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Identification of ZASC1 Encoding a Krüppel-like Zinc Finger Protein as a Novel Target for 3q26 Amplification in Esophageal Squamous Cell Carcinomas¹

Department of Molecular Cytogenetics, Medical Research Institute, Tokyo Medical and Dental University, Tokyo 113-8510, Japan [I. I., Y. Y., I. S., J. I.]; Core Research for Evolutional Science and Technology of Japan Science and Technology Corporation, Saitama 332-0012, Japan [I. I., J. I.]; and Department of Surgery, Surgically Basic Medicine, Kyoto University Graduate School of Medicine, Kyoto 606-8507, Japan [T. I., Y. S., M. I.]

	ABSTRACT

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Among the transcription factors that direct proliferation, differentiation, and death of cells, several Krüppel-like zinc finger molecules such as GLI1 and ZNF147 can become oncogenic when the genes encoding them are overexpressed by the DNA amplification mechanism. Here, we report the discovery of a novel member of this class, ZASC1, from a recently reported critical region of 3q26 amplification frequently observed in various squamous cell carcinomas, including esophageal squamous cell carcinomas (ESCs). The deduced 485-amino acid protein product of ZASC1 contained a putative nuclear localization signal; the exogenously transfected ZASC1 was translocated into cell nuclei. ZASC1 was frequently coamplified and subsequently overexpressed with PIK3CA in our panel of ESC cell lines and primary tumors. In patients with ESC, a higher mRNA expression level of ZASC1 appeared to be associated with shorter overall survival, and a multivariate analysis demonstrated that ZASC1 mRNA expression was an independent prognosticator. In addition, exogenous expression of ZASC1 promoted the growth of ESC cells, whereas down-regulation of ZASC1 expression by means of an antisense oligonucleotide suppressed the growth of ESC cells. Taken together, our results suggest that ZASC1 might be involved in the pathogenesis of ESC as one of the targets for 3q26 amplification, alone or in concert with other targets.

	Introduction

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Amplification of chromosomal DNA is one of the mechanisms capable of activating genes that are implicated in developing tumors. Several oncogenes, including CCND1, are known targets of gene amplification in ESCs³ (1) . The advent of comparative genomic hybridization has made it possible to identify additional chromosomal amplicons that are likely to harbor previously unidentified genes associated with esophageal carcinogenesis (2 , 3) .

One of the regions most frequently amplified in ESC, but poorly characterized, is the long arm of chromosome 3; in most cases, the 3q26 band seems to lie in the center of the amplicon (2 , 3) . Amplification around 3q26 is found consistently in SCCs of the cervix, lung, and head and neck (4, 5, 6, 7, 8) , suggesting that activation of certain genes in that region confers a selective advantage for cancer cells. However, the large physical size of overlapping amplified areas around 3q26, and variable minimal regions, make the precise definition of a common region to examine for target genes difficult. Recent studies of various SCCs have indicated that a few megabases around PIK3CA at 3q26.3 appear to constitute an apex within the 3q26 amplicon (7, 8, 9) , in which amplification is associated with clinical outcome (7) . Although PIK3CA is an outstanding candidate oncogene (10) , independent targets may exist within the same amplicon.

Transcription factors represent an important category of molecules that can participate in cellular cascades favoring tumor development. Among them, several genes encoding Krüppel-like zinc finger proteins, such as GLI1 (11) and ZNF217 (12) , have already been identified as being activated through gene amplification and involved in neoplastic transformation. Many additional members of Krüppel-like zinc finger proteins may be involved in the progression of human cancers (13) , but their functional roles in carcinogenesis remain largely unknown. If the genes encoding them are seen to be located in amplified regions in tumors, these proteins will become novel targets for exploration and, potentially, therapeutic strategies.

Here, we report the identification of ZASC1, which encodes a novel Krüppel-like zinc finger protein. This gene lies 200 kb telomeric to PIK3CA on 3q26.3 and within the genomic sequence that was recently identified as one of the recurrently amplified sequences by restriction landmark genomic scanning in SCC of the lung (14) , indicating it to be a separate target for 3q26 amplification in SCCs. The results of our experiments to characterize this novel gene suggest that, when ZASC1 is activated through 3q26 amplification, its consequent overexpression may be involved in the pathogenesis of ESC, either separately or in coordination with other target genes within the amplicon.

	Materials and Methods

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ESC Cell Lines and Primary Tumor Samples.
All 31 ESC cell lines examined (KYSE series) had been established from surgically resected tumors (15) . Data from comparative genomic hybridization analyses in 29 of those lines have been reported previously (3) .

Primary tumor samples were obtained and frozen at the time of surgery from 31 ESC patients who were treated at the Kyoto University Hospital, with written consent from each patient in the formal style and after approval by the local ethics committee. None of the patients was treated with such preoperative therapies as radiation, chemotherapy, or immunotherapy. The median age of the patients was 64 years (range, 49–90). Tumor stages were classified according to tumor-node-metastasis classification of the International Union Against Cancer: stage I, 1 patient; stage II, 13 patients; stage III, 11 patients; stage IV, 6 patients. The clinicopathological investigation was made on the basis of guidelines for clinical and pathological studies on carcinoma of the esophagus established by the Japanese Society for Esophageal Diseases (16) . The duration of overall survival was calculated for each patient from the date of primary surgery to the date of the last follow-up visit or death. The median follow-up periods were 52.7 months (range, 28.0–82.4) for 14 patients who are alive at the time of this writing. Total RNA was successfully extracted from all 31 tumors, and DNA from 14 of them.

Cloning and Sequencing of Human ZASC1.
We used the genomic databases archived by the National Center for Biotechnology Information⁴ or the University of California Santa Cruz Biotechnology⁵ and a similarity-search program (BLAST⁶ ) to look for genes that might encode proteins containing C2H2-type Krüppel-like zinc finger motifs. Deduced amino acid sequences of all known genes, uncharacterized transcripts, ESTs that might be part of transcripts, and computer-predicted possible transcripts in the region of interest were reviewed and/or compared with the consensus sequence (13) .

To obtain and confirm the cDNA sequences of transcripts of interest, we performed RT-PCR using a single-stranded cDNA synthesized by SuperScript First-Strand Synthesis System (Invitrogen, Carlsbad, CA) from mRNA derived from ESC cell line KYSE-1170 and cDNA from a plasmid library as templates. For construction of the cDNA plasmid library, double-stranded cDNAs were produced from mRNA of KYSE-1170 with double-strand cDNA synthesis kits (Takara, Tokyo, Japan) and cloned into pBlueScript II SK vector (Stratagene, La Jolla, CA). PCR amplifications were performed using primers generated on the basis of EST sequences, genomic sequences, and/or universal T3 and T7 priming sites in the plasmid vector, and Pyrobest DNA polymerase (Takara) according to the manufacturer’s directions. Primer sequences are available on request. PCR products were subcloned and sequenced by a 377 ABI autosequencer (Applied Biosystems, Foster City, CA). Sequences were analyzed using BLAST, Motif,⁷ and PSORT II⁸ programs.

FISH.
Metaphase chromosomes were prepared from normal male lymphocytes and from each ESC cell line. FISH analyses were performed as described previously (17) , using a bacterial artificial chromosome containing ZASC1 (RP11-255C15) as the gene-specific probe.

FIC.
Indirect FIC was performed as described previously (18) . A plasmid construct encoding an epitope-tagged form of ZASC1 (pcDNA3.1-His-ZASC1) was assembled by cloning the full coding sequence of this gene in-frame along with Xpress epitope into the pcDNA3.1/HisC vector (Invitrogen) and transfected into COS-7 cells using FuGENE6 (Roche Diagnostics, Tokyo, Japan) according to the manufacturer’s instructions. The cells were fixed with acetone/methanol (1:1 v/v) 48 h after transfection, and epitope-tagged ZASC1 protein was detected using monoclonal anti-Xpress antibody (Invitrogen) and FITC-conjugated antimouse secondary antibody (Medical & Biological Laboratories, Nagoya, Japan).

Southern and Northern Blot Hybridizations.
Southern and Northern blot analysis of genomic DNA and total RNA from each cell line, respectively, were performed as described previously (9 , 18) . To direct tissue-specific expression, a multiple tissue Northern blot (Human 12-lane MTN blot; Clontech, Palo Alto, CA) was used. Membranes were hybridized with [³²P]-dCTP-labeled ZASC1 or PIK3CA cDNA probe or with a control probe (GAPDH).

Real-Time Quantitative PCR and RT-PCR.
Quantification of genomic DNA and mRNA of ZASC1 in primary tumors was performed using a real-time fluorescence detection method as described previously (19 , 20) . Real-time quantitative PCR was performed using LightCycler (Roche Diagnostics) with CYBR Green according to the manufacturer’s protocol. ß-2-Microglobulin and GAPDH served as endogenous controls for genomic DNA and mRNA levels, respectively; the genomic DNA copy number and the expression level of ZASC1 in each sample were normalized to the respective controls. Primer sequences for each gene are available on request. PCR amplifications were performed in duplicate for each sample.

Colony Formation Assay (Anchorage-dependent Growth Assay).
Plasmid expressing ZASC1 (pcDNA3.1-His-ZASC1), its complementary strand (pcDNA3.1-His-antisense), or the empty vector (pcDNA3.1-His) were transfected using FuGENE6 (Roche Diagnostics) into KYSE-410 and KYSE-790 cells for colony formation assay as described by Lin et al. (21) , with minor modification. After 2 weeks of incubation with G418 (1.0 mg/ml), cells were fixed with 70% ethanol and stained by crystal violet solution.

ASO Experiments.
ASO experiments were performed as described previously (20) . We synthesized the following oligonucleotides containing OPTs (Espec Oligo Service Co., Tsukuba, Japan): ZASC1-AS, nucleotides 446–463 of ZASC1 cDNA (GenBank accession no. AB097862) in the antisense direction; ZASC1-IV, an inverse control for ZASC1-AS; and ZASC1-SC, a scrambled control for ZASC1-AS. OPTs were delivered into cells using Oligofectamine (Invitrogen) according to the manufacturer’s protocol.

To evaluate expression of ZASC1, 4 x 10⁴ cells were plated on 6-cm dishes, and their mRNA levels were determined 24 h after transfection by real-time quantitative RT-PCR. For measurements of cell growth and DNA synthesis, 2 x 10³ cells were seeded in 96-well plates. Viable cells were assessed 48, 72, and 96 h after transfection of various concentrations of OPTs by the colorimetric water-soluble tetrazolium salt (WST) assay (cell counting kit-8; Dojindo Laboratories, Kumamoto, Japan), whereas DNA synthesis was assessed 48, 72, and 96 h after transfection of OPTs by BrdUrd incorporation using a cell proliferation ELISA, BrdUrd colorimetric kit (Roche Diagnostics). For flow cytometric analysis, cells were trypsinized 48 h after transfection of OPTs, fixed in 70% ethanol overnight, and incubated in a staining buffer (0.1% Triton X-100 in PBS, 40 units/ml RNase A, and 20 µg/ml propidium iodide) on ice for 1 h. Cells were analyzed for DNA content using a FACSCaliber cytometer and Cell Quest software (Becton Dickinson, San Jose, CA). Experiments were repeated twice, each performed in triplicate.

Statistical Analysis.
Relationships between relative copy number ratios and expression levels of ZASC1 were analyzed using Spearman’s test to calculate CCs and associated Ps. Possible correlation between variables of the analyzed primary ESC tumors and gene expression status was tested by the ²/Fisher’s exact test. Ps of survival were calculated by the Kaplan-Meier method, and statistical differences between groups were evaluated by log-rank tests. Univariate and multivariate prognostic effects were evaluated under the Cox proportional-hazards model. One-way ANOVA with subsequent Scheffé’s tests were used to determine the significance of differences in multiple comparisons. P < 0.05 was required for significance in each case.

	Results and Discussion

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Identification of a Novel Gene, ZASC1, from the Critical Region of 3q26 Amplification.
Because one of the apices in the 3q26 amplicon has been mapped recently within a few megabases around PIK3CA (7) , we considered it feasible to search for Krüppel-like zinc finger proteins using their highly conserved motifs in silico. The critical region reported by Singh et al. (7) and information archived in the YAC-contig map database (San Antonio Genome Center⁹ ) directed our search for all possible transcripts that are located within the 2.3-megabase region between WI-4037 (YAC966F9) and WI-5357 (YAC823D8). We identified only two transcripts encoding elements of Krüppel-like zinc finger proteins, WIG1 (GenBank accession no. NM_022470) and LOC51193 (GenBank accession no. NM_0163331). The WIG1 gene is induced by wild-type p53 (22) , and its overexpression inhibits cell growth (23) ; the WIG1 is, therefore, assumed to function as a tumor suppressor. Because the LOC51193, in contrast, had never been characterized, we focused our efforts on characterizing this putative transcript. Notably, LOC51193 or genomic sequence AC007823 containing this transcript was recently identified as a recurrently amplified sequence in SCC of the lung (14) , strongly suggesting that LOC51193 locates within the core of the 3q26 amplicon. On the basis of the sequences present in LOC51193 and in Integrated Molecular Analysis and Gene Expression EST clones 3912907 and 4794621, we designed primers to amplify all coding elements as well as untranslated regions. Possible exons were connected by RT-PCR using a single-stranded cDNA as template, whereas 5' and 3' untranslated regions were determined by RT-PCR using a plasmid cDNA library as template and universal primers along with gene-specific primers. In this manner, we assembled a 3016-bp cDNA sequence encoding a putative 485-amino acid protein with a predicted molecular mass of 56.05 kDa. We designated the novel gene ZASC1 (GenBank accession no. AB097862), to indicate its nature as a Krüppel-like zinc finger protein amplified in ESCs. The genomic structure of ZASC1 was determined by comparing its cDNA sequence to the corresponding genomic sequence using the BLAST program. The ZASC1 gene consists of at least seven exons, and its genomic extent is approximately 12.5 kb (Fig. 1A) . As one would have predicted from the database, FISH using a ZASC1-containing bacterial artificial chromosome as a probe produced clear doublet signals on chromosomal band 3q26.3 in normal human lymphocytes (Fig. 1B) .

View larger version (49K): [in this window] [in a new window]   Fig. 1. A, top, deduced amino acid sequence of ZASC1 protein (GenBank accession no. AB097862). Numbers at right refer to amino acid residues. Potential tyrosine-phosphorylation sites are boxed, and nine C2H2-type zinc finger motifs are underlined. A putative nuclear localization signal is in bold. Bottom, genomic and cDNA structures of the ZASC1 gene. ATG (arrow) and TGA are the start and stop sites, respectively. White, gray, and black boxes indicate untranslated, coding, and zinc finger sequences, respectively; lines between the boxes denote introns. B, mapping of the ZASC1 gene by FISH. Typical hybridization signals appear on chromosomes 3 at band q26.3 (red signals, arrows). C, nuclear localization of ZASC1. The product of a transiently transfected, Xpress epitope-tagged full coding sequence of ZASC1 was detected in COS-7 cells by fluorescent immunocytochemistry using an anti-Xpress antibody. The preparations were counterstained with 4',6'-diamidino-2-phenylindole. An arrowhead indicates nuclear staining of the ZASC1 construct. D, expression of ZASC1 mRNA in normal human tissues. The same multi-tissue Northern blot was rehybridized with a GAPDH probe as a control for RNA loading and transfer.

Northern blot analysis of mRNA derived from various human tissues was performed to gain insight into the spatial distribution of ZASC1 mRNA. As shown in Fig. 1D , a single 4.8-kb transcript was expressed in most human tissues represented in the blot. Expression was abundant in heart and skeletal muscle and relatively high in kidney, liver, and pancreas.

Amplification and Subsequent Overexpression of ZASC1 in ESCs.
Because ZASC1 appears to lie within the critical region of 3q26 amplification of SCCs (7) , we confirmed the presence of ZASC1 in this amplicon in our panel of ESC cell lines. As shown in Fig. 2, A and B , ZASC1 was amplified in many of these cell lines. ZASC1 was always amplified in the same lines as PIK3CA, already considered a potential target for 3q26 amplification (10) ; PIK3CA lies only 200 kb away from the ZASC1 gene, on the centromeric side. We observed frequent amplification of ZASC1 in primary ESC tumors as well (Fig. 2D) . The commonly accepted criterion for describing a putative target for amplification is that amplification of the gene in question leads to its overexpression (9 , 20) . On that basis, we compared expression levels of ZASC1 with its amplification status in ESC cell lines and identified consistent overexpression of ZASC1 in lines with its amplification (Fig. 2B) . Interestingly, the expression of ZASC1 was up-regulated by 10% serum after 24 h-serum starvation (Fig. 2C) , suggesting a role for this gene in the regulation of cell growth. The positive and significant correlation between the copy number ratio and expression status of ZASC1 also pertained in primary ESC tumors (CC = 0.919 and P = 0.006; Fig. 2D ). These lines of evidence supported the view that ZASC1 might represent an independent target for 3q26 amplification in ESCs.

View larger version (58K): [in this window] [in a new window]   Fig. 2. A, representative FISH images with the ZASC1 probe hybridized to cells from ESC lines KYSE-1170 and KYSE-1190 showed remarkable copy number increases. B, representative results from Southern (top) and northern (bottom) blots using ZASC1, PIK3CA, or a control GAPDH probe in 16 ESC cell lines. Asterisks indicate cell lines showing the amplification of ZASC1. Note that ZASC1 was always coamplified with PIK3CA, and expression levels of both genes were correlated with their copy number status. C, the mRNA expression of ZASC1 is up-regulated by serum in ESC cells (KYSE-140). Cells were serum-starved for 24 h and subsequently treated with 10% serum for the indicated times. Serum clearly increased the levels of ZASC1 mRNA 8 h after treatment. D, amplification and consequent overexpression of ZASC1 in primary ESC tumors. The genomic copy number ratio of ZASC1 normalized on the basis of ß-2-microglobulin was divided by that of normal cells and recorded as a relative copy number (top). Expression levels of ZASC1 normalized on the basis of GAPDH were recorded as relative expression levels (bottom). Asterisks indicate cases showing clear increase (>2.0) in relative copy number of ZASC1 (7 of 14 cases, 50%). Significant correlation (CC = 0.919 and P = 0.006) between copy numbers and relative expression levels of ZASC1 was observed. E, Kaplan-Meier curve for overall survival rates of 31 patients with ESC. The overall survival rate for tumors with high ZASC1 mRNA expression (greater than the median value) was significantly lower than that for tumors with low ZASC1 mRNA expression (less than or equal to the median value; P = 0.0065, log-rank test).

View larger version (41K): [in this window] [in a new window]   Fig. 3. A, colony formation assay of two ESC cell lines, KYSE-410 and KYSE-790, by transfection with ZASC1. Cells were transfected with construct designed to express sense strand (pcDNA3.1-His-ZASC1) or antisense strand (pcDNA3.1-His-antisense) of ZASC1 or with empty vector (mock) and selected in medium containing 1.0 mg/ml G418 for 2 weeks. Note that transiently transfected pcDNA3.1-His-ZASC1 produced markedly more colonies than did control plasmids (pcDNA3.1-His-antisense and mock). B, inhibition of ZASC1 expression by an antisense OPT targeting the ZASC1 gene. KYSE-1170 cells were treated with 300 nM of each OPT or Oligofectamine alone (mock control), and mRNA levels of ZASC1 were determined by a real-time quantitative RT-PCR 24 h after transfection. ZASC1-AS, antisense OPT targeting the ZASC1 gene; ZASC1-IV, the inverse control OPT for ZASC1-AS; ZASC1-SC, a scramble control OPT for ZASC1-AS. The data presented are the mean ± SE of three separate experiments. Statistical analysis used the one-way ANOVA with subsequent Scheffé’s tests: a, ZASC1-AS versus ZASC1-IV; b, ZASC1-AS versus ZASC1-SC. All P < 0.05. C, effect of ZASC1-AS on the viability of KYSE-1170 cells. Cell viability was determined by WST assay 72 h after transfection with the indicated concentrations of OPTs (left) or at the indicated times after transfection with 300 nM of each OPT (right). Percentages were calculated against the absorbance of control cells treated with Oligofectamine alone (mock). D, effect of ZASC1-AS on the DNA synthesis of KYSE-1170 cells at the indicated times after transfection with 300 nM of each OPT or Oligofectamine alone (mock), as determined by BrdUrd incorporation. E, representative results of flow cytometric analyses on KYSE-1170 cells. Cells were treated with 300 nM of each OPT or Oligofectamine alone (mock) for 48 h, harvested, and subjected to the analysis of DNA profiles. The x-axis shows DNA content, and the y-axis shows the number of cells. The percentages of cells in sub-G1 phase are indicated.

These findings suggest that ZASC1 overexpression tends to promote the growth of ESC cells, although the molecular mechanisms underlying this effect remain unknown. Functional roles in the development and progression of tumors have been reported for several Krüppel-like zinc finger proteins, such as ZNF217 and ZK7 (12 , 27) . ZNF217 promotes immortalization of mammary epithelial cells by inducing increases in telomerase activity and resistance to the growth inhibition caused by transforming growth factor-ß (12) , whereas ZK7 inhibits apoptosis induced by radiation or chemotherapeutic reagents (27) . Like ZASC1, however, the detailed mechanisms remain unknown. Additional investigation will be necessary to clarify the intracellular functions and molecular targets of ZASC1, because it is possible that inactivation of ZASC1 may lead to the regression of ESC or other SCCs, and it may become an optimal molecular target for cancer therapy.

	ACKNOWLEDGMENTS

 
We thank Prof. Yusuke Nakamura (Human Genome Center, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan) for continuous encouragement. We thank Ai Watanabe and Yuri Yoshinaga for 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 Supported by Grants-in-Aid for Scientific Research on Priority Areas (C) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan and a Grant-in-Aid from Core Research for Evolutional Science and Technology of the Japan Science and Technology Corporation.

² To whom requests for reprints should be addressed, at Department of Molecular Cytogenetics, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan. Phone: 03-5803-5820; Fax: 03-5803-0244; E-mail: johinaz.cgen{at}mri.tmd.ac.jp

³ The abbreviations used are: ESC, esophageal squamous cell carcinomas; SCC, squamous cell carcinoma; EST, expressed-sequence tag; RT-PCR, reverse transcription PCR; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; OPT, oligonucleotide phosphorothioate; ASO, antisense oligonucleotide; BrdUrd, 5-bromo-2'-deoxyuridine; CC, correlation coefficient; FISH, fluorescence in situ hybridization; BLAST, Basic Local Alignment Search Tool; FIC, fluorescent immunocytochemistry.

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

⁵ Internet address: http://genome.ucsc.edu/.

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

⁷ Internet address: http://motif.genome.ad.jp/.

⁸ Internet address: http://cookie.imcb.osaka-u.ac.jp/nakai/psort.html.

⁹ Internet address: http//:apollo.utscsa.edu.

Received 5/ 5/03. Revised 7/ 6/03. Accepted 7/30/03.

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