10
Variations in the mRNA expression of poly(ADP-ribose) polymerases, poly(ADP-ribose) glycohydrolase and ADP-ribosylhydrolase 3 in breast tumors and impact on clinical outcome Ivan Bieche 1 , Vincent Pennaneach 2,3 , Keltouma Driouch 1 , Sophie Vacher 1 , Tomasz Zaremba 2,3 , Aur elie Susini 1 , Rosette Lidereau 1 and Janet Hall 2,3 1 Laboratoire d’Oncog en etique, Institut Curie-H^ opital Ren e Huguenin, 92210, Saint-Cloud, France 2 Institut Curie-Recherche, Centre Universitaire, 91405, Orsay Cedex, France 3 Inserm U612, Centre Universitaire, 91405, Orsay Cedex, France In order to assess the variation in expression of poly(ADP-ribose) polymerase (PARP) family members and the hydrolases that degrade the poly(ADP-ribose) polymers they generate and possible associations with classical pathological parameters, includ- ing long-term outcome, the mRNA levels of PARP1, PARP2, PARP3, poly(ADP-ribose) glycohydrolase (PARG) and ADP-ribosylhy- drolase 3 (ARH3) were examined using quantitative reverse transcription polymerase chain reaction in 443 unilateral invasive breast cancers and linked to hormonal status, tumor proliferation and clinical outcome. PARP1 mRNA levels were the highest among these five genes in both normal and tumor tissues, with a 2.45-fold higher median level in tumors compared to normal tissues. Tumors (34.1%) showed PARP1 overexpression (>3 fold relative to normal breast tissues) compared to underexpres- sion (<0.33 fold) in only 0.5%. This overexpression was seen in all breast tumor subgroups, with the highest fraction (51%) seen in the HR-positive/ERBB2-positive subgroup and was not highly associated with any other classical predictive factors. No correlation was seen between PARP1 mRNA and PARP-1 protein levels in a subset of 31 tumors. PARP3 was underexpressed in 10.4% of tumors, more frequently in the HR-negative tumors (25.4%) than the HR-positive tumors (5.9%). This PARP3 underexpression was mutually exclusive with a PARP1 overexpression. PARP2 levels were unchanged between normal and tumor tissues and few tumors showed overexpression of PARG (3.8%) or ARH3 (3.4%). Within the subgroup of triple negative tumors, PARG mRNA levels below the median were associated with a higher risk of developing metastases (p 5 0.039) raising the possibility this might be marker of clinical outcome. Gene expression analysis carries the potential to dissect the molecular heterogeneity of tumors and can distinguish breast cancer subclasses on the basis of differences in their gene expression profiles as demonstrated by the seminal work of Perou et al. 1 However, there is a substantial body of evidence that shows that within different classes, there is still some considerable molecular and clinical heterogeneity that needs to be investigated to identify biomarkers of clinical outcome and so that patients can be treated with the most appropriate and adapted protocols. Indeed the future development of pre- cision therapy, in which subsets of patients can be easily identified as having a disease with distinct genetic profiles, Key words: PARP, PARG, ARH3, breast cancer Abbreviations: ARH3: ADP-ribosylhydrolase 3; DSB: double strand break; ER: estrogen receptor; HR: hormone receptor; IR: ionizing radiation; MFS: metastasis free survival; NHEJ: nonhomologous end-joining; PAR: polymers of ADP-ribose; PARP: poly(ADP-ribose) polymerase; PARG: poly(ADP-ribose) glycohydrolase; PR: progesterone receptor; qRT-PCR: quantitative reverse transcription–polymerase chain reaction; SB: strand break; SSB: single strand break; TBP: TATA box-binding protein; TNBC: triple-negative breast cancer Additional Supporting Information may be found in the online version of this article. Tomasz Zaremba’s present address is: AstraZeneca Pharma Poland Sp. z o.o.ul., Poste R pu 18, Warsaw, 02-676, Poland Grant sponsors: Fondation Pierre-Gilles de Gennes pour la Recherche (FPGG), Institut Curie’s Postdoctoral Fellowship Scheme, Institut Curie (Medical Section), The Comit eD epartemental des Hauts-de-Seine de la Ligue Nationale Contre le Cancer, Association d’Aide a la Recherche Canc erologique de Saint-Cloud (ARCS), INSERM, Institut Curie (Research Section) DOI: 10.1002/ijc.28304 History: Received 17 Feb 2013; Accepted 6 May 2013; Online 4 Jun 2013 Correspondence to: Janet Hall, Inserm U612, Institut Curie-Recherche, B^ at. 110-112, Centre Universitaire, 91405 Orsay Cedex, France, Tel.: 133-0169863061, Fax: 133-0169075327, E-mail: [email protected] Cancer Cell Biology Int. J. Cancer: 133, 2791–2800 (2013) V C 2013 UICC International Journal of Cancer IJC

Variations in the mRNA expression of poly(ADP-ribose) polymerases , poly(ADP-ribose) glycohydrolase and ADP-ribosylhydrolase 3 in breast tumors and impact on clinical outcome

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Page 1: Variations in the mRNA expression of poly(ADP-ribose) polymerases , poly(ADP-ribose) glycohydrolase and ADP-ribosylhydrolase 3 in breast tumors and impact on clinical outcome

Variations in the mRNA expression of poly(ADP-ribose)polymerases, poly(ADP-ribose) glycohydrolase andADP-ribosylhydrolase 3 in breast tumors and impact onclinical outcome

Ivan Bieche1, Vincent Pennaneach2,3, Keltouma Driouch1, Sophie Vacher1, Tomasz Zaremba2,3, Aur�elie Susini1,

Rosette Lidereau1 and Janet Hall2,3

1 Laboratoire d’Oncog�en�etique, Institut Curie-Hopital Ren�e Huguenin, 92210, Saint-Cloud, France2 Institut Curie-Recherche, Centre Universitaire, 91405, Orsay Cedex, France3 Inserm U612, Centre Universitaire, 91405, Orsay Cedex, France

In order to assess the variation in expression of poly(ADP-ribose) polymerase (PARP) family members and the hydrolases that

degrade the poly(ADP-ribose) polymers they generate and possible associations with classical pathological parameters, includ-

ing long-term outcome, the mRNA levels of PARP1, PARP2, PARP3, poly(ADP-ribose) glycohydrolase (PARG) and ADP-ribosylhy-

drolase 3 (ARH3) were examined using quantitative reverse transcription polymerase chain reaction in 443 unilateral invasive

breast cancers and linked to hormonal status, tumor proliferation and clinical outcome. PARP1 mRNA levels were the highest

among these five genes in both normal and tumor tissues, with a 2.45-fold higher median level in tumors compared to normal

tissues. Tumors (34.1%) showed PARP1 overexpression (>3 fold relative to normal breast tissues) compared to underexpres-

sion (<0.33 fold) in only 0.5%. This overexpression was seen in all breast tumor subgroups, with the highest fraction (51%)

seen in the HR-positive/ERBB2-positive subgroup and was not highly associated with any other classical predictive factors. No

correlation was seen between PARP1 mRNA and PARP-1 protein levels in a subset of 31 tumors. PARP3 was underexpressed

in 10.4% of tumors, more frequently in the HR-negative tumors (25.4%) than the HR-positive tumors (5.9%). This PARP3

underexpression was mutually exclusive with a PARP1 overexpression. PARP2 levels were unchanged between normal and

tumor tissues and few tumors showed overexpression of PARG (3.8%) or ARH3 (3.4%). Within the subgroup of triple negative

tumors, PARG mRNA levels below the median were associated with a higher risk of developing metastases (p 5 0.039) raising

the possibility this might be marker of clinical outcome.

Gene expression analysis carries the potential to dissect themolecular heterogeneity of tumors and can distinguish breastcancer subclasses on the basis of differences in their geneexpression profiles as demonstrated by the seminal work ofPerou et al.1 However, there is a substantial body of evidencethat shows that within different classes, there is still some

considerable molecular and clinical heterogeneity that needsto be investigated to identify biomarkers of clinical outcomeand so that patients can be treated with the most appropriateand adapted protocols. Indeed the future development of pre-cision therapy, in which subsets of patients can be easilyidentified as having a disease with distinct genetic profiles,

Key words: PARP, PARG, ARH3, breast cancer

Abbreviations: ARH3: ADP-ribosylhydrolase 3; DSB: double strand break; ER: estrogen receptor; HR: hormone receptor; IR: ionizing

radiation; MFS: metastasis free survival; NHEJ: nonhomologous end-joining; PAR: polymers of ADP-ribose; PARP: poly(ADP-ribose)

polymerase; PARG: poly(ADP-ribose) glycohydrolase; PR: progesterone receptor; qRT-PCR: quantitative reverse transcription–polymerase

chain reaction; SB: strand break; SSB: single strand break; TBP: TATA box-binding protein; TNBC: triple-negative breast cancer

Additional Supporting Information may be found in the online version of this article.

Tomasz Zaremba’s present address is: AstraZeneca Pharma Poland Sp. z o.o.ul., PosteRpu 18, Warsaw, 02-676, Poland

Grant sponsors: Fondation Pierre-Gilles de Gennes pour la Recherche (FPGG), Institut Curie’s Postdoctoral Fellowship Scheme, Institut

Curie (Medical Section), The Comit�e D�epartemental des Hauts-de-Seine de la Ligue Nationale Contre le Cancer, Association d’Aide �a la

Recherche Canc�erologique de Saint-Cloud (ARCS), INSERM, Institut Curie (Research Section)

DOI: 10.1002/ijc.28304

History: Received 17 Feb 2013; Accepted 6 May 2013; Online 4 Jun 2013

Correspondence to: Janet Hall, Inserm U612, Institut Curie-Recherche, Bat. 110-112, Centre Universitaire, 91405 Orsay Cedex, France, Tel.:

133-0169863061, Fax: 133-0169075327, E-mail: [email protected]

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cerCellBiology

Int. J. Cancer: 133, 2791–2800 (2013) VC 2013 UICC

International Journal of Cancer

IJC

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will rely on the identification and validation of biomarkersthat will predict cancer cell sensitivity or resistance to thedrugs that target those changes.

It has been known for over 30 years that poly(ADP)ribosepolymerase (PARP) inhibitors can be used in combinationwith DNA damaging agents such as alkylating and platinumagents, topoisomerase poisons or ionizing radiation exposureto increase cytotoxicity.2 Poly(ADP)ribosylation is ubiquitousin mammalian cells and modulates many cellular responsesincluding transcription, regulation of chromatin dynamics,differentiation and cell death,3 in addition to playing a keyrole in the response to DNA damage. This posttranslationalmodification is carried out by several members of the PARPfamily, of which PARP-1 is the most prevalent. PARP-1,PARP-2 and PARP-3 are activated by DNA strand breaks(SBs) and play a role in DNA single and/or double SBrepair.4,5 Once activated, the enzymes rapidly catalyze thetransfer of successive ADP-ribose moieties from NAD1 toacceptor proteins resulting in the attachment of linear orbranched polymers of ADP-ribose (PAR). In addition to thisdirect covalent modification, some proteins have a high affin-ity for the polymers themselves and this is exploited in somesettings, for instance in DNA repair, for the control of theirlocalization and function. The status quo is restored by theactivity of poly(ADP-ribose) glycohydrolase (PARG) andADP-ribosylhydrolase 3 (ARH3, also known as ADPRHL2)that cleave the polymers chains.6

In order to investigate whether variation in the expressionof these genes involved in PAR metabolism in response toDNA damaging agents is associated with classical pathologi-cal parameters, including long-term outcome, we haveassessed the mRNA expression patterns of PARP1, PARP2,PARP3, PARG and ARH3 in a series of 443 patients withunilateral invasive breast cancer. The transcript levels of thefive genes, as well as that of the cell proliferation markerMKI-67, were determined using real time quantitative reversetranscription-polymerase chain reaction (qRT-PCR). For asubset of tumors where frozen tissue was available, PARP-1protein levels were assessed by Western blot and the correla-tion between protein and mRNA expression analyzed.

Material and MethodsPatients and samples

The study was conducted in accordance with national lawand institutional guidelines under a protocol approved by the

local Ethics Committee. The study included 443 primary uni-lateral invasive primary breast tumors excised from patientswho were referred for surgery to Institut Curie’s Ren�eHuguenin Hospital (Saint-Cloud, France) between 1978 and2008. Immediately following surgery, the tumor samples werestored in liquid nitrogen until RNA extraction. The sampleswere examined histological for the presence of tumor cells. Atumor sample was considered suitable for this study if theproportion of tumor cells was more than 70%.

The patients (mean age, 61.7 years; range, 31–91) met thefollowing criteria: primary unilateral nonmetastatic breastcarcinoma on which complete clinical, histological and bio-logical data were available; no radiotherapy or chemotherapybefore surgery; and full follow-up at Institut Curie/Ren�eHuguenin Hospital (Supporting Information Table 1S).

Two hundred eighty-three patients (63.9%) underwentmodified radical mastectomy and 160 (36.1%) had breast-conserving surgery with locoregional radiotherapy. Patientsunderwent physical examinations and routine chest radiogra-phy every 3 months for 2 years, then annually. Mammo-grams were done annually. Three hundred fifty-eight patientsreceived adjuvant therapy, consisting of chemotherapy alonein 90 cases, hormone therapy alone in 175 cases and combi-nation of both treatments in 93 cases. The histological typeand the number of positive axillary nodes were established atthe time of surgery. The malignancy of infiltrating carcino-mas was scored according to Bloom and Richardson’s histo-prognostic system.7

Estrogen receptor (ER), progesterone receptor (PR) andERBB2 status was determined at the protein level by biochem-ical methods (dextran-coated charcoal method, enzymaticimmunoassay or immunohistochemistry) and confirmed byERa, PR and ERBB2 real-time qRT-PCR assays.8,9 Using hor-mone receptor (HR)(ERa and PR) and ERBB2 status, we sub-divided our study population (n 5 443) into four subgroups:HR positive (HR1) (ER positive (ER1) or PR positive(PR1))/ERBB21 (n 5 51), HR1 (ER1 or PR1)/ERBB2negative (2) (n 5 283), HR2 (ER2 and PR2)/ERBB21 (n5 46) and HR2 (ER2 and PR2)/ERBB22 (n 5 63). Stand-ard prognostic factors of this tumor set are presented in Sup-porting Information Table 1S. The median of follow-up was 9years (range, 6 months to 29 years). Tumors of 168 patientsdeveloped metastases before 180 months.

Ten specimens of adjacent normal breast tissue from10 breast cancer patients or normal breast tissue from

What’s new?

Gene expression profiling to distinguish between different types of breast cancers stands to bring major benefits to the clini-

cal management of the disease. However, progress toward this end has been hindered by the realization that substantial het-

erogeneity exists among breast tumors, even within subclasses. This investigation advances knowledge of gene expression

profiles in breast cancer by revealing that PARP1 mRNA overexpression is common in all subclasses and is mutually exclusive

with PARP3 mRNA underexpression. In triple negative tumors, low PARG mRNA levels were linked to increased risk of metasta-

sis, suggesting that it may be a marker of clinical outcome.

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women undergoing cosmetic breast surgery were used assources of RNA for comparative purposes.

RNA extraction

Total RNA was extracted from breast samples by using theacid-phenol guanidinum method. The quality of the RNAsamples was determined by electrophoresis through agarosegels and staining with ethidium bromide. The 18S and 28SRNA bands were visualized under ultraviolet light.

Quantitative real-time-PCR

Primers for TBP (Genbank accession NM_003194), encodingfor the TATA box-binding protein (a component of theDNA-binding protein complex TFIID) and used as an endog-enous RNA control, and the five target genes were chosenwith the assistance of the computer programs Oligo 5.0(National Biosciences, Plymouth, MN, USA). We conductedsearches in dbEST and nr databases to confirm the genespecificity of the nucleotide sequences chosen for the primersand the absence of single nucleotide polymorphisms. Thenucleotide sequences of the oligonucleotide hybridization pri-mers of TBP, MKI67 and the five targets genes are shown inSupporting Information Table 2S. To avoid amplification ofcontaminating genomic DNA, one of the two primers wasplaced at the junction between two exons and in the case ofPARG were placed in two different exons separated by a 9-kbintron. Agarose gel electrophoresis was used to verify thespecificity of PCR amplicons. The cDNA synthesis and PCRreaction conditions are described elsewhere10 and the effi-ciency of PCR (E) was checked to be higher than 90% for thedifferent target gene qRT-PCR assays (using the formula E 5

101/jmj21, where m is the slope of a standard curve con-structed with fourfold serial dilutions of cDNA from a poolof breast tissues.

Using this experimental set-up, quantitative values for thedifferent transcript levels were obtained from the cycle num-ber (Ct value) at which the increase in fluorescent signalassociated with an exponential growth of PCR products startsto be detected by the laser detector of the ABI Prism 7900Sequence Detection System (Perkin-Elmer Applied Biosys-tems, Foster City, CA, USA) using the PE Biosystems analysissoftware according to the manufacturer’s manuals. The Ctvalues from the qRT-PCR analysis shows an inverse relation-ship with the level of transcript.

Results, expressed as N-fold differences in target geneexpression relative to the TBP gene, termed “Ntarget,” weredetermined by the formula: Ntarget 5 2DCtsample, where DCtvalue of the sample was determined by subtracting the aver-age Ct value of the target gene from the average Ct value ofthe TBP gene. The Ntarget values of the samples were subse-quently normalized such that the median of the 10 normalbreast tissue Ntarget values was 1.

Tumor biopsy fragments were lysed in buffer containing50 mM Tris pH6.8, 2% (w/v) SDS, 5% (v/v) glycerol, 2 mMDTT, 2.5 mM EDTA, 2.5 mM EGTA, phosphatase inhibitors

(Sigma–Aldrich, St. Louis, MO, USA), protease inhibitors(Sigma-Aldrich, St.Louis, USA), 4 mM sodium orthovanadateand 20 mM sodium fluoride. Protein samples in Laemlibuffer were loaded onto a 10% sodium dodecyl sulfate-polyacrylamide gel for electrophoresis under reducing condi-tions and then transferred to nitrocellulose membrane. Pro-teins of interest were revealed after overnight incubation withthe monoclonal anti-PARP-1 (clone C2-10, 1:2000, Trevigen,Gathersburg, MD, USA) antibody that detects the 114-kDaPARP-1 holoenzyme, the 85 kDa apoptosis related and thenecrosis-related 50/62/74 kDa cleavage fragments at 4�C.This step was followed by incubation with IRDye 680RD-conjugated anti-mouse secondary antibody (1:10,000, LI-CORBiosciences, Lincoln, NE, USA), and signal detection andquantification of the 114 kDa PARP-1 holoenzyme with theOdyssey imaging system (LI-COR Biosciences, Lincoln, NE,USA). Actin levels, detected using an anti-actin rabbit poly-clonal antibody (1:5000, Sigma-Aldrich, St. Louis, MO, USA),were used to control protein loading.

Statistical analysis

The distributions of the gene mRNA levels were characterizedby their median values and ranges. Relationships betweenmRNA levels of the different target genes and comparisonbetween the target gene mRNA levels and the clinical parame-ters were estimated using nonparametric tests: the Mann-Whitney U test (link between one qualitative parameter andone quantitative parameter) and the Spearman rank correla-tion test (link between two quantitative parameters). Differen-ces between the two populations were judged significant atconfidence levels greater than 95% (p < 0.05). Metastasis-freesurvival (MFS) was determined as the interval between diagno-sis and detection of the first relapse metastases. Survival distri-butions were estimated by the Kaplan-Meier method,11 and thesignificance of differences between survival rates was ascer-tained using the log-rank test.12 The correlation betweenPARP1 mRNA fold increase and protein level was analyzedwith Spearman nonparametric test using GraphPad Prism 5.0bsoftware (GraphPad Software Inc., La Jolla, CA, USA).

ResultsIn this study, 443 individuals with primary breast tumors anda mean patient follow-up of 15 years were investigated, Sup-porting Information Table 1S shows the tumors characteristicsand the number of metastases. The classical predictive parame-ters of higher grade, involvement of lymph nodes at time ofdiagnosis and tumor size were all highly significantly correlatedwith a risk of metastasis in this study population.

The transcripts for PARP1, 2 and 3, PARG and ARH3were detected in the RNA extracted from the 10 normalbreast tissues. In terms of the relative amounts of each tran-script in the normal tissues examined, the PARP1 transcriptwas found at the highest levels while the levels of the fourother transcripts were all approximately fourfold less (varia-tion in the Ct value of �2) (Table 1). The Ntarget values of

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the target in the 10 normal breast RNA samples were consis-tently between 0.52 and 1.50 (PARP1 from 0.68 to 1.50;PARP2 from 0.63 to 1.22; PARP3 from 0.52 to 1.26; PARGfrom 0.76 to 1.27; ARH3 from 0.62 to 1.31). Based on theseresults, Ntarget values of 3 or more were considered to repre-sent overexpression, and values of 0.33 or less were consid-ered to represent underexpression of these five genes intumor RNA samples.13

The mRNAs of the five target genes were detected in over99% of the 443 breast tumors analyzed (Table 1). The medianlevel of PARP1 mRNA was 2.45-fold higher in the tumorscompared to the normal tissues with a substantial range of lev-els being detected with over a 250-fold difference between thelowest and the highest (median, 2.45; 0.08–21.94 relative to thenormal tissues). 34.1% of the tumors (151/443) showed PARP1mRNA overexpression while only 0.5% showed PARP1 under-expression. Higher median values for PARG and ARH3mRNAs were also noted compared to the normal tissues but ina lower proportion of tumors (PARG 3.8% and ARH3 3.4%).The median PARP3 levels were reduced compared to the nor-mal tissues with 10.4% of the tumors showing a PARP3 under-expression. The median level of PARP2 in the tumors was verysimilar to that found in the normal tissues with few of thetumors either showing an overexpression (2.9%, 13/443) orunderexpression (1.6%, 7/443).

Using the RH (ERa and PR) and ERBB2 status, we subdi-vided our total population (n 5 443) into four subgroups,

HR1/ERBB21 (n 5 51), HR1/ERBB22 (n 5 283), HR2/ERBB21 (n 5 46) and HR2/ERBB22 (n 5 63) (Table 2).Based on this molecular classification, although the PARP1transcript was consistently overexpressed in all the sub-groups, the intersubgroup comparison showed that the HR1/ERBB21 subgroup had a higher PARP1 expression profilethan the other subgroups. The PARP1 mRNA overexpressionwas not highly associated with any other classical predictivefactors (Supporting Information Table 3S). In contrast, theunderexpression of the PARP3 transcript was more frequentlyfound in the HR-negative subgroup (p 5 <1027): 25.7% (28/109) of the two HR-negative subgroups (HR2/ERBB21 andHR2/ERBB2-) vs. 5.4% (18/334) of the two HR-positive sub-groups (HR1/ERBB21 and HR1/ERBB22) (Table 2).PARP3 underexpression was also observed more frequentlyin high Scarff Bloom Richardson (SBR) histopathologicalgrade tumors (Supporting Information Table 4S) with only1.9% (1/54) of the grade I tumors underexpressing PARP3compared with 20.1% (31/154) of the grade III tumors. Theoverexpression of the PARG and ARH3 transcripts were themost frequent changes noted for these transcripts but wereobserved in only <5% of the tumors examined. Thesechanges were not highly associated with any specific patho-logical subgroups (Supporting Information Tables 5S and 6S).Similarly very few tumors showed either an over or underex-pression of PARP2; however, the rare cases of overexpressionwere seen in high-grade tumors and ER- and PR-negative

Table 1. mRNA levels of target genes in the 443 breast tumors

Genes

Normal breasttissues (n 5 10)

Normal breasttissues (n 5 10)

All breasttumors (n 5 443)

Ct values mRNA levels mRNA levels

PARP1 28.25 (27.08–29.50)1 1.0 (0.68–1.50)2 2.45 (0.08–21.94)2

Number with under-expression (%)3 0 (0) 2 (0.5)

Number with over-expression (%)4 0 (0) 151 (34.1)

PARP2 29.78 (28.97–30.95) 1.0 (0.63–1.22) 1.02 (0.00–10.62)

Number with under-expression (%) 0 (0) 13 (2.9)

Number with over-expression (%) 0 (0) 7 (1.6)

PARP3 29.77 (28.73–31.24) 1.0 (0.52–1.26) 0.88 (0.06–8.71)

Number with under-expression (%) 0 (0) 46 (10.4)

Number with over-expression (%) 0 (0) 6 (1.4)

PARG 30.88 (29.71–32.09) 1.0 (0.76–1.27) 1.38 (0.00–6.67)

Number with under-expression (%) 0 (0) 3 (0.7)

Number with over-expression (%) 0 (0) 17 (3.8)

ARH3 30.37 (28.93–31.51) 1.0 (0.62–1.31) 1.37 (0.00–11.84)

Number with under-expression (%) 0 (0) 3 (0.7)

Number with over-expression (%) 0 (0) 15 (3.4)

1Median (range) of gene Ct values.2Median (range) of gene mRNA levels; the mRNA values of the samples were normalized such that the median of the 10 normal breast tissue mRNAvalues was 1.3mRNA values of 0.33 or less were considered to represent under-expression.4mRNA values of 3 or more were considered to represent over-expression.

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2794 PARPs, PARG and ARH3 expression in breast tumors

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Tab

le2

.m

RN

Ale

vels

of

targ

et

ge

ne

sin

the

fou

rb

rea

sttu

mo

rsu

bty

pe

s

Ge

ne

s

No

rma

lb

rea

stti

ssu

es

(n5

10

)

All

bre

ast

tum

ors

(n5

44

3)

HR

1/E

RB

B2

1tu

mo

rs(n

55

1)

HR

1/E

RB

B2

2tu

mo

rs(n

52

83

)

HR

2/E

RB

B2

1tu

mo

rs(n

54

6)

HR

2/E

RB

B2

2tu

mo

rs(n

56

3)

HR

1/E

RB

B2

1vs

.H

R2

an

dH

R1

/E

RB

B2

2(5

1vs

.3

92

)

HR

1/

ER

BB

22

vs.

HR

2a

nd

HR

1/E

RB

B2

1(2

83

vs.

16

0)

HR

2/

ER

BB

21

vs.

HR

2/E

RB

B2

2a

nd

HR

1(4

6vs

.3

97

)

HR

2/E

RB

B2

2vs

.H

R2

/ER

BB

21

an

dH

R1

(63

vs.

38

0)

HR

2vs

.H

R1

(10

9vs

.3

34

)

PA

RP

11

.0(0

.68

–1

.50

)12

.45

(0.0

8–

21

.94

)13

.00

(1.1

9–

7.6

2)

2.4

6(0

.08

–2

1.9

4)

2.3

2(0

.32

–1

3.5

5)

2.2

3(0

.69

–6

.25

)

Nu

mb

er

wit

ho

ver-

exp

ress

ion

(%)2

0 (0)

15

1(3

4.1

)2

6(5

1.0

)9

6(3

3.9

)1

3(2

8.3

)1

6(2

5.4

)0

.00

68

30

.92

(NS

)0

.38

(NS

)0

.12

(NS

)0

.05

8(N

S)

PA

RP

21

.0(0

.63

–1

.22

)1

.02

(0.0

0–

10

.62

)1

.07

(0.2

8–

10

.62

)0

.93

(0.0

0–

2.2

9)

1.1

7(0

.57

–3

.21

)1

.19

(0.4

9–

5.1

5)

Nu

mb

er

wit

hu

nd

er-

exp

ress

ion

(%)4

0 (0)

13

(2.9

)1 (2

.0)

12

(4.2

)0 (0

)0 (0

)1

.00

(NS

)0

.06

1(N

S)

0.4

3(N

S)

0.2

8(N

S)

0.0

78

(NS

)

PA

RP

31

.0(0

.52

–1

.26

)0

.88

(0.0

6–

8.7

1)

0.8

0(0

.20

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tumors while those tumors having an underexpression havethe opposite profile (histological grade I and ER- and PR-positive tumors) (Supporting Information Table 7S).

The statistical analysis of the correlation between theexpression of the PARP1 and PARP3 transcript levels showeda mutual exclusion of PARP1 overexpression and PARP3underexpression. Indeed, PARP3 underexpression wasobserved only in 4.6% (7/151) of the PARP1 overexpressingtumors as compared with 13.4% (36/292) of the PARP1 nor-mal expressing tumors (p 5 0.0043; Table 3). Of note, thevast majority of the small percentage of tumors overexpress-ing PARG (17/443) and ARH3 (15/443) were found intumors with an overexpression of PARP1 (p < 1027 and p 5

0.00014, respectively; Table 3).Based on in vitro experimental evidence,14,15 it has been

suggested that the greatest therapeutic benefit a PARP inhibi-tor combined with radiotherapy would be seen in cells thatare rapidly dividing as the greatest relative impact on cellkilling from this combined treatment was seen in S-phasecells. In order to investigate whether transcript levels weremodulated under these circumstances, we studied their corre-lation with the transcript level of the nuclear proliferativemarker MKI67. While significant associations were seenbetween the most frequent change seen for each transcriptand MKI67 levels, the positive association between PARP1over-expression and MKI67 levels was highly significant(Table 4).

The statistical comparison of the levels of the expressionof the five genes in the primary breast tumors and the risk ofmetastasis found no association with the most frequentchange seen for each transcript (Supporting InformationTable 8S). However, it should be noted that the population

was heterogeneous and had different treatments which is alimitation of the study. As the triple negative (HR2/ERBB22) subgroup of tumors have a particularly bad prog-nosis, we examined whether the expression pattern of any ofthe five transcripts was associated with the risk of metastasiswithin this subgroup of 63 individuals, 27 of whom hadrelapses/metastasis. No correlation was seen with other classi-cal predictive factors (data not shown) but the individualswith a low PARG mRNA expression (level below the medianin the primary tumor) had a significantly higher risk ofmetastasis suggesting that the expression level of this tran-script could be a marker of bad prognosis within this sub-group (Supporting Information Table 9S and Fig. 1).

Finally, in order to evaluate whether levels of the PARP-1full-length protein and mRNA levels were correlated, PARP-1 protein levels were measured using a Western blotapproach in protein extracts prepared from a small panel ofbreast tumors (n 5 31) for which frozen tissue was available.While some PARP-1 cleavage products were detected insome tumor protein extracts, no correlation (R2 5 0.01, p 5

0.572) between PARP-1 full-length protein and mRNA levelswas noted (Fig. 2).

DiscussionGene expression analysis carries the potential to dissect themolecular heterogeneity of tumors and can distinguish breastcancer subclasses on the basis of differences in their geneexpression, and several studies have reported expression pro-files that are associated with clinical outcome.16

It is recognized though that tumor samples from patientscan exhibit significant tumor heterogeneity and differ in theproportion of tumor subpopulations and that the proportion

Table 3. Relationship between target gene mRNAlevels

PARP1 expression status

Total number p1Number ofoverexpression (%)

Number of normalexpression (%)

PARP3 expression status

Number with underexpression (%)2 7 (4.6) 39 (13.4) 46 0.0043

Number with normal expression (%)3 144 (95.4) 253 (86.7) 397

Total number 151 292

PARG expression status

Number with overexpression (%)4 16 (10.6) 1 (0.3) 17 0.0000001

Number with normal expression (%) 135 (89.4) 291 (99.7) 426

Total number 151 292

ARH3 expression status

Number with overexpression (%) 12 (7.9) 3 (1.0) 15 0.00014

Number with normal expression (%) 139 (92.1) 289 (99.0) 428

Total number 151 292 443

1Chi-square2 test.2mRNA values of 0.33 or less were considered to represent underexpression.3mRNA values between 0.34 and 2.99 were considered to represent normal expression.4mRNA values of 3 or more were considered to represent overexpression.

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of healthy tissue in a sample can vary and could also influ-ence expression profiles. In this study, a clear trend toward agreater variation in the transcript expression levels of all fivegenes examined in the tumors compared to the normal tis-sues was observed with the highest percentage of alterationbeing found for PARP1 with 34.1% of tumors showing anoverexpression. This PARP1 mRNA profile is in agreementwith three smaller studies from the same treatment centre17–19 and several other recent reports. For instance, Ossovskayaet al.20 found that 30% of breast infiltrating ductal carcinomasamples showed an upregulation of PARP1 mRNA comparedwith 2.9% of the normal tissues examined. A recent meta-analysis21 performed on 2485 breast cancer tumors in 12publically available oligonucleotide microarray data sets alsofound a twofold overexpression of PARP1 mRNA in 58% oftumors. It was also shown that this increase was often seenin tumors with a PARP1 gain or amplification compared totumors displaying a normal gene copy number in agreementwith Bieche et al.22 who observed a trend toward a linkbetween copy number gain of the 1q41-q44 locus, containingthe PARP1 coding region, and the PARP1 mRNA level. Theseresults suggest that deregulation of PARP1 mRNA expressioncould be partly due to a gene dosage effect, but this is notthe sole mechanism as mRNA overexpression was also foundin tumors without any PARP1 gene copy number gain.

The variation in PARP1 mRNA expression in differenthistological subgroups of sporadic breast tumors has beeninvestigated in several studies. Ossovskaya et al.20 showed ahigher incidence of PARP1 upregulation in TNBC tumors

compared with receptor positive samples while Goncalveset al.21 observed PARP1 mRNA overexpression in basalbreast cancer but also in other subtypes. In our study, PARP1transcript levels were higher in HR1/ERBB21 tumors com-pared to other subtypes but as reported by Goncalves et al.21

overexpression of PARP1 compared with normal tissue wasobserved in all subtypes. PARP1 overexpression has been cor-related with high grade and tumor size in the studies ofOssovskaya et al.20 and Goncalves et al.21 although we didnot observe these associations in this present study. However,in agreement with Ossovskaya et al.,20 we noted a correlationbetween PARP1 levels and the proliferation marker MKI67expression. Negroni et al.23 showed that mitogen-stimulatedhuman lymphocytes had 20-fold higher levels of PARP1mRNA compared to quiescent lymphocytes. More recently,Carbone et al.24 demonstrated that PARP-1 activity wasinduced by mitogen stimulation and contributed to the G0-G1 cell cycle transition via the induction of immediate-earlygenes such as c-Myc and c-Fos. In addition, there is strongevidence in the literature showing that PARP expression andactivity are affected by differentiation and cell proliferationwith generally higher PARP activity and expression in prolif-erating cells.25 It has also been found that PARP1 expressiongradually increases in non-atypical and atypical endometrialhyperplasia compared to normal endometrial epithelium26

and that in endometrial cancers PARP-1 protein and PRexpression are also positively correlated (p < 0.0001).27 Thus,the increased level of PARP1 mRNA in PR1 breast tumorsmight be linked to PARP-1’s role in the regulation of hor-mone responsive genes. An alternative, nonexclusive explana-tion is that the increase in PARP1 mRNA expression in theHR1 tumors might be a response to the increased level ofspontaneous DNA lesions forming in the context of highlyproliferating tumor cells.28,29 Whether this increase in PARP1mRNA expression is concomitant with an increase in proteinlevels and enzymatic activity remains to be established. ForPARP1 there is limited data on this relationship. Nosignificant correlation between PARP activity and PARP-1

Table 4. Relationship between MKI67 and target gene mRNA levels

Number oftumors (%)

MKI67expression p1

PARP1

Overexpression2 151 (34.1) 14.6 (0.8-94.5)3 0.00000036

Non overexpression 292 (65.9) 11.5 (0.9-117)

PARP3

Underexpression4 46 (10.4) 15.7 (3.2-79.5) 0.020

Non underexpression 397 (89.6) 12.1 (0.8-117)

PARG

Overexpression 17 (3.8) 24.3 (0.8-62.6) 0.015

Non overexpression 426 (96.2) 12.5 (0.9-117)

ARH3

Overexpression 15 (3.4) 17.5 (6.8-117) 0.024

Non overexpression 428 (96.6) 12.3 (0.8-106)

1Kruskall–Wallis’s H test.2mRNA values of 3 or more were considered to representoverexpression.3Median (range) of gene mRNA levels; the mRNA values of the sampleswere normalized such that the median of the 10 normal breast tissuemRNA values was 1.4mRNA values of 0.33 or less were considered to representunderexpression.

Figure 1. Survival of patients with triple negative breast tumors

depending on PARG expression. Survival distributions, estimated

by the Kaplan-Meier method, for patients with PARG mRNA expres-

sion below (low) or above (high) the median in the primary breast

tumor.

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protein levels was found in a panel of human tumor celllines,30 while a weak correlation was reported in peripheralblood mononuclear cells (PBMCs) from cancer patients (R2

5 0.06, p 5 0.01) and a stronger correlation (R2 5 0.19, p5 0.003) in PBMCs from cancer patients.31 In the small sub-set of 31 tumors for which frozen tissues were available inthis present study, no correlation was noted between PARP1mRNA and the level of full length PARP-1 protein butunfortunately suitable samples were not available from thisretrospective series to assess PARP activity. The assessmentof such correlations is further confounded by the fact thatthe PARP-1 protein can undergo posttranslational modifica-tion that impacts on its activity. For instance, it has beenrecently shown that while PARP-1 enzymatic activity israpidly enhanced by progestin treatment of breast cancercells through a mechanism involving the phosphorylation of

PARP-1 by CDK2,32 PARP-1 activity is downregulated afterphosphorylation by CDK5.33

There is very little published information on the expres-sion patterns in breast tumors of the other four transcriptsstudied here. We noted little variation in PARP2 mRNA lev-els in agreement with the findings of Ossovskaya et al.20 anda similar profile was seen for PARG or ARH3 expressionwith a few rare cases of overexpression. A downregulation ofPARP3 expression was observed in 10.4% of our breast tumorseries, and more particularly in the HR-negative subgroups(25.7%) as compared to the HR-positive subgroups (5.4%).PARP-3 has been implicated in the cellular response to DNADSBs34 and was found to accelerate the rate of classical non-homologous end-joining (NHEJ) by stabilizing the XRCC4/DNA Ligase 4 complex to the chromatin.35 On the otherhand, the overexpression of PARP1 could contribute toinhibit canonical NHEJ via PARP-1 blocking the binding ofthe Ku proteins to double strand breaks.36 The observationthat PARP1 overexpression and PARP3 downexpression aremutually exclusive would suggests that breast tumors becomemore reliant on homologous recombination.

In terms of the prognostic impact of transcript changes ofgenes associated with PAR metabolism, PARP1 expressionhas been shown in a multivariate analysis to be an independ-ent prognostic value for metastasis free survival but only inpatients untreated with any adjuvant chemotherapy.21 In thepresent study, no such association was observed; however, inthe subgroup analysis undertaken, it was noted that the triplenegative breast tumors with PARG mRNA levels below themedian were associated with a higher risk of developingmetastases (p 5 0.039). Human PARG is encoded by a singlegene but is present within the cell as different protein iso-forms found in various subcellular localizations. In vitrocoupled transcription and translation of human PARG cDNAgenerates several proteins suggesting the usage of alternativetranslation sites.37 Depletion of all PARG protein isoformsresulted in embryonic lethality in a mouse model38 and thedeletion of only the nuclear isoform in mouse embryonicfibroblasts resulted in defects in the repair of DNA damagecaused by various genotoxic agents and increased genomicinstability.39 Irradiated PARG-deficient human cells alsoshow increase radiosensitivity and centrosome amplificationwhich was associated with induced polyploidy or cell deathby mitotic catastrophe.40 It is thus tempting to speculate thatthe reduced levels of PARG transcript might be associatedwith an increased genetic instability and could be linked tothe higher risk of developing metastases seen in the TNBCpatients. The balance between PARP and PARG activity hasalso been shown to be critical for PARP-1-dependent celldeath (Parthanatos) that involves the mitochondrial oxidore-ductase apoptosis-inducing factor which is a high-affinitypoly(ADP)ribose-binding protein41 and PARG may also beimportant for the production of monomeric ADP-ribose,which is a trigger of TRPM2-mediated cellular Ca21 influxwhich induces cell death.42 Recently, Le May et al. 43 have

Figure 2. Correlation analysis between PARP1 mRNA and PARP-1

protein level in 31 tumors. (a) The level of the 114 kDa PARP-1

holoenzyme was analyzed by Western blotting, the actin protein

signal was used as a loading control, sample marked with (*) indi-

cate that the PARP-1 protein level in this sample was set to 1 for

comparison purpose. (b) Correlation between PARP-1 protein fold

increase and PARP1 mRNA fold increase.

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shown that PARG regulates retinoic acid receptor-mediatedgene expression through a mechanism that involves chroma-tin remodeling at the promoters of retinoic acid receptorresponsive genes. Thus, whether a defect in the DNA damageresponse, an altered balance in the level of polymers or analtered PARG-dependent gene expression profile is theunderlying biochemical cause of the higher risk of developingmetastases in patients with TNBC tumors with lower PARGtranscript levels remains to be established. Whether theexpression of the PARG transcript is a useful marker of clini-cal outcome and can be used to subdivide this group ofalready poor prognosis tumors also needs to be validated inlarger studies.

AcknowledgementsWe thank the staff of Centre Ren�e Huguenin for their assis-tance in specimen collection and patient care. T.Z. was sup-ported by a fellowship from the Fondation Pierre-Gilles deGennes pour la Recherche (FPGG) and Institut Curie’s post-doctoral fellowship scheme. Work in the Laboratoired’Oncog�en�etique, Institut Curie-Hopital Ren�e Huguenin, wassupported by Institut Curie (Medical Section), the Comit�ed�epartemental des Hauts-de-Seine de la Ligue NationaleContre le Cancer and the Association d’Aide �a la RechercheCanc�erologique de Saint-Cloud (ARCS). Work in InsermU612 was supported by INSERM and Institut Curie(Research Section).

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