Genomic and Expression Analysis of the 8P11–12 Amplicon in Human Breast Cancer Cell Lines

[CANCER RESEARCH 64, 40–47, January 1, 2004] Genomic and Expression Analysis of the 8p11–12 Amplicon in Human Breast Cancer Cell Lines

Michael E. Ray,1 Zeng Quan Yang,1 Donna Albertson,4 Celina G. Kleer,3 Joseph G. Washburn,3 Jill A. Macoska,2 and Stephen P. Ethier1 1Departments of Radiation Oncology and 2Urology, 3Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, Michigan, and 4Cancer Research Institute, University of California San Francisco, San Francisco, California

ABSTRACT In our panel of 11 breast cancer cell lines, 3 lines (SUM-44, SUM-52, and SUM-225) have an amplification in the 8p11-p12 Gene amplification is an important mechanism of oncogene activation region (8, 14, 15). However, unlike FGFR2, we have been unable to in breast and other cancers. Characterization of amplified regions of the find direct evidence that FGFR1 is the driving oncogene in these cell genome in breast cancer has led to the identification of important oncogenes including erbB-2/HER-2, C-MYC, and fibroblast growth factor re- lines. We have undertaken a detailed genomic and expression analysis ceptor (FGFR) 2. Chromosome 8p11-p12 is amplified in 10–15% of of the 8p11-p12 amplicon in these three cell lines, using array com- human breast cancers. The putative oncogene FGFR1 localizes to this parative genomic hybridization (CGH), Southern blot analysis, North- region; however, we show evidence that FGFR inhibition fails to slow ern blot analysis, and chromosome 8-specific cDNA microarrays. growth of three breast cancer cell lines with 8p11-p12 amplification. We These experiments have identified new candidate oncogenes and have present a detailed analysis of this amplicon in three human breast cancer provided support for the hypothesis that genes other than FGFR1 may cell lines using comparative genomic hybridization, traditional Southern play a causal role in breast cancer progression in tumors with ampli- and Northern analysis, and chromosome 8 cDNA microarray expression fication in the 8p11–12 region. profiling. This study has identified new candidate oncogenes within the 8p11-p12 region, supporting the hypothesis that genes other than FGFR1 may contribute to oncogenesis in breast cancers with proximal 8p ampli- MATERIALS AND METHODS fication. Bacterial Artificial Chromosome (BAC) Array CGH. Array CGH was INTRODUCTION carried out using arrays of 2464 BAC clones each printed in triplicate (Hum- Array1.14) according to published protocols (16, 17). Briefly, cell line and In a significant subset of human breast cancers (HBC), as in other normal male reference DNA (300 ng each) were labeled by random priming in solid tumors, gene amplification and overexpression is an important separate 50-␮l reactions to incorporate Cy3 and Cy5, respectively. The labeled mechanism for oncogene activation. In breast cancer, a number of DNAs were combined with 100 ␮g human Cot-1 DNA and hybridized to the discrete amplicons have been identified including 17q12 (erbB-2/ BAC arrays for ϳ48 h at 37°C. After posthybridization washes, the arrays HER-2), 8q24 (C-MYC), 11q13 (CCND1/PRAD1), and 10q26 [fibro- were mounted in a solution containing 90% glycerol, 10% PBS, and 1 ␮M blast growth factor receptor (FGFR) 2)]. The 17q12 amplicon is well 4Ј,6-diamidino-2-phenylindole, and sealed with a coverslip. A custom built ϫ Ј characterized, and overexpression of erbB-2 plays an important causal CCD camera system was used to acquire 16 bit 1024 1024 pixel 4 ,6- diamidino-2-phenylindole, Cy3 and Cy5 images (18). Image analysis was role in breast cancer development (1–3). However, other genes within carried out using University of California San Francisco SPOT software (19). the 17q12 amplicon, such as Grb7, are also overexpressed (4) and The log ratio of the total integrated Cy3 and Cy5 intensities for each spot after may play a role in breast cancer progression, possibly interacting with 2 background subtraction was calculated, normalized to the median log2ratio of erbB-2 in mediating transformed phenotypes (5). all of the clones on the array, and the average of the triplicates calculated using The 10q26 amplicon includes FGFR2, which is amplified and a second custom program, SPROC. Automatic data filtering to reject data overexpressed in 4–12% of breast cancers (6, 7) as well as the points based on low 4Ј,6-diamidino-2-phenylindole intensity, low correlation SUM-52 cell line developed in our laboratory (8). We have shown that between Cy3 and Cy5 within each segmented spot, and low reference/4Ј,6- expression of FGFR2 in human mammary epithelial (HME) cells is diamidino-2-phenylindole signal intensity was also carried out using SPROC. transforming.5 Data files were subsequently manually edited by rejecting clones for which Amplification of 8p11-p12, including the FGFR1 region, has been only one spot of the triplicate survived after SPROC analysis and for which the SD of the log ratio of the triplicate was Ͼ0.2. For each tumor, the data are reported in 10–15% of breast cancers (6, 9, 10), and may be associ- 2 plotted as the mean log ratio of the triplicate spots for each clone normalized ated with a poor prognosis, particularly for node-positive patients 2 to the genome median log2ratio. The clones are ordered by position in the (11). Amplification of the 8p11–12 region has been associated with genome according to the University of California-Santa Cruz Biotechnology estrogen receptor-positive tumors and lobular histology (12). FGFR1 (UCSC) Human Genome Working Draft.6 amplification is associated with overexpression in a subset of those Cell Lines and FGFR Inhibitor Growth Experiments. The isolation and tumors (10), but in the others, FGFR1 expression is comparable with culture of the SUM-44, SUM-52, and SUM-225 HBC cell lines have been levels in normal breast tissue (9). Furthermore, FGFR1 is excluded described in detail previously (14, 20, 21). SUM-44 cells were grown under from the amplicon in some tumors (13). serum-free conditions using a base medium of Ham’s F-12 supplemented with 0.1% BSA, insulin (5 ␮g/ml), hydrocortisone (1 ␮g/ml), 5 mM ethanolamine, 10 mM HEPES, transferrin (5 ␮g/ml), triiodothyronine (10 ␮g/ml), 50 ␮M sodium Received 4/14/03; revised 10/30/03; accepted 10/31/03. ␮ ␮ Grant Support: NIH Grants CA70354 (S. P. E.), and CA60948 and CA46592 selenite, gentamicin (5 g/ml), and fungizone (0.5 g/ml). SUM-52 and SUM-225 (J. A. M.). cells were routinely cultured in Ham’s F-12 supplemented with 5% fetal bovine The costs of publication of this article were defrayed in part by the payment of page serum, insulin (5 ␮g/ml), hydrocortisone (1 ␮g/ml), gentamicin (5 ␮g/ml), and charges. This article must therefore be hereby marked advertisement in accordance with fungizone (0.5 ␮g/ml). Detailed information regarding the SUM series of HBC 18 U.S.C. Section 1734 solely to indicate this fact. 7 Note: M. E. Ray and Z. Q. Yang contributed equally to this work. cell lines is accessible online. The MCF10A cell line, a spontaneously immor- Requests for reprints: Stephen Ethier, 7312 CCGC, 1500 East Medical Center Drive, talized normal mammary epithelial cell line (22), was cultured in the same Ann Arbor, MI, 48109-0948. Phone: (734) 763-1317; Fax: (734) 647-9480; E-mail: serum-free medium supplemented with epidermal growth factor (10 ng/ml). [email protected]. 5 A. Moffa, S. L. Tannheimer, N. Grewal, and S. P. Ethier. Transforming potential of alternatively spliced variants of fibroblast growth factor receptor 2 in human mammary 6 Internet address: http://genome.ucsc.edu. epithelial cells, submitted for publication. 7 Internet address: http://www.cancer.med.umich.edu/breast_cell/production/index.html. 40

For the FGFR inhibitor experiments, cells were grown in their normal for ratio calculation. For purposes of displaying the expression profiling data growth medium supplemented with the inhibitor PD 173074 (Ref. 23; dis- of the amplicon region, the clones localizing to 8p11–12 were listed in order, solved in DMSO) at a final concentration of 1 ␮M. PD173074 was provided by Dr. and ratios of medians Ͼ2.0 were colored red whereas ratio of medians Ͻ0.5 Wilbur Leopold and Pfizer Pharmaceuticals (Ann Arbor, MI). Growth media for were colored green. control cells were supplemented with equivalent volumes of DMSO with no Primary HBC Tissue Microdissection, and Quantitative Genomic and inhibitor. Cells were counted at the indicated time points using a Coulter Counter Reverse Transcription-PCR (RT-PCR). Primary breast cancer tissues were (Beckman Coulter, Miami, Florida) per the manufacturer’s instructions. obtained from the Surgical Pathology files at the University of Michigan with Southern and Northern Analyses. Known genes and expressed sequence Institutional Review Board approval. Laser capture microdissection was per- tags (ESTs) from the 8p11–12 region were selected from the databases of the formed on 32 specimens for isolation of pure cancer cell populations using a UCSC and the National Center for Biotechnology Information. cDNA clones of SL␮Cut Laser Microdissection system (Nikon). Genomic DNA was extracted these genes and ESTs were purchased from the ResGen Invitrogen Corporation. using the QIAamp DNA micro kit (Qiagen) according to the manufacturer’s Plasmid DNA was isolated using a standard plasmid isolation protocol (QIAspin; protocol. Genomic DNA and total RNA were also extracted from frozen Qiagen), and digested with the appropriate restriction endonuclease. Digested metastatic lymph node tissue from the same patient whose cancer gave rise to DNA was electrophoresed through an agarose gel, and checked for consistent the SUM-52 cell line. Real-time quantitative PCR and RT-PCR experiments banding patterns and/or directly sequenced to verify the clone identity. The insert used TaqMan probes and the Applied Biosystems Prism 7900HT Sequence DNA was extracted and purified (QIAquick gel extraction kit; Qiagen), and Detection System. The TC-1, FGFR1, LSM1, FLJ14299, and GAPDH TaqMan prepared as probes for Southern blot and Northern blot hybridization. probe and primer mix, as well as the TaqMan Universal PCR master mix, were Cellular DNA and total RNA was prepared from cultured HBC cell lines purchase from Applied Biosystems. The data were analyzed using Sequence and the HME cell line (MCF10A) by standard methods. For Southern blot Detector System v2.1 (Applied Biosystems) and Microsoft Excel software. analysis, 10-␮g aliquots of EcoRI-digested DNA were electrophoresed on 0.8% agarose gels and transferred to nylon membranes (Hybond). For North- RESULTS ern blot experiments, 20-␮g aliquots of total RNA were electrophoresed in 1.0% agarose/0.67 M formaldehyde gels and transferred to positively CGH: 8p11–12 Amplification in SUM HBC Cells. When the charged nylon membranes (Hybond). Each membrane was hybridized with SUM-44 and SUM-52 cell lines were developed several years ago, we [␣-32P]dCTP-labeled cDNA probes, which were labeled using the Mega- observed in Southern blot experiments that both of these lines had an prime DNA labeling system (Amersham Pharmacia Biotech). Hybridiza- amplification of the FGFR1 gene (8). Subsequent chromosome CGH tions were performed using ExpressHyb hybridization solution (Clontech), studies performed with our panel of cell lines confirmed the presence according to the manufacturer’s instructions. Blots were exposed for auto- of a focal area of gene amplification at 8p11-p12 (14, 15). radiography for 8–36hatϪ80°C as necessary to achieve optimal exposure before developing. More recently, we performed BAC array CGH studies using our Expression Profiling of Human Chromosome 8 Using a Custom cDNA entire panel of breast cancer cell lines. The results of these experi- Microarray. A custom chromosome 8 cDNA array was produced at the ments confirmed and extended the previous results, and demonstrated University of Michigan Comprehensive Cancer cDNA and Affymetrix Mi- that a third cell line in our panel, SUM-225, also has focal areas of croarray Core (24). This array consisted of PCR products amplified from 677 gene amplification in the 8p11 region. The chromosome 8 BAC array Research Genetics (Huntsville, AL) human cDNA clones of which the se- CGH results for SUM-44, -52, and -225 are shown in Fig. 1. The array quences mapped to chromosome 8 according to information from the National CGH data suggest that the three cell lines have some overlapping Center for Biotechnology Information Ensemble database8 and the UCSC areas of gene amplification, whereas each cell line also has uniquely Genome Bioinformatics database.9 A complete list of cDNAs spotted onto the amplified regions. The SUM-44 cells appear to have a single focal chromosome 8 array can be obtained on the internet.10 region of amplification. In contrast, SUM-52 cells appear to have two Total RNA was prepared from SUM-44, SUM-52, and SUM-225 breast distinct peaks of amplification, one centered around the FGFR1 locus, cancer cell lines, and from MCF10A HME cells using TRIzol reagent (In- and a second, more telomeric peak. The SUM-225 cell line differs vitrogen Life Technologies, Inc., Carlsbad, CA) following the manufacturer’s from the other two lines in that it has two distinct peaks of amplifi- instructions. The RNA was additionally purified using RNeasy reagents (Qia- cation. Interestingly, the 8p11 peak is more centromeric from the gen, Valencia, CA). RNA quality was confirmed by denaturing agarose gel FGFR1 gene, and the second peak is across the centromere extending electrophoresis. Twenty ␮g total RNA was labeled with Cy3 or Cy5 reagent using the CyScribe First-Strand cDNA Labeling kit and purified on AutoSeq into the 8q11 region. Thus, the array CGH data, taken together with G50 columns (Amersham Biosciences, Piscataway, NJ) following the manu- previous findings, indicate that the SUM-44, -52, and -225 cell lines facturer’s protocols. Cy-3- and Cy-5-labeled samples were pooled and lyoph- all have areas of gene amplification in the proximal 8p region. ilized to a volume of ϳ5 ␮l. Microarray slides were prehybridized for 45 min Furthermore, the amplicon structure is complex, prompting additional in 5ϫ SSC, 0.1% SDS, 25% formamide, and 1% BSA at 42°C. Slides were studies to more carefully characterize this region of the genome in the then rinsed with ddH2O, dried, and preheated to 68°C. SlideHyb #1 hybrid- three cell lines. ization buffer (75 ␮l; Ambion, Houston, TX) was preheated to 68°C and added Fluorescence in situ hybridization analysis using BAC probes from to the pooled labeled cDNAs. The probe was incubated at 68°C and added to various parts of the 8p11 region demonstrated that all three of the the microarray surface under a lifterslip (Erie Scientific, Portsmouth, NH). The breast cancer cell line exhibited homogeneously staining regions. The slides were hybridized in a dark, humidified chamber at 42°C overnight. Slides were washed the following day by rotating in 2ϫ SSC, 0.5% SDS at 45°C for results of these experiments will be described in detail elsewhere. 10 min followed by 0.5ϫ SSC, 0.5% SDS at 45°C for 10 min. The slides were Assessment of the Candidate Oncogene FGFR1 in SUM HBC then briefly rinsed in water, dried, and stored in the dark until scanning with Cells. Within the 8p11–12 amplicon, most attention has focused on a GenePix 4000A Axon laser scanner and associated software (Axon Instru- the candidate oncogene FGFR1 (9, 11, 13). We have shown previ- ments, Burlingame, CA). Photomultiplier tube settings were manually adjusted ously that FGFR1 is amplified in SUM-44 and SUM-52 cells (8). to achieve red (Cy5) to green (Cy3) normalization factors close to 1. Each However, FGFR1 is only overexpressed at the message level in experiment was performed in duplicate using a color switch. Microsoft Excel SUM-44 cells (Fig. 2A). Thus, of the three lines in our panel that have data spreadsheets were generated for data analysis. Chromosome 8 clones were copy number increases in the 8p11–12 region, FGFR1 is only ampli- ordered using the April 2003 freeze of the UCSC Human Genome Working fied in two lines (SUM-44 and SUM-52) and overexpressed in only Draft.6 Array elements with signal sum of medians Ͻ1000 and defective data points flagged by the scanning software were excluded from analysis. For each one cell line (SUM-44). Furthermore, we have detected no evidence duplicate experiment, the element with the greatest sum of medians was chosen of FGFR1 protein overexpression or activation in SUM-44 cells (data not shown). To additionally assess the importance of FGFR signaling in these 8 Internet address: http://www.ensemble.org/homo_sapiens. 9 Internet address: http://genome.cse.ucsc.edu/. cells, we used the FGFR-specific small molecule tyrosine kinase 10 Internet address: http://www-personal.umich.edu/ϳjcoska/Chrom8Array.xls. inhibitor PD 173074 (23). This compound had no effect on the 41

Fig. 1. A, genomic copy number profiles of chromosome 8 with array CGH in SUM-44, -52, and -225. The log 2 ratios for chromosome 8 in order from pter to qter are plotted from left to right. B, the detailed map and log 2 ratios around 8p11-p12 region were recapitulated based on the April 2003 freeze of the UCSC Genome Working Draft.6 proliferation of SUM-44 or SUM-225 cells (Fig. 2B). In contrast, the On the basis of the Southern blot studies in the three cell lines, the FGFR inhibitor did slow proliferation of SUM-52 cells, which over- smallest common region of gene amplification in the three cell lines express FGFR2 at the message and protein level (8). These results does not include FGFR1 (Table 1), supporting the hypothesis that suggest that whereas FGFR2 amplification and overexpression can genes other than FGFR1 from the 8p11–12 amplicon may play lead to a transformed phenotype, FGFR1 does not appear to be a important roles in breast cancer development. driving oncogene in any of the SUM breast cancer cell lines contain- To confirm that the amplicon structure as detected in the three ing the 8p11–12 amplicon. breast cancer cell lines is consistent with 8p11–12 amplifications Detailed Mapping of the 8p11–12 Amplicon. Because we were that occur in primary breast cancers, quantitative PCR analysis was unable to find direct evidence implicating FGFR1 as the target onco- carried out using genomic DNA obtained from microdissected gene for the 8p11–12 amplicon, we sought to define the amplification breast cancer specimens derived from our frozen breast cancer and expression status of other genes on the 8p11–12 amplicon. To map bank. The PCR experiments were performed using primers specific the amplified region in these three cell lines in detail, and to define the for FLJ14299, LSM1, FGFR1, and TC-1, as these genes span the minimal common regions of amplification, the copy number for known amplicon detected in the cell lines. Of the 32 breast cancers genes and ESTs distributed across bands 8p11-p12 was determined by examined, 8 showed evidence of high level amplification (Ͼ4- Southern blot analysis. Representative examples are shown in Fig. 3. fold) in at least part of the 8p11–12 region. Interestingly, TC-1 and A total of 33 known genes or ESTs were found to be amplified in FLJ14299 were most commonly amplified, whereas FGFR1 was at least one of the three cell lines. Of these, 11 known genes were only found to be Ͼ4-fold amplified in 1 of 32 primary breast cancers amplified in only one of the three cell lines (NRG, MGC1136, ASH2L, (Table 2). These results are consistent with those obtained from the cell STAR, ADAM18, SFRP1, ZNF220, IKK-␤, POLB, VDAC3, and SLC20A2), whereas 15 known genes or ESTs were amplified in two lines, which suggest that regions of 8p11–12 flanking the FGFR1 locus of the three cell lines (FLJ14299, PROSC, ADRB3, LSM1, BAG4, may be of greater significance in breast cancer. These results also suggest KIAA0725, WHSC1L1, FGFR1, FLJ25409, ADAM2, Hs.170537, that genes flanking the FGFR1 locus may be amplified in a higher FLJ13842, ANK1, BRF2, and EIF4EBP1). Only 5 known genes proportion of breast cancers than has been published previously. (HTPAP, TACC1, INDO, TC-1, and RCP) and two ESTs (Hs.156542 Gene Expression Analyses of the 8p11–12 Amplicon: Chromo- and Hs.188833) were amplified in all three of the cell lines. some 8 cDNA Microarray Expression Profiling, Northern Analy- There was significant heterogeneity in the copy number gains sis, and Quantitative RT-PCR. To rapidly determine whether genes detected by Southern analysis, ranging from dramatic, high level from 8p11–12 were overexpressed in association with amplification, amplification such as HTPAP in SUM-44 cells, to subtle low level we performed expression profiling using a custom human chromo- copy number gain such as HTPAP in SUM-225 cells. some 8 cDNA microarray. The microarray was designed by querying 42

ments, with 939 clones localizing to chromosome 8 and 116 clones localizing to 8p11–12. Expression profiling of breast cancer cell lines SUM-44, SUM-52, and SUM-225 versus the HME cell line MCF10A as control revealed that several genes from the amplicon region were overexpressed in the SUM cell lines (Fig. 4). In particular, cDNAs for FLJ14299, PROSC, RCP, EIF4EBP1, LSM1, HTPAP FGFR1, and LOC51125 were up- regulated Ͼ3-fold in SUM-44 cells. SUM-225 cells also showed significant up-regulation of FLJ14299 and RCP. SUM-52 cells showed more moderate up-regulation of RCP and EIF4EBP1, but showed more significant up-regulation of C8ORF4 (TC-1) and LOC51125. To validate the expression profiling data and to extend our gene expression analysis to include genes not represented on the cDNA microarray, we also performed Northern hybridizations comparing the SUM HBC cell lines and HME cell controls. Representative results of Northern blot hybridizations are shown in Fig. 5, and the gene overexpression results are summarized in Table 1. The Northern results demonstrated a high level of qualitative agree- ment with cDNA microarray results; however, the microarray data appeared to quantitatively underestimate the fold-differences in gene expression observed by Northern analysis. In a few instances, North- ern hybridization detected gene expression patterns inconsistent with the cDNA microarray analysis. This was true in particular for TACC1 expression, which was found to be overexpressed by Northern (Fig. 5) as well as Western (data not shown) analysis. We note that many of the TACC1 cDNA clones on the cDNA microarray were from un- translated portions of the gene, whereas the Northern probe was the full-length coding sequence. We postulate that alternative mRNA splicing within certain cell types may occasionally limit the sensitivity Fig. 2. A, Northern blot analysis of FGFR1 shows overexpression in SUM-44 but not of microarrays composed of cDNA fragments from spliced portions of SUM-52 or SUM-225. GAPDH is shown as a control. B, treatment of SUM-44, -52, and certain genes. -225 cells with the fibroblast growth factor receptor (FGFR) inhibitor PD 173074 shows inhibition of SUM-52 growth but no inhibition of SUM-44 or SUM-225. This inhibitor is Several genes from the amplicon show a close correlation between active against both FGFR1 and FGFR2 receptors. We have shown that SUM-52 cells are DNA amplification and mRNA expression (Figs. 3–5). This correla- characterized by FGFR2 amplification and overexpression, which is transforming in HME tion is noticeable for FLJ14299, LSM1, RCP, and HTPAP. FLJ14299 cells. and RCP showed particularly high levels of overexpression in SUM-44 and SUM-225 cells (Figs. 4 and 5). Interestingly, C8ORF4 the human chromosome 8 Unigene database (National Center for (TC-1) was found to be overexpressed only in SUM-52 cells although it Biotechnology Information) to identify clones from a sequence-veri- is amplified in all three of the cell lines (Figs. 3–5). Two genes, HTPAP fied Research Genetics cDNA library, which localized to chromosome and TACC1, show amplification and overexpression in all three of the cell 8. These clones were assembled into a sublibrary, and cDNA microar- lines with the amplicon, making them particularly interesting candidate rays were constructed. The microarray contains a total of 1134 ele- genes that warrant further study for their roles in HBC.

Fig. 3. Southern blot hybridization analysis using cDNA probes from the 8p11-p12 region shows gene amplification in three breast cancer cell lines: SUM-44, -52, and -225. The GAPDH control shown at the bottom of C applied to A and B as well. Southern blots of genes showing amplification in one of the three cell lines (A), in two of the three cell lines (B), and in all three cell lines (C) are displayed.

Table 1 Summary of 8p11–12 gene amplification and overexpression in SUM-44, in this study (14). Our results, derived from chromosome CGH and BAC SUM-52, and SUM-225 as determined by Southern and Northern analyses array CGH analysis of the 11 breast cancer cell lines in our panel, are a Genes and ESTs within 8p11–p12 region are ordered from telomere to centromere consistent with the idea that 8p11-p12 is a common region of amplifica- based on the UCSC Genome Working Draft (April 2003). tion, and could, therefore, harbor important breast cancer oncogenes. Amplification Overexpression The FGFR1 gene has long been considered to be the best candidate Map (Mb) Gene symbol 44 52 225 44 52 225 oncogene at this locus. However, the exact involvement of this receptor 32.26 NRG1 ϪϩϪϪϪϪ in the progression of breast cancer is unclear, because it is not consistently 33.31 MGC1136 ϪϩϪϪϩϪ present in the amplification unit and is not always overexpressed when 37.41 FLJ14299 ϩϪϩϩϪϩ amplified. The results obtained with our cell lines are not consistent with 37.48 PROSC ϩϪϩϩϪϪ 37.56 BRF2 (FLJ11052) ϩϪϩϩϪϩ a dominant, transforming role for FGFR1. Among our three cell lines 37.58 RCP ϩϩϩϩϪϩ with the amplicon, FGFR1 is overexpressed at the message level in only 37.70 ADRB3 ϩϩϪϪϪϪ one, and even in this line, the FGFR1 receptor is not overexpressed at the Chrom. 8b EIF4EBP1 ϩϩϪϩϩϪ 37.71 ASH2L ϩϪϪϪϪϪ protein level. Thus, it was not surprising that an FGFR-specific tyrosine 37.74 STAR ϩϪϪϪϪϪ kinase inhibitor did not influence the growth of these cells. 37.76 LSM1 ϩϩϪϩϩϪ TACC1 is another gene from the region that has been implicated 37.78 BAG4 ϩϩϪϩϪϪ 37.86 KIAA0725 ϩϩϪϩϪϪ in breast cancer development (34). The TACC gene family encodes 37.87 HTPAP ϩϩϩϩϩϩ centrosomal proteins that may play roles in microtubule regulation 37.91 WHSC1L1 ϩϩϪϪϪϪ and spindle function, and, thus, may be an important driver of ϩϩϪϪϪϪ 37.98 FLJ25409 genomic instability in cancer cells (35–39). Still et al. (34) dem- 38.01 FGFR1 ϩϩϪϩϪϪ 38.14 EST (Hs. 156542) ϩϩϩϪϪϪ onstrated that TACC1 is overexpressed in some breast cancers, and 38.36 EST (Hs. 188833) ϩϩϩϪϩϩ it is transforming in NIH3T3 cells. The data from our work are 38.39 TACC1 ϩϩϩϩϩϩ consistent with a possible transforming role for TACC1, because it 39.18 ADAM18 ϩϪϪϪϪϪ 39.34 ADAM2 ϪϩϩϪϪϪ is the only transcribed gene present within the smallest common region of 39.51 INDO ϩϩϩϪϪϪ gene amplification in the three SUM lines with the amplicon. Further- 39.75 C8ORF4 (TC-1) ϩϩϩϪϩϪ more, TACC1 is overexpressed at the mRNA level in these cells. How- 39.91 EST (Hs. 170537) ϩϪϩϪϪϪ 40.13 FLJ13842 ϩϩϪϪϪϪ ever, other reports suggest that the TACC proteins are lost or down- 40.86 SFRP1 ϪϩϪϪϪϪ regulated in breast cancers and propose that they might be tumor 41.09 LOC51125 (HSPC041) ND ND ND ϩϩϪ suppressor genes, which play a role in mRNA homeostasis (40, 41). 41.25 ANK1 ϩϩϪϪϪϪ 41.53 ZNF220(RUNXBP2) ϪϩϪϪϪϪ Interpretation of this result is complicated by the fact that most of 8p is 41.88 IKBKB ϪϩϪϪϪϪ lost in a high proportion of breast cancers making definitive identification 41.94 POLB ϪϩϪϪϪϪ of the tumor suppressor gene at this locus difficult. Clearly, additional 41.99 VDAC3 ϪϩϪϪϪϪ 42.01 SLC20A2(GLVR2) ϪϩϪϪϩϪ work will be required to precisely define the transforming function, if 42.35 CHRNA6 ϪϪϪND ND ND any, of TACC1 in HBC. 42.43 FLJ10477 ϪϪϪND ND ND Several other genes are present within the smallest common am- a EST, expressed sequence tag; UCSC, University of California-Santa Cruz Biotech- plification unit of the three SUM lines, providing circumstantial nology; BAC, bacterial artificial chromosome; FISH, fluorescence in situ hybridization; evidence for roles for these genes in carcinogenesis. However, in ND, not determined. b EIF4EBP1 is currently shown as mapping randomly to chromosome 8; however, addition to the small common region of amplification, each cell line BAC clone RP11-701H6 contains both EIF4EBP1 and ADRB3, so we believe that also has unique regions of gene amplification containing several EIF4EBP1 maps close to 37.7 Mb on Chromosome 8. Furthermore, FISH studies confirm interesting and highly transcribed genes. Thus, it remains possible that that the BAC maps cytogenetically to 8p11. the 8p11–12 region, like other “hot spots” for gene amplification in breast cancer, contains multiple genes, which contribute to cell trans- To confirm that results obtained with the breast cancer cell lines formation. For this reason, we have carefully defined the amplifica- at the expression level were consistent with expression patterns in tion and expression pattern of genes in the 8p11–12 region in three uncultured breast cancer specimens, quantitative RT-PCR experi- cells lines with the amplicon and have identified several candidate ments were carried out using RNA isolated from frozen metastatic genes that need to be studied further. lymph node tissue obtained from the same patient whose pleural In addition, we found that in the SUM-225 cells, the region of gene effusion specimen gave rise to the SUM-52 cell line. As indicated above, amplification may extend across the chromosome 8 centromere into the TC-1 is both highly amplified and overexpressed in the SUM-52 cell line. 8q11 region. The pericentromeric region is transcript poor, and currently Quantitative genomic PCR and RT-PCR experiments on the lymph node there are few candidate genes from this region. However, this region is specimen confirmed similar levels of amplification and overexpression of worthy of further study because, among all of the amplified regions of TC-1, as well as other genes from the amplicon. Thus, at both the gene chromosome 8 in these cell lines, the 8q11 BAC array CGH peak in amplification and gene expression levels, data from these breast cancer SUM-225 showed the highest copy number increase. cell lines appear highly consistent with those observed in primary, un- Several known genes and ESTs from the amplicon were found to be cultured breast cancer tissues. overexpressed in one, two, or all three of the cell lines. The known genes include LSM1, TC-1, RCP, FLJ14299, EIF4EBP1, and HTPAP. LSM1, also termed cancer-associated Sm-like protein, was originally DISCUSSION cloned from human pancreatic cancers as an up-regulated gene (42). Aberrations of chromosome 8 have been observed in many human Antisense LSM1 RNA is able to alter the transformed phenotype of pancreatic cancer cells by reducing their ability to form large colonies cancers, and several studies have shown that chromosome 8 is a frequent target for gene amplification and gene loss in breast cancer. In particular, copy number increases are commonly observed in distal 8q (12, 25–29) and in the 8p11-p12 region (6, 9). The 8p region is also Table 2 Greater than 4-fold copy number increase assessed by quantitative genomic PCR analysis of microdissected primary breast cancer specimens commonly associated with copy number decreases in breast cancer (30–33). Indeed, gene amplification at 8p11-p12 often occurs in a FLJ14299 LSM1 FGFR1 TC-1 background of gene loss, and that is the case with the cell lines used 5/32 3/32 1/32 6/32 44

Fig. 4. Chromosome 8 cDNA microarray gene expression profile of the amplified regions of 8p11–12 in SUM-44, -52, and -225 cells versus MCF10A HME control cells. The gene symbol, clone identification number, chromosome band, chromosome map location based on the April 2003 freeze of the UCSC Genome Working Draft6 and the expression ratios for the three cell lines are displayed. Values Ͼ2.0 are highlighted in red, whereas values Ͻ0.5 are in green. Defective data points or those with a sum of median signals Ͻ1000 are omitted.

ASH2L, LSM1, BAG4, KIAA0725, WHSC1L1, FGFR1, TACC1, C8ORF4(TC-1), FLJ13842, and SFRP1. One of 19 additional sporadic breast cancer specimens also showed up-regulation of PROSC, EIF4EBP1, ASH2L, LSM1, BAG4, KIAA0725, and WHSC1L1. These data are consistent with previously published 10–15% prevalence of the 8p11–12 amplicon in HBC (6, 9, 10) and indicate that the expression profiles of these cell lines are highly consistent with primary HBCs that harbor the amplicon. Our results show that the 8p11–12 amplicon resembles other amplicons that have been identified in breast cancer, such as those found on 17q, 11q13, and 20q13, in several ways. Each of these amplicons occurs nonrandomly in a significant fraction of breast cancer, and each region harbors several genes that have been implicated in breast cancer development. The observation that all of the well-defined breast cancer amplicons contain several candidate genes suggests that these regions may be “hot spots” for amplicon formation precisely because they may contain multiple genes with oncogenic potential. This view is in contrast to the assumption held previously that each amplicon contains only a single dominant oncogene. This distinction is important because Fig. 5. Northern blot analysis using cDNA probes from the 8p11-p12 region shows past work has relied on the identification of commonly amplified gene overexpression in SUM-44, -52, and -225 cell lines. GAPDH is shown as a control. and overexpressed genes within large panels of cancers to provide Notice that FLJ 14299 showed the highest level of overexpression in SUM-44 and evidence for transforming function, implicating genes as candidate SUM-225, and also that TC-1 was only overexpressed in SUM-52. oncogenes. However, if amplicons, by their very nature, contain more than one functional oncogene, then identifying oncogenes on the basis of common amplification and overexpression patterns in soft agar when compared with untransfected cells and can decrease would be likely to miss, or even rule out, genes that actually have pancreatic tumor growth in vivo (43). functional significance in cancer progression. TC-1 is a novel gene that was originally cloned from suppression For this reason, we used an alternate approach by using three subtractive hybridization between a papillary thyroid carcinoma and well-characterized cell lines developed in our laboratory, and identi- its surrounding normal thyroid tissue (44). Overexpression of TC-1 in fied candidate oncogenes based on amplification and expression pat- thyroid cancer was found in 15 of 16 paired primary samples. In terns in each of the cell lines. These candidate genes can now be tested addition, serial analysis of gene expression of normal mammary for transforming function either by overexpressing the genes in im- epithelial cells, and in situ, invasive, and metastatic breast carcinomas mortalized HME cells, or by disrupting their function in the breast demonstrated high levels of TC-1 expression in intermediate-grade/ cancer cells themselves using antisense- or RNA-interference-based estrogen receptor-positive breast cancers (45). approaches. This direct, functional assessment of the transforming RCP has been shown to interact with Rab4 and Rab11, small GTPases potential of these candidate oncogenes will suggest whether these belonging to the Ras superfamily, and may function in regulation of the genes represent good targets for novel therapeutics. membrane trafficking of endosomal receptor recycling (46, 47). FLJ14299 is a novel gene that contains a C2H2 motif. C2H2 zinc finger domains are nucleic acid-binding protein structures that are also ACKNOWLEDGMENTS present in several tumor-related genes such as BCL6 (48), ZNF217 (49), and GLI (50). FLJ14299 is of particular interest, because it is expressed We thank Navdeep Grewal for TACC1 northern data, Rick Segraves for at moderate levels in MCF-10A normal HME cells and is overexpressed array CGH hybridizations, Michele Dziubinski for FGFR inhibitor cell growth to very high levels in the breast cancer cells with the amplicon. experiments, Rohit Mehra for primary human breast cancer tissue microdis- HTPAP is a novel gene containing a type 2 phosphatidic acid section, and Steven Kronenberg for assistance in preparing the figures. phosphatase domain. Finally, the product of EIF4EBP1 is in the mTOR pathway, participating in the regulation of the formation of eIF4F complex and acting to negatively regulate translational initia- REFERENCES tion (51). 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Michael E. Ray, Zeng Quan Yang, Donna Albertson, et al.

Cancer Res 2004;64:40-47.

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