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Promoter Methylation of the PTEN Gene Is a Common Molecular Change in Breast Cancer

Jose M.Garc?′a,1Javier Silva,1Cristina Pen?a,1Vanesa Garcia,1Rufo Rodr?′guez,2Miguel A.Cruz,3Blanca Cantos,1 Mariano Provencio,1Pilar Espan?a,1and Felix Bonilla1*

1Department of Medical Oncology,Hospital Universitario Puerta de Hierro,Madrid,Spain

2Department of Pathology,Hospital Virgen de la Salud,Toledo,Spain

3Department of Medical Oncology,Hospital Virgen de la Salud,Toledo,Spain

About25–50%of women with Cowden disease,a syndrome associated with germ-line mutations of the PTEN gene(at10q23), develop breast cancer(BC),but PTEN mutations have been found in only5%of sporadic BCs.However,29–48%of BCs display loss of heterozygosity in10q23,and about40%of BCs show a decrease or absence of PTEN protein levels at the time of diagnosis.Promoter hypermethylation has been identi?ed as an alternative mechanism of tumor-suppressor gene inacti-vation,but its importance in PTEN silencing in sporadic BC is unknown.We investigated PTEN promoter hypermethylation in 90sporadic BCs and its correlations with11molecular and pathologic parameters,including mRNA levels of PTEN.The study, a methylation-speci?c PCR assay,was carried out with methylated speci?c primers designed in a region with scarce homology with the psiPTEN pseudogene.Expression was analyzed by real-time PCR.We found that the PTEN promoter was hypermethylated in43BCs(48%).PTEN hypermethylation was associated with ERBB2overexpression,larger size,and higher histologic grade(P?0.012,0.03,and0.009,respectively).We concluded that PTEN promoter hypermethylation is a common event in sporadic BC,correlating with other well-established prognostic factors of this malignancy.Additionally,PTEN mRNA expression was lower in tumors with aberrant methylation.?2004Wiley-Liss,Inc.

INTRODUCTION

Breast carcinoma(BC)is the most common ma-lignancy of women in Western countries.Almost 10%of all patients with BC report a family history, and about30–70%of these familial cases have been associated with germ-line mutations of the BRCA1or BRCA2tumor-suppressor genes (Wooster et al.,1995;Newman et al.,1998). Cowden disease(CD)is an autosomal-dominant cancer predisposition syndrome associated with an elevated risk for tumors of the thyroid,skin,and breast.Approximately25–50%of women with CD develop BC(Lloyd et al.,1963;Mallory et al., 1995).Linkage analyses have determined that a single locus within10q23is likely to be responsible for CD(Nelen et al.,1996).Independent groups identi?ed a novel tumor-suppressor gene from this region called PTEN(phosphatase and tensin ho-mologue deleted on chromosome10;Li et al., 1997)and MMAC1(mutated in multiple advanced cancers1;Steck et al.,1997).The gene is also known as TEP1(transforming growth factor TGF-?-regulated and epithelial cell–enriched phospha-tase;Li et al.,1997).

The product encoded by PTEN is a protein that has sequence homology with dual-speci?city phos-phatases,which can dephosphorylate serine/threo-nine and tyrosine residues.It also has extensive homology with auxilin and tensin,cytoskeletal pro-teins that interact with actin?laments(Li et al., 1997;Li and Sun,1997;Steck et al.,1997). Along with being implicated in the development of CD,mutations in this gene have been found in other,related human autosomal-dominant disor-ders,such as Lhermitte–Duclos disease(LDD; Liaw et al.,1997)and Bannayan–Zonana syndrome (BZS;Marsh et al.,1997),as well as in sporadic cancers such as glioblastoma,prostatic,endome-trial,breast,lung,kidney,bladder,testicular,colon, and head and neck cancers,malignant melanoma, and lymphoma(Li et al.,1997;Steck et al.,1997; Tashiro et al.,1997;Teng et al.,1997;Okami et al., 1998;Sakai et al.,1998;Wang et al.,1998;Guanti et al.,2000).

Despite the low mutation frequency found in sporadic BC(Rhei et al.,1997;Steck et al.,1997; Supported by:Fundacio′n Banco Santander Central Hispano; Grant number:CAM08.1/0069/2000.2;SEOM;Aventis Pharma, S.A.

*Correspondence to:Felix Bonilla,MD,PhD,Department of Medical Oncology,Hospital Universitario Puerta de Hierro,C/San Martin de Porres4,E-28035Madrid,Spain.E-mail:felixbv@stnet.es Received24November2003;Accepted27April2004

DOI10.1002/gcc.20062

Published online10June2004in

Wiley InterScience(https://www.sodocs.net/doc/689388931.html,).

GENES,CHROMOSOMES&CANCER41:117–124(2004)

?2004Wiley-Liss,Inc.

Teng et al.,1997),10q23has a frequency of LOH that ranges from 29%to 48%(Steck et al.,1997;Stingh et al.,1998;Garc?′a et al.,1999),and the presence of 10q23loss has been associated with a poor prognosis and tumor progression (Bose et al.,1998;Stingh et al.,1998).In BC cells it has been demonstrated that the protein encoded by PTEN can induce cell-cycle arrest,apoptosis,or anoikis (Lu et al.,1999;Weng et al.,1999),and loss of PTEN expression in primary ductal adenocarci-noma of the breast also has been identi?ed (Perren et al.,1999;Bose et al.,2002).

The high frequency of LOH in 10q23and the loss of protein expression without a comparable mutational status suggest there should be other inactivation mechanisms of the PTEN gene.Epi-genetic events,such as hypermethylation of cy-tosine-guanine (CpG)sites in the promoter region,could be one mechanism.Indeed,PTEN has been found to be methylated in its promoter region in endometrial,gastric,and non–small cell lung can-cer (Salvesen et al.,2001;Kang et al.,2002;Soria et al.,2002).

Here we report aberrant methylation in the pro-moter region of the PTEN gene after discrimina-tion of the pseudogene psiPTEN (Dahia et al.,1998;Zysman et al.,2002)in sporadic BC,showing that this event is correlated with some clinicopath-ologic characteristics,such as tumor size,histologic grade,and ERBB2expression.

MATERIALS AND METHODS

Tumor Samples and DNA and RNA Extraction

The present study,approved by the Research Ethics Board of our hospital,was based on a series of invasive BCs with corresponding normal tissues obtained from 90patients after mastectomy.Tis-sue samples were taken from macroscopically healthy breast parenchyma consisting of glandular

and fat components.Both normal and tumor tissues were snap-frozen in liquid nitrogen immediately after resection and stored until processing.All specimens underwent histological examination to con?rm the diagnosis of adenocarcinoma and to verify that all tumor samples studied showed at least 75%tumor cells.DNA of all cases was ex-tracted using a nonorganic method (S-4520kit,Oncor,Inc.,Gaithersburg,MD),and total RNA of 20normal and tumor samples was extracted using an RNeasy Mini Kit (Quiagen Inc.,Hilden,Ger-many)and quanti?ed spectrophotometrically.

Pathological Parameters Analyzed

The following parameters were obtained from the medical records of the 90patients:birth date,tumor size,lymph node metastases,presence of steroid receptors (estrogen and progesterone),his-tologic type,vascular invasion,pathological stage,histologic grade,proliferation index,ERBB2ex-pression,P14ARF expression,and TP53immuno-staining status.Pathological stage was assessed us-ing the tumor-node-metastases (TNM)classi?cation.The steroid receptor content was de-termined by an immunohistochemical procedure.The proliferative index was calculated using Ki-67antigen expression (Inmunotech,Westbrook,ME).ERBB2expression was evaluated by a monoclonal mouse antibody (CB11;Novocastra Lab.,Ltd.,Newcastle,UK).P14ARF expression was analyzed by multiplex-PCR in a semiquantitative assay.In-munohistochemistry of TP53was analyzed with the cl 1801mouse monoclonal antibody (Oncogene Science,Manhasset,NY).

Promoter Hypermethylation

PTEN promoter methylation was analyzed in tumor and normal DNA from 90patients.DNA methylation patterns in the CpG islands of the gene were determined by methylation-speci?c PCR (MSP)as described elsewhere (Herman et al.,1996).Brie?y,1?g of DNA from both tumor and normal tissues was denatured by NaOH (?nal con-centration 0.3M)and subsequently treated with hydroquinone (?nal concentration 7mM)and so-dium bisul?te,pH 5(?nal concentration 3.7M),at 53°C for 16hr.Modi?ed DNA samples were puri-?ed using Wizard DNA puri?cation resin following the manufacturer’s instructions (Promega,Madi-son,WI)and eluted into 50?l of water.NaOH (?nal concentration 0.3M)was added to complete the modi?cation,followed by ethanol precipitation and ?nally resuspension into 30?l of water.Pla-cental DNA,treated with SssI

methyltransferase

Figure 1.Photograph of gels taken under normal light showing

results of the methylation-speci?c PCR assay (MSP).PL ,placental DNA treated with SssI methylase and sodium bisul?te (positive methylation control);LF,normal lymphocytes treated with sodium bisul?te (nega-tive methylation control);T4and N4,tumor and normal DNA,respec-tively,of a patient treated with sodium bisul?te;M,DNA ampli?ed with methylation-speci?c primers;U,DNA ampli?ed with unmethylation-speci?c primers.

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(SssI methylase,New England Biolabs,Inc.,MA)and subsequently treated with sodium bisul?te,was used as a positive control for methylated al-leles,and DNA from normal lymphocytes treated with sodium bisul?te was used as a negative control for the unmethylated gene (Fig.1).To establish positive and negative controls,2?g of placental DNA and lymphocyte DNA,respectively,were incubated for 2hr at 37°C in the presence of S-adenosylmethionine (?nal concentration 160?M),1?NEBuffer 2,and 4units of SssI methyl-ase.The reliability of the cytosine modi?cation reaction was tested by direct sequencing of the DNA before treatment with sodium bisul?te.

PCR was performed in 25-?l volumes using 0.2units of Taq Gold DNA polymerase and 1?PCR buffer (Promega,Madison,WI),200?M dNTPs,30pmol of each primer,and 1.5mM of MgCl 2.PCR was carried out in a thermocycler (Perkin Elmer Cetus,Foster City,CA).Each sample was denatured at 94°C for 11min and subjected to 30cycles (denaturation at 94°C for 30sec,annealing at 69°C for 30sec,and elongation at 72°C for 30sec)followed by a ?nal 12min extension at 72°C.The ampli?ed products were mixed with 6?L of load-ing buffer (0.02%xylene cyanol,0.02%bromophe-nol blue),and electrophoresed on nondenaturing 8%polyacrylamide gels at 450v for 2hr and were visualized by silver staining (Fig.1).

Methylated and unmethylated primers,summa-rized in Table 1,were designed in a promoter region with scarce homology with the psiPTEN sequence (AL356489and AF029308;Fig.2),where the reverse primers,which conduct the speci?c ampli?cation reaction,lack homology with the cited pseudogene,especially at the 3?end of the primer,ensuring that they anneal only with the gene.We chose this region because it is the nearest region to that predicted by Han et al.(2003)as a strong promoter in which there is consistent differ-ence between gene and pseudogene that facilitates the identi?cation of each.Ten of the cases positive for methylation were sequenced in an ABI Prism ?377DNA sequencer apparatus (PE Applied Bio-systems,Foster City,CA)in order to exclude the existence of unspeci?c ampli?cation either of the pseudogene or of the unmethylated sequence,in which all the cytosines (C)of the amplicon

not

Figure 2.Sequences of the PTEN gene (above the line,ref:AF406618or AF143312)and the psiPTEN pseudogene (below the line,ref:AL356489or AF029308).The base pairs in bold show the differences between the two sequences.PCR primers for methylated alleles are underlined in the corresponding sequence.Note the low homology between the sequence of the gene and the pseudogene in the region of the reverse primer,especially at the 3?end of the primer,which ensures that it anneals only with the PTEN gene.The shaded region represents the fragment ampli?ed with primers speci?c for methylation and corre-sponds to the sequence obtained after sequencing.For clarity,the sequence of unmethylated primer is not marked (the position of the primer in the genome is shown in Table 1).

TABLE 1.Primers Used in Methylation-Speci?c Assay

Primers Sequence

Genomic position a Size (bp)MF GGCGGCGGTCGCGGTTC ?751,?73571MR GACTCCCCGAAAACGCTAC ?681,?699UF GAGAGATGGTGGTGGTTGT ?758,?74078

UR

AACTCCCCAAAAACACTACC

?681,

?700

a

De?ned by the location of the 5?and 3?nucleotides of each primer from the translational site of PTEN (Gene Bank accession number AF143312).

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followed by a guanine (G)should be modi?ed to thymine (T)and C followed by G should remain a C after treatment with bisul?te (Figs.3and 4).

Real-Time PCR

PTEN mRNA levels were calculated using a relative quanti?cation approach in which the amount of the target,PTEN ,in each sample is expressed as a ratio of PTEN relative to a geometric mean of three reference housekeeping genes,GAPDH (glyceraldehyde-3-phosphate dehydroge-nase),UBC (ubiquitin C),and SDHA (succinate dehydrogenase complex,subunit A)(Fig.5).The relative concentrations of the target and the refer-ence genes were calculated by interpolation with a standard curve of each of the respective genes generated with a serial dilution of a cDNA of nor-mal tissue.We considered the expression of PTEN to be altered in tumor tissue when there was at least a 2-fold decrease or increase in its level com-

pared to that in the normal counterpart.The prim-ers used for PTEN expression were sense 5?-CAATGTTCAGTGGCGGAACTTG-3?and anti-sense 5?-GAACTTGTCTTCCCGTCGTGTG-3?;for GAPDH expression,sense 5?-CATCTTCTTT-TGCGTCGCC-3?and antisense 5?-AAAAG-CAGCCCTGGTGAC-3?;for UBC,sense 5?-AT-TTGGGTCGCGGTTCTTG-3?and antisense 5?-TGCCTTGACATTCTCGATGGT 3?;and for SDHA ,sense 5?-TGGGAACAAGAGGGCA-TCTG-3?and antisense 5?-CCACCACTGCAT-CAAATTCATG-3?.For the synthesis of ?rst-strand cDNA,250ng of total RNA was retro-tran-scribed using the Gold RNA PCR Core Kit (Applied Biosystems)according to the manufactur-er’s protocol.

Real-time PCR was performed in a light-cycler apparatus (Roche Diagnostics,Mannheim,Ger-many)using the LightCycler-FastStart DNA Mas-ter SYBR Green I Kit (Roche Diagnostics).Each reaction was performed in a ?nal volume of 20?l containing 2?l of the cDNA product sample,3mM MgCl 2,0.5?M of each primer,as well as 1?reaction mix including FastStar DNA polymerase,reaction buffer,dNTPs,and SYBR green.Thermal cycling for both genes was initiated with a dena-turation step of 95°C for 10min and subjected to 30cycles (denaturation at 94°C for 0sec,annealing at 56°C for 5sec,and elongation at 72°C for 5sec,in which ?uorescence was acquired).At the end of the PCR cycles,melting curve analyses,as well as electrophoresis of the products of PCR on non-denaturing 8%polyacrylamide gels,together with a marker of molecular weight,were performed to validate the generation of the expected speci?c PCR product.

To check for the absence of psiPTEN pseudo-gene DNA ampli?cation together with the mRNA of the PTEN gene,all samples in each assay were ampli?ed with FastStar DNA polymerase without previous retrotranscription.

Statistical Analysis

The variables analyzed were compared using the chi-square test with Yates correction,or,when any of the expected frequencies was less than 5,Fish-er’s exact test and Wilcoxon test.Two-tailed P values of ?0.05were considered statistically sig-ni?cant.Statistical analyses were performed using the SPSS Version 10.0package.

RESULTS

Among the 90cases tested,we found 43cases (48%)with PTEN promoter hypermethylation

in

Figure 3.Fragments of sequence obtained in an ABI Prism ?377

DNA sequencer apparatus (PE Applied Biosystems)from a methylated tumor sample after sodium bisul?te treatment.The wild-type PTEN promoter nucleotide sequence (shaded fragment in Fig.2)is replaced by the sequence shown in Figure 4(sequence after treatment with bisul-?te).Upper panel shows the sequence obtained with the forward primer.Lower panel shows the fragment obtained with the reverse primer.Underlined T in the sense strand (and underlined A in the antisense strand)represent the C modi?ed by treatment with sodium bisul?te,whereas the C*in the sense strand (G*in the antisense strand)corresponding to the CpG,remains a C,as expected in a methylated region.

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the tumor tissues and none in the normal counter-part.Of the molecular characteristics studied,only ERBB2expression levels correlated with PTEN promoter hypermethylation (P ?0.012):22of 33(67%)cases with high ERBB2levels showed pro-moter methylation versus 21of 57(37%)with no ERBB2expression.Of the pathological character-istics tested,tumor size and histologic grade corre-lated with promoter hypermethylation:T3(P ?0.03)and histologic grade III (P ?0.009)were associated with PTEN promoter methylation.The distribution of promoter methylation in each group is shown in Table 2.

Direct sequencing showed that all 10cases ana-lyzed had PTEN promoter hypermethylation,not nonspeci?c ampli?cation of unmethylated samples or ampli?cation of pseudogene sequence (Fig.3).All but one of the 43tumor tissue samples pos-itive for methylation also showed ampli?cation with the primers speci?c for the unmethylated se-quence,probably re?ecting the existence of con-tamination of normal tissue (Fig.1).

Of the 20patients tested for PTEN gene expres-sion,8showed promoter methylation with a me-dian gene expression in the tumor tissue 0.6-fold of that found in healthy tissue (mean,0.38-fold).Among the 12patients without promoter methyl-ation,the median expression level was 0.37-fold (mean,0.16-fold;P ?0.09,Wilcoxon test).Among the 8patients with promoter methylation,5(63%)showed decreased levels of PTEN mRNA,whereas only 4of the 12patients without methylation showed similar decreased levels.Finally,whereas none of the patients with promoter methylation showed signi?cant increases of PTEN mRNA in their tumor tissues,four of the patients (33%)with-out PTEN promoter methylation showed a signi?-cant increase.

None of the 20samples ampli?ed without pre-vious retrotranscription showed ampli?cation in the real-time PCR assay,con?rming the absence of

both DNA contamination and psiPTEN DNA am-pli?cation.

DISCUSSION

In this report,we show an increased number of BC patients (48%)with PTEN promoter hyper-methylation.This is similar to the BRCA1gene,which rarely is mutated in sporadic cases but which may display hypermethylation in its promoter (Es-teller et al.,2000).Moreover,we provide evidence of a link between decreased expression of PTEN and promoter hypermethylation.

The vast majority of PTEN mutations in CD kindred appear in exons 5and 7.Keeping in mind that glioblastomas and endometrial carcinoma present distinct mutational PTEN spectra and that,depending on the tissue type,the gene appears to be involved in the initiation or the progression of cancer,it is feasible to consider two inactivation mechanisms in BC,depending on whether it is sporadic or inherited (Ali et al.,1999).

Di Cristofano et al.(1998)found that mice with only one inactivated PTEN allele developed hyper-plastic-dysplastic changes characteristic of CD,LDD,and BZS,as well as tumors of various histological origins.Moreover,they found remarkable differences among tumors originating from cells without PTEN activity and cells with only one allele inactivated.In our study,all but one positive sample also showed ampli?cation with the unmethylated primers.We cannot be sure whether those unmethylated alleles correspond to tumor cells with only one allele meth-ylated or to normal contaminating cells.Nevertheless,it is possible that hypermethylation of just one allele in?uences the PTEN dosage in the cells and,conse-quently,tumor development,as was reported by Di Cristofano et al.(1998).

Recent research examined the possible misinter-pretation of methylation assays in the PTEN pro-moter region,attributable to the high homology with a pseudogene,psiPTEN ,on chromosome

9

Figure 4.Sequence of one methylated allele obtained after treatment with sodium bisul?te,where all unmethylated C (the line above),is transformed to U (uracil),which is read as T (below)in the sequence assay.

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(Dahia et al.,1998;Zysman et al.,2002).However,the high speci?city of the reverse primers for the methylated allele and the results of the sequencing assay that the methylated sequence that we de-tected corresponds to the promoter of the PTEN gene,not to the pseudogene.On the other

hand,

Figure 5.Representative example of

real-time PCR assay showing results ob-tained for patient 1,with PTEN promoter methylation,and patient 2,without PTEN promoter methylation (N,normal sam-ples;T,tumor samples).The X axis shows the ?uorescence intensity and the Y axis shows the number of cycles.The higher the starting quantity of a target sequence,the earlier a signi?cant increase in ?uores-cence was detected.Note that the house-keeping (GAPDH )expression pro?les (lower part of ?gure)are similar in the N and T samples of both patients,suggesting that the difference in PTEN expression (upper part of ?gure)in the T and N samples of patient 1was not a result of differences in the quality or quantity of total RNA from each patient.

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that promoter hypermethylation was not detected in normal tissue indicates that this event is a tumor-speci?c change,at least in BC.

Here we have reported the preferential loss of PTEN expression at a level close to having statis-tical signi?cance (P ?0.09),despite the small num-ber of samples,among patients with promoter hy-permethylation.The transcription but not the translation of the PTEN pseudogene in several tissues and cell lines has been described (Fujii et al.,1999;Yokoyama et al.,2000;Zhang et al.,2000).So,the presence of PTEN mRNA in breast tumor tissue,despite the promoter hypermethylation,could be explained by the expression of the psiPTEN pseudogene.Thus,the number of tumor samples with decreased gene product among those with promoter hypermethylation could be higher than the number reported here.However,other authors have shown data that argue against the expression of the pseudogene (Hamilton et al.,2000).

In conclusion,in this report we showed that PTEN promoter hypermethylation is a common event in sporadic BC and that this correlates with pathological

TABLE 2.Molecular and Pathologic Parameters of the Breast Tumors Studied and Correlation Analysis with Methylation Status Characteristics Methylated

%Unmethylated

%P

Tumors 4347.84752.2TP53

Positive 1125.601634NS Negative 3274.403166P14ARF

Overexpressed 614.001123.40NS Not altered 2558.102655.32Decreased 1227.901021.28ERBB2Positive 2251.161123.400.012Negative 2148.843676.60Ki-67High 3376.742961.70NS Low 1023.261838.30Estrogen R Positive 3172.103880.85NS Negative 1227.90919.15Progesterone R Positive 2455.812859.57NS Negative 1944.191940.43Histologic types IDC

3376.743989.98NS Non IDC 1023.26817.02LNM ?32762.803472.34NS ?31637.201327.66Tumor size T1920.931225.530.03

T22148.843165.96T3

1330.2348.51Histologic grade I 2 4.651123.400.009

II 1637.212144.68III 2558.141531.92Stage I 511.63714.90NS

II 2148.842859.57III 1739.531225.53VI Yes 1944.192757.45NS No 2455.812042.55Age ?501227.901225.53NS

?50

31

72.10

35

74.47

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PTEN HYPERMETHYLATION IN BREAST CANCER

characteristics such as histologic grade(P?0.009) and tumor size(P?0.03)and with high levels of ERBB2expression(P?0.012),all considered well-established prognostic factors for breast cancer.

ACKNOWLEDGMENTS

We thank M.M.Garcia and R.Alcalde for help with the collection of tissue samples and Robin Rycroft for assistance with the English manuscript.

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