[CANCER RESEARCH 60, 5340–5344, October 1, 2000]

Advances in Brief

Multiple Genes at 17q23 Undergo Amplification andOverexpression in Breast Cancer1

Maarit Barlund, Outi Monni, Juha Kononen, Robert Cornelison, Joachim Torhorst, Guido Sauter,Olli-P. Kallioniemi, and Anne Kallioniemi 2

Laboratory of Cancer Genetics, Institute of Medical Technology, University of Tampere and Tampere University Hospital, FIN-33101 Tampere, Finland [M. B.]; Cancer GeneticsBranch, National Human Genome Research Institute, NIH, Bethesda, Maryland 20892-4470 [O. M., J. K., R. C., O-P. K., A. K.]; and Institute of Pathology, University of Basel,CH-4003 Basel, Switzerland [J. T., G. S.]

Abstract

Studies by comparative genomic hybridization imply that amplificationof the chromosomal region 17q22-q24 is common in breast cancer. Here,amplification and expression levels of six known genes located at 17q23were examined in breast cancer cell lines. Four of them (RAD51C,S6K,PAT1, and TBX2) were found to be highly amplified and overexpressed.To investigate the involvement of these genesin vivo, fluorescencein situhybridization analysis of a tissue microarray containing 372 primarybreast cancers was used.S6K,PAT1, andTBX2 were coamplified in about10% of tumors, whereasRAD51C amplification was seen in only 3% oftumors. Expression analysis in 12 primary tumors showed thatRAD51Cand S6K were consistently expressed in all cases in which they wereamplified and also in some tumors without amplification. These datasuggest that 17q23 amplification results in simultaneous up-regulation ofseveral genes, whose increased biological activity may jointly contribute tothe more aggressive clinical course observed in patients with 17q23-amplified tumors.

Introduction

Gene amplification plays an important role in the progression andinitiation of many solid tumors, including breast cancer. To date, atleast 20 genes, includingHER-2, CCND1, EMS1, MYC, EGFR,FGFR1,FGFR2, andAIB1, have been shown to be amplified in breastcancer. Some of these amplifications, such asHER-2at 17q12 as wellas CCND1 at 11q13 andAIB1 at 20q12, have been linked to poorprognosis of the patients. Studies by CGH3 have shown that DNAamplifications are very common in breast cancer and often involveregions of the genome that were not previously known to be ampli-fied. One of these novel amplified regions is at 17q22-q24, which hasbeen shown to be amplified in about 20% of primary breast tumors byCGH (1, 2). Recently, we and others showed that the 17q22-q24amplification in breast cancer is due to high-level amplification of atleast two separate regions and localized one amplified region moredistinctly to 17q23 (3, 4). Based on the Human Gene Map4 and ourown physical and transcript mapping efforts,5 the 17q23 region isrelatively gene rich. In the present study, we evaluated the possible

role of six known genes (RAD51C,S6K,SIGMA1B,PAT1,NACA, andTBX2) that we localized to 17q23 in breast cancer cell lines anddetermined their amplification frequencies in primary breast tumorsusing the recently developed tissue microarray technology (5).

Materials and Methods

Cell Lines. Breast cancer cell lines BT-474, HBL-100, MCF-7, and MDA-436 were obtained from American Type Culture Collection (Manassas, VA),and KPL-1 was obtained from the German Collection of Microorganisms andCell Cultures (Braunschweig, Germany). The ZR-75-1wt strain was obtainedfrom Dr. Jeff Moscow (National Cancer Institute, Bethesda, MD), andSUM-52 was obtained from Dr. Stephen P. Ethier (University of Michigan,Ann Arbor, MI). Cell lines were grown under the recommended cultureconditions. Interphase cell preparations from cell lines and normal peripheralblood lymphocytes were done according to routine protocols.

Primary Tumors. A total of 372 ethanol-fixed primary breast cancerswere obtained from the Institute of Pathology, University of Basel (Basel,Switzerland). The tumor samples were reviewed by one pathologist (J. T.) andincluded 69.6% ductal carcinomas, 14% lobular carcinomas, 2.4% medullarycarcinomas, 1.6% mucinous carcinomas, 0.8% cribriform carcinomas, 0.8%tubular carcinomas, 0.5% papillary carcinomas, 4% ductal carcinomasin situ,and 6.1% of other rare histological subtypes or unclassified carcinomas. Thegrade distribution was 24% grade 1, 40% grade 2, and 36% grade 3. The pTstage was pT1 in 29% of patients, pT2 in 54% of patients, pT3 in 9% ofpatients, and pT4 in 8% of patients. The average age of the patients was 60years (range, 28–92 years); 45% of patients had node-negative disease, and55% of patients had node-positive disease. All specimens evaluated wereanonymous, archival tissue specimens. The use of these tissue specimens inretrospective analyses was approved by the Ethics Committee of the Universityof Basel on November 4, 1997, and the use of such specimens for tissuemicroarray analysis was approved by the NIH Institutional Review Board(exemption day, March 4, 1998).

Tissue Microarray Construction. The tissue microarrays were con-structed as described previously (5). Briefly, a representative tumor area wasmarked from H&E-stained sections of each tumor. The blocks and the corre-sponding histological slides were overlayed for tissue microarray sampling. Atissue microarray instrument (Beecher Instruments, Silver Spring, MD) wasused to create holes in a recipient paraffin block, to obtain cylindrical tissuebiopsies with a diameter of 0.6 mm from the donor paraffin blocks, and totransfer these biopsies to the recipient block at defined array positions. Mul-tiple 5-mm sections were cut from the tissue microarray block using a mi-crotome with an adhesive-coated tape sectioning system (Instrumedics, Hack-ensack, NJ).

Physical Mapping. RAD51C,S6K, SIGMA1B,PAT1, NACA, and TBX2were mapped against a panel of Centre d’Etude du Polymorphisme HumainYACs by PCR. The PCR primer sequences were obtained from the Unigenedatabase.6 The chromosomal localization of the YACs was verified by FISH tonormal metaphase chromosomes.

DNA Probes for FISH. Gene-specific BAC clones were obtained byscreening a human BAC library (Genome Systems, St. Louis, MO) using PCRwith gene-specific primers. BAC probes were labeled with SpectrumOrange

Received 10/28/99; accepted 8/15/00.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby markedadvertisementin accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported in part by grants from The Research and Science Foundation of Farmos,the Medical Research Fund of Tampere University Hospital, The Finnish Medical Foun-dation, The Irja Karvonen Cancer Foundation, and the Swiss National Science Foundation(Grant 81BS-052807).

2 To whom requests for reprints should be addressed, at Cancer Genetics Branch,National Human Genome Research Institute, NIH, 49 Convent Drive, Room 4B24,Bethesda, MD 20892-4470. Phone: (301) 402-6048; Fax: (301) 402-7957; E-mail:akallion@nhgri.nih.gov.

3 The abbreviations used are: CGH, comparative genomic hybridization; FISH, fluo-rescencein situhybridization; BAC, bacterial artificial chromosome; YAC, yeast artificialchromosome; RT-PCR, reverse transcription-PCR.

4 World Wide Web address: http://www.ncbi.nlm.nih.gov/genemap/.5 Unpublished observations. 6 World Wide Web address: http://www.ncbi.nlm.nih.gov/UniGene/.

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using random priming. SpectrumGreen-labeled chromosome 17 centromereprobe (Vysis Inc.) was used as a reference.

Copy Number Analysis by FISH. Interphase FISH to breast cancer celllines was done as described previously (3). The hybridizations were evaluatedusing a Zeiss fluorescence microscope, and approximately 20 nonoverlappingnuclei with intact morphology based on the 49,6-diamidino-2-phenylindolecounterstain were scored to determine the mean number of hybridizationsignals for each test and reference probe. For the tissue microarrays, FISH wasperformed as described previously (5). Briefly, consecutive sections of thearray were deparaffinized, dehydrated in ethanol, denatured at 74°C for 5 minin 70% formamide/23SSC, and hybridized with test and reference probes.The specimens containing tight clusters of signals or a.3-fold increase in thenumber of test probe signals as compared with the chromosome 17 centromeresignals in at least 10% of the tumor cells were considered as amplified.

Northern Hybridization. Total RNA was extracted from breast cancer celllines, and the Northern hybridization was performed using standard methods.Briefly, 10 mg of total RNA were transferred on a Nytran membrane(Schleicher & Schuell, Keene, NH). The blot was prehybridized for 1 h at 68°Cin Express Hybridization solution (Clontech, Palo Alto, CA) together withboiled sheared DNA (10mg/ml; Research Genetics, Huntsville, AL). PCRproducts or sequence-verified cDNA inserts were labeled with32P by randompriming (Prime-It; Stratagene, La Jolla, CA). Hybridization was performed inthe prehybridization solution at 68°C overnight. The membrane was washedseveral times with 23SSC/0.05% SDS at 30°C and then washed in 0.13SSC/0.1% SDS at 50°C. Hybridized probe was detected autoradiographicallyor by using a Molecular Dynamics PhosphorImager. After removal of thebound probe, the membrane was rehybridized with glyceraldehyde-3-phos-phate dehydrogenase probe to confirm equal loading among samples.

Expression Analyses in Primary Breast Tumors.Total RNA was ex-tracted from 12 primary breast tumors using the RNeasy kit (Qiagen, Inc.,Valencia, CA). The PCR analyses were performed using the LightCyclersystem (Roche Diagnostics Corp., Indianapolis, IN). Briefly, PCR was per-formed using 2ml of LightCycler RT-PCR Reaction Mix SYBR Green I, 0.5mM of each of the 39and 59primers, 0.4ml of LightCycler RT-PCR EnzymeMix, 500 ng of RNA, and H2O to a final volume of 20ml. The MgCl2concentration was optimized separately for each primer set and was 5 mM forS6K, 6 mM for PAT1, and 7 mM for RAD51C and TBX2. Assays wereperformed using total RNA from MCF-7 and HBL-100 breast cancer cell linesas positive and negative controls, respectively. Reverse transcription was doneat 55°C for 10 min followed by inactivation at 95°C for 30 s. Amplificationwas done in three steps (denaturation at 95°C for 1 s with a temperature transitionrate of 20°C/s, annealing at 58°C for 10 s with a temperature transition rateof 20°C/s, and extension at 72°C for 10 s with a temperature transition rate of20°C/s) for 45 cycles. Melting curve analysis was performed to discriminatebetween nonspecific and specific products. The PCR products were denaturedat 95°C for 0 s, and then the temperature was dropped quickly to 58°C for 20 sand raised slowly to 90°C at 0.2°C/s. The amount of the SYBR Greenfluorescence was measured simultaneously and reflects the amount of double-stranded DNA. The rate of fluorescence change (2dF/dT) was plotted as afunction of temperature.

Results

Six genes (RAD51C,S6K, SIGMA1B,PAT1, NACA, andTBX2)were localized to Centre d’Etude de Polymorphisme Humain YACsby PCR.RAD51Cmapped to YAC 898E7;S6K,SIGMA1B, andPAT1mapped to YAC 913D6; andNACA and TBX2 mapped to YAC948C8. According to the Whitehead database,7 these YACs are con-tiguous, and we mapped them to 17q23 by FISH. Based on theinformation in the Human Gene Map,4 all six genes are located withina 14-cR interval corresponding to about 2 Mb.

Copy number changes ofRAD51C,S6K,SIGMA1B,PAT1,NACA,andTBX2were studied by FISH in seven breast cancer cell lines. Fourcell lines (BT-474, KPL-1, MCF-7, and ZR-75-1wt) were previouslyknown to have amplification or gain at 17q22-q24 by CGH, whereasthree cell lines (SUM-52, HBL-100, and MDA-436) did not show any

copy number increase at this region (3, 6). All six genes were foundto be highly amplified (8–19-fold relative to the chromosome 17centromere) in three cell lines (KPL-1, MCF-7, and ZR-75-1wt; Table1; Fig. 1). In addition, 4–5-fold amplification ofS6K,SIGMA1B, andPAT1was seen in BT-474, and 4–5-fold amplification ofNACAandTBX2was seen in SUM-52 (Table 1).

Northern analysis was performed to determine whether the ampli-fication led to elevated expression of these genes (Fig. 2).NACAwasubiquitously expressed in all cell lines, andSIGMA1Bshowed ele-vated expression only in BT-474 and HBL-100, indicating that theexpression of these two genes did not correlate with amplificationlevels. The expression ofRAD51C,S6K, andPAT1was elevated in allcell lines with amplification (Fig. 2). Interestingly,RAD51Cwas alsooverexpressed in BT-474, which did not show amplification of thisgene.TBX2 was expressed in the three cell lines with high-levelamplification but was not detectable in the SUM-52 cell line, whichhad low-level amplification.

To survey whether amplifications of these four genes (RAD51C,S6K,PAT1, andTBX2) also occur in primary breast cancers, we usedFISH analysis on a tumor tissue microarray containing 372 primarybreast tumors (Fig. 3). Thirty-nine tumors (10.5%) showed amplifi-cation of at least one of the genes.RAD51Cwas amplified mostinfrequently (in 3.1% of cases), and it was always coamplified withthe other three genes.S6K amplification was seen in 10.2% of thetumors, PAT1 amplification was seen in 8.9% of the tumors, andTBX2amplification was seen in 8.6% of the tumors. In a majority ofthe cases (62%),S6K, PAT1, andTBX2 were amplified simulta-neously. However, amplification of a single gene was observed in 12tumors (31%) [S6Kin 5 tumors,PAT1 in 4 tumors, andTBX2 in 3tumors].

The expression ofRAD51C,S6K,PAT1, andTBX2was analyzed in12 primary breast tumors by LightCycler RT-PCR (Fig. 4).RAD51Cwas expressed in all four cases withRAD51Camplification and wasalso expressed in two of eight tumors without amplification. Simi-larly, S6K was consistently expressed in all nine tumors withS6Kamplification and in one of three tumors without amplification.PAT1was expressed in five of eight amplified tumors, andTBX2 wasexpressed in three of eight amplified tumors and in none of the fournonamplified primary breast cancers.

Discussion

In this study, we examined amplification and expression levels ofsix genes (RAD51C,S6K, SIGMA1B,PAT1, NACA, andTBX2) lo-cated at 17q23 to determine their role as putative target genes for the17q23 amplification in breast cancer. High-level amplification of allsix genes was seen in three breast cancer cell lines. Four of the genes(RAD51C,S6K, PAT1, andTBX2) showed a correlated pattern ofamplification and expression in breast cancer cell lines, suggestingthat these genes could play a role as amplification target genes at17q23. The lack of correlation between amplification and overexpres-sion forSIGMA1BandNACAexcludes them from being target genesfor the 17q23 amplification.

7 World Wide Web address: http://www-genome.wi.mit.edu/.

Table 1 Amplification levels of six 17q23 genes relative to chromosome 17 centromerein breast cancer cell lines by FISH

Cell line RAD51C S6K SIGMA1B PAT1 NACA TBX2

MCF-7 18.3 11.7 13 10 16.7 10ZR-75-1wt 19.2 10.8 11.2 7.7 19.2 11.5KPL-1 15 10.5 15.8 13.3 16.7 13.3BT-474 1.8 5.3 3.9 5.4 1.4 1.2SUM-52 NDa 2.4 1.9 1.4 4.9 4.3HBL-100 1 1 1 1.3 1 1.3MDA-436 1 1 1 1.5 1 1.5a ND, not determined.

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Using the high-throughput tissue microarray technology, we exam-ined the involvement ofRAD51C,S6K,PAT1, andTBX2in a large setof primary breast tumors. Analysis of amplifications of these fourgenes in the series of 372 primary tumors could be accomplished injust four consecutive FISH experiments (resulting in about 1500observations). Amplifications ofS6K,PAT1, andTBX2were seen inabout 10% of the tumors, whereasRAD51C was clearly less fre-quently amplified. AlthoughS6K,PAT1, andTBX2were occasionallyfound to be independently amplified, coamplification ofS6K,PAT1,andTBX2was the predominant pattern in up to 62% of the tumors. Itis unclear whetherS6K,PAT1, andTBX2are part of a single ampliconor whether they represent separate amplicons at 17q23. However,considering the relatively small distance of these genes from oneanother, we believe that in most cases they are located on a singleamplicon. Similar coamplifications have been reported previously inbreast cancer. For example, multiple putative target genes, such asEMS1 and CCND1, have been identified at 11q13, and target genesBTAK, ZNF217, and NABC have been identified at 20q13 (7, 8).

AlthoughRAD51Cwas amplified less often in primary tumors thanthe other genes, it was clearly overexpressed in all cell lines andprimary tumors with amplification and also in one cell line (BT-474)and two primary breast tumors without amplification. Thus, overex-

pression ofRAD51Ccould play a role in breast cancer developmentand progression.RAD51Cis the sixth member of the RecA/RAD51gene family that encodes strand-transfer proteins involved in bothrecombinational repair of DNA damage and meiotic recombination(9). RAD51 protein interacts with the tumor suppressor protein TP53(p53) as well as with the breast cancer susceptibility gene (BRCA1and BRCA2) products (10, 11). RAD51C shows a 27% sequenceidentity to RAD51, and the homology is at the region of the proteinthat is involved in the protein-protein interaction (9). It is thus possiblethat RAD51C also interacts with p53 and BRCA1/BRCA2, making itan interesting candidate for an amplification target gene. Such inter-actions between oncogenes and tumor suppressor genes,e.g., MDM2and p53 as well asMYCandBRCA1, have been previously implicatedto play a role in human cancer (12, 13).

PAT1 and TBX2 were shown to be more frequently amplified inprimary breast tumors than RAD51C, but they were not consistentlyexpressed in all cases with amplification. However, the fact that theywere sometimes expressed in 17q23-amplified primary breast cancerssuggests that they may modify the phenotype of these tumors.PAT1(also known asAPPBP2) is a cytoplasmic protein that is involved inthe translocation of amyloid precursor protein along microtubulestoward the cell surface (14) and has not been previously linked tocancer.TBX2 is a member of a gene family of transcription factorsnamed T-box genes (15). Members of the T-box gene family playimportant roles in developmental gene regulation.TBX2 is normallyexpressed in the milk ridge, thickened ridge of underlying mesen-chyme during the development of the duct system of the mammarygland in mouse (16) and could therefore play a role in breast cancerby mediating mesenchymal/epithelial cell interactions.

Of the four genes identified in this study, the ribosomal protein S6kinase (S6K) has been previously implicated in breast cancer (17, 18).S6Kwas most frequently amplified in primary breast tumors and wasexpressed in all cases with amplification as well as in one tumorwithout amplification.S6Kencodes for a critical mediator involved inG1 to S-phase progression and is possibly also involved in the controlof cell size (19, 20). Therefore, based on its biological role, it alsorepresents an ideal candidate for an amplification target gene. Fur-thermore, we recently showed using another set of tumors withclinical follow-up information that amplification and overexpression

Fig. 1. High-level amplification ofTBX2 in (A) KPL-1 and (B) MCF-7 breast cancer cell lines by interphase FISH.

Fig. 2. Expression levels ofS6K, TBX2, PAT1, RAD51C,NACA, andSIGMA1Binbreast cancer cell lines by Northern analysis. The cell lines analyzed are indicatedabovethe lanes:Lane 1, MCF-7;Lane 2, ZR-75-1wt;Lane 3, KPL-1;Lane 4, BT-474;Lane 5,SUM-52;Lane 6, MDA-436; andLane 7, HBL-100. The size of each transcript is shownon theleft sideof the corresponding picture. Hybridization of glyceraldehyde-3-phosphatedehydrogenase probe was used to confirm equal loading among samples.

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of S6Kare associated with poor prognosis of the patients independ-ently of HER-2 amplification at 17q12 (18).

In summary, our findings indicate that the frequent amplification of17q23 in breast cancer leads to up-regulation of at least four genes,RAD51C,S6K,PAT1, andTBX2, suggesting that their simultaneous

activation may contribute to the genesis and the progression of breastcancer. Further functional analyses of the genes reported here willhave to be undertaken to define which of them play the most impor-tant roles in breast cancer progression. Based on the Human GeneMap,4 the 17q23 region is a relatively gene-rich region of the genome.

Fig. 3. Analysis of gene amplification in primary breast tumors by FISH to a tissue microarray containing 372 tumor samples. Part of the 49,6-diamidino-2-phenylindole-stainedtissue microarray is shown, illustrating the structure of the array with cylindrical tissue samples. Theinsetshows a selected area from a tumor with high-level amplification ofTBX2.

Fig. 4. S6K expression in primary breast tumors by LightCycler RT-PCR. The melting curve analysis for MCF-7 and HBL-100 cell lines and two primary breast tumors is shown.The rate of fluorescence change is blotted as a function of temperature [2d(F1)/dT]. Specific products identified based on the higher melting temperature (at 80°C to 84°C) indicatethat S6K is highly expressed in MCF-7 cells and in tumor 5271. No expression is seen in tumor 6684.

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Thus, it is possible and even likely that other genes mapping to thisregion will also be affected by the amplification. Therefore, we arecurrently undertaking a full expression survey of all transcripts fromthe 17q23 amplicon (altogether, about 200 clones) using cDNA mi-croarray technologies to further evaluate the hypothesis that multiplegenes in DNA amplicons play important roles in cancer progression.

Acknowledgments

We thank Nasser Z. Parsa for excellent technical assistance.

Note Added in Proof

We recently cloned a genomic rearrangement with the exact samebreakpoint in the MCF7, KPL-1, and ZR-75-1wt cell lines indicatingthat these cell lines may be clonal variants of one another. Genotypingwith highly polymorphic markers showed that KPL-1 and ZR-75-1wtare both likely to have derived from the MCF7 cell line. Despite thisclonal relationship, the three cell lines do possess unique character-istics, such as several distinct genetic alterations by CGH and avariable gene expression pattern by cDNA microarray analysis. TheNorthern analysis shown in Fig. 2 also illustrates that these three celllines (Lanes 1–3) are not exactly identical. In this study, the resultsobtained from cell lines were extensively validatedin vivo in uncul-tured tumors, and, therefore, this new information does not impact theconclusions of our study. However, these findings further emphasizethe critical need for technologies and strategies utilized in this study:validation of genes discovered from model systemsin vivo usingtissue microarrays.

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2000;60:5340-5344. Cancer Res   Maarit Bärlund, Outi Monni, Juha Kononen, et al.   Overexpression in Breast CancerMultiple Genes at 17q23 Undergo Amplification and

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