression of homologousrecombination may lead to genetic instability However nothing is yet known about the involvement of BRCA2 in hypoxicconditions in breast cancer Initially a cell proliferation assay allowed us to hypothesize that hypoxia could negatively regulate thebreast cancer cell growth in short term in vitro studies Subsequently we analyzed gene expression in breast cancer cell lines exposedto hypoxic condition bymicroarray analysis Interestingly genes involved inDNAdamage repair pathways such asmismatch repairnucleotide excision repair nonhomologous end-joining and homologous recombination repair were downregulated In particularwe focused on the BRCA2 downregulation which was confirmed at mRNA and protein level In addition breast cancer cells weretreatedwith dimethyloxalylglycine (DMOG) a cell-permeable inhibitor of both proline and asparaginyl hydroxylases able to induceHIF-1120572 stabilization in normoxia providing results comparable to those previously described These findings may provide newinsights into themechanisms underlying genetic instabilitymediated by hypoxia and BRCA involvement in sporadic breast cancers

1 Introduction

Breast cancer (BC) is one of the leading causes of cancer-related death among women worldwide [1] About 5ndash10 offamilial breast cancers can be attributed to two autosomaldominant genes with high penetrance BRCA1 and BRCA2[2] Carriers of germline mutations in BRCA1 and BRCA2have a predisposition for developing breast andor ovariancancer [3] In addition it has been reported that BRCA1expression was reduced or undetectable in the majority ofhigh-grade ductal carcinomas suggesting that absence ofBRCA1 may contribute to the pathogenesis of a significantpercentage of sporadic breast cancers [4 5] BRCA playsan important role in DNA repair activation of cell-cyclecheckpoints andmaintenance of chromosome stability [6 7]In the last years several authors reported that the tumor

microenvironment can contribute to genetic instability andalter the overall DNA repair [8ndash14]

Mammalian cells are extremely intolerant to prolongedexposure to hypoxia contrariwise tumor cells are tolerant toanoxia and many tumors contain hypoxic regions [15 16]Intratumoral hypoxia is an adverse clinical prognostic factorassociated with decreased disease-free survival for manycancers such as the prostate cervix breast and head and neck[17ndash19] Hypoxic tumor cells can be locally and systematicallyaggressive with a decreased sensitivity to apoptotic and othercell death signals increased angiogenesis increased prolifer-ation and increased capacity for systemic metastasis [20 21]It is now well known that hypoxia causes the stabilizationof HIF-1120572 monomer that translocates to the nucleus whereit heterodimerizes with HIF-1120573 and HIF-1 complex bindsto the hypoxia responsive element (HRE) on the promoter

2 BioMed Research International

regions of target genes in order to promote tumor survivalinvasion andmetastasis [22ndash25] Recently it has been shownthat under severe hypoxia conditions the mismatch repair(MMR) genes MLH1 and MSH2 are downregulated in p53-and HIF-1a-dependent way in many tumor cell lines thusinducing genetic instability [10 26] Furthermore Mihaylovaet al reported that cells exposed to hypoxia- and low pHwerefound to have a diminished capacity for DNA repair com-pared with controls [26] In 2005 an important study showedthat hypoxia-induced decreases in BRCA1 expression and theconsequent suppression of homologous recombination maylead to genetic instability [27] However nothing is yet knownabout the involvement of BRCA2 in hypoxic conditions inbreast cancer

Here we aim to analyze gene expression in breast cancercell lines exposed to hypoxic condition with a focus on genesinvolved in DNA damage repair (DDR) especially BRCA2

2 Materials and Methods

21 Cell Cultures Human BC cell lines MCF-7 MDA-MB-231 and SKBr3 purchased from the American TypeCulture Collection (Rockville MD USA) were cultured inDulbeccorsquos Modified Eagle Medium (DMEM F12) supple-mented with 10 fetal bovine serum (FBS) and antibiotics(100UmL penicillin and 50mgmL streptomycin) (Invitro-gen Carlsbad CA USA) Eighty percent of confluent celllines were cultured in normoxic atmosphere of 16 O

2 79

N2 and 5 CO

2(by volume) for 24 h Then medium was

changed and cells were further cultured under normoxiaor hypoxia (3 O

2 87 N

2 5 CO

2 by volume) at two

different time-points 24 h and 48 h Furthermore cells wereincubated in the absence (normoxia) or presence (hypoxia) ofHIF hydroxylase inhibitor dimethyloxalylglycine (DMOG)(Sigma-Aldrich St Louis MO USA) for 24 h and 48 h atfinal concentration of 1mM

22 Cell Proliferation Assay To analyze cell proliferativeactivity a 5-bromo-21015840-deoxyuridine (BrdU) assay was per-formed using BrdU Cell Proliferation Assay Kit accordingto the manufacturerrsquos instructions (Cell Signaling Technol-ogy Euroclone Milano Italy) Cells were seeded at 1 times104 cellswell in a 96-well plate and incubated overnight To

assess proliferative activity under hypoxia cells were directlyincubated under normoxia and hypoxia (for 24 48 72 and96 hours) Ten 120583MBrdU was added to the plate and the cellswere incubated for 4 hours The absorbance at a wavelengthof 450 nm was measured using an ELISA microplate readerAll experiments were performed in triplicate

23 Microarray Analysis Microarray analysis was performedas previously described [28] Total RNA was extractedaccording to the manufacturerrsquos protocol (Affymetrix SantaClara CA USA) Fragmented cRNA was hybridized usinga human oligonucleotide array U133 Plus 20 (GenechipAffymetrix Santa Clara CA USA) Washing and stain-ing were performed through Affymetrix GeneChip FluidicStation 450 Probe arrays were scanned using Affymetrix

GeneChipScanner3000 G7enabled for high-resolution scan-ning Two biological replicates were performed for eachexperimental condition Images were extracted with theGeneChip Operating Software (Affymetrix GCOS v14)Quality control of the arrays was performed using theAffyQCReport software [29]

24 Statistical Analysis For statistical analysis the back-ground subtraction and normalization of probe set intensitieswere performed using themethod of robustmultiarray analy-sis (RMA) described by Irizarry et al [30] To identify DEGsgene expression intensity was compared using a moderated119905-test and a Bayes smoothing approach developed for a lownumber of replicates [31] To correct for the effect of multipletesting the false discovery rate was estimated from 119875 valuesderived from the moderated 119905-test statistics [32]The analysiswas performed using the affylmGUIGraphical User Interfacefor the limma microarray package (Bioconductor Software)[33] Significant differences were determined by Studentrsquos 119905-test 119875 value lt005 was considered to be statistically signifi-cant

25 Microarray Data Analysis Hierarchical cluster and heatmap analyses (HCA) were performed using the MultiExper-iment Viewer (MeV v48) program of TM4 Microarray Soft-ware Suite Gene Set Analysis Toolkit was used to investigatethe biological significance of a set of genes represented by thespecific expression pattern inDNArepairmechanismsDEGswere analyzed according to predefined pathways annotatedby KEGG [34] and Biocarta bioinformatic resources For anoverrepresented KEGG or Biocarta pathway a cut-off119875 valueof 001 was selected All showed values are in logarithm scale

26 Quantitative Real-Time PCR (qRT-PCR) and RT-PCRTotal cellular RNA was extracted using RNeasy Mini Kit(Qiagen Inc Valencia CA USA)Then RNAwas controlledthrough 2100 Bioanalyzer (Agilent Technologies Santa ClaraCA USA) and quantified through the spectrophotometerNanoDrop ND-1000 (CELBIO) For BRCA1 BRCA2 andMLH1 mRNAs detection 2 ng of total RNA was reversetranscribed into single-stranded cDNA using High CapacitycDNAReverse Transcription Kit (Applied Biosystems FosterCity CA USA) according to vendorrsquos instructions Gene-primers for BRCA1 BRCA2 and MLH1 were purchasedfrom Applied Biosystems (TaqMan gene expression assay)Quantitative real-time PCR (qRT-PCR) was performed withthe ABI PRISM 7900 sequence detection system (AppliedBiosystems Foster City CA USA) using SDS software ver-sion 21 The reactions were performed in triplicate and theresults were normalized using Human 120573-actin PredevelopedTaqMan assay reagents (Applied Biosystems) Changes in thetarget mRNA content were determined using a comparativeCT method (ABI User Bulletin number 2) An average CTvalue for each RNA was obtained for triplicate reactions

27 Western Blotting (WB) Cells were lysed using completeLysis-M reagent set (Roche Mannheim Germany) Proteinconcentration was measured using Quick Start Bradford

BioMed Research International 3

5

4

3

2

1

0

0

MCF-724h 48h 72h 96h

(a)

6

5

4

3

2

1

00

SKBr324h 48h 72h 96h

(b)

6

5

4

3

2

1

0

0

MDA-MB-231

NormoxiaHypoxia

24h 48h 72h 96h

(c)

Figure 1 Role of hypoxia in cell proliferation Cell proliferative activity was evaluated by BrdU incorporation assay Data represent means ofdifferent culture experiments each performed in triplicate and are presented as fold changes plusmn SD Cell proliferative activity at time 0 was setto 1 MCF-7 (a) SKBr3 (b) and MDA-MB-231 (c) cell lines were cultured under normoxia or hypoxia for 24 48 72 and 96 hours

(Bio-Rad Laboratories Hercules CA USA) 80ndash100mg oftotal protein lysate was separated on 10 polyacrylamidegel under denaturing conditions and immunoblotted intonitrocellulose membrane The following antibodies (Abs)were used anti-BCRA2 goat (Santa Cruz BiotechnologySanta Cruz CA USA) anti-GAPDH(6C5) (Santa CruzBiotechnology Santa Cruz CA USA) anti-HIF-1a rabbit(Bethyl Laboratories Montgomery USA)

3 Results

31 Effects of Hypoxia on Cell Proliferation To analyze theeffects of hypoxia on cell proliferation a BrdU incorporationassay was performed and proliferative activity was evaluatedin BC cells incubated under normoxia and hypoxia atdifferent times Evaluation of BrdU incorporation percentageshowed that hypoxia reduces cell proliferation in MCF-7SKBr3 and MDA-MB-231 cells (Figure 1) It is interestingto note that hypoxia appears to have a greater effect onproliferation in the first 48 hours The percentage increase

in fact after 48 hours appears to be comparable in bothconditions (normoxia and hypoxia) in all BC cell lines

32 Gene Expression Profiling in Breast Cancer Cells underHypoxia Since hypoxia is a condition which during tumorgrowth influences the expression of several genes involvedin angiogenesis proliferation cell cycle control and DNAdamage repair (DDR) mechanisms we first performed amicroarray analysis using Affymetrix platform in orderto compare differential gene expression profiles in MCF-7MDA-MB-231 and SKBr3 human breast cancer cell linesin response to hypoxic exposure for 24 and 48 hoursrespectively This work was carried out in order to evaluatethe involvement of some genes inmolecular pathways relatedto tumor pathogenesis This analysis has allowed to obtainfor each examined BC cell line two lists of differentiallyexpressed genes (DEGs) in hypoxia for 24 and 48 hourscompared to normoxia (control) In particular for this studythe lists were screened considering as significant only thegenes with fold change (119872) gt |05| (logarithm scale) and


119875 lt 005 Thus we obtained six lists of DEGs with respectto normoxia including after 24 h of hypoxia 10437 genes(5958 downregulated and 4479 upregulated) for MCF-711267 genes (6801 downregulated and 4466 upregulated)for MDA-MB-231 and 14280 genes (8450 downregulatedand 5830 upregulated) for SKBr3 and after 48 h of hypoxia11205 genes (5955 downregulated and 5250 upregulated)for MCF-7 12756 genes (5656 downregulated and 7100upregulated) for MDA-MB-231 and 17098 genes (9180downregulated and 7918 upregulated) for SKBr3

Then six lists of geneswere comparedwith each other andanalyzed in order to identify a set of DEGs common to threeBC cell lines Using Venn diagrams we found 270 sharedgenes (81 downregulated and 189 upregulated) that showeda gene expression variation in hypoxia (24 h and 48 h) withrespect to normoxia

33 Molecular Pathways Deregulated in Hypoxia A furtherseries of studies was carried out on the microarray analysisdata in order to identify any genes of particular interestSignificant gene expression variations were detected in genesinvolved in angiogenesis proliferation cell cycle progressionmitosis genomic stability and response to hypoxiaThe inte-grated analysis resulting from KEGG and Biocarta databasesallowed us to identify the main molecular pathways alteredin BC cell lines after hypoxic exposure for 24 h and 48 hThis analysis indicated that the genes showing a significantvariation in expression levels are included in the followingpathways DNA damage repair (37 genes) cell cycle regula-tion (35 genes) HIF-1120572 network (15 genes) and mitosis reg-ulation (12 genes) A considerable number of genes involvedin proliferation and cell cycle control through the G1S andG2M transitions showed a significantly deregulated expres-sion in all three BC cell lines Among the DEGs in hypoxiarequired for G1S transition CDC6 CCND1 CCNE2 andCDK2 were downregulated whereas CDKN1A (p21Cip1) wasupregulated Instead CDC25A was downregulated in MCF-7 and SKBr3 cells and upregulated in MDA-MB-231 cellsMoreover we found that in MCF-7 and SKBr3 cells hypoxiainduces downregulation of several genes required for G2Mtransition such as CCNA2 CCNB1 CCNB2 CDC2 (CDK1)and CDC25CThe same results were shown byMDA-MB-231cells under hypoxic conditions for 24 h

In addition significant increases in expression levels werealso observed in key genes involved in HIF-1120572 network(Figure 2(b)) such as VEGF-A SLC2A1 JUN FOS andother genes encoding for glycolytic enzymes (PGK1 PFKFB3HK2 and ALDOA) Finally microarray analysis showed asignificant variation in expression levels of some genes thatregulate the mitosis included in AURKA signaling pathwayOther genes involved in this pathway such as AURKB JUB(ajuba) andTPX2 are downregulated in all three BC cell linesunder hypoxic conditions

However since several studies reported that the hypoxiaof tumor microenvironment can contribute to genetic insta-bility [35] our attention was focused mainly on the DEGsinvolved in DNA damage repair (DDR) pathways mismatchrepair (MMR) nucleotide excision repair (NER) nonhomol-ogous end-joining (NHEJ) and homologous recombination

00 6386284 8069622

MMR

NER

NHEJ

HRR

MLH1MLH3MSH2MSH5MSH6RFC1RFC2RFC3RFC4RFC5PMS1XPCRPA1RPA2RPA4POLSGTF2H1RAD23BERCC1ERCC2ERCC3XRCC4XRCC5XRCC6NHEJ1PRKDCDCLRE1CPOLMLIG4RAD50

RAD51RAD51CRAD52RDM1ATRIP

N H24 H48 N H24 H48 N H24 H48

MCF-7 MDA-MB-231 SKBr3

BRCA1BRCA2

Figure 2 Heat maps of DEGs involved in DNA damage repair(DDR) pathways deregulated in hypoxia Clustering of DEGsinvolved in DNA damage repair (DDR) pathways mismatch repair(MMR) nucleotide excision repair (NER) nonhomologous end-joining (NHEJ) and homologous recombination repair (HRR)Theheat maps were generated from microarray data reflecting geneexpression values inMCF-7MDA-MB-231 and SKBr3 cells exposedto hypoxia (3 O

2) for 24 h and 48 h in comparison to control

cells cultured under normoxic conditions (16 O2 119872 gt |1| and

119875 lt 005) Each row represents the expression levels for a single genetested for different experimental conditions Each column showsthe expression levels for the genes tested for a single experimentalcondition The absolute expression value (log scale) of each geneis derived from the mean of two biological replicates The colorscale bar on the top represents signal intensity variations rangingfromgreen (poorly expressed genes) to red (highly expressed genes)Black boxes indicate intermediate expression values N = normoxiaH24 = hypoxia for 24 h H48 = hypoxia for 48 h

repair (HRR) (Figure 2) In general the expression of genesinvolved in these pathways was found downregulated (seeTable S1 in Supplementary Material available online athttpdxdoiorg1011552013746858) The most significantchanges were observed in the HRR pathway In particularin HRR we observed a significant decrease in expressionlevels of BRCA1 BRCA2 RAD51 and RAD50 Furthermorethere were significant variations in key genes involved inMMR In particular we found a significant decrease in theexpression levels ofMLH1MSH2 andMSH6 Moreover wedemonstrated a more global effect on DNA damage repairpathways as a result of the hypoxic exposure

34 Downregulation of BRCA2 Expression under Hypoxia Inorder to validate microarray analysis data we have evaluated


12

1

08

06

04

02

0

lowast

lowast lowast lowast

lowastlowast

C C CRela

tive B

RCA1

mRN

A le

vels

BCRA1

MCF-7 MDA-MB-231SKBr324h 48h 24h 48h 24h 48h

(a)

12

1

08

06

04

02

0

lowast

lowastlowast

lowast

lowastlowast

BRCA2

Rela

tive B

RCA2

mRN

A le

vels

C C CMCF-7 MDA-MB-231SKBr324h 48h 24h 48h 24h 48h

(b)

C

12

1

08

06

04

02

0C C C

MCF-7 MDA-MB-231SKBr3

lowastlowast lowast

lowast

MLH1

Relat

ive M

LH1

mRN

A le

vels

24h 48h 24h 48h 24h 48h

(c)

Figure 3 Effects of hypoxia on BRCA2 expression in breast cancer cell lines Validation of microarray data by quantitative real-time PCRanalysis Changes in BRCA1 (a) BRCA2 (b) and MLH1 (c) mRNA expression levels were determined in MCF-7 SKBr3 and MDA-MB-231cells exposed to hypoxia (3 O

2) for 24 and 48 hours with respect to control condition (normoxia 16 O

2) Relative transcript levels were

determined using the 2minusΔΔCt method and normalized to 120573-actin mRNA (endogenous control) Normoxic condition values are taken as 1 andhypoxic condition values represent fold decrease relative to control condition Data are presented as fold changes plusmn SDs Significant differencehypoxia for 24 h (H24) or for 48 h (H48) versus normoxia (N) lowast119875 lt 005

the BRCA2 mRNA expression levels in the same BC celllines under hypoxic conditions using qRT-PCR Moreoverwe assessed BRCA1 andMLH1mRNA expression levels alsoinvolved in DNA repair and for which a downregulationin BC cell lines under hypoxic conditions has been alreadyobserved The qRT-PCR analysis confirmed that hypoxiainduces BRCA2 downregulation in all three BC cell linesused In particular BRCA2 downregulation was more pro-nounced in MDA-MB-231 cells (01-fold in both conditions)in MCF-7 cells the reduction was 04-fold after 24 h ofhypoxia and 03-fold after 48 h of hypoxia exposure Theseresults are comparable to those obtained in SKBr3 cells (03-fold at 24 h 02-fold at 48 h) (Figure 3(b)) As previouslyreported we also observed a downregulation of BRCA1 andMLH1 mRNA levels under hypoxic conditions (Figures 3(a)and 3(c)) In particularBRCA1was 02-fold 04-fold and 03-fold after 24 hours of hypoxia in MCF-7 SKBr3 and MDA-MB-231 respectively and 04-fold 04-fold and 02-fold after48 hours of hypoxia in MCF-7 SKBr3 and MDA-MB-231respectively (Figure 3(a)) MLH1 was 04-fold 08-fold and05-fold after 24 hours of hypoxia in MCF-7 SKBr3 andMDA-MB-231 respectively and 05-fold 075-fold and 06-fold after 48 hours of hypoxia in MCF-7 SKBr3 and MDA-MB-231 respectively (Figure 3(c))

Interestingly in parallel to BRCA2 mRNA downregu-lation we observed through western blot (WB) analysisa reduction of BRCA2 protein levels in MCF-7 SKBr3and MDA-MB-231 cells after 24 h and 48 h of hypoxiacompared to control condition (normoxia) (Figure 4) HIF-1120572 protein levels were also evaluated At the same timeBC cells were treated with dimethyloxalylglycine (DMOG)a cell-permeable inhibitor of both proline and asparaginylhydroxylases able to induceHIF-1a stabilization in normoxiaThus treatment of MCF-7 MDA-MB-231 and SKBr3 cellswith a PHD inhibitor (for 24 h and 48 h) caused the activationof the HIF-1 pathway in normoxia In fact WB analysisprovided results comparable to those previously describedleading to an increase in HIF-1120572 expression levels andconcomitant reduction of BRCA2 expression in presence ofDMOG (Figure 4) Taken together these data suggest thathypoxia could be the main reason for BRCA2 downregula-tion

4 Discussion

Breast cancer is the most common malignancy of themammary gland and its incidence increases with ageNowadays although oncologists have several available


MCF-7 MDA-MB-231

Hypoxia

DMOG

BRCA2

HIF-1120572

GAPDH

BRCA2

HIF-1120572

GAPDH

SKBr3N H24 H48 N H24 H48 N H24 H48

Figure 4 Effects of hypoxia on BRCA2 and HIF-1120572 proteinexpression levels in MCF-7 SKBr3 and MDA-MB-231 cells Proteinexpression was examined by western blot analysis with the indicatedantibodies using the enhanced chemiluminescence reagent (ECL)The GAPDH house-keeping protein was used as loading control(N = normoxia H24 = hypoxia for 24 h H48 = hypoxia for 48 h)The experimentswere performed at least three different timesMCF-7 SKBr3 and MDA-MB-231 cells were also incubated with 1mMDMOG for 24 h (H24) and 48 h (H48)

options (chemotherapy hormone therapy and biologicagents such as antiangiogenic and anti-HER2 drugs) BC isstill responsible for a significant percentage of cancer deathsin women [1 36] 80ndash85 of breast cancer is sporadic while15ndash20 shows a familial history About 5ndash10 of cancerscan be attributed to two autosomal dominant genes withhigh penetrance BRCA1 and BRCA2 BRCA2 is a proteinof about 3000 amino acids which can bind directly toDNA by helix-loop-helix domain This protein is importantwith BRCA1 in homologous recombination repair (HRR)mechanism of double-strand DNA breaks BRCA2 interactsdirectly with RAD51 at the C-terminal region and seems tobe also involved in its transport to the nucleus

Hypoxia is a typical feature of microenvironment ofseveral solid tumors and is associated with poor prognosisin several cancer types including BC [37] Under conditionsof severe hypoxia several cancer cell lines exhibit geneticinstability showing downregulation of MLH1 and MSH2expressions in a p53- and HIF-1120572-dependent manner Sincetumor hypoxia is known to be an important factor for theexpression of many genes involved in tumorigenesis cellcycle regulation and genetic instability [38] we performeda microarray-based gene expression analysis in order todetermine different expression profiles inMCF-7 MDA-MB-231 and SKBr3 BC cells in response to hypoxia for 24 h and48 h respectively In addition cell proliferation assays byBrdU allowed us to hypothesize that hypoxia could negativelyregulate the BC cell growth in short term in vitro studiesThismicroarray study allowed us to identify a set of 270 DEGsin hypoxia (81 downregulated and 189 upregulated) commonto three BC cell lines (fold change gt |05| and 119875 lt 005)Our attention was focused mainly on the genes showing asignificant variation in expression levels involved in prolif-eration cell cycle progression and regulation mitosis DDRmechanisms and response to hypoxia After hypoxia for

24 h and 48 h the following main molecular pathways werefound altered in BC cell lines DNA damage repair cell cycleregulation HIF-1120572 network and mitosis control Amongthe DEGs involved in proliferation and cell cycle controlCDKN1A (p21Cip1) that can elicit the G1S checkpoint wasupregulated while genes that further determine entry fromG1 to S-phase including CDC6 CCND1 CCNE2 and CDK2were decreased in expression Concomitant with decreasedG1 to S-phase progression a reduced expression of genesthat regulate the passage of cells through G2M includingCCNA2 CCNB1 CCNB2 CDC2 (CDK1) and CDC25C wasalso found in MCF-7 and SKBr3 cells exposed to hypoxiafor 24 h and 48 h and in MDA-MB-231 cells under hypoxiafor 24 h In addition among the DEGs involved in HIF-1a network VEGF-A SLC2A1 JUN FOS and other genesencoding for glycolytic enzymes (PGK1 PFKFB3 HK2 andALDOA) were upregulated in all three analyzed BC cell linesFinally microarray analysis showed a significant downregu-lation of expression of the DEGs involved in DNA damagerepair (DDR) pathways mismatch repair (MMR) nucleotideexcision repair (NER) nonhomologous end-joining (NHEJ)and homologous recombination repair (HRR) The mostsignificant gene expression variations were observed in theHRR pathway In particular in HRR we found a significantdecrease in expression levels of BRCA1 BRCA2 RAD51 andRAD50 Also other genes involved inMMRmechanismwerefound downregulated (MLH1MSH2 andMSH6)

In a previous work Meng et al reported that hypoxiadownregulates the expression of DNA double-strand break(DNA-dsb) repair genes involved in HHR mechanismincluding BRCA1 and BRCA2 in prostate cancer cell lines[39]

For the first time our microarray study showed thathypoxia induces a noteworthy downregulation of BRCA2expression that could involve an important pathophysiologi-cal role in BC Quantitative real-time PCR and western blotanalyses confirmed microarray results BRCA2 downregu-lation by hypoxia may represent an interesting mechanismof functional BRCA inactivation in the absence of geneticmutations

5 Conclusions

Since BRCA2 is an important regulator of homologousrecombination process in mammalian cells its downregula-tion could play a critical role inDNAdamage repair providinginnovative approaches for the development of novel possibletherapeutic strategies against BC These findings may pro-vide new insights into the mechanisms underlying geneticinstability mediated by hypoxia and BRCA involvement insporadic breast cancers However further studies will beneeded to better understand the mechanisms underlying thehypoxia-induced BRCA downregulation

Conflict of Interests

The authors declare no conflict of interests


Authorsrsquo Contribution

Daniele Fanale and Viviana Bazan contributed equally to thiswork

References

[1] C Desantis R Siegel P Bandi and A Jemal ldquoBreast cancerstatistics 2011rdquo CA Cancer Journal for Clinicians vol 61 no 6pp 409ndash418 2011

[2] P Apostolou and F Fostira ldquoHereditary breast cancer the eraof new susceptibility genesrdquo BioMed Research International vol2013 Article ID 747318 11 pages 2013

[3] N Mavaddat A C Antoniou D F Easton and M Garcia-Closas ldquoGenetic susceptibility to breast cancerrdquo MolecularOncology vol 4 no 3 pp 174ndash191 2010

[4] C A Wilson L Ramos M R Villasenor et al ldquoLocalization ofhuman BRCA1 and its loss in high-grade non-inherited breastcarcinomasrdquo Nature Genetics vol 21 no 2 pp 236ndash240 1999

[5] V Birgisdottir O A Stefansson S K Bodvarsdottir HHilmarsdottir J G Jonasson and J E Eyfjord ldquoEpigeneticsilencing and deletion of the BRCA1 gene in sporadic breastcancerrdquo Breast Cancer Research vol 8 no 4 article R38 2006

[6] C G Murphy andM E Moynahan ldquoBRCA gene structure andfunction in tumor suppression a repair-centric perspectiverdquoCancer Journal vol 16 no 1 pp 39ndash47 2010

[7] M Jasin ldquoHomologous repair of DNA damage and tumorigen-esis the BRCA connectionrdquo Oncogene vol 21 no 58 pp 8981ndash8993 2002

[8] R S Bindra and PM Glazer ldquoGenetic instability and the tumormicroenvironment towards the concept of microenvironment-induced mutagenesisrdquo Mutation Research vol 569 no 1-2 pp75ndash85 2005

[9] R Fan T S Kumaravel F Jalali P Marrano J A Squireand R G Bristow ldquoDefective DNA strand break repair afterDNA damage in prostate cancer cells implications for geneticinstability and prostate cancer progressionrdquo Cancer Researchvol 64 no 23 pp 8526ndash8533 2004

[10] M Koshiji K K-W To S Hammer et al ldquoHIF-1120572 inducesgenetic instability by transcriptionally downregulating MutS120572expressionrdquoMolecular Cell vol 17 no 6 pp 793ndash803 2005

[11] T Y Reynolds S Rockwell and P M Glazer ldquoGenetic instabil-ity induced by the tumor microenvironmentrdquo Cancer Researchvol 56 no 24 pp 5754ndash5757 1996

[12] E M Hammond M J Dorie and A J Giaccia ldquoATRATMtargets are phosphorylated by ATR in response to hypoxiaand ATM in response to reoxygenationrdquo Journal of BiologicalChemistry vol 278 no 14 pp 12207ndash12213 2003

[13] A Coquelle F Toledo S Stern A Bieth and M DebatisseldquoA new role for hypoxia in tumor progression induction offragile site triggering genomic rearrangements and formationof complex DMs and HSRsrdquo Molecular Cell vol 2 no 2 pp259ndash265 1998

[14] JW Evans S B Chernikova L A Kachnic et al ldquoHomologousrecombination is the principal pathway for the repair of DNAdamage induced by tirapazamine in mammalian cellsrdquo CancerResearch vol 68 no 1 pp 257ndash265 2008

[15] G L Semenza ldquoCancer-stromal cell interactions mediated byhypoxia-inducible factors promote angiogenesis lymphangio-genesis andmetastasisrdquoOncogene vol 32 pp 4057ndash4063 2012

[16] H H W Chen W-C Su P-W Lin H-R Guo and W-Y LeeldquoHypoxia-inducible factor-1120572 correlates with MET and metas-tasis in node-negative breast cancerrdquo Breast Cancer Researchand Treatment vol 103 no 2 pp 167ndash175 2007

[17] A Ben Lassoued N Beaufils J P Dales and J GabertldquoHypoxia-inducible factor-1alpha as prognostic markerrdquo ExpertOpinion on Medical Diagnostics vol 7 no 1 pp 53ndash70 2013

[18] M Nordsmark S M Bentzen V Rudat et al ldquoPrognosticvalue of tumor oxygenation in 397 head and neck tumors afterprimary radiation therapyAn internationalmulti-center studyrdquoRadiotherapy and Oncology vol 77 no 1 pp 18ndash24 2005

[19] J-P Dales N Beaufils M Silvy et al ldquoHypoxia induciblefactor 1120572 gene (HIF-1120572) splice variants potential prognosticbiomarkers in breast cancerrdquo BMC Medicine vol 8 article 442010

[20] N Chan M Milosevic and R G Bristow ldquoTumor hypoxiaDNArepair and prostate cancer regression new targets andnewtherapiesrdquo Future Oncology vol 3 no 3 pp 329ndash341 2007

[21] N Chaudary and R P Hill ldquoHypoxia and metastasisrdquo ClinicalCancer Research vol 13 no 7 pp 1947ndash1949 2007

[22] M Yoshimura S Itasaka H Harada and M HiraokaldquoMicroenvironment and radiation therapyrdquo BioMed ResearchInternational vol 2013 Article ID 685308 13 pages 2013

[23] P Van Der Groep A Bouter F H Menko E Van Der Walland P J Van Diest ldquoHigh frequency of HIF-1120572 overexpressionin BRCA1 related breast cancerrdquo Breast Cancer Research andTreatment vol 111 no 3 pp 475ndash480 2008

[24] P Vaupel ldquoThe role of hypoxia-induced factors in tumorprogressionrdquo Oncologist vol 9 supplement 5 pp 10ndash17 2004

[25] G L Semenza ldquoHypoxia clonal selection and the role of HIF-1 in tumor progressionrdquo Critical Reviews in Biochemistry andMolecular Biology vol 35 no 2 pp 71ndash103 2000

[26] V T Mihaylova R S Bindra J Yuan et al ldquoDecreasedexpression of the DNA mismatch repair gene Mlh1 underhypoxic stress in mammalian cellsrdquo Molecular and CellularBiology vol 23 no 9 pp 3265ndash3273 2003

[27] R S Bindra S L Gibson A Meng et al ldquoHypoxia-induced down-regulation of BRCA1 expression by E2Fsrdquo Can-cer Research vol 65 no 24 pp 11597ndash11604 2005

[28] M Federico C E Symonds L Bagella et al ldquoR-Roscovitine(Seliciclib) prevents DNA damage-induced cyclin A1 upregu-lation and hinders non-homologous end-joining (NHEJ) DNArepairrdquoMolecular Cancer vol 9 article 208 2010

[29] L Gautier L Cope B M Bolstad and R A Irizarry ldquoaffymdashanalysis of Affymetrix GeneChip data at the probe levelrdquoBioinformatics vol 20 no 3 pp 307ndash315 2004

[30] RA Irizarry BHobbs F Collin et al ldquoExploration normaliza-tion and summaries of high density oligonucleotide array probelevel datardquo Biostatistics vol 4 no 2 pp 249ndash264 2003

[31] G K Smyth ldquoLinear models and empirical bayes methods forassessing differential expression in microarray experimentsrdquoStatistical Applications in Genetics and Molecular Biology vol3 no 1 article 3 2004

[32] Y Benjamini D Drai G Elmer N Kafkafi and I Golani ldquoCon-trolling the false discovery rate in behavior genetics researchrdquoBehavioural Brain Research vol 125 no 1-2 pp 279ndash284 2001

[33] J M Wettenhall K M Simpson K Satterley and G K SmythldquoaffylmGUI a graphical user interface for linear modeling ofsingle channel microarray datardquo Bioinformatics vol 22 no 7pp 897ndash899 2006


[34] M Kanehisa and S Goto ldquoKEGG kyoto encyclopedia of genesand genomesrdquo Nucleic Acids Research vol 28 no 1 pp 27ndash302000

[35] R Avni B Cohen and M Neeman ldquoHypoxic stress andcancer imaging the axis of evil in tumor metastasisrdquo NMR inBiomedicine vol 24 no 6 pp 569ndash581 2011

[36] P van der Groep P J van Diest Y H Smolders et al ldquoHIF-1alpha overexpression in ductal carcinoma in situ of the breastin BRCA1 and BRCA2 mutation carriersrdquo PLoS One vol 8 no2 Article ID e56055 2013

[37] E Favaro S Lord A L Harris and F M Buffa ldquoGeneexpression and hypoxia in breast cancerrdquoGenomeMedicine vol3 article 55 no 8 2011

[38] M Kunz and S M Ibrahim ldquoMolecular responses to hypoxiain tumor cellsrdquoMolecular Cancer vol 2 article 23 2003

[39] A XMeng F Jalali A Cuddihy et al ldquoHypoxia down-regulatesDNA double strand break repair gene expression in prostatecancer cellsrdquo Radiotherapy and Oncology vol 76 no 2 pp 168ndash176 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

ObesityJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawi Publishing Corporation httpwwwhindawicom Volume 2013

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of

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Evidence-Based Complementary and Alternative Medicine

Volume 2013Hindawi Publishing Corporationhttpwwwhindawicom

OphthalmologyJournal of



Computational and Mathematical Methods in Medicine

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BioMed Research International


International Journal of

EndocrinologyHindawi Publishing Corporationhttpwwwhindawicom

Volume 2013

ISRN Addiction


Hindawi Publishing Corporationhttpwwwhindawicom

OncologyJournal of

Volume 2013

ISRN AIDS



Oxidative Medicine and Cellular Longevity

Diabetes ResearchJournal of


Clinical ampDevelopmentalImmunology


Volume 2013


Gastroenterology Research and Practice


ISRN Biomarkers

PPARRe sea rch



regions of target genes in order to promote tumor survivalinvasion andmetastasis [22ndash25] Recently it has been shownthat under severe hypoxia conditions the mismatch repair(MMR) genes MLH1 and MSH2 are downregulated in p53-and HIF-1a-dependent way in many tumor cell lines thusinducing genetic instability [10 26] Furthermore Mihaylovaet al reported that cells exposed to hypoxia- and low pHwerefound to have a diminished capacity for DNA repair com-pared with controls [26] In 2005 an important study showedthat hypoxia-induced decreases in BRCA1 expression and theconsequent suppression of homologous recombination maylead to genetic instability [27] However nothing is yet knownabout the involvement of BRCA2 in hypoxic conditions inbreast cancer

Here we aim to analyze gene expression in breast cancercell lines exposed to hypoxic condition with a focus on genesinvolved in DNA damage repair (DDR) especially BRCA2

2 Materials and Methods

21 Cell Cultures Human BC cell lines MCF-7 MDA-MB-231 and SKBr3 purchased from the American TypeCulture Collection (Rockville MD USA) were cultured inDulbeccorsquos Modified Eagle Medium (DMEM F12) supple-mented with 10 fetal bovine serum (FBS) and antibiotics(100UmL penicillin and 50mgmL streptomycin) (Invitro-gen Carlsbad CA USA) Eighty percent of confluent celllines were cultured in normoxic atmosphere of 16 O

2 79

N2 and 5 CO

2(by volume) for 24 h Then medium was

changed and cells were further cultured under normoxiaor hypoxia (3 O

2 87 N

2 5 CO

2 by volume) at two

different time-points 24 h and 48 h Furthermore cells wereincubated in the absence (normoxia) or presence (hypoxia) ofHIF hydroxylase inhibitor dimethyloxalylglycine (DMOG)(Sigma-Aldrich St Louis MO USA) for 24 h and 48 h atfinal concentration of 1mM

22 Cell Proliferation Assay To analyze cell proliferativeactivity a 5-bromo-21015840-deoxyuridine (BrdU) assay was per-formed using BrdU Cell Proliferation Assay Kit accordingto the manufacturerrsquos instructions (Cell Signaling Technol-ogy Euroclone Milano Italy) Cells were seeded at 1 times104 cellswell in a 96-well plate and incubated overnight To

assess proliferative activity under hypoxia cells were directlyincubated under normoxia and hypoxia (for 24 48 72 and96 hours) Ten 120583MBrdU was added to the plate and the cellswere incubated for 4 hours The absorbance at a wavelengthof 450 nm was measured using an ELISA microplate readerAll experiments were performed in triplicate

23 Microarray Analysis Microarray analysis was performedas previously described [28] Total RNA was extractedaccording to the manufacturerrsquos protocol (Affymetrix SantaClara CA USA) Fragmented cRNA was hybridized usinga human oligonucleotide array U133 Plus 20 (GenechipAffymetrix Santa Clara CA USA) Washing and stain-ing were performed through Affymetrix GeneChip FluidicStation 450 Probe arrays were scanned using Affymetrix

GeneChipScanner3000 G7enabled for high-resolution scan-ning Two biological replicates were performed for eachexperimental condition Images were extracted with theGeneChip Operating Software (Affymetrix GCOS v14)Quality control of the arrays was performed using theAffyQCReport software [29]

24 Statistical Analysis For statistical analysis the back-ground subtraction and normalization of probe set intensitieswere performed using themethod of robustmultiarray analy-sis (RMA) described by Irizarry et al [30] To identify DEGsgene expression intensity was compared using a moderated119905-test and a Bayes smoothing approach developed for a lownumber of replicates [31] To correct for the effect of multipletesting the false discovery rate was estimated from 119875 valuesderived from the moderated 119905-test statistics [32]The analysiswas performed using the affylmGUIGraphical User Interfacefor the limma microarray package (Bioconductor Software)[33] Significant differences were determined by Studentrsquos 119905-test 119875 value lt005 was considered to be statistically signifi-cant

25 Microarray Data Analysis Hierarchical cluster and heatmap analyses (HCA) were performed using the MultiExper-iment Viewer (MeV v48) program of TM4 Microarray Soft-ware Suite Gene Set Analysis Toolkit was used to investigatethe biological significance of a set of genes represented by thespecific expression pattern inDNArepairmechanismsDEGswere analyzed according to predefined pathways annotatedby KEGG [34] and Biocarta bioinformatic resources For anoverrepresented KEGG or Biocarta pathway a cut-off119875 valueof 001 was selected All showed values are in logarithm scale

26 Quantitative Real-Time PCR (qRT-PCR) and RT-PCRTotal cellular RNA was extracted using RNeasy Mini Kit(Qiagen Inc Valencia CA USA)Then RNAwas controlledthrough 2100 Bioanalyzer (Agilent Technologies Santa ClaraCA USA) and quantified through the spectrophotometerNanoDrop ND-1000 (CELBIO) For BRCA1 BRCA2 andMLH1 mRNAs detection 2 ng of total RNA was reversetranscribed into single-stranded cDNA using High CapacitycDNAReverse Transcription Kit (Applied Biosystems FosterCity CA USA) according to vendorrsquos instructions Gene-primers for BRCA1 BRCA2 and MLH1 were purchasedfrom Applied Biosystems (TaqMan gene expression assay)Quantitative real-time PCR (qRT-PCR) was performed withthe ABI PRISM 7900 sequence detection system (AppliedBiosystems Foster City CA USA) using SDS software ver-sion 21 The reactions were performed in triplicate and theresults were normalized using Human 120573-actin PredevelopedTaqMan assay reagents (Applied Biosystems) Changes in thetarget mRNA content were determined using a comparativeCT method (ABI User Bulletin number 2) An average CTvalue for each RNA was obtained for triplicate reactions

27 Western Blotting (WB) Cells were lysed using completeLysis-M reagent set (Roche Mannheim Germany) Proteinconcentration was measured using Quick Start Bradford


5

4

3

2

1

0

0

MCF-724h 48h 72h 96h

(a)

6

5

4

3

2

1

00


(b)

6

5

4

3

2

1

0

0

MDA-MB-231

NormoxiaHypoxia

24h 48h 72h 96h

(c)



3 Results










00 6386284 8069622

MMR

NER

NHEJ

HRR



N H24 H48 N H24 H48 N H24 H48


BRCA1BRCA2








12

1

08

06

04

02

0

lowast


lowastlowast

C C CRela

tive B

RCA1

mRN

A le

vels

BCRA1


(a)

12

1

08

06

04

02

0

lowast

lowastlowast

lowast

lowastlowast

BRCA2

Rela

tive B

RCA2

mRN

A le

vels


(b)

C

12

1

08

06

04

02

0C C C


lowastlowast lowast

lowast

MLH1

Relat

ive M

LH1

mRN

A le

vels

24h 48h 24h 48h 24h 48h

(c)







4 Discussion



MCF-7 MDA-MB-231

Hypoxia

DMOG

BRCA2

HIF-1120572

GAPDH

BRCA2

HIF-1120572

GAPDH

SKBr3N H24 H48 N H24 H48 N H24 H48







5 Conclusions







References











































ObesityJournal of





of

ISRN Anesthesiology








ISRN Allergy






Volume 2013

ISRN Addiction



OncologyJournal of

Volume 2013

ISRN AIDS








Volume 2013




ISRN Biomarkers

PPARRe sea rch



5

4

3

2

1

0

0

MCF-724h 48h 72h 96h

(a)

6

5

4

3

2

1

00


(b)

6

5

4

3

2

1

0

0

MDA-MB-231

NormoxiaHypoxia

24h 48h 72h 96h

(c)



3 Results










00 6386284 8069622

MMR

NER

NHEJ

HRR



N H24 H48 N H24 H48 N H24 H48


BRCA1BRCA2








12

1

08

06

04

02

0

lowast


lowastlowast

C C CRela

tive B

RCA1

mRN

A le

vels

BCRA1


(a)

12

1

08

06

04

02

0

lowast

lowastlowast

lowast

lowastlowast

BRCA2

Rela

tive B

RCA2

mRN

A le

vels


(b)

C

12

1

08

06

04

02

0C C C


lowastlowast lowast

lowast

MLH1

Relat

ive M

LH1

mRN

A le

vels

24h 48h 24h 48h 24h 48h

(c)







4 Discussion



MCF-7 MDA-MB-231

Hypoxia

DMOG

BRCA2

HIF-1120572

GAPDH

BRCA2

HIF-1120572

GAPDH

SKBr3N H24 H48 N H24 H48 N H24 H48







5 Conclusions







References











































ObesityJournal of





of

ISRN Anesthesiology








ISRN Allergy






Volume 2013

ISRN Addiction



OncologyJournal of

Volume 2013

ISRN AIDS








Volume 2013




ISRN Biomarkers

PPARRe sea rch








00 6386284 8069622

MMR

NER

NHEJ

HRR



N H24 H48 N H24 H48 N H24 H48


BRCA1BRCA2








12

1

08

06

04

02

0

lowast


lowastlowast

C C CRela

tive B

RCA1

mRN

A le

vels

BCRA1


(a)

12

1

08

06

04

02

0

lowast

lowastlowast

lowast

lowastlowast

BRCA2

Rela

tive B

RCA2

mRN

A le

vels


(b)

C

12

1

08

06

04

02

0C C C


lowastlowast lowast

lowast

MLH1

Relat

ive M

LH1

mRN

A le

vels

24h 48h 24h 48h 24h 48h

(c)







4 Discussion



MCF-7 MDA-MB-231

Hypoxia

DMOG

BRCA2

HIF-1120572

GAPDH

BRCA2

HIF-1120572

GAPDH

SKBr3N H24 H48 N H24 H48 N H24 H48







5 Conclusions







References











































ObesityJournal of





of

ISRN Anesthesiology








ISRN Allergy






Volume 2013

ISRN Addiction



OncologyJournal of

Volume 2013

ISRN AIDS








Volume 2013




ISRN Biomarkers

PPARRe sea rch



12

1

08

06

04

02

0

lowast


lowastlowast

C C CRela

tive B

RCA1

mRN

A le

vels

BCRA1


(a)

12

1

08

06

04

02

0

lowast

lowastlowast

lowast

lowastlowast

BRCA2

Rela

tive B

RCA2

mRN

A le

vels


(b)

C

12

1

08

06

04

02

0C C C


lowastlowast lowast

lowast

MLH1

Relat

ive M

LH1

mRN

A le

vels

24h 48h 24h 48h 24h 48h

(c)







4 Discussion



MCF-7 MDA-MB-231

Hypoxia

DMOG

BRCA2

HIF-1120572

GAPDH

BRCA2

HIF-1120572

GAPDH

SKBr3N H24 H48 N H24 H48 N H24 H48







5 Conclusions







References











































ObesityJournal of





of

ISRN Anesthesiology








ISRN Allergy






Volume 2013

ISRN Addiction



OncologyJournal of

Volume 2013

ISRN AIDS








Volume 2013




ISRN Biomarkers

PPARRe sea rch



MCF-7 MDA-MB-231

Hypoxia

DMOG

BRCA2

HIF-1120572

GAPDH

BRCA2

HIF-1120572

GAPDH

SKBr3N H24 H48 N H24 H48 N H24 H48







5 Conclusions







References











































ObesityJournal of





of

ISRN Anesthesiology








ISRN Allergy






Volume 2013

ISRN Addiction



OncologyJournal of

Volume 2013

ISRN AIDS








Volume 2013




ISRN Biomarkers

PPARRe sea rch





References











































ObesityJournal of





of

ISRN Anesthesiology








ISRN Allergy






Volume 2013

ISRN Addiction



OncologyJournal of

Volume 2013

ISRN AIDS








Volume 2013




ISRN Biomarkers

PPARRe sea rch











ObesityJournal of





of

ISRN Anesthesiology








ISRN Allergy






Volume 2013

ISRN Addiction



OncologyJournal of

Volume 2013

ISRN AIDS








Volume 2013




ISRN Biomarkers

PPARRe sea rch


hypoxia and human genome stability: downregulation of ... · showed that hypoxia reduces cell...

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