Silencing of tumor suppressor genes RASSF1A, SLIT2, and WIF1 by promoter hypermethylation in hereditary breast cancer

MOLECULAR CARCINOGENESIS

Silencing of Tumor Suppressor GenesRASSF1A, SLIT2, and WIF1 by PromoterHypermethylation in Hereditary Breast Cancer

Carolina Alvarez,1 Teresa Tapia,1 Valeria Cornejo,2 Wanda Fernandez,2 Alex Munoz,3 Mauricio Camus,4

Manuel Alvarez,5 Luigi Devoto,3 and Pilar Carvallo1*1Departamento de Biologıa Celular y Molecular, Facultad de Ciencias Biologicas, Pontificia Universidad Catolica de Chile,Santiago, Chile2Unidad de Anatomıa Patologica, Hospital San Borja Arriaran, Santiago, Chile3Instituto de Investigaciones Materno Infantil (IDIMI); Facultad de Medicina; Universidad de Chile Santiago, Chile4Centro de Cancer, Facultad de Medicina, Pontificia Universidad Catolica de Chile, Santiago, Chile5Centro Clınico del Cancer, Clınica Las Condes, Santiago, Chile

Promoter hypermethylation is gaining strength as one of the main mechanisms through which tumor suppressorgenes are silenced during tumor progression. Three tumor suppressor genes are frequently found methylated in their

promoter, in concordance with absence of expression, RASSF1A, SLIT2, and WIF1. In addition, a previous array-CGHanalysis from our group showed that these genes are found in deleted genomic regions observed in hereditary breastcancer tumors. In the present work we analyzed the methylation status of these three tumor suppressor gene pro-moters in 47 hereditary breast cancer tumors. Promoter methylation status analysis of hereditary breast tumors

revealed high methylation frequencies for the three genes (67% RASSF1A, 80% SLIT2, and 72% WIF1). Additionally,the presence of methylated PCR products was associated with absence of protein expression for the three genes andstatistically significant for RASSF1A and WIF1. Interestingly, methylation of all the three genes was found in 4 out of 6

grade I invasive ductal carcinoma tumors. Association between RASSF1A methylation and DCIS tumors was found.These results suggest that silencing of these tumor suppressor genes is an early event in hereditary breast cancer, andcould be a marker for pre-malignant phenotypes. � 2012 Wiley Periodicals, Inc.

Key words: hereditary breast cancer; BRCAX; biopsies; tumor suppressor gene; promoter hypermethylation; protein

expression

INTRODUCTION

Breast cancer is the second cause of death bycancer among Chilean women, with a mortalityrate of 13.8 (in 100,000 women) (Departamento deEstadısticas e Informacion de Salud (Department ofStatistics and Health Information), Ministerio deSalud (Ministry of Health)) [1]. Several genetic andepigenetic processes that affect tumor suppressorgenes and proto-oncogenes occur during cancerdevelopment and progression. Most of these alter-ations correlate with changes in gene expression,so the detection of these events helps us to under-stand tumor progression. Participation of tumorsuppressor genes in cancer progression involvesthe inactivation of both alleles; either by gene de-letion, somatic mutation and promoter hyperme-thylation, or a combination of these mechanisms[2]. Promoter hypermethylation is gaining strengthas the main mechanism through which gene si-lencing occurs for several tumor suppressor genes.One tumor suppressor gene may be hypermethy-lated in several types of cancer, but in differentpercentages [3]. For instance, p16INK4a presentshypermethylation frequencies from 1% in leuke-mia to 48% in lymphomas [3]. On the other hand,

analyzing the same tumor type, for example breastcancer, one can clearly observe that differentgenes are hypermethylated in different amount oftumors [3].In a parallel study analyzing hereditary breast

tumors by array comparative genomic hybridiza-tion (CGH), we found several loci deleted in thehereditary non-BRCA1/BRCA2 tumors (BRCAX

Additional supporting information may be found in the onlineversion of this article.

Abbreviation: BRCA1, breast cancer 1, early onset; BRCA2,breast cancer 2, early onset; BRCAX, breast cancer X; CGH, com-parative genomic hybridization; DCIS, in situ ductal carcinoma; ER,estrogen receptor; ERBB2, v-erb-b2 erythroblastic leukemia viraloncogene homolog 2; M, methylated; MS-PCR, methylation specif-ic PCR-based; p16INK4a, cyclin-dependent kinase inhibitor 2A;RASSF1A, Ras association (RalGDS/AF-6) domain family member 1;ROBO1 roundabout, axon guidance receptor, homolog 1 (Drosoph-ila); SLIT2, slit homolog 2 (Drosophila); WIF1, WNT inhibitory factor1; UM, unmethylated.

*Correspondence to: Departamento de Biologıa Celular y Molec-ular Facultad de Ciencias Biologicas Pontificia Universidad Catolicade Chile Casilla 114-D Santiago, Chile.

Received 5 May 2011; Revised 26 October 2011; Accepted 10January 2012

DOI 10.1002/mc.21881

Published online in Wiley Online Library(wileyonlinelibrary.com).

� 2012 WILEY PERIODICALS, INC.

tumors) (manuscript in preparation), which con-tain tumor suppressor candidate genes that havebeen described to be silenced by hypermethylationin several types of cancers. We observed 3p21 locusdeleted in 10% BRCAX tumors, which contains atumor suppressor gene cluster including RASSF1Aas one of the main components. RASSF1A isexpressed in all epithelial cells and this expressionis frequently lost in several types of tumors, suchas lung and breast cancer [4,5]. RASSF1A is partof the RAS-association domain family of proteins,and its tumor suppressor activity relies on its abili-ty to bind and stabilize microtubules [6]. Eventhough deletion of RASSF1A locus occurs frequent-ly, the main mechanism by which RASSF1A issilenced seems to be promoter hypermethylation[4,7]. Promoter hypermethylation of RASSF1A ispostulated to occur early during tumor develop-ment as suggested by the finding of methylatedRASSF1A in apparently normal tissue adjacent totumor cells in gastric cancer [7].

Another gene identified in our array CGH studyis SLIT2, localized at chromosome 4p15. This geneencodes a secreted protein that forms part of thefamily of ligands for ROBO receptors, whose path-way is involved in repulsive axon guidance duringneural development [8]. It has been previouslydemonstrated SLIT2 expression in developing andadult mammary gland, as well as in other non-neuronal tissues, suggesting a role different fromaxon guidance [9–11]. SLIT2 tumor suppressoractivity was described by Dallol et al. [9], in a studytesting the participation of the ROBO pathwaymembers in cancer. They demonstrated that SLIT2promoter is hypermethylated in lung and sporadicbreast tumors and that this hypermethylation washighly correlated with loss of SLIT2 expression [9].In a parallel study, we found SLIT2 locus deletedin 3 BRCAX tumors, and ROBO1-2 locus deleted inall BRCA2 tumors (manuscript in preparation), sug-gesting that SLIT-ROBO pathway participates inhereditary breast cancer.

Finally, a deletion localized at chromosome12p14 includes WIF1, a gene that codifies a Wntpathway inhibitor. WIF1 is a secreted protein thatcompetes with the Wnt ligands in the binding toFrizzled receptor [12]. WIF1 expression in breast,lung and urinary bladder tissue has been previous-ly described by Wissmann et al. [13]. Additionallyin Wissmann’s work, WIF1 was found to befrequently down regulated in several cancers asprostate, lung, bladder, and breast. This down reg-ulation was later correlated with WIF1 promoterhypermethylation, by Mazieres et al. [14] andothers [15,16].

Silencing of different tumor suppressor genesmay indicate distinct pathways contributing tobreast cancer development and progression. Deter-mining which pathways are affected in hereditary

tumors could lead to a deeper comprehensionof the molecular mechanisms involved intumorigenesis.For some tumor suppressor genes, it has been

described different hypermethylation frequenciesbetween sporadic and hereditary breast carcino-mas. For instance, BRCA1 has been describedhypermethylated in about 10–30% of sporadicbreast cancer tumors [17–20], while it has beenfound hypermethylated at a higher frequency(51%) in hereditary breast tumors [21]. In anotherscenario, p16INK4a has methylation frequenciesthat varies from 18 to 36% in sporadic tumors[22–24] and only slightly lower in hereditary breastcarcinomas (15–26%) [25]WIF1 and SLIT2 genes have only been described

methylated in sporadic breast tumors [9,13,26,27],and to our knowledge, no correlation with proteinexpression has been informed until today. In thecase of RASSF1A, there are only two reports relatedto hereditary cases, both directed to study breastfluids and not solid tumors. The first study, byVasilatos et al., [28] was performed in asymptomat-ic women at increased risk for breast cancer usingnipple aspirates as a sample. The second work pub-lished by Antill et al. 2010 [29] was performedonly in BRCA1/BRCA2 mutation carriers thatwere asymptomatic or had unilateral breast cancer,taking ductal fluid as a sample of the healthybreast.The present work is the first study specially

directed to analyze and associate promoter methyl-ation status of RASSF1A, SLIT2, and WIF1 and theirprotein expression in hereditary breast cancertumors.

MATERIALS AND METHODS

Tumor Samples

We analyzed a total of 47 formalin-fixed paraffinembedded tumor biopsies from women withhereditary breast cancer. All patients had been pre-viously screened for BRCA1 and BRCA2 germlinemutations. Two of them carried BRCA2 mutations,3 BRCA1 mutations, and 44 BRCAX (SupplementaryTable). Families and patients were selected usingthe standard criteria for hereditary breast cancer[30]. All patients signed an informed consent, andthis protocol was approved by the Ethics Commit-tee of the Faculty of Medicine, Pontificia Universi-dad Catolica de Chile. The histological type andgrade of the tumors were determined according tothe World Health Organization criteria and paraf-fin sections were processed according to standardprotocols. All 47 biopsies were previously analyzedby immunohistochemistry (IHC) for the expres-sion of estrogen receptor (ER), progesterone recep-tor, and ERBB2 (Supplementary Table).

2 ALVAREZ ET AL.

Molecular Carcinogenesis

DNA Isolation and Modification

Formalin fixed-paraffin embedded biopsies weresliced and placed in a sterile tube. DNA wasextracted by Proteinase K digestion (0.4 mg/mlProteinase K, 1 mM EDTA, 0.02 M Tris, 0.5% Tween20) for 48 h at 378C in a water bath under gentleshaking, followed by ethanol precipitation.DNA was chemically modified with sodium bisul-

fite as described previously [31]. This modificationconverts all unmethylated cytosines to uracils andmaintains methylcytosines unaltered allowing spe-cific detection of methylated or unmethylatedDNA. Briefly, DNA (2 mg) in a volume of 20 ml wasdenatured with NaOH (0.3 M final concentration)for 15 min at 378C, followed by 2 min at 908C.Hydroquinone (0.8 mM final concentration) andsodium bisulfite at pH 5.0 (1.2 M final concentra-tion) were added and samples were incubated at558C for 4 h. Modified DNA was purified using theWizard DNA Clean-Up System (Promega, Madison,WI, Cat. A7280), and eluted with 50 ml of sterilewater. DNA modification was completed by NaOHtreatment (0.3 M final concentration) for 15 min at378C followed by ethanol precipitation. Modifiednormal peripheral blood DNA was used as a positivecontrol for unmethylated alleles. Normal peripheralblood DNA was methylated in vitro with M.SssImethyltransferase (New England Biolabs, Ipswich,MA, Cat. # M0226S) modified by sodium bisulfite asdescribed above, and used as a positive control formethylation. The integrity of tumor DNA was deter-mined in a 1% agarose gel before bisulfate modifica-tion. Unmodified and modified DNA was measuredusing NanoDrop spectrophotometer.

Methylation Specific PCR (MS-PCR)

We selected a promoter region for each genebased on the literature [4,9,16]. Methylation analy-sis was done using a methylation specific PCR-based (MS-PCR) approach as previously described[32]. For each gene, MS-PCR was performed asfollows:RASSF1A: the reaction was performed in 25 ml

reaction volume containing 1� GeneAmp PCRbuffer (Applied Biosystems), 0.2 mM of each deox-ynucleoside triphosphate, 1.5 mM MgCl2, 0.5 mMof each primer, and 1 U of AmpliTaq Gold1 DNAPolymerase (Applied Biosystems, Foster City, CA,Cat. # N808-0240).SLIT2: the reaction was performed in 25 ml re-

action volume containing 1� PCRx buffer (PCRxEnhancer System Invitrogen, Frederick, MD, Cat. #11495-017), 50 mM of each deoxynucleoside tri-phosphate, 1.5 mM MgCl2, 0.2 mM of each primer,and 2 U of Taq Platinum DNA Polymerase (Invitro-gen Cat. # 10966-034).WIF1: a nested PCR was performed in order to

amplify WIF1 promoter. A first PCR (WIF1 PAN)

was carried out in 50 ml reaction volume containing1� PCRx buffer, 0.5� PCRx Enhancer (Invitrogen),50 mM of each deoxynucleoside triphosphate,1.5 mM MgSO4, 0.24 mM of each primer, and 2.5 Uof Taq Platinum1 DNA Polymerase (Invitrogen).One microliter of each WIF1PAN product was usedas template for a second PCR. This reaction wasperformed in 25 ml containing 1� PCR buffer(Fermentas, Burlington, Ontario, Canada, with KCl),0.2 mM of each deoxynucleoside triphosphate,1.5 mM MgCl2, 0.4 mM of each primer, and 1 U ofTaq DNA Polymerase (Fermentas Cat. # EP0402).Each gene promoter was amplified with two set

of primers. For primers recognizing methylatedand modified DNA the M suffix was used, and forprimers identifying unmethylated and modifiedDNA UM suffix was used. For WIF1, a first PCRreaction was performed using primers directed tosequences flanking the CpG island. Then, the MS-PCR was performed using the specific primers todetect methylated or unmethylated modified DNA.Localization of each pair of primers for MS-PCR atthe analyzed promoters is illustrated in Figure 1.Primer sequences and PCR product features aredescribed in Table 1. All amplified products wereanalyzed by electrophoresis in 2% agarose gels andvisualized in a UV transilluminator.

Sequencing MS-PCR Products

MS-PCR products were confirmed by directsequencing using the forward and reverse PCRprimers (Macrogen, Seoul, Korea).

Immunohistochemistry

We evaluated the expression of RASSF1A, SLIT2,and WIF1 proteins by immunohistochemistry in47 paraffin embedded breast tumors. A 4 mm sec-tion from each tumor was stained with hematoxy-lin–eosin for morphological assessment, and thefollowing sections were used for immunostainingwith the biotin-streptavidin peroxidase technique(Histostain-Plus kit Invitrogen, Cat. # 859673). Theslides were first heated at 708C for 30 min, and lat-er deparaffinized and re-hydrated with a series ofethanol dilutions (100–35%). Endogenous peroxi-dase was inactivated by incubation with 3% H2O2

in methanol for 20 min. In order to expose the an-tigen each slide was treated with an antigenunmasking solution (10 mM sodium citrate, pH6.0, with Tween 20) at 908C for 15 min in a micro-wave oven. The slides were cooled in the same so-lution and washed three times in 1� PBS (2 mineach time). After blocking for 1 h (blocking solu-tion A), the slides were drained. Slides were thenincubated overnight at 48C in a humidity chamberwith the primary antibodies: RASSF1A, mousemonoclonal antibody clone 3F3 (Abcam Cat. #AB23950, 1:150 dilutions); SLIT2, rabbit polyclonalantibody (Santa Cruz biotechnologies, Santa Cruz,

RASSF1A, SLIT2, AND WIF1 SILENCING IN BREAST CANCER 3


CA, Cat. # SC-28945, 1:50 dilutions); WIF1, mousemonoclonal antibody clone 133015 (R&D Systems,Minneapolis, MN, Cat. # MAB134-500, 1:100 dilu-tions). After draining the primary antibody, slideswere incubated with a universal secondary biotiny-lated antibody (broad spectrum antibody, B) for1 h. Slides were then washed with 1� PBS 3 times,2 min each, and incubated with streptavidin–per-oxidase conjugate (C) for 30 min at room tempera-ture. The chromogenic reaction was developedby incubation of the slides with 3-amino-9-

ethilcarbazol single solution chromogen (D) andcounterstaining with Hematoxylin (Invitrogen) be-fore mounting the slides. Negative controls, wereprepared omitting the primary antibody. Breastsamples from healthy women undergoing reduc-tion surgery were used as positive controls (Supple-mentary Figure). The analysis of the slides wasdone independently by two pathologists. Positivestaining in more than 10% of tumor cells in theexamined area was considered. Using Image-Prosoftware (Media Cybernetics, Inc., Bethesda, MD)

Figure 1. Promoter region of RASSF1A (a), SLIT2 (b), and WIF1 (c) analyzed by MS-PCR. For each region thepicture shows CpG sites, the transcription start site, and the location of the primers for the methylated and theunmethylated condition, proportionally distributed in the DNA sequence.

Table 1. MS-PCR Features and Primer Sequences for Each Analyzed Gene Promoter

Gene Primers

T8annealing

(8C)

PCRproductsize (bp) Reference

RASSF1A M Forward: 50-GGGTTTTGCGAGAGCGCG-30 64 169 Burbee et al. [4]Reverse: 50-GCTAACAAACGCGAACCG-30

RASSF1A UM Forward: 50-GGTTTTGTGAGAGTGTGTTTAG-30 59 169Reverse: 50-CACTAACAAACACAAACCAAAC-30

SLIT2 M Forward: 50-CGGTTTAGGTTGCGGCGGAGTCGAGGGC-30 68 160 Dallol et al. [9]Reverse: 50-CGCGAAAACCCAACGAACCCGTAACAAAACGCG-30

SLIT2 UM Forward: 50-TGGTTTAGGTTGTGGTGGAGTTGAGGGT-30 59 160Reverse: 50-CACAAAAACCCAACAAACCCATAACAAAACACA-30

WIF1 PAN Forward: 50-TAGGGGTTTTTGAGTGTTT-30 50 404 Urakamiet al. [16]Reverse: 50-ACCTAAATACCAAAAAACCTAC-30

WIF1 M Forward: 50-CGTTTTATTGGGCGTATCGT-30 57 145Reverse: 50-ACTAACGCGAACGAAATACGA-30

WIF1 UM Forward: 50-GGGTGTTTTATTGGGTGTATTGT-30 52 154Reverse: 50-AAAAAAACTAACACAAACAAAATACAAAC-30

4 ALVAREZ ET AL.


we calculated a score (mean intensity � % area)for each tumor as follows: weak <100, moderate100–200, and strong >200. Then a score equal orover 100 was considered positive expression, andbellow 100 considered as significant loss ofexpression.

Statistical Analysis

All comparisons for statistical significancewere performed with Fisher’s two-tailed exact test(GraphPad software), a P-value <0.05 was consid-ered statistically significant.

RESULTS

The methylation status and protein expressionof RASSF1A, SLIT2, and WIF1—strong candidatesto be involved in tumor progression—was deter-mined. Each promoter region (Figure 1) was PCRamplified with specific primers, in all 47 biopsies.Due to low DNA quality of some biopsies weobtained only 42 MS-PCR products for RASSF1A,40 for SLIT2, and 32 for WIF1. As shown inFigure 2, some tumors yielded PCR products thatindicate the presence of methylated (M) andunmethylated promoters (UM). This is the case ofT1 and T48 (Figure 2a), tumors T6, T26 and T33(Figure 2b), and T25 (Figure 2c). The presence ofboth products, M and UM, may be the result ofhypermethylation of only one allele in the tumorcells or to the presence of DNA from nontumorcells, unmethylated, admixed with the tumor

DNA. In the case of tumors giving only one classof product, UM or M, we can clearly state thatboth alleles are either methylated or unmethylatedfor a particular gene; with the exception of tumorsT9 and T49 carrying genomic deletion at RASSF1Alocus presenting only M or UM product, respec-tively; and tumor T20 with a genomic deletion atSLIT2 region, showing only M product. Due to therelevance of tumor suppressor genes BRCA1 andBRCA2, in breast cancer, we analyzed 5 tumorswith germline mutations (2 with BRCA2 mutationsand 3 with BRCA1 mutations). In general, tumorspresenting BRCA1 or BRCA2 germline mutationsdid not show any differences with the BRCAXtumors regarding methylation status at eachpromoter.

Hypermethylation and Expression of RASSF1A

One product of 169 bp was amplified for theanalysis of RASSF1A promoter for the hypermethy-lated and unmethylated conditions. It is importantto notice that six tumors, T1, T9, T25, T28, T35,and T49, presented a deletion in the locus con-taining RASSF1A, detected in a parallel arrayCGH study (manuscript in preparation). Promoterhypermethylation of RASSF1A was observed in 28/42 tumors (67%) (Supplementary Table). Nine ofthe 28 tumors presented only the hypermethylatedproduct suggesting that both RASSF1A alleles weresilenced due to methylation, or to gene deletionand methylation in the case of tumor T9. TumorsT1, T25, T28, and T35 presenting a deletion of the

Figure 2. Products amplified by methylation specific PCR (MS-PCR) of (a) RASSF1A, (b) SLIT2, and (c) WIF1promoter regions. The hypermethylated product is designated M and the unmethylated UM. Positive controlsare referred as Cþ. Two percent Agarose gel electrophoresis is shown. Molecular size marker corresponds topBR322 digested with Hinf I.



RASSF1A locus, showed M and UM products, prob-ably corresponding to the presence of differentcells in the tumor, since those having RASSF1A de-leted have only one allele remaining. In this sense,one possibility is that cells not having a RASSF1Adeletion may have one allele methylated and oneallele unmethylated. In the case of tumor T49,having one allele deleted, only the UM productwas observed. In relation to tumors with no dele-tion of RASSF1A we obtained only M products in 8tumors, M and UM products in 15 tumors, andonly UM products in 13 samples.

Sequencing of methylated products from 25%of the tumors revealed that 100% of CpG sitesin this promoter region were methylated (Table 2).Expression analysis detected by immunohis-tochemistry showed different intensities in stain-ing, indicating variable expression levels amongsamples. As shown in Figure 3, RASSF1A wasdetected in the cytoplasm according to its cellularfunction (A, B, C, and D). Tumors T24 and T43,where only UM products were observed (Supple-mentary Table), showed moderate cytoplasmicexpression (score 100–200) (Figure 3A and B).Tumors T44, T33, and T34 with scores <100(Figure 3C, D, and E) showed different levels of ex-pression for RASSF1A, all in agreement to promotermethylation status in each sample. We found a sig-nificant association between RASSF1A promoterhypermethylation status and its protein expression(P ¼ 0.0063) (Table 3).

We observed that 75% of RASSF1A methylatedtumors expressed the estrogen receptor. In

concordance, 78% of ER positive tumors are meth-ylated in RASSF1A promoter, contrasting with the47% of ER negative tumors found methylated. Wealso found RASSF1A promoter methylation in thefive informative ‘‘in situ’’ carcinomas (DCIS) ana-lyzed, which in addition were ER positive (Supple-mentary Table and Table 4). Moreover, we foundthat five out of six informative grade I ductal carci-nomas presented methylation at RASSF1A promot-er, which added to the finding in DCIS tumors,suggest an early inactivation of this gene in breastcancer.

Hypermethylation and Expression of SLIT2

A product of 160 bp for both methylated andunmethylated conditions was obtained for 40 outof 47 tumors. Three tumors, T4, T20, and T48, pre-sented deletion in SLIT2 locus, and only one, T20,was informative for the methylation analysis.Hypermethylated products were observed in 80%of the informative tumors. We observed that fivetumors led to only methylated products, includingtumor T20 with deletion at this locus. In relationto tumors with no deletion of SLIT2 we obtainedonly M products in 4 tumors, M and UM productsin 27 tumors, and only UM products in 8 samples.Sequencing of 20% of methylated PCR productsfrom SLIT2 promoter, showed over 87% of CpGsites methylated (Table 2). In this region between9% and 43% of CpG sites were partially methylat-ed, revealing a C and T in these positions. Partialmethylation of CpG sites was not associated withdistinct protein expression, indicating that 100%

Table 2. Frequency of CpG Sites Methylated at Each Promoter, Amplified by MSPCR for Methylated DNA

Gene/promoter Sample % Of methylated CpG sites Protein expression

RASSF1A T3 100% (17/17) þþT5 100% (17/17) þT8 100% (17/17) �T25 100% (17/17) þþT26 100% (17/17) þþT35 100% (17/17) �T40 100% (17/17) �

SLIT2 T1 100% (23/23) �T3 100% (23/23) �T11 87% (20/23) �T12 100% (23/23) þT18 100% (23/23) �T34 87% (20/23) �

WIF1 T17 39% (7/18) þT25 50% (9/18) þT26 61% (11/18) þT31 39% (7/18) þT32 39% (7/18) �T33 39% (7/18) þþT35 39% (7/18) �

Level of expression: �, negative; þ, weak; þþ, moderate; þþþ, strong.

6 ALVAREZ ET AL.


methylation is not necessary for silencing gene ex-pression. In addition, we observed that 2 out of 4informative DCIS and all (6) informative grade Iductal carcinomas presented methylation of SLIT2promoter, suggesting an early inactivation of thisgene in breast cancer.SLIT2 expression, as shown in Figure 3F and G

(T24 and T29), was present mostly in the

membrane and also in the cytoplasm, with moder-ate T24 (score 100–200) or weak intensity T29(score <100). These two samples (T24 and T29)gave M and UM products indicating that, at least,one allele was methylated and therefore, silenced.Figure 3H and I (T44 and T17) show a weak SLIT2staining. In the methylation analysis T44 (H) gaveonly UM product and T17 (I) UM and M products.

Figure 3. Immunohistochemistry analysis of RASSF1A (A–E), SLIT2 (F–J) and WIF1 (K–O) proteins. Correspond-ing methylation status is shown under each image. Photographs were taken at objective 40�.



Finally T36 in Figure 3J did not express SLIT2 andshowed both M and UM products. In summary90% of tumors with at least one hypermethylatedallele, showed weak or null protein expression(score <100). Nevertheless, a high percentage ofSLIT2 unmethylated tumors (7/8 tumors) also havea diminished or null expression of the protein(Table 3). No association was found between SLIT2promoter methylation and protein expression(Table 3). These results suggest that silencing ofSLIT2 expression is relevant event in breast cancer,and it is caused by different mechanisms includingpromoter methylation.

Hypermethylation and expression of WIF1

A 145 bp methylated product and 154 bp unme-thylated product were obtained for WIF1 promot-er. Four tumors, T4, T10, T11, and T20, presenteddeletion at WIF1 locus, and none of them were in-formative for the promoter methylation analysis.In general, only 32 tumors gave at least one prod-uct, out of which 23 (72%) were methylated. Forthis promoter, only one tumor (T28) presentedexclusively the methylated product, suggestinghypermethylation of both alleles, 22 presented Mand UM products, and 9 tumors showed only UMproducts. In addition, we observed that 3 out of3 informative DCIS and 5 out of six informativeductal carcinomas presented methylation at WIF1promoter, suggesting an early inactivation of thisgene in breast cancer.

Overall, sequencing of 30% of methylated WIF1promoter products revealed methylation of 39 to61% of all CpG sites (Table 2). Tumor T28, show-ing only M products (Figure 3O) gave no expres-sion of WIF1. For tumor T29 (Figure 3K), WIF1expression was detected strongly (score >200) inthe cytoplasm and in the membrane, correspond-ing to its localization as a secreted protein. Con-cordantly, T29 showed only the UM product.

Tumor T27 (Figure 3L) showed moderate staining(score 100–200) and both M and UM products.Two other tumors T39 and T43 (Figure 3M and N)showed a weak WIF1 stain (score <100) in all cells,presenting T39 both MS-PCR products, M andUM. In total, 76% of the methylated tumors had asignificant low WIF1 expression (score <100) and67% of unmethylated tumors had strong or mod-erate (score >100) expression of WIF1. For WIF1we found a significant association between pro-moter hypermethylation and protein expression(P ¼ 0.042) (Table 3).

DISCUSSION

In our sample of hereditary breast tumors,RASSF1A was found to be methylated in 67% ofbiopsies suggesting a relevant role in tumor pro-gression. Concordant with its methylation status,we found down regulated expression of this tumorsuppressor gene in 64% of the methylated tumors(45.4% considering all tumors). This finding indi-cates that silencing of one or both alleles is an im-portant mechanism in hereditary breast cancer,and suggests that promoter hypermethylation isa relevant molecular mechanism in inhibitingRASSF1A expression. To our knowledge, only oneprevious report described RASSF1A protein expres-sion analyses in breast cancer tumors [33]. Thiswork published by Li et al. in Chinese womenwith breast cancer, showed similar frequency ofRASSF1A promoter methylation, 66% compared to67% in our tumors. However 90% of Li et al. meth-ylated tumors presented a decreased protein ex-pression, much higher than the 64% shown in thisstudy. Li et al. report a reduced or absent RASSF1Aexpression, in 26 out of the 36 total cases studied(72%), higher than the 45.4% observed in ourwork. This difference indicates that RASSF1A si-lencing occurs more frequently in Chinese sporad-ic breast tumors than in our group of hereditary

Table 3. Association Between Promoter Methylation Status of RASSF1A, SLIT2, and WIF1 Genes, and Protein Expres-sion in Hereditary Breast Cancer Tumors

Gene/proteinname

Methylationstatus

Protein expression Association

Score <100N/total (%)

Score �100N/total (%) OR 95% CI P-value

RASSF1A M 16/25 (64.0) 9/25 (36.0) 10.67 1.94–58.72 0.0063�UM 2/14 (14.3) 12/14 (85.7)

SLIT2 M 27/30 (90) 3/30 (10) 1.29 0.11–14.34 1UM 7/8 (87.5) 1/8 (12.5)

WIF1 M 16/21 (76.2) 5/21 (23.8) 6.40 1.16–35.45 0.0419�UM 3/9 (33.3) 6/9 (66.7)

Two sided Fisher’s exact test.ELS score: <100: loss of expression, �100: positive expression.M, tumors with promoter hypermethylation; UM, tumors with no promoter hypermethylation; OR, odd ratio; CI, confidence interval.�Significant P-value �0.05.

8 ALVAREZ ET AL.


Table

4.AssociationofRASSF1A,SLIT2,andWIF1MethylationStatuswithClinicopathologicalFeaturesoftheTumors

Tumor’scharacteristics

RASSF1A

SLIT2

WIF1

Methylated(%

)Unmethylated(%

)P

Methylated(%

)Unmethylated(%

)P

Methylated(%

)Unmethylated(%

)P

Estrogenreceptor

Negative

7(46.7)

8(53.3)

0.085

13(76.5)

4(23.5)

0.703

6(60.0)

4(40.0)

0.407

Positive

21(77.8)

6(22.2)

19(82.6)

4(17.4)

17(77.3)

5(22.7)

Progesteronereceptor

Negative

8(50.0)

8(50.0)

0.098

12(75.0)

4(25.0)

0.691

8(72.7)

3(27.3)

1.000

Positive

20(76.9)

6(23.1)

20(83.3)

4(16.7)

15(71.4)

6(28.6)

BRCA1expression

Negative

12(57.1)

9(42.9)

0.326

19(86.4)

3(13.6)

0.430

13(76.5)

4(23.5)

0.699

Positive

16(76.2)

5(23.8)

13(72.2)

5(27.8)

10(66.7)

5(33.3)

Tumorgrade

I5(83.3)

1(16.7)

0.361

6(100.0)

00.565

5(83.3)

1(16.7)

0.640

II-III

14(53.8)

12(46.2)

20(76.9)

6(23.1)

15(68.2)

7(31.8)

Tumortype

Lobular

4(80.0)

1(20.0)

0.650

4(100.0)

00.566

1(100.0)

01.000

Ductal

24(64.9)

13(35.1)

28(77.8)

8(22.2)

23(74.2)

8(25.8)

Insitu

8(100)

00.037�

5(71.4)

2(28.6)

0.611

3(100.0)

00.555

Invasive

20(58.8)

14(41.8)

27(81.8)

6(18.2)

21(72.4)

8(27.6)

� SignificantPvalue,tw

osidedFisher’sexact

test.



tumors, possibly associated to a different geneticbackground of the tumors due to the ethnicity orto the hereditary condition of our tumors. In thiscontext, differences in expression between sporad-ic and hereditary tumors have been described forBRCA1 [21]. Another possibility for the observeddifferences might be the proportion of ER positivetumors constituting Li et al. cohort. As describedin this research, we found a trend of ER tumors tobe more frequently methylated than ER negativetumors. In this regard, Li et al. do not describe hor-mone receptor status of their tumors, neverthelessif they would have more ER positive tumors, itcould explain in part why they observed moretumors with RASSF1A silenced expression. Finallyand in addition, Li et al. might have a higher fre-quency of tumors showing only M products, caus-ing the silencing of RASSF1A alleles in a greaterproportion of breast tumors.

RASSF1A has been reported to be methylated in40% of osteosarcomas, 70% of prostate tumors,and 60% of sporadic cases of breast cancer [34]. Inother types of cancer, as head and neck, and colona lower frequency of RASSF1A promoter methyla-tion was reported (20% or lower). Recently Karray-Chouayekh et al. [35] reported in a sample ofTunisian women, that a high percentage (78%) ofinvasive breast carcinomas is methylated in RASSF1Apromoter. In addition, this report describes anassociation of RASSF1A methylation with the ageat diagnosis and a poor prognosis. In our study, wedid not find a correlation between RASSF1A meth-ylation status and tumor grade (Table 4), howeverwe found association of RASSF1A methylation withthe noninvasiveness of the tumor (Table 4). All‘‘in situ’’ carcinomas of ductal origin showed meth-ylation at RASSF1A promoter and most of thema significant decrease in protein expression (Sup-plementary Table), suggesting that inactivation ofthis tumor suppressor may occur in early stages ofcarcinogenesis as well as in pre-malignant lesions,and not only in more advanced cancer stages.A similar observation has been published byLehmann et al. [36] and Pascuali et al. [37],describing RASSF1A methylated in pre-malignantlesions such as epithelial hyperplasia, and DCIS. Inour study all DCIS showed expression of ER, whichcould be the reason of observing promoter methyl-ation in all these tumors, since we observed in oursample set a trending of ER tumors of being morefrequently methylated in RASSF1A promoter.Although we must consider that the DCIS sampleinvolves only six tumors.

As mentioned, we observed that 78% of ER posi-tive tumors are methylated in RASSF1A promoter,contrasting with the 47% of methylated ER nega-tive tumors. In agreement with this study, a signif-icant association between RASSF1A methylation

and ER expression has been described previouslyby Sunami et al. [38].The second tumor suppressor candidate gene,

SLIT2, is methylated in 80% of our hereditarytumors, and 90% of these tumors show a signifi-cant decrease in expression. SLIT2 was foundmethylated in a greater rate that RASSF1A andWIF1 in this study, and in good correlation withexpression down regulation. Sequencing of pro-moter region revealed that more than 87% ofall CpG sites were found methylated and SLIT2expression was strongly decreased in all thesequenced tumors (Table 2). This observation hasbeen previously described for gliomas, Wilm’stumors, neuroblastomas, renal cell carcinomas,and leukemias [9,41,42].Besides methylated/nonexpressing tumors, we

observed a high percentage of unmethylatedtumors that did not express SLIT2. These resultsstrongly indicate alternative mechanisms to silencethis gene in hereditary tumors, such as somaticmutations or postrancriptional regulation. It hasbeen reported previously that SLIT2 is down regu-lated in 83% of hepatocellular carcinomas incorrelation with promoter hypermethylation. Inaddition, down regulation of SLIT2 was found tobe associated with lymph node metastasis [43]. Inrelation to breast cancer, SLIT2 has been reportedto be hypermethylated in 43% of primary breasttumors in correlation to its down regulation [9], inaddition a second study demonstrated hyperme-thylation of SLIT2 in 58% of sporadic breasttumors [27]. To our knowledge, ours is the firststudy describing SLIT2 protein expression in breasttumors. Our group of hereditary breast cancershowed a higher percentage of tumors with pro-moter methylation of SLIT2 than the describedsporadic cases, suggesting that SLIT2 could bea relevant tumor suppressor for hereditary breastcarcinogenesis.WIF1 promoter analysis showed that 72% of

the hereditary tumors were methylated and 76%of these presented weak (9/21) or no expression(score <100) of WIF1 protein (7/21) (Supplementa-ry Table). Among 23 tumors with WIF1 hyper-methylation, 17 (74%) expressed the estrogenreceptor. In concordance 77.3% of ER positivetumors are methylated, however 60% of ER nega-tive tumors are also methylated, giving no signifi-cant difference in WIF1 methylation among ERpositive or negative tumors. No association wasfound between WIF1 methylation and expression,with grade or other tumor characteristics (Table 4).In gastrointestinal cancers, it has been describedthat in 74–83% of tumors WIF1 expression isdown regulated in concordance with promotermethylation [39]. In addition, the absence of ex-pression of WIF1 occurred in 72.7% of colorectaladenomas suggesting an early event in this type

10 ALVAREZ ET AL.


of cancer [39]. As described by Ai et al. [26], insporadic breast cancer WIF1 mRNA expression iscommonly diminished when compared with nor-mal tissues, and this decrease in expression is high-ly correlated with its promoter hypermethylation,which is found in 67% of tumors. To our knowl-edge, only one previous study reported the loss ofWIF1 protein in breast cancer tumors. This work,published by Wissmann et al. [13] showed that60% of the breast tumors presented absent or re-duced WIF1 protein expression similar to the 67%found in our study (Table 3). The fact that WIF1could be equally silenced in sporadic and heredi-tary tumors indicates its role as a key tumor sup-pressor in the mammary gland, which expressionneeds to be silenced in order that tumor continuesgrowing.The WIF1 promoter region analyzed in this

study includes nucleotide �333 to nucleotide�189 relative to WIF1 transcription initiation site.Sequencing of this region showed methylation of39–61% of the 17 CpG sites, being the most meth-ylated CpG sites localized close to the transcriptioninitiation site. The analyzed region overlaps withthe one described by Licchesi et al. [40] in a studyof nasopharyngeal carcinomas. These authorsdescribed a methylated region which is criticalfor WIF1 silencing in these tumors. This regionincludes nucleotides �295 and �95, overlappingthe sequence analyzed in our study (�333 to�189), and the most frequent methylated CpGsites in breast tumors. Urakami et al. [16] foundthat WIF1 promoter is methylated in bladder can-cer, but 50% or more of the CpG sites were partial-ly or not methylated. These tumors presentingpartial methylation of CpG sites showed down-reg-ulation of WIF1 expression. In our study, sequen-ces in the promoter region presented between 39and 61% of total CpG sites methylated, and therespective tumors showed weak or negative expres-sion of WIF1 (Table 2), accordingly to the previousreport. These results indicate that methylation of100% of sites is not necessary for down regulatingWIF1 expression, and indicate that there are keyCpG sites that need to be methylated to silenceWIF1 transcription.Promoter hypermethylation is one of the main

molecular mechanisms of tumor suppressor genesilencing in cancer and it has been suggested byseveral studies as an early event in tumor develop-ment. In our study, we have six informativegrade I tumors all methylated in SLIT2 promoter,five methylated in WIF1 and five methylatedin RASSF1A. Moreover in four out of six grade Itumors, promoter CpG islands were hypermethy-lated in the three genes. These results suggest thatthe hypermethylation of RASSF1A, SLIT2, andWIF1 promoters is an early event in hereditary

breast cancer, and maybe participants of keymolecular pathways in tumor progression.Taken together, these results clearly indicate

that promoter hypermethylation of these threegenes, RASSF1A, SLIT2, and WIF1, is a relevantmechanism for silencing gene expression in hered-itary breast cancer, even though other molecularmechanisms might be involved. In this regard, wefound several tumors with no evidence for promot-er methylation and not expressing the respectiveprotein.The silencing of gene expression may also be

explained by gene deletion or point mutations.Tumors having a deletion of RASSF1A, and pre-senting M and UM PCR products, show a signifi-cant loss of expression of RASSF1A protein(Supplementary Table). Tumors T1 and T25 repre-sent a controversy because, in addition to the ge-nomic deletion, they present M and UM productsand weak or moderate expression of the protein,respectively. We observed in both cases some cellswith no expression of RASSF1A and others withweak expression of the protein for T1, or moderatefor T25, (Supplementary Figure 2). Since DNA fromthese tumors led M and UM products, we mayexpect that cells not expressing the protein haveboth alleles silenced, either both methylated orone deleted and one methylated. In addition, cellspresenting a weak or moderate expression ofRASSF1A should have only one allele silenced,either by methylation or deletion. This interpreta-tion is supported by the evidence of a high cellularheterogeneity widely mentioned in several studies.Tumor T35, lacking expression of RASSF1A, pre-sented M and UM products suggesting that in tu-mor cells one allele is methylated and the other isdeleted and that the UM product may came fromthe stroma cells or adipocytes, well represented inthis piece of tumor. For tumor T28 it was not pos-sible to have IHC results due to deficient tissuequality. On the other hand, tumor T9 presentedonly M product and negative expression ofRASSF1A protein in agreement with the presenceof one methylated allele and loss of the other al-lele. And finally, tumor T49 presented only theUM product and a weak expression consistent withthe loss of at least one allele in all tumor cells.In relation to tumors carrying a deletion at

SLIT2 locus (T4, T20, and T48), we could onlyhave data for both MS-PCR and IHC for tumor T20(Supplementary Table). Tumor T20 showed only Mproduct and absence of SLIT2 protein expressionin agreement with the presence of one hyperme-thylated allele and loss of its locus in the secondallele. For tumors T4 and T48 we only obtainedresults for IHC, not discarding that promotermethylation could occur in these tumors. TumorsT4 and T48 showed negative and weak proteinexpression, respectively, suggesting at least the



silencing of SLIT2 by deletion of one allele, andperhaps silencing of the second allele by methyla-tion or a different mechanism.

Interestingly, 90% of the hypermethylatedtumors showed weak or null protein expressionand SLIT2 deletion was found in only one of thesetumors, suggesting that gene deletion is not themain molecular mechanism for silencing the sec-ond allele. Sequencing of SLIT2 in these tumorsmay reveal inactivating mutations affecting geneexpression.

In the case of WIF1 locus two out of four tumorspresenting deletion were informative for IHCshowing a significant decrease in WIF1 protein ex-pression. Unfortunately no MS-PCR products couldbe obtained from any of the four tumors probablydue to the greater size of the first PCR product forWIF1 (WIFPAN) in relation to RASSF1A and SLIT2promoter regions being tougher to analyze usinglow quality DNA.

In this study we were able to demonstrate a sig-nificant association between gene promoter hyper-methylation and expression for RASSF1A andWIF1, two relevant tumor suppressor genes in-volved in different types of cancer, including spo-radic breast tumors. These tumor suppressor genes,in addition to BRCA1 that was previously demon-strated to be hypermethylated in 51% and silencedin 67% of this same group of tumors [21], consti-tute good candidates as drivers of breast tumorprogression. Due to the relevance of tumor sup-pressors BRCA1 and BRCA2 for breast cancer weincluded in our analysis five tumors containinggermline mutations in either gene. Tumors pre-senting germline mutations in BRCA1 or BRCA2(Supplementary Table) did not show differenceswith BRCAX tumors in relation to the methylationstatus or expression of the three genes in thisstudy. On this regard, silencing of RASSF1A, SLIT2,and WIF1 may be independent of BRCA1 orBRCA2 status in the tumor cells.

It has been recently stated that the real targets incancer progression are the molecular pathwaysrather than specific genes, so, it would be interest-ing to analyze other genes involved in RASSF1A,SLIT2, and WIF1 pathways in breast tumors. Inthis regard, ROBO1 that codifies for one of thereceptors for SLIT2 was found deleted in our paral-lel array CGH study, in some hereditary breasttumors, and moreover it also has been postulatedas a candidate tumor suppressor gene for lung [44]and cervical cancer [45]. These findings, in addi-tion to the high frequency of SLIT2 down regula-tion in our tumors, suggest the relevance of theSLIT-ROBO pathway in hereditary breast cancer.Understanding which molecular pathways partici-pate in hereditary tumor development will helpus to find new candidate genes to this complexdisease.

ACKNOWLEDGMENTS

We are grateful to FONDECYT grant 1080595and to CONICYT for providing scholarships toCarolina Alvarez.

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Silencing of tumor suppressor genes RASSF1A, SLIT2, and WIF1 by promoter hypermethylation in hereditary breast cancer