Novel targets for the 18p11.3 amplification frequently observed in esophageal squamous cell carcinomas
Koichi Nakakuki1,2,
Issei Imoto1,
Atiphan Pimkhaokham1,2,
Yoji Fukuda1,
Yutaka Shimada3,
Masayuki Imamura3,
Teruo Amagasa2 and
Johji Inazawa1,4
1 Department of Molecular Cytogenetics, Medical Research Institute, Tokyo Medical and Dental University,
2 Maxillofacial Surgery, Graduate School, Tokyo Medical and Dental University and
3 Department of Surgery, Surgically Basic Medicine, Kyoto University Graduate School of Medicine, Japan
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Abstract
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Amplification of DNA in certain chromosomal regions, with consequent over-expression of specific genes within these amplicons, plays a crucial role in the development and progression of human cancer. Since our previous comparative genomic hybridization (CGH) study revealed frequent amplifications at 18p in esophageal squamous cell carcinomas (ESC) cell lines, we focused on the identification of genetic target(s) within the 18p amplicon. In four cell lines having remarkable copy-number amplification with homogeneously staining region (HSR) pattern by fluorescence in situ hybridization (FISH), the smallest common region of overlapping covered ~3.5 Mb at 18p11.3. We screened 29 ESC cell lines to discern amplifications and expression levels of 14 known genes and 21 uncharacterized transcripts within the amplicon. Only four known genes, YES1, TYMS, HEC and TGIF showed amplification and consequent over-expression. These genes were amplified in several of primary ESCs. Moreover, resistance to transforming growth factorß (TGFß)-induced growth inhibition was enhanced in four cell lines with amplification and expression of TGIF, which encodes the repressor for TGFß-activated transcription, appears to be involved in the progression of ESC. Taken together, these results suggest that YES1, TYMS, HEC and TGIF are likely to be candidate targets for 18p11.3 amplification and be associated with esophageal tumorigenesis.
Abbreviations: BACs, bacterial artificial chromosomes; CGH, comparative genomic hybridization; ESC, esophageal squamous cell carcinomas; 5FU, 5-fluorouracil; SCC, squamous cell carcinomas; TGFß, transforming growth factorß; TS, thymidylate synthase.
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Introduction
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Amplification of DNA in certain chromosomal regions is a mechanism allowing for activation of critical genes involving the development and progression of various human tumors, including esophageal squamous cell carcinoma (ESC) (1). Considerable efforts have been made to explore amplified regions and genes in ESC. To date, several genes, such as MYC at 8q24, EGFR at 7q32 and CCND1 at 11q13, have been successfully identified in this manner, and shown to be associated with malignant phenotype of this type of tumors (24). Furthermore, cumulative results of recent comparative genomic hybridization (CGH) studies demonstrated that additional amplification targets are yet to be identified in ESC (59). In order to provide novel insights into the pathogenesis of ESC, exploring and characterizing these target genes is indispensable.
Among regions implicated by CGH, 18p is of interest because of its relatively frequent amplification in ESC cell lines as well as primary ESC tumors. For example, copy-number gains have been detected in 37.9% [11/29 (8)] and 33.3% [4/12 (6)] of ESC lines and, in 35.3% [6/17 (7)] and 13.8% [4/29 (6)] of primary ESC tumors. In addition, this amplification has also been reported in other types of tumor, including squamous cell carcinomas (SCC) of the larynx and pharynx (10), malignant fibrous histiocytomas (11), ovarian cancers (12) and small-cell lung cancers (13). Taken together, it is strongly suggested that this chromosomal region may harbor one or more target genes affecting carcinogenesis regardless of the specific tissues.
Two known genes, YES1 and TYMS, which are very closely located each other on 18p11.3, have been shown to be amplified and over-expressed in human cancers. YES1 is a proto-oncogene homologous to Yamaguchi sarcoma virus oncogene [v-yes (1416)] and TYMS is a gene encoding thymidylate synthase that is a metabolic target of 5-fluorouracil (5FU) (17,18). However, the amplification of both genes has not yet been shown in ESC. In addition, the process of gene amplification usually involves co-amplification of extensive genomic DNA materials that share a common origin, and other genes affecting ESC may be present within the 18p amplicon. Accordingly, the analysis of this frequently recurring amplification on 18p may lead us to the identification of a series of target genes involved in the tumorigenesis of ESC.
Here, we report a defined mapping of the 18p amplicon in affected ESC cell lines, and identify two novel potential target genes, HEC and TGIF, that were co-amplified with YES1 and TYMS and consequently over-expressed. In addition, we investigated transforming growth factorß1 (TGFß1) responsiveness of ESC cell lines exhibiting amplification and over-expression of TGIF to clarify whether the resistance to TGFß-induced cell growth inhibition was enhanced in these lines.
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Materials and methods
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ESC cell lines and preparation of metaphase slides
A total of 29 ESC cell lines (KYSE series) examined had been established from surgically resected tumors (19). Copy-number aberrations of all these lines are reported elsewhere (8). Metaphase-chromosome slides were prepared and employed in FISH experiments as described earlier (20).
For dot-blot analysis, ESC tumor samples from 44 independent patients were provided by the Kyoto University Hospital, with written consent from each patient in the formed style and after approval by the local ethics committee. All of the tumors were at advanced TMN stage and surgically resected.
Fluorescence in situ hybridization (FISH) analysis
FISH analysis using bacterial artificial chromosomes (BACs) as probes was performed as described previously (20,21). The location of 22 BACs (683L23, 324G2, 14P20, 145B19, 78F17, 16P11, 2911G24, 21K5, 133O18, 351P1, 55N14, 840H14, 106J7, 359P13, 352L24, 340P19, 70G19, 167M1, 309J17, 286N3, 119P12 and 263O14) within the region of interest was compiled from information archived by the University of California, Santa Cruz (UCSC, http://genome.ucsc.edu/) and the National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov/). The probes were labeled by nick-translation with biotin (BIO)-16-dUTP or digoxigenin (DIG)-11-dUTP (Roche Diagnostics, Tokyo, Japan) and hybridized to metaphase and interphase chromosomes. Fluorescent signals specific for BIO- and DIG-labeled probes were detected with FITC-avidin and anti-DIG-rhodamine, respectively. The copy-number and molecular organization of the region of interest were assessed according to the hybridization patterns observed on both metaphase and interphase chromosomes. Hybridizations to normal lymphocyte nuclei were performed as controls to ascertain that each probe recognized a single-copy target.
Southern-, dot- and northern-blot hybridizations
A total of 35 cDNA clones in the 18p11.3 region (Table I
), chosen from the UCSC (http://genome.ucsc.edu/) and NCBI (http://www.ncbi.nlm.nih.gov/) database, were purchased from Incyte Genomics (Palo Alto, CA, USA) and used as probes for Southern, northern and dot-blot analyses. For YES1 only, we prepared a 1.5 kb PCR product using primers for YES1 (F299: 5'-ATTATGGAGCAGAACCCACTACAGTGTCAC-3' and R1726: 5'-TCTTTCATCAGGGTCCTTCTTCCAACACAG-3').
For Southern analyses, 10 µg aliquots of EcoRI-digested DNA extracted from each cell line or from normal lymphocytes were electrophoresed in 0.8% agarose gels and transferred to nylon membranes (BIODYNE B, Nihon Pall, Tokyo, Japan). For dot-blots, 2 µg of DNA from each tumor, cell line, or normal lymphocyte was denatured with 0.4 N NaOH, and then transferred to a nylon membrane (BIODYNE B, Nihon Pall). For northern analyses, 10 µg samples of total RNA extracted from each cell line were size-fractionated in 1.0% agarose/0.67 M formaldehyde gels and transferred to positively charged nylon membranes (Hybond-N+, Amersham Pharmacia Biotech, Tokyo, Japan). Each membrane was hybridized with [
-32P]dCTP-labeled cDNA or PCR-fragment probes under appropriate conditions, washed, and then exposed to Kodak XO-mat film as described elsewhere (22). The human glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) was used as a control probe.
Cell proliferation assay
KYSE cell lines were plated onto 96 well microtiter plates at a density of 4x103 cells/well in RPMI-1640 with 10% FBS, then medium was changed to RPMI-1640 containing 0.5% FBS 12 h later. After 24 h, cells were incubated in the 0.5% FBS-containing fresh medium with or without 1100 pM of TGFß1 (R&D Systems, Minneapolis, MN, USA), and the number of cells was determined 72 h later using the colorimetric assay (Cell-counting kit 8, Dojindo Laboratories, Tokyo, Japan).
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Results
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Definition of the 18p11 amplicon by FISH
Our previous CGH analysis had shown that a gain of copy-number on 18p was detected in 11 of the 29 ESC cell lines examined (37.9%), and five of those (KYSE70, 350, 510, 520 and 790) showed high-level gains (HLGs) indicative of gene amplifications (8). By preliminarily performed FISH analysis using BACs (RP11-145B19) mapped on 18p11.3, four (KYSE70, 350, 510 and 790) of these five lines revealed a pattern of homogeneously staining regions (HSRs) with markedly increased FISH signals (Figure 1
). KYSE520 also demonstrated the increased FISH signals ranged from 1012, but no HSR pattern was evident (data not shown). In order to generate a defined map of the 18p11.3 amplicon, we determined the extent of the HSRs in four ESC cell lines (KYSE70, 350, 510 and 790) by FISH using 22 BACs, because of easy detection of amplified signals. Relative positions of BACs on a map of the 18p11.3 amplicon are indicated in Figure 2
.

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Fig. 1. Representative image of FISH analyses on metaphase chromosomes from KYSE70 (A) and 350 (B). BAC145B19 shows a HSR pattern on marker chromosomes (arrows) of both cell lines. Similar amplification patterns with BAC145B19 were observed in KYSE510 and 790 (data not shown).
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Fig. 2. Amplicon map at 18p11.3. BACs used for FISH analysis, STS markers corresponding to BACs and positions of 14 known genes located within commonly amplified region (see below) are represented on the left side. BACs, STS markers and genes were positioned according to the information archived by the University of California, Santa Cruz (UCSC, http://genome.ucsc.edu/) and the National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov/). Thick bars on the right side represent the extent of HSR determined by FISH analyses on metaphase chromosomes from four ESC cell lines, KYSE70, 350, 510 and 790, which have amplification of 18p11.3 with HSR pattern. The commonly amplified region (the smallest region of overlap; SRO) was defined between BAC145B19 and 352L24.
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In all lines, 12 BACs between 145B19 and 352L24 produced the increased number of signals as HSRs (Figure 2
). In contrast, no HSR pattern and less FISH signals were detected with five BACs (683L23, 309J17, 286N3, 119P12 and 263O14) (Figure 2
) in all cell lines, suggesting those BACs are located outside the amplicon. The remaining five BACs (324G2, 14P20, 340P19, 70G19 and167M1) demonstrated the different pattern of signals between KYSE70 and the other three cell lines (KYSE350, 510 and 790) (Figure 2
). Taken together, as summarized in Figure 2
, we defined the smallest common amplified region of overlapping (SRO) between BAC145B19 and 352L24. The extent of this region was estimated at 3.5 Mb at 18p11.3 according to the genome database archived by the NCBI and UCSC. Target genes involved in ESC are most likely to lie within this SRO.
Positional candidate gene analysis within the 18p11.3 amplicon
To explore candidate target genes involved in 18p11.3 amplification, 35 ESTs were selected based on the database to represent transcripts from the SRO of this amplicon (Table I
). Then we examined amplification and expression status for those ESTs in our panel of 29 ESC cell lines.
Representative results are shown in Figure 3
. Among 35 ESTs, only four known genes (YES1, TYMS, HEC and TGIF) were consistently over-expressed in cell lines showing amplification, strongly suggesting that these genes are potential targets within the SRO of 18p11.3. Another 10 genes and 21 uncharacterized transcripts located within the same amplicon either exhibited no visible signals or appeared to be expressed equally or inconsequently in cell lines examined. To determine whether any of these four genes were also amplified in primary tumors, we examined 44 primary ESCs by dot-blot analysis. As shown in Figure 4
, TGIF and HEC were both amplified in seven of 44 tumors (15.9%) and in all seven cases these two genes were co-amplified.

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Fig. 3. Southern- and northern-blot analyses of the YES1, TYMS, HEC and TGIF genes in ESC cell lines. (A) Representative results of Southern blot analyses using YES1, TYMS, HEC and TGIF probes and control (GAPDH) probe in ESC cell lines. Four genes were clearly amplified in KYSE70, 350, 510 and 790 with HSR pattern in FISH and KYSE520 without HSR pattern. By FISH, amplicon of KYSE520 was larger than those of the other four lines (data not shown). N, DNA derived from peripheral blood lymphocytes from a healthy donor. (B) Representative results of northern-blot analyses using YES1, TYMS, HEC and TGIF probes and control (GAPDH) probe in ESC cell lines. Note the close correlation of the expression level of each gene with the DNA copy-number status.
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Fig. 4. Representative dot-blot analyses of TGIF, HEC and control GAPDH in primary ESC tumors. TGIF and HEC were amplified together in seven out of 44 primary ESCs examined. Arrowheads indicate amplified spots detected in representative cases. N, DNA derived from peripheral blood lymphocytes from a healthy donor.
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ESC cell lines with the amplification of TGIF exhibited the enhanced resistance to TGFß-induced cell growth inhibition
TGIF is a transcriptional co-repressor that interacts with Smads to negatively regulate the TGFßSmad response in a cell (23). As TGFß is a potent growth inhibitor in epithelial cells (24), it is possible that cells over-expressing TGIF may acquire the resistance to TGFß-induced growth inhibition. To test this hypothesis, we investigated whether ESC cell lines with the amplification of TGIF are resistant to TGFß1-mediated growth inhibition as compared with cell lines without over-expression of TGIF. Although all ESC cell lines examined showed poor responsiveness to growth suppression by TGFß1, TGFß1 responsiveness of KYSE 70, 350, 510 and 790 was significantly lower than that of KYSE30, 150, 200 and 220, in which TGIF was not amplified and over-expressed (Figure 5
).

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Fig. 5. ESC cell lines having amplification of TGIF exhibit enhanced TGFß1 resistance. ESC cell lines were treated with 0, 1, 10, 100 pM of TGFß1 for 72 h. Cell number was determined by colorimetry, and results were presented as percentage inhibition of cell proliferation by TGFß1. Results are shown as the mean ± SEM from three separate experiments each performed in triplicate. Note that ESC cell lines with amplification of TGIF (KYSE70, 350, 510 and 790) showed lower responsiveness to TGFß1 than those without amplification (KYSE30, 150, 200 and 220), although all lines respond poorly to TGFß1.
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Discussion
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In this study, we characterized the amplicon at 18p11.3 that was detected in multiple ESC cell lines and appears to play an important role in the development and/or progression of ESC. We first delineated a precise amplicon map using four cell lines showing 18p11.3 amplification with HSR pattern (Figure 2
). The commonly amplified region among these cell lines was located between BACs 145B19 and 352L24, which lie ~3.5 Mb apart. To identify the target genes involved, next we examined the amplification and expression status of 35 ESTs mapped to this commonly amplified region in 29 ESC cell lines. Based on the recent estimation of the number of human genes (25), the number of examined ESTs appears to be reasonable to explore the targets within this region. The expression of four known genes (YES1, TYMS, HEC and TGIF) was concordant with their amplification, especially in cell lines with HSR (Figure 3
). The fact that so few of the transcripts (four of 35) within the amplicon showed over-expression suggests that specific genes involved in esophageal carcinogenesis can be activated through the amplification in this region. We also confirmed that two genes, TGIF and HEC, were amplified also in primary ESC tumors. Therefore, the 18p11.3 amplicon, by harboring multiple target genes, seems to play an important role in the pathogenesis of ESC.
TGIF is a transcriptional repressor, which can interact with TGFß-activated Smads and repress expression of TGFß target genes through its ability to recruit other transcriptional co-repressors, including HDACs and CtBP (23,26). TGFß is a potent growth inhibitor in epithelial cells (24). In ESC, tumor cell lines are frequently resistant to TGFß-mediated growth inhibition (27), although little is known about the mechanism of this resistance. Mutation or inactivation of genes encoding components in the TGFß-signaling pathway, such as SMAD4 and TGFBR2, has been reported in various tumors but appear to be rare in ESC (28). In fact, no ESC cell lines examined in this study have mutations in SMAD4 and TGFBR2 (29). These results indicate that alterations of other components may exist to disrupt TGFßSmad signaling and contribute to the esophageal tumorigenesis. From this point of view, TGIF is likely to be a candidate molecule; the activated TGIF may have oncogenic potential through the inhibition of TGFßSmad response (23). This hypothesis may be supported by our initial preliminary finding that ESC cell lines with the amplification of TGIF exhibited the enhanced resistance to TGFß-induced growth inhibition, although all cell lines have been already resistant to TGFß and this finding should be confirmed by further studies, e.g. experiments using antisense oligonucleotides or inducible constructs for regulating TGIF expression. Therefore, the amplification of TGIF is one of the mechanisms to increase the expression level of this gene and acquire the resistance to TGFß-mediated growth inhibition in cancer cells. Besides the amplification, the phosphorylation of TGIF by epidermal growth factor (EGF) signaling via the RasMek pathway has been demonstrated to stabilize TGIF protein, accelerate the formation of Smad2TGIF co-repressor complexes, and modulate sensitivity to TGFß-mediated growth inhibition (30).
Recently we have identified a TGIF-related gene, TGIF2, which is amplified and over-expressed in ovarian cancer cell lines (21). Like TGIF, TGIF2 has been reported to interact with TGFß-activated Smads and repress TGFß-responsive transcription as well (31). Therefore, both genes are activated through gene amplification and involved in the tumorigenesis via a similar mechanism, such as decreasing the ability of TGFß signals to prevent cell cycle progression.
TYMS encodes thymidylate synthase (TS), which is a rate-limiting enzyme in the DNA synthetic pathway and also the critical target of 5FU, one of the most widely used chemotherapeutic agents for treating tumors of epithelial origin (18,32). Cells can acquire resistance to 5FU, when TS is over-produced as a result of gene amplification of TYMS (33), although other mechanisms for over-production of this protein have been reported as well (34). The cell lines used in the present study were established from patients without the history of treatment with 5FU or its derivatives before surgery. Therefore, TYMS amplification may have occurred spontaneously in those tumors, i.e. without reagent-induced selective pressure, suggesting that gene amplification is one of the mechanisms of innate resistance to 5FU in ESC.
The putative proto-oncogene YES1 is a homologue of v-yes, a component of Yamaguchi sarcoma virus, and encodes cytoplasmic, membrane-associated protein tyrosine kinase activity. For the related Src kinase, a close correlation exists between elevated tyrosine kinase activity and cell transformation (35). Although amplification of YES1 in gastric cancers (14) and its over-expression in various types of human cancers (15,16) have been reported, little is known about the actual role of this gene in carcinogenesis of esophagus and other tissues. The amplification of YES1 clearly warrants further investigation into its potential contribution to the development and progression of ESC.
HEC, highly expressed in cancer, encodes a protein containing 642 amino acids and long series of leucine heptad repeats at its C-terminal region (36). This protein is expressed most abundantly in the S and M phases of actively dividing cells including cancer cells but not in terminally differentiated cells. Inactivation of HEC protein severely disturbs chromosome distribution. The data together with its distribution to the centromere during mitosis suggest that this protein may play an important role in cell proliferation. Therefore, over-expression caused by amplification of HEC is of interest with respect to its possible involvement in the pathogenesis of ESC, although it is unclear whether its over-expression is a cause or a consequence of cellular proliferation in cancer cells.
Genomic amplification in tumors does not usually contain only the core domain but may extend several hundred kilobases to several megabases flanking the selected genes (22). Co-activated genes via co-amplification may be factors that influence the phenotype and/or clinical outcome of tumors. It is still unclear whether the target genes within a given amplicon are just physically linked by sharing the same origin, or whether there is any functional correlation among the co-amplified genes in the same amplicon. In the 18p11.3 amplicon, further study is necessary to clarify the individual and cooperative functional role of over-expressed target genes in the esophageal carcinogenesis.
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Notes
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4 To whom correspondence should be addressed E-mail: johinaz.cgen{at}mri.tmd.ac.jp 
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Acknowledgments
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We thank Professor Yusuke Nakamura for his continuous encouragement, and Drs Yasuhiro Yuki and Itaru Sonoda for their useful discussion. We also thank Ms Tomoko Usami, Chika Kato and Ai Watanabe for their technical assistance in carrying out this study. The work was supported by Grants-in-Aid from the Ministry of Health and Welfare, the Ministry of Education, Science, Sports and Culture, and the Organization for Pharmaceutical Safety and Research (OPSR).
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Received August 10, 2001;
revised September 13, 2001;
accepted September 13, 2001.