Studies by comparative genomic hybridization imply that amplification of the chromosomal region 17q22-q24 is common in breast cancer. Here,amplification and expression levels of six known genes located at 17q23 were 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 genes in vivo, fluorescence in situ hybridization analysis of a tissue microarray containing 372 primary breast cancers was used. S6K, PAT1, and TBX2 were coamplified in about 10% of tumors, whereas RAD51C amplification was seen in only 3% of tumors. Expression analysis in 12 primary tumors showed that RAD51C and S6K were consistently expressed in all cases in which they were amplified and also in some tumors without amplification. These data suggest that 17q23 amplification results in simultaneous up-regulation of several genes,whose increased biological activity may jointly contribute to the more aggressive clinical course observed in patients with 17q23-amplified tumors.

Gene amplification plays an important role in the progression and initiation of many solid tumors, including breast cancer. To date, at least 20 genes, including HER-2, CCND1, EMS1, MYC, EGFR, FGFR1, FGFR2, and AIB1, have been shown to be amplified in breast cancer. Some of these amplifications, such as HER-2 at 17q12 as well as CCND1 at 11q13 and AIB1 at 20q12, have been linked to poor prognosis of the patients. Studies by CGH3have shown that DNA amplifications are very common in breast cancer and often involve regions of the genome that were not previously known to be amplified. One of these novel amplified regions is at 17q22-q24,which has been shown to be amplified in about 20% of primary breast tumors by CGH (1, 2). Recently, we and others showed that the 17q22-q24 amplification in breast cancer is due to high-level amplification of at least two separate regions and localized one amplified region more distinctly to 17q23 (3, 4). Based on the Human Gene Map4and our own physical and transcript mapping efforts,5the 17q23 region is relatively gene rich. In the present study, we evaluated the possible role of six known genes (RAD51C, S6K, SIGMA1B, PAT1, NACA,and TBX2) that we localized to 17q23 in breast cancer cell lines and determined their amplification frequencies in primary breast tumors using the recently developed tissue microarray technology(5).

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 and Cell Cultures (Braunschweig, Germany). The ZR-75-1wt strain was obtained from Dr. Jeff Moscow (National Cancer Institute, Bethesda,MD), and SUM-52 was obtained from Dr. Stephen P. Ethier (University of Michigan, Ann Arbor, MI). Cell lines were grown under the recommended culture conditions. Interphase cell preparations from cell lines and normal peripheral blood lymphocytes were done according to routine protocols.

Primary Tumors.

A total of 372 ethanol-fixed primary breast cancers were obtained from the Institute of Pathology, University of Basel (Basel, Switzerland). The tumor samples were reviewed by one pathologist (J. T.) and included 69.6% ductal carcinomas, 14% lobular carcinomas, 2.4%medullary carcinomas, 1.6% mucinous carcinomas, 0.8% cribriform carcinomas, 0.8% tubular carcinomas, 0.5% papillary carcinomas, 4%ductal carcinomas in situ, and 6.1% of other rare histological subtypes or unclassified carcinomas. The grade distribution was 24% grade 1, 40% grade 2, and 36% grade 3. The pT stage was pT1 in 29% of patients, pT2 in 54% of patients, pT3 in 9%of patients, and pT4 in 8% of patients. The average age of the patients was 60 years (range, 28–92 years); 45% of patients had node-negative disease, and 55% of patients had node-positive disease. All specimens evaluated were anonymous, archival tissue specimens. The use of these tissue specimens in retrospective analyses was approved by the Ethics Committee of the University of Basel on November 4, 1997,and the use of such specimens for tissue microarray analysis was approved by the NIH Institutional Review Board (exemption day, March 4,1998).

Tissue Microarray Construction.

The tissue microarrays were constructed as described previously(5). Briefly, a representative tumor area was marked from H&E-stained sections of each tumor. The blocks and the corresponding histological slides were overlayed for tissue microarray sampling. A tissue microarray instrument (Beecher Instruments, Silver Spring, MD)was used to create holes in a recipient paraffin block, to obtain cylindrical tissue biopsies with a diameter of 0.6 mm from the donor paraffin blocks, and to transfer these biopsies to the recipient block at defined array positions. Multiple 5-μm sections were cut from the tissue microarray block using a microtome with an adhesive-coated tape sectioning system (Instrumedics, Hackensack, NJ).

Physical Mapping.

RAD51C, S6K, SIGMA1B, PAT1, NACA, and TBX2 were mapped against a panel of Centre d’Etude du Polymorphisme Humain YACs by PCR. The PCR primer sequences were obtained from the Unigene database.6The chromosomal localization of the YACs was verified by FISH to normal metaphase chromosomes.

DNA Probes for FISH.

Gene-specific BAC clones were obtained by screening a human BAC library(Genome Systems, St. Louis, MO) using PCR with gene-specific primers. BAC probes were labeled with SpectrumOrange using random priming. SpectrumGreen-labeled chromosome 17 centromere probe (Vysis Inc.) was used as a reference.

Copy Number Analysis by FISH.

Interphase FISH to breast cancer cell lines was done as described previously (3). The hybridizations were evaluated using a Zeiss fluorescence microscope, and approximately 20 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. For the tissue microarrays, FISH was performed as described previously(5). Briefly, consecutive sections of the array were deparaffinized, dehydrated in ethanol, denatured at 74°C for 5 min in 70% formamide/2× SSC, and hybridized with test and reference probes. The specimens containing tight clusters of signals or a >3-fold increase in the number of test probe signals as compared with the chromosome 17 centromere signals in at least 10% of the tumor cells were considered as amplified.

Northern Hybridization.

Total RNA was extracted from breast cancer cell lines, and the Northern hybridization was performed using standard methods. Briefly, 10 μg of total RNA were transferred on a Nytran membrane (Schleicher & Schuell,Keene, NH). The blot was prehybridized for 1 h at 68°C in Express Hybridization solution (Clontech, Palo Alto, CA) together with boiled sheared DNA (10 μg/ml; Research Genetics, Huntsville, AL). PCR products or sequence-verified cDNA inserts were labeled with 32P by random priming (Prime-It; Stratagene, La Jolla, CA). Hybridization was performed in the prehybridization solution at 68°C overnight. The membrane was washed several times with 2× SSC/0.05% SDS at 30°C and then washed in 0.1× SSC/0.1%SDS at 50°C. Hybridized probe was detected autoradiographically or by using a Molecular Dynamics PhosphorImager. After removal of the bound probe, the membrane was rehybridized with glyceraldehyde-3-phosphate dehydrogenase probe to confirm equal loading among samples.

Expression Analyses in Primary Breast Tumors.

Total RNA was extracted from 12 primary breast tumors using the RNeasy kit (Qiagen, Inc., Valencia, CA). The PCR analyses were performed using the LightCycler system (Roche Diagnostics Corp., Indianapolis, IN). Briefly, PCR was performed using 2 μl of LightCycler RT-PCR Reaction Mix SYBR Green I, 0.5 μm of each of the 3′ and 5′primers, 0.4 μl of LightCycler RT-PCR Enzyme Mix, 500 ng of RNA, and H2O to a final volume of 20 μl. The MgCl2 concentration was optimized separately for each primer set and was 5 mm for S6K, 6 mm for PAT1, and 7 mm for RAD51C and TBX2. Assays were performed using total RNA from MCF-7 and HBL-100 breast cancer cell lines as positive and negative controls, respectively. Reverse transcription was done at 55°C for 10 min followed by inactivation at 95°C for 30 s. Amplification was done in three steps (denaturation at 95°C for 1 s with a temperature transition rate of 20°C/s, annealing at 58°C for 10 s with a temperature transition rate of 20°C/s, and extension at 72°C for 10 s with a temperature transition rate of 20°C/s) for 45 cycles. Melting curve analysis was performed to discriminate between nonspecific and specific products. The PCR products were denatured at 95°C for 0 s, and then the temperature was dropped quickly to 58°C for 20 s and raised slowly to 90°C at 0.2°C/s. The amount of the SYBR Green fluorescence was measured simultaneously and reflects the amount of double-stranded DNA. The rate of fluorescence change (−dF/dT) was plotted as a function of temperature.

Six genes (RAD51C, S6K, SIGMA1B, PAT1, NACA, and TBX2) were localized to Centre d’Etude de Polymorphisme Humain YACs by PCR. RAD51C mapped to YAC 898E7; S6K, SIGMA1B, and PAT1 mapped to YAC 913D6; and NACA and TBX2 mapped to YAC 948C8. According to the Whitehead database,7these YACs are contiguous, and we mapped them to 17q23 by FISH. Based on the information in the Human Gene Map,4 all six genes are located within a 14-cR interval corresponding to about 2 Mb.

Copy number changes of RAD51C, S6K, SIGMA1B, PAT1, NACA, and TBX2 were studied by FISH in seven breast cancer cell lines. Four cell lines (BT-474, KPL-1, MCF-7, and ZR-75-1wt) were previously known to have amplification or gain at 17q22-q24 by CGH, whereas three 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 found to be highly amplified (8–19-fold relative to the chromosome 17 centromere) in three cell lines (KPL-1, MCF-7, and ZR-75-1wt; Table 1; Fig. 1). In addition, 4–5-fold amplification of S6K, SIGMA1B, and PAT1 was seen in BT-474, and 4–5-fold amplification of NACA and TBX2 was seen in SUM-52 (Table 1).

Northern analysis was performed to determine whether the amplification led to elevated expression of these genes (Fig. 2). NACA was ubiquitously expressed in all cell lines, and SIGMA1B showed elevated expression only in BT-474 and HBL-100, indicating that the expression of these two genes did not correlate with amplification levels. The expression of RAD51C, S6K, and PAT1 was elevated in all cell lines with amplification (Fig. 2). Interestingly, RAD51C was also overexpressed in BT-474, which did not show amplification of this gene. TBX2 was expressed in the three cell lines with high-level amplification but was not detectable in the SUM-52 cell line, which had low-level amplification.

To survey whether amplifications of these four genes(RAD51C, S6K, PAT1, and TBX2) also occur in primary breast cancers, we used FISH analysis on a tumor tissue microarray containing 372 primary breast tumors (Fig. 3). Thirty-nine tumors (10.5%) showed amplification of at least one of the genes. RAD51C was amplified most infrequently (in 3.1%of cases), and it was always coamplified with the other three genes. S6K amplification was seen in 10.2% of the tumors, PAT1 amplification was seen in 8.9% of the tumors, and TBX2 amplification was seen in 8.6% of the tumors. In a majority of the cases (62%), S6K, PAT1, and TBX2 were amplified simultaneously. However, amplification of a single gene was observed in 12 tumors (31%) [S6K in 5 tumors, PAT1 in 4 tumors, and TBX2 in 3 tumors].

The expression of RAD51C, S6K, PAT1,and TBX2 was analyzed in 12 primary breast tumors by LightCycler RT-PCR (Fig. 4). RAD51C was expressed in all four cases with RAD51C amplification and was also expressed in two of eight tumors without amplification. Similarly, S6K was consistently expressed in all nine tumors with S6Kamplification and in one of three tumors without amplification. PAT1 was expressed in five of eight amplified tumors, and TBX2 was expressed in three of eight amplified tumors and in none of the four nonamplified primary breast cancers.

In this study, we examined amplification and expression levels of six genes (RAD51C, S6K, SIGMA1B, PAT1, NACA, and TBX2) located at 17q23 to determine their role as putative target genes for the 17q23 amplification in breast cancer. High-level amplification of all six genes was seen in three breast cancer cell lines. Four of the genes(RAD51C, S6K, PAT1, and TBX2) showed a correlated pattern of amplification and expression in breast cancer cell lines, suggesting that these genes could play a role as amplification target genes at 17q23. The lack of correlation between amplification and overexpression for SIGMA1B and NACA excludes them from being target genes for the 17q23 amplification.

Using the high-throughput tissue microarray technology, we examined the involvement of RAD51C, S6K, PAT1, and TBX2 in a large set of primary breast tumors. Analysis of amplifications of these four genes in the series of 372 primary tumors could be accomplished in just four consecutive FISH experiments(resulting in about 1500 observations). Amplifications of S6K, PAT1, and TBX2 were seen in about 10% of the tumors, whereas RAD51C was clearly less frequently amplified. Although S6K, PAT1, and TBX2 were occasionally found to be independently amplified,coamplification of S6K, PAT1, and TBX2was the predominant pattern in up to 62% of the tumors. It is unclear whether S6K, PAT1, and TBX2 are part of a single amplicon or whether they represent separate amplicons at 17q23. However, considering the relatively small distance of these genes from one another, we believe that in most cases they are located on a single amplicon. Similar coamplifications have been reported previously in breast cancer. For example, multiple putative target genes, such as EMS1 and CCND1, have been identified at 11q13, and target genes BTAK, ZNF217, and NABC have been identified at 20q13 (7, 8).

Although RAD51C was amplified less often in primary tumors than the other genes, it was clearly overexpressed in all cell lines and primary tumors with amplification and also in one cell line(BT-474) and two primary breast tumors without amplification. Thus,overexpression of RAD51C could play a role in breast cancer development and progression. RAD51C is the sixth member of the RecA/RAD51 gene family that encodes strand-transfer proteins involved in both recombinational 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 (BRCA1 and BRCA2) products (10, 11). RAD51C shows a 27% sequence identity to RAD51, and the homology is at the region of the protein that is involved in the protein-protein interaction (9). It is thus possible that RAD51C also interacts with p53 and BRCA1/BRCA2, making it an interesting candidate for an amplification target gene. Such interactions between oncogenes and tumor suppressor genes, e.g.,MDM2 and p53 as well as MYC and BRCA1, have been previously implicated to play a role in human cancer (12, 13).

PAT1 and TBX2 were shown to be more frequently amplified in primary breast tumors than RAD51C, but they were not consistently expressed in all cases with amplification. However, the fact that they were sometimes expressed in 17q23-amplified primary breast cancers suggests that they may modify the phenotype of these tumors. PAT1 (also known as APPBP2) is a cytoplasmic protein that is involved in the translocation of amyloid precursor protein along microtubules toward the cell surface(14) and has not been previously linked to cancer. TBX2 is a member of a gene family of transcription factors named T-box genes (15). Members of the T-box gene family play important roles in developmental gene regulation. TBX2is normally expressed in the milk ridge, thickened ridge of underlying mesenchyme during the development of the duct system of the mammary gland in mouse (16) and could therefore play a role in breast cancer by mediating mesenchymal/epithelial cell interactions.

Of the four genes identified in this study, the ribosomal protein S6 kinase (S6K) has been previously implicated in breast cancer(17, 18). S6K was most frequently amplified in primary breast tumors and was expressed in all cases with amplification as well as in one tumor without amplification. S6K encodes for a critical mediator involved in G1 to S-phase progression and is possibly also involved in the control of cell size (19, 20). Therefore, based on its biological role, it also represents an ideal candidate for an amplification target gene. Furthermore, we recently showed using another set of tumors with clinical follow-up information that amplification and overexpression of S6K are associated with poor prognosis of the patients independently of HER-2 amplification at 17q12 (18).

In summary, our findings indicate that the frequent amplification of 17q23 in breast cancer leads to up-regulation of at least four genes, RAD51C, S6K, PAT1, and TBX2, suggesting that their simultaneous activation may contribute to the genesis and the progression of breast cancer. Further functional analyses of the genes reported here will have to be undertaken to define which of them play the most important roles in breast cancer progression. Based on the Human Gene Map,4 the 17q23 region is a relatively gene-rich region of the genome. Thus, it is possible and even likely that other genes mapping to this region will also be affected by the amplification. Therefore, we are currently undertaking a full expression survey of all transcripts from the 17q23 amplicon(altogether, about 200 clones) using cDNA microarray technologies to further evaluate the hypothesis that multiple genes in DNA amplicons play important roles in cancer progression.

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

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

            
3

The abbreviations used are: CGH, comparative genomic hybridization; FISH, fluorescence in situhybridization; BAC, bacterial artificial chromosome; YAC, yeast artificial chromosome; 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/.

      
7

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

Table 1

Amplification levels of six 17q23 genes relative to chromosome 17 centromere in breast cancer cell lines by FISH

Cell lineRAD51CS6KSIGMA1BPAT1NACATBX2
MCF-7 18.3 11.7 13 10 16.7 10 
ZR-75-1wt 19.2 10.8 11.2 7.7 19.2 11.5 
KPL-1 15 10.5 15.8 13.3 16.7 13.3 
BT-474 1.8 5.3 3.9 5.4 1.4 1.2 
SUM-52 NDa 2.4 1.9 1.4 4.9 4.3 
HBL-100 1.3 1.3 
MDA-436 1.5 1.5 
Cell lineRAD51CS6KSIGMA1BPAT1NACATBX2
MCF-7 18.3 11.7 13 10 16.7 10 
ZR-75-1wt 19.2 10.8 11.2 7.7 19.2 11.5 
KPL-1 15 10.5 15.8 13.3 16.7 13.3 
BT-474 1.8 5.3 3.9 5.4 1.4 1.2 
SUM-52 NDa 2.4 1.9 1.4 4.9 4.3 
HBL-100 1.3 1.3 
MDA-436 1.5 1.5 
a

ND, not determined.

Fig. 1.

High-level amplification of TBX2 in(A) KPL-1 and (B) MCF-7 breast cancer cell lines by interphase FISH.

Fig. 1.

High-level amplification of TBX2 in(A) KPL-1 and (B) MCF-7 breast cancer cell lines by interphase FISH.

Close modal
Fig. 2.

Expression levels of S6K, TBX2, PAT1, RAD51C, NACA, and SIGMA1B in breast cancer cell lines by Northern analysis. The cell lines analyzed are indicated above the 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; and Lane 7, HBL-100. 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.

Fig. 2.

Expression levels of S6K, TBX2, PAT1, RAD51C, NACA, and SIGMA1B in breast cancer cell lines by Northern analysis. The cell lines analyzed are indicated above the 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; and Lane 7, HBL-100. 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.

Close modal
Fig. 3.

Analysis of gene amplification in primary breast tumors by FISH to a tissue microarray containing 372 tumor samples. Part of the 4′,6-diamidino-2-phenylindole-stained tissue microarray is shown,illustrating the structure of the array with cylindrical tissue samples. The inset shows a selected area from a tumor with high-level amplification of TBX2.

Fig. 3.

Analysis of gene amplification in primary breast tumors by FISH to a tissue microarray containing 372 tumor samples. Part of the 4′,6-diamidino-2-phenylindole-stained tissue microarray is shown,illustrating the structure of the array with cylindrical tissue samples. The inset shows a selected area from a tumor with high-level amplification of TBX2.

Close modal
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 [−d(F1)/dT]. Specific products identified based on the higher melting temperature (at 80°C to 84°C) indicate that S6K is highly expressed in MCF-7 cells and in tumor 5271. No expression is seen in tumor 6684.

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 [−d(F1)/dT]. Specific products identified based on the higher melting temperature (at 80°C to 84°C) indicate that S6K is highly expressed in MCF-7 cells and in tumor 5271. No expression is seen in tumor 6684.

Close modal

We thank Nasser Z. Parsa for excellent technical assistance.

Note Added in Proof: We recently cloned a genomic rearrangement with the exact same breakpoint in the MCF7, KPL-1, and ZR-75-1wt cell lines indicating that these cell lines may be clonal variants of one another. Genotyping with highly polymorphic markers showed that KPL-1 and ZR-75-1wt are both likely to have derived from the MCF7 cell line. Despite this clonal relationship, the three cell lines do possess unique characteristics,such as several distinct genetic alterations by CGH and a variable gene expression pattern by cDNA microarray analysis. The Northern analysis shown in Fig. 2 also illustrates that these three cell lines(Lanes 1–3) are not exactly identical. In this study, the results obtained from cell lines were extensively validated in vivo in uncultured tumors, and, therefore, this new information does not impact the conclusions of our study. However, these findings further emphasize the critical need for technologies and strategies utilized in this study: validation of genes discovered from model systems in vivo using tissue microarrays.

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