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New Amplified and Highly Expressed Genes Discovered in the ERBB2 Amplicon in Breast Cancer by cDNA Microarrays¹

Laboratory of Cancer Genetics, Institute of Medical Technology, University of Tampere and Tampere University Hospital, FIN-33101 Tampere, Finland [P. K., M. B.], and Cancer Genetics Branch, National Human Genome Research Institute, NIH, Bethesda, Maryland 20892 [O. M., A. K.]

	ABSTRACT

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Amplification of the ERBB2 oncogene at 17q12 has been well documented in breast cancer and has been shown to contribute to a poor clinical outcome. However, systematic surveys of copy number and expression levels of all genes within the 17q12 region have not been performed. Here, we used cDNA and comparative genomic hybridization microarray technologies to undertake a broad survey of genes involved in the 17q12 amplification in breast cancer. A chromosomal region-specific cDNA microarray containing 217 expressed sequence tag (EST) clones from 17q12 was constructed and used for parallel analysis of gene copy numbers and expression levels in seven breast cancer cell lines allowing direct identification of genes whose expression is elevated because of an increase in copy number in this chromosomal region. The copy number and expression survey identified 12 transcripts that showed a consistent pattern of increased copy number and expression in three or more of the 17q12-amplified cell lines. As expected, these included ERBB2 as well as the GRB7 and MLN64 genes previously shown to be coamplified with ERBB2. In addition, five other known genes and four uncharacterized ESTs were also found to be consistently activated by amplification in these breast cancer cell lines. Amplicon mapping by fluorescence in situ hybridization revealed a minimal common region of amplification containing four highly expressed genes, ERBB2, GRB7, MLN64, and an uncharacterized EST 48582. Furthermore, several other genes, although not located in the minimal common region of amplification, showed a correlated pattern of amplification and expression indicating that they might play a role in breast cancers with the 17q12 amplification. In conclusion, parallel analysis of gene copy number and expression levels by cDNA microarray can be used to directly identify candidate target genes involved in amplifications. Our results show that the 17q12 amplification in breast cancer leads to the simultaneous elevation of expression levels of several genes.

	INTRODUCTION

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Gene amplification plays an important role in the progression and initiation of many solid tumors, including breast cancer. Amplification and overexpression of the ERBB2 oncogene is found in 10–34% of primary human breast tumors, and various studies have linked ERBB2 amplification to poor clinical outcome (1) , thus establishing an essential role for ERBB2 in breast cancer. The ERBB2 oncogene has recently received additional attention as a target for antibody-based therapies and as a possible predictor of response to adjuvant chemotherapy (1, 2, 3, 4, 5) . However, modification of treatment strategies based on the ERBB2 status is still very controversial, and identification of additional genetic alterations that might influence the chemosensitivity of ERBB2-positive tumors are needed (6 , 7) .

Traditionally, gene amplifications have been attributed to the effects of a single target gene. However, recent evidence suggests that ERBB2 is not the only gene that is activated by the 17q12-q21 amplification in breast cancer. Coamplification of ERBB2 and several other genes, such as THRA/ERBA1, RARA, TOP2A, GRB7, MLN50, MLN51, MLN62/TRAF4, MLN64, and PPARBP genes, have been reported (8, 9, 10, 11, 12, 13) . Interestingly, studies on breast tumors by cDNA microarrays have identified a distinct set of genes with an expression pattern similar to ERBB2 (14 , 15) . Most of the genes in this so called ERBB2 overexpression cluster were located at 17q12 and were also amplified by CGH³ microarray analysis (14 , 15) . These findings imply the possibility that activation of several genes in the ERBB2 amplicon may contribute to breast cancer development and progression.

In the present study, we describe a systematic characterization of the 17q12 amplification in breast cancer. A full expression and copy number profiling of the amplified region was performed using a custom-made cDNA microarray containing 217 transcripts from the 17q12-q21 region as well as all known genes mapping to chromosome 17. In addition, we used the human genome sequence information for detailed evaluation of the 17q12-q21 amplicon structure in breast cancer cell lines.

	MATERIALS AND METHODS

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Cell Lines.
Seventeen breast cancer cell lines were used in this study. BT-474, HBL-100, HCC202, HCC1419, HCC1954, MCF7, MDA-361, MDA-436, MDA-453, SK-BR-3, ZR-75-30, UACC-732, UACC-812, UACC-893, and UACC-3133 were obtained from American Type Culture Collection (Manassas, VA). MX-1 and EFM-192A came from the German Collection of Micro-organisms and Cell Cultures (Braunschweig, Germany). Normal HMECs were obtained from Clonetics (Walkersville, MD). Cells were grown under recommended culture conditions.

Chromosome 17-specific cDNA Microarray.
The construction of the chromosome 17-specific cDNA microarray has been described (16) . Briefly, clones for the cDNA microarray were selected on the basis of the information in the GeneMap’99⁴ and included all known genes on chromosome 17 as well as all EST clones from intervals D17S933-D17S930 (293–325 cR; the 17q12-q21 region; ERBB2 locus) and D17S791-D17S795 (333–435 cR; the 17q23-q24 region). In addition, clones representing the ERBB2, RARA, BRCA1, MLN50, MLN51, MLN62/TRAF4, MLN64, and NF1 genes, which were not included in the radiation hybrid map, were placed on the array. Our array consisted of a total of 636 clones, including 217 clones from the 17q12-q21 region. The preparation and printing of the cDNA clones on glass slides were performed as described (17 , 18) . All clones were printed on the array in duplicate.

Expression Analyses by cDNA Microarrays.
Total RNA (MDA-361 and UACC-893) or mRNA (BT-474, HCC202, MDA-436, MDA-453, SK-BR-3, UACC-812, and HMEC) was extracted by using the FastTrack 2.0 kit (Invitrogen, Carlsbad, CA). The breast cancer cell line MDA-436 with no copy number increase at 17q12-q21 (16 , 19) was used as a standard reference in all experiments. Sixteen µg of MDA-436 mRNA was labeled with Cy5-dUTP (Amersham Pharmacia Biotech, Piscataway, NJ), and 4 µg of test mRNA or 80 µg of total RNA was labeled with Cy3-dUTP by use of oligo(dT)-primed polymerization by SuperScript II reverse transcriptase (Life Technologies, Inc., Rockville, MD). The labeled cDNAs were hybridized on microarrays as described previously (20 , 21) . The fluorescence intensities at the targets were measured by using a laser confocal scanner (Agilent Technologies, Palo Alto, CA). The fluorescent images from the test and control hybridizations were scanned separately, and the data were analyzed using the DEARRAY software (22) . The expression levels in the breast cancer cell lines were determined relative to a common reference (MDA-436) with no copy number increase in the 17q12-q21 region. Hybridization of HMECs against MDA-436 was used to exclude transcripts that were underrepresented in the common reference. Clones that showed a ratio >3 in the breast cancer cell lines, without a similar increase in the HMECs, were considered to be highly expressed.

Copy Number Analyses by cDNA Microarrays.
Genomic DNA was extracted from breast cancer cell lines BT-474, SK-BR-3, UACC-812, and UACC-893. Normal placental DNA was used as a reference in all experiments. CGH microarray analysis was done as described previously (15) , with slight modifications (16) . Briefly, genomic DNA was digested for 14–18 h with AluI and RsaI restriction enzymes (Life Technologies, Inc.) and purified by phenol/chloroform extraction. Six µg of digested cell-line DNA was labeled with Cy3-dUTP and 8 µg of placental DNA was labeled with Cy5-dUTP using Bioprime Labeling kit (Life Technologies, Inc.). Hybridization was done according to the protocol by Pollack et al. (15) , and posthybridization washes was done as described (20 , 21) . Analysis of copy number ratios was done using the DEARRAY software (16 , 22) . Clones that showed a copy number ratio 1.5 were considered to be amplified.

FISH.
Gene-specific BAC or PAC clones were identified by performing sequence similarity searches against the nonredundant and high throughput genomic sequence databases using the blastn⁵ program. The specificity of the probes was confirmed by PCR with gene-specific primers. BAC and PAC probes were labeled with SpectrumOrange-dUTP (Vysis, Inc., Downers Grove, IL) using random priming, and a chromosome 17-specific centromere probe labeled with FITC-12-dCTP (NEN, Boston, MA) was used as a reference. FISH to normal metaphase chromosomes was done to verify that the probes recognized a single copy target at 17q12-q21. Dual-color interphase FISH to breast cancer cell lines was done as described (23) . Hybridization signals were evaluated using an Olympus BX50 epifluorescence microscope equipped with a x63 oil-immersion objective (NA 1.4). A dual band-pass fluorescence filter (Chromatechnology, Brattleboro, VT) was used for simultaneous visualization of the FITC and SpectrumOrange signals. Approximately 50 nonoverlapping nuclei with intact morphology based on the 4,6-diamidino-2-phenylindole counterstain were scored to determine the mean number of hybridization signals for each test and reference probe. Both absolute and relative (ratio of test:reference) copy numbers were determined.

Northern Hybridization.
Total RNA was extracted from breast cancer cell lines by using TRIzol Reagent (Life Technologies, Inc.). Northern hybridization was performed using standard methods. Briefly, 15 µg of total RNA was transferred on a Nytran membrane (Schleicher & Schuell, Keene, NH). The blot was prehybridized for a 1 h at 42°C in NorthernMax Prehybridization/Hybridization Buffer (Ambion, Inc., Austin, TX) together with denaturated Herring Sperm DNA (10 µg/ml; Sigma Chemical Co., St. Louis, MO). PCR products or sequence-verified cDNA inserts were labeled with ³²P by random priming (rediprime II, Amersham Pharmacia). Hybridization was performed in the prehybridization solution at 45°C-48°C overnight. The membrane was washed several times with 2x SSC-0.05% SDS at room temperature and then twice in 0.1x SSC-0.1% SDS at 55°C. The hybridized probe was detected by using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). After removal of the bound probe, the membrane was rehybridized with a glyceraldehyde-3-phosphate dehydrogenase probe (CLONTECH, Palo Alto, CA) to confirm equal loading of the samples.

	RESULTS

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We performed an expression and copy number profiling across the 17q12-q21 region using a custom-made cDNA microarray containing 636 cDNA clones. Expression analysis was done in seven breast cancer cell lines (BT-474, HCC202, MDA-361, MDA-453, SK-BR-3, UACC-812, UACC-893) known to have gain or amplification at 17q12-q21 by chromosomal CGH (19 , 24) . Four cell lines (BT-474, SK-BR-3, UACC-812, and UACC-893) were analyzed by CGH microarray to determine the copy number of all of the 636 chromosome 17-specific transcripts. Plotting of the expression and copy number ratios as a function of the location of the clones in the radiation hybrid map identified a distinct region of increased expression and copy number around 300 cR, corresponding to the ERBB2 locus at 17q12-q21 (Fig. 1, A–B) . Focused examination of expression levels of the 217 clones from this region distinguished 20 transcripts, representing 11 known genes and 9 uncharacterized ESTs, which were highly expressed in three or more of the cell lines (Table 1) . Four transcripts (ERBB2, GRB7, MLN64, and an EST clone 48582) were highly expressed in all seven cell lines, and an additional two transcripts (EST clones 244062 and 1623927) showed increased expression levels in six cell lines (Table 1) . Comparison of the expression data with the copy number results in four cell lines indicated that 12 of these 20 transcripts (CDC6, ERBB2, GRB7, MLN51, MLN64, ZNF144, as well as ESTs 48582, 244062, 418240, 767775, 1623927, and 810305) were always highly expressed when amplified (Table 1 ; Fig. 1C ). This result allowed exclusion of eight of the transcripts as putative target genes for the 17q12-q21 amplification because they did not always show increased expression when amplified.

View larger version (24K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. cDNA microarray analysis of expression (A) and copy number (B) changes of chromosome 17-specific genes in the BT-474 breast cancer cell line. The expression and copy number ratio data are plotted as a function of the position of the clones in the radiation hybrid map in cR scale. Individual data points are connected with a line. A moving average of three (a mean copy number ratio of three adjacent clones) is shown in B. C, the copy number ratio as a function of the expression ratio in the BT-474 breast cancer cell line. Oval indicates a set of genes that are both highly amplified and overexpressed.

View larger version (83K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. A, Physical map of the 17q12-q21 region. BAC and PAC clones are symbolized with horizontal lines and the corresponding genes/transcripts are shown on the top. The map is not drawn in scale. B, schematic representation of amplicon mapping in 11 breast cancer cell lines by FISH. Left, cell lines; top, the 11 BAC/PAC clones used for FISH. Red boxes, 3-fold amplification; white boxes, no copy number increase. C, copy number profile of 11 BAC/PAC clones in the HCC1419, MDA-361, and UACC-812 breast cancer cell lines. The absolute mean copy number/cell is plotted for each probe. D, interphase FISH with BAC clone 390P24 (red) and chromosome 17 centromere probe (green) to the HCC1419 breast cancer cell line.

All four transcripts located within the BAC clone 2019C10, the ERBB2, GRB7, and MLN64 genes, as well as an uncharacterized EST 48582 (Fig. 2A) , were highly expressed by our cDNA microarray analysis (Table 1) . Northern hybridization in 14 breast cancer cell lines (BT-474, EFM-192A, HBL-100, HCC1419, HCC1954, MCF7, MDA-361, MDA-436, MDA-453, MX-1, SK-BR-3, UACC-812, UACC-893, and ZR-75-30) confirmed the cDNA microarray results, except for MDA-453, and indicated that the expression of ERBB2, MLN64, GRB7, and EST 48582 was consistently elevated in cell lines with amplification as compared with the normal mammary epithelial cells (Fig. 3) . For an unknown reason, MDA-453 showed increased expression by cDNA microarray but not by Northern analysis.

View larger version (85K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. Expression levels of ERBB2, GRB7, MLN64, and EST 48582 in 14 breast cancer cell lines and normal HMECs by Northern analysis. The cell lines are indicated on the top. The size of each transcript is shown on the left side of the corresponding picture. Hybridization of glyceraldehyde-3-phosphate dehydrogenase probe was used to confirm equal loading among samples.

	DISCUSSION

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The expression survey implicated a total of 20 transcripts that were highly expressed in at least three of the breast cancer cell lines known to have gain or amplification affecting the ERBB2 locus. As expected, these included genes that had been previously shown to be coamplified with ERBB2. In addition, five other known genes, CCR7, CDC6, MLLT6, PPP1R1B, and ZNF144 as well as nine uncharacterized ESTs were also frequently found to be highly expressed. A parallel copy number analysis by CGH microarray indicated that 12 of these 20 transcripts were consistently highly expressed when amplified and thus represent genes that might play a role in the 17q12-q21 amplification in breast cancer.

We then used the information from the human genomic sequence to localize these transcripts more accurately within the 17q12-q21 region and to obtain large-insert-size genomic clones for absolute copy number analysis. FISH analysis in a large set of breast cancer cell lines narrowed down the minimal common region of amplification to a single BAC clone (2019C10, AC040933). According to the human genomic sequence annotation⁶ and our own sequence analysis, the minimal-region BAC clone contains seven transcripts. Four of these, the ERBB2, GRB7, and MLN64 genes and EST 48582, were amplified and highly expressed by cDNA microarray in all breast cancer cell lines examined, and the elevated expression was also confirmed by Northern analysis. The three remaining genes, NEUROD2, PNMT, and ZNFN1A3, were not included in our cDNA microarray because they were not present in the radiation hybrid map. The expression levels of these three genes need to be studied to evaluate their possible involvement in the ERBB2 amplicon.

Our cDNA microarray analysis confirmed the previous data by showing that GRB7 and MLN64 are coamplified and overexpressed with ERBB2 (9 , 12) . GRB7 is a SH2 domain-containing signaling protein and a member of the growth factor receptor tyrosine kinases (25) . Overexpression of GRB7 has been shown to have a role in cell migration and invasion (26 , 27) , and coexpression of ERBB2 and GRB7 has been shown to relate to the depth of tumor invasion in esophageal carcinoma (28) . It is therefore possible that GRB7 may be directly involved in tumor progression. MLN64 shows significant homology to the steroidogenic acute regulatory protein, StAR, and is likely to be involved in cholesterol transport (29 , 30) . Cholesterol is a precursor of all steroid hormones, and it has been suggested that MLN64 might have a role in the intratumoral biosynthesis of steroid hormones (31) .

In addition to these previously known genes, our expression and copy number analysis identified a highly expressed EST (48582) that was located in the minimal common region of amplification. Evaluation of the possible role of this novel gene in breast cancer pathogenesis is clearly warranted. Furthermore, several other genes, such as CDC6, MLN51, ZNF144, and ESTs 244062, 418240, 767775, and 810305, although not located in the minimal common region of amplification, showed a correlated pattern of amplification and expression indicating that they might play a role in breast cancers with the 17q12-q21 amplification.

Although the cDNA microarray-based CGH method was published more than a year ago (15) , the correlation of copy number ratios with actual gene copy numbers has not been fully explored. In this study, we performed a direct comparison between CGH microarray data and actual gene copy numbers determined by FISH. An excellent concordance (89%) was observed between these two techniques in determining the amplification status of genes. However, as already indicated by Pollack et al. (15) , CGH microarray clearly underestimates the real DNA amplification levels. The data presented here illustrate the applicability of CGH microarray for successful identification of high-level amplifications. However, additional studies are needed to establish the utility of this technique for the detection of low-level amplifications.

In conclusion, parallel analysis of gene copy numbers and expression levels by cDNA microarray allows efficient identification of genes whose expression levels are elevated because of increased copy number. Analysis of the ERBB2 amplicon identified all of the previously reported amplified and overexpressed genes from this region, illustrating the utility of this approach for the detection of amplification target genes in cancer. Our results indicate that, in addition to ERBB2, several other genes are activated through the 17q12-q21 amplification in breast cancer. The biological relevance and possible clinical significance of these other amplified genes in breast cancer need to be evaluated further.

	ACKNOWLEDGMENTS

 
We thank Kati Rouhento for excellent technical assistance.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This study was supported in part by grants from the Sigrid Juselius Foundation, the Academy of Finland, the Medical Research Fund of Tampere University Hospital, and the Nordic Cancer Union.

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 20892-4465. Phone: (301) 402-6048; Fax: (301) 402-7957; E-mail: akallion{at}nhgri.nih.gov

³ The abbreviations used are: CGH, comparative genomic hybridization; HMEC, human mammary epithelial cell; EST, expressed sequence tag; cR, centiRay; FISH, fluorescence in situ hybridization; BAC, bacterial artificial chromosome; PAC, P1 artificial chromosome.

⁴ Internet address: http://www.ncbi.nlm.nih.gov/genemap.

⁵ Internet address: http://www.ncbi.nlm.nih.gov/BLAST/.

⁶ Internet address: http://www.ncbi.nlm.nih.gov/genome/guide/.

Received 4/10/01. Accepted 9/14/01.

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