Frequent MYC coamplification and DNA hypomethylation of multiple genes on 8q in 8p11-p12-amplified breast carcinomas

Abstract

Genetic and epigenetic (DNA methylation, histone modifications, microRNA expression) crosstalk promotes inactivation of tumor suppressor genes or activation of oncogenes by gene loss/hypermethylation or duplications/hypomethylation, respectively. The 8p11-p12 chromosomal region is a hotspot for genomic aberrations (chromosomal rearrangements, amplifications and deletions) in several cancer forms, including breast carcinoma where amplification has been associated with increased proliferation rates and reduced patient survival. Here, an integrative genomics screen (DNA copy number, transcriptional and DNA methylation profiling) performed in 229 primary invasive breast carcinomas identified substantial coamplification of the 8p11-p12 genomic region and the MYC oncogene (8q24.21), as well as aberrant methylation and transcriptional patterns for several genes spanning the 8q12.1-q24.22 genomic region (ENPP2, FABP5, IMPAD1, NDRG1, PLEKHF2, RRM2B, SQLE, TAF2, TATDN1, TRPS1, VPS13B). Taken together, our findings suggest that MYC activity and aberrant DNA methylation may also have a pivotal role in the aggressive tumor phenotype frequently observed in breast carcinomas harboring 8p11-p12 regional amplification.

Citation: Oncogenesis (2014) 3, e95; doi:10.1038/oncsis.2014.8& 2014 Macmillan Publishers Limited All rights reserved 2157-9024/14

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SHORT COMMUNICATION

Frequent MYC coamplication and DNA hypomethylation of multiple genes on 8q in 8p11-p12-amplied breast carcinomas

TZ Parris1, A Kovcs2, S Hajizadeh2, S Nemes3,4, M Semaan1, M Levin1, P Karlsson1 and K Helou1

Genetic and epigenetic (DNA methylation, histone modications, microRNA expression) crosstalk promotes inactivation of tumor suppressor genes or activation of oncogenes by gene loss/hypermethylation or duplications/hypomethylation, respectively. The 8p11-p12 chromosomal region is a hotspot for genomic aberrations (chromosomal rearrangements, amplications and deletions) in several cancer forms, including breast carcinoma where amplication has been associated with increased proliferation rates and reduced patient survival. Here, an integrative genomics screen (DNA copy number, transcriptional and DNA methylation proling) performed in 229 primary invasive breast carcinomas identied substantial coamplication of the 8p11-p12 genomic region and the MYC oncogene (8q24.21), as well as aberrant methylation and transcriptional patterns for several genes spanning the 8q12.1-q24.22 genomic region (ENPP2, FABP5, IMPAD1, NDRG1, PLEKHF2, RRM2B, SQLE, TAF2, TATDN1, TRPS1, VPS13B). Taken together, our ndings suggest that MYC activity and aberrant DNA methylation may also have a pivotal role in the aggressive tumor phenotype frequently observed in breast carcinomas harboring 8p11-p12 regional amplication.

Oncogenesis (2014) 3, e95; doi:http://dx.doi.org/10.1038/oncsis.2014.8

Web End =10.1038/oncsis.2014.8 ; published online 24 March 2014

Subject Categories: Molecular oncology

Keywords: breast cancer; DNA amplication; DNA methylation; 8p11-p12; MYC

INTRODUCTIONGenomic instability and epigenetic modulations, that is, DNA methylation, histone modications, microRNA expression, contribute to the neoplastic phenotype by deregulating key gene functions that permit cells to bypass regulatory mechanisms controlling and maintaining normal cellular physiology.1 Recently, genetic and epigenetic crosstalk has shown to be one of several major driving forces behind tumor initiation and progression.26 However, DNA methylation is considered by some to be a secondary event which locks genes in their inactive/active states only after gene silencing/activation has been achieved by other means.710

Several well-characterized DNA regions have been investigated extensively in breast cancer for their role in genetic modulations, interactions in molecular pathways and association with unfavorable clinical outcome. These include the 8p11-p12, 8q24 (MYC), 11q13 (CCND1), 17q12 (ERBB2, GRB7, STARD3) and 20q13 (ZNF217, MYBL2, STK6) amplicons, some of which have become major molecular targets for breast cancer treatment. Regional amplication of the 8p11-p12 genomic region is a common genetic event in solid tumors, for example, breast carcinoma,1113 pleuro-pulmonary blastoma,14 lung cancer and esophageal squamous cell carcinomas,1518 urinary bladder cancer,19,20 osteosarcoma21 and pancreatic adenocarcinoma.17 In breast cancer cell lines, the initiation site and structure of the 8p11-p12 DNA rearrangement involved different mechanisms of gene activation, thereby resulting in the activation of different combinations of candidate genes.22

To further dene the role, 8p11-p12 regional amplication may have on breast cancer pathophysiology, we examined genome-wide copy number alterations, DNA methylation patterns and transcriptional changes in 229 primary invasive breast tumors.Here, we demonstrate that B50% of 8p11-p12-amplied tumors also harbor MYC amplication, as well as, hypomethylation of genes located in close proximity to the MYC gene.

RESULTS AND DISCUSSIONAmplication of the 8p11-p12 genomic region is a common genetic event in breast carcinoma with clinical implications. To assess aberrant transcriptional and DNA methylation patterns in invasive breast carcinomas harboring the 8p11-p12 amplicon, an integrative analysis was performed using DNA copy number, DNA methylation and transcriptome data from 229 primary invasive breast cancer samples previously presented in our work,23,24 including our own unpublished data. The DNA copy number analysis using array-comparative genomic hybridization data showed recurrent copy number alterations on chromosome bands 8p11-p12 in 83 tumors (36%), including 47/83 high-level gains/amplications, 20/83 low-level gains and 16/83 heterozygous losses. Copy number alterations were conrmed using a set of overlapping BAC clones building a contig over the 8p11-p12 genomic region. On average, there was a ve-fold increase in the number of amplications observed in lesions containing the 8p11-p12 amplicon compared with those lacking the amplicon (P 1.8E 13). In general, amplication of the 8p11-p12 genomic

1Department of Oncology, Institute of Clinical Sciences, Sahlgrenska Cancer Center, Sahlgrenska Academy at University of Gothenburg, Gothenburg, Sweden; 2Department of Clinical Pathology and Cytology, Sahlgrenska University Hospital, Gothenburg, Sweden; 3Division of Clinical Cancer Epidemiology, Department of Oncology, Institute of Clinical Sciences, Sahlgrenska Academy at Gothenburg University, Gothenburg, Sweden and 4Department of Oncology, Regional Cancer Center (West), Western Sweden Health Care Region, Sahlgrenska University Hospital, Gothenburg, Sweden. Correspondence: Dr TZ Parris, Department of Oncology, Institute of Clinical Sciences, Sahlgrenska Cancer Center, Sahlgrenska Academy at University of Gothenburg, Medicinaregatan 1G, 6th oor, Gothenburg 41390, Sweden.

E-mail: mailto:[email protected]

Web End [email protected] Received 10 December 2013; revised 22 January 2014; accepted 27 January 2014

Genetic and epigenetic changes in 8p11-p12-amplied breast tumors

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region was predominantly coamplied with 1q, 8q, 11q, 12p, 16p, 17q or 20q, but also occurred as the sole region of amplication in two cases. Notably, 53% (n 24) of 8p11-p12-amplied tumors

were coamplied with the MYC gene, whereas only 20% (n 9)

and 18% (n 8) were coamplied with the CCND1 and ERBB2

genes, respectively (Figure 1). Extensive research has been carried out on the coamplication of 8p11-p12 and CCND1, but few studies have investigated 8p11-p12 and MYC interactions.22,25

In accordance with published studies, genetic aberrations of the 8p11-p12 region (gain, loss and amplication; P 5.0E 6),

including amplication (P 4.0E 5) or loss (P 0.005), were

associated with reduced overall survival rates.26 Conversely, genomic gain was not indicative of unfavorable patient outcome (P 0.08). The amplicon was most prevalent in tumors

of large pathologic size (P 0.0002), high genomic grade index

status (P 0.0004) and high S-phase fraction (P 0.002; Table 1).

There was no signicant difference in histologic type, axillary lymph node status, estrogen/progesterone receptor status, human epidermal growth factor receptor 2 (HER2)/neu receptor status, triple negative status or molecular breast cancer subtype. These ndings are consistent with previous reports showing high cell proliferation (high Ki-67) and high tumor grade in breast carcinomas harboring the 8p11-p12 amplicon. However, Gelsi Boyer et al. 26 did not nd a connection with amplication and tumor size. Recently, several studies have found an association between the luminal B molecular subtype and DNA amplication of two genes (ZNF703 and FGFR1) within the 8p11-p12 amplicon. Interestingly, tumors harboring these genetic alterations were also resistant to endocrine therapy.2730 However, we show that B80%

of the breast tumors analyzed here were luminal B subtype/ estrogen receptor-positive regardless of 8p11-p12 amplicon status. Furthermore, ZNF703 was generally upregulated in breast

carcinomas, particularly in estrogen receptor-positive tumors, compared with normal breast tissue.24 Functional studies have provided additional evidence for biological effects in vitro and in vivo using small-interfering RNA-mediated knockdown of candidate genes within the 8p11-p12 genomic region.2733 Eight genes (BAG4, C8orf4, DDHD2, ERLIN2, LSM1, PPAPDC1B, WHSC1L1 and ZNF703) have thereby emerged as targets with oncogenic potential.

To delineate whether aberrant methylation patterns may also has a role in the evolution of breast tumors harboring the 8p11-p12 amplicon, we performed genome-wide DNA methylation analysis on 22/229 tumors (11 tumors harboring the amplicon and 11 tumors lacking the amplicon) using the 450k Innium Methylation Beadchip (Illumina Inc., San Diego, CA, USA). Of the 382 815 cytosine sites remaining after ltering, p1% (n 2066)

were differentially methylated in tumors harboring the 8p11-p12 amplicon compared with samples lacking the amplicon. Eighty-nine percent of aberrantly-methylated cytosine sites were hypermethylated (n 1847) and 11% (n 219) of sites were

hypomethylated. The promoter regions (200 and 1500 bp upstream transcriptional start sites, 50 untranslated region and the rst exon) were tightly linked with hypermethylation (n 352

sites, 92%), whereas fewer methylation events occurred further downstream in the body of genes and at the 30 untranslated region region. The highest number of aberrantly-methylated cytosine sites surrounded CpG islands (n 408) with fewer sites

found in the CpG shores (up to 2 kb from CpG islands, n 172)

and shelves (24 kb from CpG islands, n 48). The majority of

aberrant methylation patterns occurred within genes and inter-genic regions, whereas few microRNA transcripts were found (Figure 2). We found that differential methylation occurred on all chromosomes including the X chromosome in 8p11-p12-amplied

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Figure 1. Array-CGH genomic proles showing recurrent DNA amplication of the 8p11-p12 genomic region in breast carcinoma. The top panel shows focal amplication (log2 ratio40.5) of the 8p11-p12 region in two breast tumors. Black dots depict BAC clones spanning chromosome 8 for tumor 8931 and gray dots for tumor 9493. The bottom panel shows amplication of the 8p11-p12 and 8q regions. Black dots depict BAC clones spanning chromosome 8 for tumor 11248 and gray dots for tumor 8138. The x-axis shows chromosome 8 from the 8p telomere to the 8q telomere. The y-axis shows the log2 ratio value for each BAC clone (tumor gDNA versus normal control gDNA).

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Table 1. Correlation between 8p11-p12 DNA amplication and clinicopathological features in breast carcinoma

Characteristics Number of tumors (%)

Total tumors (n 229)

Neutral DNA dosagea (n 71)

DNA amplicationa (n 45)

P-value

Age

Mean 59 60 60 Range 3088 3779 3088

Histologic type 0.7

Ductal 136 (59) 52 (73) 21 (47) Lobular 22 (10) 7 (10) 4 (9) Other 26 (11) 12 (17) 3 (7) Not available 45 (20) 0 (0) 17 (38)

Axillary lymph node status 0.2 pN0 82 (36) 38 (54) 12 (27)pN1 84 (37) 33 (46) 19 (42)

Not available 63 (28) 0 (0) 14 (31)

Pathologic tumor size 0.0002 pT1 51 (22) 22 (31) 4 (9)pT2 89 (39) 35 (49) 12 (27)pT3 51 (22) 11 (15) 20 (44)pT4 6 (3) 3 (4) 0 (0)

Not available 32 (14) 0 (0) 9 (20)

S-phase fraction 0.002 p6.1 112 (49) 59 (83) 18 (40)

46.1 69 (30) 12 (17) 16 (36) Not available 48 (21) 0 (0) 11 (24)

GGI status 0.0004

Low 45 (20) 31 (44) 6 (13) High 73 (32) 26 (37) 29 (64) Not available 111 (48) 14 (20) 10 (22)

Estrogen receptor 0.8

Negative 60 (26) 14 (20) 10 (22) Positive 166 (72) 57 (80) 34 (76) Not available 3 (1) 0 (0) 1 (2)

Progesterone receptor 0.7

Negative 108 (47) 31 (44) 21 (47) Positive 118 (52) 40 (56) 23 (51) Not available 3 (1) 0 (0) 1 (2)

HER2/neu status 0.6

Negative 199 (87) 61 (86) 37 (82) Positive 30 (13) 10 (14) 8 (18) Not available 0 (0) 0 (0) 0 (0)

Triple negative status 1.0

Yes 41 (18) 9 (13) 5 (11) No 186 (81) 62 (87) 39 (87) Not available 2 (1) 0 (0) 1 (2)

Subtype 0.9

Luminal subtype A

2 (1) 1 (1) 0 (0)

Luminal subtype B/

HER2-

101 (44) 47 (66) 31 (69)

Luminal subtype B/

HER2

13 (6) 8 (11) 4 (9)

HER2/ER- 18 (8) 10 (14) 5 (11) Basal-like 16 (7) 5 (7) 5 (11) Normal-like 0 (0) 0 (0) 0 (0) Not available 79 (34) 0 (0) 0 (0)

Abbreviations: GGI status, genomic grade index; HER2, human epidermal growth factor receptor 2. P-values were calculated using the Fishers exact test (neutral DNA dosage versus DNA amplication). aTumor specimens included in the analysis with both array-CGH and gene expression microarray data are available.

tumors, where hypermethylation ranged from 7298% and the highest hypomethylation rates were found on chromosomes 8 and 9 with 28% and 24%, respectively.

Few of the methylation events resulted in aberrant gene expression patterns in 8p11-p12-amplied tumors (n 61, 4.5% of

aberrantly-methylated coding RNAs), although disparate methylation-transcriptional patterns were observed for 23/61 genes (38%); 20/23 genes were hypermethylated and overexpressed and 3/23 genes were hypomethylated and underexpressed (Table 2). Univariate Cox regression analysis showed that aberrant transcriptional patterns for 47/61 genes inuenced overall survival rates. In addition, only one gene located at 8p11-p12 showed differential methylation and gene expression patterns, that is, BRF2 was hypermethylated but overexpressed owing to BRF2 gene amplication in 7/11 cases. Gene Ontology enrichment analysis of the genes with aberrant DNA methylation and gene expression patterns revealed several cancer-related processes, for example, cell differentiation, DNA replication, cell migration and cell adhesion (Table 3).

Interestingly, 11 genes spanning the 8q12.1-q24.22 genomic region were differentially methylated and expressed, of which nine genes (IMPAD1, NDRG1, PLEKHF2, RRM2B, SQLE, TAF2, TATDN1, TRPS1, VPS13B) were hypomethylated and overexpressed, the ENPP2 gene was hypermethylated and underexpressed and the FABP5 gene was hypermethylated but overexpressed. As the 11 genes were also coamplied with the 8p11-p12 region in at least one tumor specimen, we examined whether DNA copy number, DNA methylation or both had an impact on gene expression (Figure 3). We found that hypomethylation alone frequently enhanced gene expression patterns. However, hypomethylation and DNA amplication of the same transcript further enhanced expression levels. These ndings suggest that genes in the 8q region are frequently targeted by more than one mechanism for activation in breast tumors harboring 8p11-p12 amplication. Consequently, ENPP2 was the only example showing lower expression levels when hypermethylated (at four different cytosine sites in the promoter region) despite amplication of the gene in 2/11 samples harboring the 8p11-p12 amplicon. These results indicate that aberrant methylation patterns may be a secondary event to further lock genes in their inactive or active states only after they have already been silenced or activated by other means.710 The ENPP2 gene was an exception to this phenomenon because hypermethylation occurred at four different cytosine sites in the promoter region of the gene, resulting in lower transcriptional levels despite amplication of the gene in 2/11 samples harboring the 8p11-p12 amplicon. However, 8/11 genes (FABP5, NDRG1, PLEKHF2, RRM2B, SQLE, TAF2, TATDN1, TRPS1) may not be distinctive of 8p11-p12 amplication as they were also differentially regulated in MYC coamplied tumors.

Several of the aberrantly-methylated genes spanning the 8q arm have been previously associated with cancer-related processes. In addition to gene amplication shown in the present study, VPS13B frameshift mutations have also been identied in gastric and colorectal cancers, as well as TRPS1-LASP1, PLEC1-ENPP2 and TATDN1-GSDMB fusion genes in breast carcinoma.3436 Recently, gain of TRPS1, TATDN1 and SQLE DNA copy numbers in estrogen receptor-positive, ERBB2-amplied breast tumors have been reported, and elevated SQLE levels were associated with distant metastasis-free survival.3739 TRPS1, a transcription factor that belongs to the GATA gene family, in which, protein expression is inhibited by androgens via the androgen receptor in prostate cancer and demonstrates high expression levels in both normal breast and tumor tissue. In breast tumors, TRPS1 expression is associated with ER, PgR, GATA3, HER2/neu expression and favorable clinical outcome.40,41 Elevated NDRG1 protein levels have been associated with shorter disease-free and overall survival, cell differentiation and breast cancer progression.4244 In contrast, Han et al.45 demonstrated that NDRG1 methylation in breast cancer is associated with a more aggressive phenotype. Interestingly, substantial NDRG1 phosphorylation is found in Akt inhibitor-resistant breast cancer cell lines,

& 2014 Macmillan Publishers Limited Oncogenesis (2014), 1 8

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Hypermethylation Hypomethylation Downregulation Upregulation

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Island 408 regions

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Shelf 48 regions

Coding RNAs 1361 sites

Non-coding RNAs 20 sites

Intergenic 694 sites

CpG and neighborhood context

Functional genomic distribution Associated RNA transcripts

Number of differentially expressed genes corresponding

to cytosine sites/regions

Figure 2. DNA methylation patterns in 8p11-p12-amplied tumors. The distribution of aberrant methylation (hyper- and hypomethylation, Qo0.05) and gene expression patterns (downregulation and upregulation, Qo0.01) among the 2066 differentially-methylated cytosine sites in 8p11-p12-amplied tumors. Transcripts were categorized into functional genomic regions (promoter region (between 200 and 1500 bp upstream of transcriptional start sites, 50 untranslated region, rst exon), gene body and 30 untranslated region region) and regions surrounding CpG islands (CpG islands, 2 kb from CpG islands (CpG shores) and 24 kb from CpG islands (CpG shelves)).

which can be reversed by the mTORC1/2 inhibitor, MLN0128, in breast cancer xenograft models.46,47 p53-mediated induction of DNA damage-associated genes, such as RRM2B, can promote resistance of cancer cells to genotoxic therapy, which can be prevented by inhibiting histone deacetylases that can in turn inhibit ataxia telangiectasia-mutated kinase and p53 activation and their downstream targets.4850 The TAF2 gene is involved in general transcription processes and is the DNA binding component of the transcription factor II D transcription factor complex.51

In summary, we have identied the enrichment of MYC amplication and hypomethylation of genes on cytoband 8q in 8p11-p12-amplied tumors. These ndings indicate that the aggressive phenotype observed in invasive breast tumors harboring the 8p11-p12 amplicon may not only be a consequence of altered activity of amplied genes in the genomic region, but also a result of MYC coamplication and aberrant DNA methylation patterns on chromosome 8q.

MATERIALS AND METHODSTumor specimensPrimary invasive breast carcinoma specimens (n 229) corresponding to

185 patients diagnosed from 19881999 were obtained from the fresh-frozen tumor bank at the Sahlgrenska University Hospital Oncology Lab in accordance with the Declaration of Helsinki and approved by the Medical Faculty Research Ethics Committee (Gothenburg, Sweden). The 229 cases were compiled from three independent array-comparative genomic hybridization microarray datasets, including two published (138/229 tumors) and one unpublished (91/229 tumors) studies.23,24 The clinicopathological features of the 229 cases are shown in Table 1. Each tumor specimen was assessed for the presence of malignant cells using MayGrnwald Giemsa staining (Chemicon International, Temecula, CA, USA) on touch preparations. Highly representative specimens containing 470% neoplastic cell content were included in the microarray and uorescence in situ hybridization analyses.

Genomic and transcriptome prolingGenomic proling of the tumor specimens was performed using whole-genome tiling 38K array-comparative genomic hybridization microarrays, as previously described.23,24 Data preprocessing, normalization and data analysis were performed as previously described using log2 ratio thresholds set at 0.2, X 0.5, 0.2 and p 1.0 for low-level gain,

high-level gain/amplication, heterozygous loss and homozygous deletion

(henceforth referred to as gain, amplication, loss and deletion), respectively.24 Total RNA samples from 150/229 tumor specimens were isolated and proled using Illumina HumanHT-12 Beadchips (Illumina Inc.) as previously described.24 Enriched gene ontology terms associated with differentially regulated genes were set to Po0.05, analyzed further using the gene ontology database (http://www.geneontology.org

Web End =http://www.geneontology.org). The dataset was stratied into the molecular breast cancer subtypes using the ve centroids (normal-like, basal-like, luminal subtype A, luminal subtype B and human epidermal growth factor receptor 2/estrogen receptor-negative (HER2/ER )) and genomic grade index (high, low), as previously

described.5254 Luminal subtype B was further stratied according HER2 status as determined by array-comparative genomic hybridization; HER2

was set to log2 ratio X 0.5 and HER2 was set to log2 ratio o 0.5.55

Univariate Cox proportional hazard models were calculated for statistically signicant genes using overall survival.

Fluorescence in situ hybridizationProbe labeling and hybridization were performed as described elsewhere56 using locus-specic bacterial articial chromosome (BAC; BACPAC Resources, Oakland, CA, USA) probes to verify gene amplication. Dual-color uorescence in situ hybridization was performed on touchprint and metaphase preparations using cohybridized biotin-16-deoxyuridine triphosphate (dUTP) and dioxigenin-11-dUTP-labeled probes. Analysis was performed using a Leica DMRA2 uorescent microscope (Leica, Wetzler, Germany) equipped with an ORCA Hamamatsu CCD (charged-couple devices) camera (Hamamatsu City, Japan) and lter cubes specic for uorescein isothiocyanate, Rhodamine and ultraviolet for DAPI visualization. Digitalized black and white images were acquired using the Leica CW4000 software package (Leica).

DNA methylation prolingIn total, 22/229 tumor samples harboring (n 11) or lacking the 8p11-p12

amplicon (n 11) were proled using Illumina Innium Human Methyla

tion 450 Beadchips (Illumina Inc) according to the manufacturers instructions. The estimated methylation level for specic cytosine sites (average beta) was calculated as a ratio between the intensities of methylated and unmethylated alleles and ranged from 0 (null methylated) to 1 (completely methylated). Delta beta values were calculated using (average beta values8p11p12-amplied tumorsaverage beta values8p11-p12 nonamplied tumors). Cytosine sites located on the Y chromosome or containing single-nucleotide polymorphisms were removed. Differential DNA methylation was determined using the IMA package in R/ Bioconductor (Bioconductor, FHCRC, Seattle, WA, USA) with thresholds set at: X0.14 delta beta value and Bonferroni adjusted at Po0.05.57

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DNA copy number 8p11-p12-amplied tumors (n 11)e

amplication/ loss/normal

5

Table 2. Differentially-methylated genes in 8p11-p12-amplied breast tumors

Gene symbol Chromosome Delta beta valuea Gene expression(n 22)b

Gene expression (n 150)c

Cox coefcient (n 150)d

DNA copy number 8p11-p12 nonamplied tumors (n 11)f

amplication/ loss/normal

BAMBI 10p12.1 Hypomethylated Overexpressed NS 0/0/11 0/0/11

CXCL12 10q11.21 Hypermethylated Underexpressed 0.498 3.76E 05 0/0/11 0/0/11

HTRA1 10q26.13 Hypermethylated Underexpressed 0.400 1.57E 04

CRYAB;HSPB2 11q23.1 Hypermethylated Underexpressed Underexpressed NS 0/4/7 0/0/11 PTHLH 12p11.22 Hypermethylated Underexpressed Underexpressed NS 1/0/10 0/0/11 HOXC13 12q13.13 Hypermethylated Overexpressed 0.397 3.96E 04 0/1/10 0/0/11

PABPC3 13q12.13 Hypermethylated Overexpressed 0.643 1.12E 06 0/4/7 0/0/11

TRAPPC6B 14q21.1 Hypermethylated Overexpressed 0.866 1.80E 05 0/2/9 0/0/11

BMP4 14q22.2 Hypermethylated Underexpressed 0.379 0.001 0/3/8 0/0/11

BATF 14q24.3 Hypomethylated Underexpressed 0.502 0.002 0/4/7 0/0/11

ELL3 15q15.3 Hypomethylated Overexpressed 0.432 0.009 0/0/11 0/0/11 SEPHS2 16p11.2 Hypomethylated Overexpressed 0.495 0.003 0/0/11 0/0/11 SPN 16p11.2 Hypermethylated Overexpressed 0.254 0.064 0/0/11 0/0/11 SPAG9 17q21.33 Hypermethylated Overexpressed Overexpressed 0.628 0.000 1/0/10 0/0/11 DHX40 17q23.1 Hypermethylated Overexpressed 0.599 0.004 2/0/9 0/0/11 CCDC47 17q23.3 Hypomethylated Overexpressed 0.671 0.001ICAM2 17q23.3 Hypermethylated Underexpressed Underexpressed 0.441 0.012 1/0/10 0/0/11

CYGB 17q25.1 Hypermethylated Underexpressed 0.433 0.013 0/0/11 0/0/11

NFIX 19p13.2 Hypermethylated Underexpressed 0.289 0.023 0/0/11 0/0/11

NFIC 19p13.3 Hypermethylated Underexpressed NS 0/1/10 0/0/11 CACNG6 19q13.42 Hypermethylated Overexpressed NS 0/0/11 0/0/11 PHGDH 1p12 Hypermethylated Underexpressed 0.313 0.002 0/0/11 0/0/11 CHI3L2 1p13.3 Hypermethylated Underexpressed NS 0/0/11 0/0/11 COL11A1 1p21.1 Hypermethylated Overexpressed NS 0/1/10 0/0/11 AGL 1p21.2 Hypomethylated Overexpressed Overexpressed 0.337 0.013 0/1/10 0/0/11 PODN 1p32.3 Hypermethylated Underexpressed 0.366 0.001 0/2/9 0/0/11

MMP23A;MMP23B 1p36.33 Hypermethylated Underexpressed 0.415 0.003 0/1/10 0/0/11

MMP23B 1p36.33 Hypermethylated Underexpressed 0.415 0.003 0/1/10 0/0/11

EXOC8 1q42.2 Hypomethylated Overexpressed Overexpressed 0.734 2.58E 05 1/1/9 0/0/11

SYCP2 20q13.33 Hypomethylated Overexpressed Overexpressed 0.447 3.15E 05 2/0/9 0/0/11

GREB1 2p25.1 Hypomethylated Overexpressed 0.268 0.050 0/2/9 0/1/10

C2orf40 2q12.2 Hypermethylated Underexpressed Underexpressed 0.268 0.005

SATB2 2q33.1 Hypermethylated Overexpressed NS 0/0/11 0/0/11 KIF1A 2q37.3 Hypermethylated Overexpressed NS 0/0/11 0/0/11 TF 3q22.1 Hypermethylated Underexpressed NS 0/1/10 0/0/11 TAPT1 4p15.32 Hypomethylated Overexpressed NSSORBS2 4q35.1 Hypomethylated Underexpressed Underexpressed 0.326 0.004

PIK3R1 5q13.1 Hypermethylated Overexpressed 0.536 3.96E 04 0/0/11 0/0/11

CARTPT 5q13.2 Hypermethylated Underexpressed NS

PCSK1 5q15 Hypermethylated Underexpressed 0.326 0.002 0/1/10 0/0/11

PAM 5q21.1 Hypermethylated Underexpressed Underexpressed 0.340 0.024 0/0/11 0/0/11

DMXL1 5q23.1 Hypermethylated Overexpressed Overexpressed 0.620 9.99E 06 0/0/11 0/0/11

H2AFY 5q31.1 Hypermethylated Overexpressed Overexpressed 0.831 3.15E 07 0/0/11 0/0/11

DOCK2 5q35.1 Hypomethylated Underexpressed NS 0/0/11 0/0/11 SCGB3A1 5q35.3 Hypermethylated Underexpressed 0.240 0.001 0/0/11 0/0/11

USP49 6p21.1 Hypermethylated Overexpressed 0.310 0.031 1/0/10 0/0/11 SCAND3 6p22.1 Hypermethylated Overexpressed 0.492 0.015ID4 6p22.3 Hypermethylated Overexpressed 0.492 1.95E 05 0/0/11 0/0/11

RARS2;ORC3L 6q15 Hypomethylated Overexpressed 0.822 1.85E 05 2/1/8 0/0/11

LRP11 6q25.1 Hypomethylated Overexpressed 0.638 3.05E 04 0/1/10 0/0/11

C7orf28A 7p22.1 Hypomethylated Overexpressed 0.775 1.85E 05 0/0/11 0/0/11

LFNG 7p22.3 Hypermethylated Underexpressed 0.668 4.55E 06 0/0/11 0/0/11

PON3 7q21.3 Hypermethylated Underexpressed Underexpressed 0.305 3.19E 05 1/0/10 0/0/11

NRCAM 7q31.1 Hypomethylated Overexpressed 0.336 0.024 0/0/11 0/0/11 NDUFA5 7q31.32 Hypermethylated Overexpressed 0.771 1.48E 04 0/1/10 0/0/11

LEP 7q32.1 Hypermethylated Overexpressed 0.357 0.003 0/0/11 0/0/11 RARRES2 7q36.1 Hypermethylated Underexpressed 0.272 0.020 0/0/11 0/0/11

BRF2 8p11.23 Hypermethylated Overexpressed Overexpressed NS 7/2/2 0/0/11 IMPAD1 8q12.1 Hypomethylated Overexpressed Overexpressed 1.067 2.70E 07

FABP5 8q21.13 Hypermethylated Overexpressed NS 1/0/10 0/0/11 PLEKHF2 8q22.1 Hypomethylated Overexpressed Overexpressed 0.393 4.47E 04 3/0/8 0/0/11

VPS13B 8q22.2 Hypomethylated Overexpressed 0.878 7.06E 06

RRM2B 8q22.3 Hypomethylated Overexpressed Overexpressed 0.452 0.001 3/0/8 0/0/11 TRPS1 8q23.3 Hypermethylated Overexpressed Overexpressed 0.320 0.008 6/0/5 0/0/11 TRPS1 8q23.3 Hypomethylated Overexpressed Overexpressed 0.320 0.008 6/0/5 0/0/11 ENPP2 8q24.12 Hypermethylated Underexpressed Underexpressed 0.258 0.017 2/0/9 0/0/11

TAF2 8q24.12 Hypomethylated Overexpressed 1.164 1.34E 06 2/0/9 0/0/11

SQLE 8q24.13 Hypomethylated Overexpressed Overexpressed 0.566 2.95E 07 2/0/9 0/0/11

TATDN1 8q24.13 Hypomethylated Overexpressed Overexpressed 0.783 2.89E 06 5/0/6 0/0/11

NDRG1 8q24.22 Hypomethylated Overexpressed 0.843 5.03E 11 2/1/8 0/0/11

WDR44 Xq24 Hypomethylated Overexpressed 0.937 4.75E 06 3/0/8 0/0/11

Abbreviation: NS, not statistically signicant. Genes not correlating between DNA methylation and transcriptional patterns are shown in bold text. aDelta beta value (8p11-p12-amplied tumors versus nonamplied tumors) 40.14 are indicated by hypermethylation and o 0.14 are indicated by hypomethylation;

Bonferroni adjusted P-value Po0.05. bGene expression microarray log2 ratio for the 22 tumors (8p11p12-amplied tumors versus nonamplied tumors) 40.58 are indicated by overexpression and o 0.58 are indicated by underexpression. cGene expression microarray log2 ratio for the 150 tumors (8p11-p12-

amplied tumors versus nonamplied tumors) 4 0.58 are indicated by overexpression and o 0.58 are indicated by underexpression. dUnivariate Cox

proportional hazard regression models using the gene expression data for the 150 tumors and overall survival rates. eArray-CGH log2 ratio thresholds set at X 0.5, 0.2 and between 0.5 and 0.2 for amplication, loss and normal copy number, respectively. fArray-CGH log2 ratio thresholds set at X 0.5, 0.2

and between 0.5 and 0.2 for amplication, loss and normal copy number, respectively.

& 2014 Macmillan Publishers Limited Oncogenesis (2014), 1 8

Cox P-value (n 150)d

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Table 3. Signicantly enriched Gene Ontology (GO) terms identied by integrated DNA methylation and expression proling in 8p11-p12-amplied breast tumors

Category GO term P-value Gene count

Biological processGO:0032099 Negative regulation of appetite 8.32E 05 2

GO:0045671 Negative regulation of osteoclast differentiation 2.48E 04 2

GO:0045060 Negative thymic T-cell selection 2.48E 04 2

GO:0008343 Adult feeding behavior 4.93E 04 2

GO:0006935 Chemotaxis 0.00203 4 GO:0007281 Germ cell development 0.002871 2 GO:0006260 DNA replication 0.003236 4 GO:0007420 Brain development 0.003323 3 GO:0030335 Positive regulation of cell migration 0.007046 2 GO:0006366 Transcription from RNA polymerase II promoter 0.014531 3 GO:0016337 Cell-cell adhesion 0.030651 2 GO:0008544 Epidermis development 0.042861 2

Molecular functionGO:0043169 Cation binding 0.011569 2 GO:0008083 Growth factor activity 0.013173 3 GO:0001104 RNA polymerase II transcription factor activity 0.013842 3 GO:0005179 Hormone activity 0.021768 2 GO:0004252 Serine-type endopeptidase activity 0.030247 3 GO:0006351 Transcription regulator activity 0.038615 2

Cellular componentGO:0005615 Extracellular space 3.46E 05 10

GO:0005576 Extracellular region 2.89E 04 17

GO:0043005 Neuron projection 0.002871 2 GO:0009897 External side of plasma membrane 0.079513 2 GO:0005634 Nucleus 0.081344 22 GO:0005794 Golgi apparatus 0.08346 6 GO:0005768 Endosome 0.096152 2 GO:0005578 Proteinaceous extracellular matrix 0.107112 3 GO:0005737 Cytoplasm 0.33593 19

ENPP2 gene expression

10

7

11

9

9

SYCP2gene expression

6

9

10

8

8

7

6

hypermethylated

normal CNA

hypermethylated

amplification

hemimethylated

normal CNA

hypomethylated

normal CNA

SQLE gene expression

8

hypermethylated

normal CNA

hemimethylated

normal CNA

hypomethylated

normal CNA

hypermethylated

amplification

hypermethylated

normal CNA

hemimethylated

normal CNA

hypomethylated

normal CNA

hypermethylated

amplification

Figure 3. The effect of aberrant DNA copy number and DNA methylation on gene expression. Box plots showing the relationship between DNA copy number (CNA), methylation status and gene expression for three candidate genes (ENPP2, SQLE and SYCP2) in 22 tumor samples. X-axis, methylation and CNA status; Y-axis, gene expression signal intensity.

CONFLICT OF INTEREST

The authors declare no conict of interest.

ACKNOWLEDGEMENTS

This work was supported by grants from the Swedish Cancer Society (KH), King Gustav V Jubilee Clinic Cancer Research Foundation (KH), the Wilhelm and Martina Lundgren Research Foundation (TZP), Serena Ehrenstrm Foundation for Cancer Research/Torsten

and Sara Jansson Research Foundation (TZP), Assar Gabrielsson Research Foundation for Clinical Cancer Research (TZP) and Lars Hiertas Memorial Research Foundation (TZP).

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