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The FEZ1 Gene at Chromosome 8P22 Encodes a Leucine-Zipper Protein, and Its Expression Is Altered in Multiple Human Tumors

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The FEZ1 Gene at Chromosome 8P22 Encodes a Leucine-Zipper Protein, and Its Expression Is Altered in Multiple Human Tumors Proc. Natl. Acad. Sci. USA Vol. 96, pp. 3928–3933, March 1999 Medical Sciences The FEZ1 gene at chromosome 8p22 encodes a leucine-zipper protein, and its expression is altered in multiple human tumors HIDESHI ISHII*, RAFFAELE BAFFA*, SHIN-ICHIRO NUMATA*, YOSHIKI MURAKUMO*, SHASHI RATTAN*, HIROSHI INOUE†,MASAKI MORI†,VINCENZO FIDANZA*, HANSJUERG ALDER*, AND CARLO M. CROCE*‡ *Kimmel Cancer Institute, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA 19107; and †Medical Institute of Bioregulation, Kyushu University, Beppu 874, Japan Communicated by Sidney Weinhouse, Jefferson Medical College, Philadelphia, PA, January 13, 1999 (received for review November 27, 1998) ABSTRACT Alterations of human chromosome 8p occur been studied in cancer, because of their carcinogen- frequently in many tumors. We identified a 1.5-Mb common metabolizing action (29, 30). The results showed no abnor- region of allelic loss on 8p22 by allelotype analysis. cDNA mality in cancer cells (29, 30). selection allowed isolation of several genes, including FEZ1. Esophageal cancer occurs worldwide, and its incidence is The predicted Fez1 protein contained a leucine-zipper region increasing in the Western world (31–33). We performed with similarity to the DNA-binding domain of the cAMP- allelotyping to identify a common region of loss at 8p in responsive activating-transcription factor 5. RNA blot analy- primary esophageal squamous cell carcinomas. We identified sis revealed that FEZ1 gene expression was undetectable in a common region of LOH at 8p22 around marker D8S261, more than 60% of epithelial tumors. Mutations were found in which overlaps with the target region in other tumors, includ- primary esophageal cancers and in a prostate cancer cell line. ing prostate and breast cancer (6–9, 15). This region is .2Mb Transcript analysis from several FEZ1-expressing tumors centromeric to the MSR region (23–26). To clone the genes revealed truncated mRNAs, including a frameshift. Alteration present in this region, cDNA selection, CpG island cloning, and inactivation of the FEZ1 gene may play a role in various and shotgun sequencing were carried out. Analysis of cDNAs human tumors. in human tumors and tumor-derived cell lines indicates that we cloned a gene, FEZ1, which is altered in many tumors, Frequent loss of heterozygosity (LOH) at specific chromo- including esophageal, prostate, and breast cancer. somal regions in certain tumors implies the presence of suppressor genes (1–5). Recent allelotyping studies have MATERIALS AND METHODS shown that allelic losses on the short arm of chromosome 8, particularly at bands 21–22, frequently are associated with Cell Lines and Tissues. Esophageal cancer cell lines were various tumors, including prostate cancer (6, 7), breast cancer cultured in RPMI 1640 with 10% FBS (34). The other cancer (8), head and neck squamous cell carcinoma (9–11), urinary cell lines were obtained from the American Type Culture bladder carcinoma (12), hepatocellular carcinoma (13), and Collection and maintained. Seventy two primary esophageal hematological malignancies (14). A recent genome-wide cancer samples and flanking normal portions, as well as 39 search for LOH in breast cancer showed that 8p is one of the breast, 24 prostate, and 8 ovarian cancers, were obtained most frequently altered chromosome regions (15); LOH at surgically from patients with their informed consent. 8p21–22 was associated with the invasive behavior of breast LOH Study. PCR amplifications using 59-fluorescein phos- cancer (16, 17). Loss at 8p21.3–22 has been shown to be phoramidite- or 59-tetrachloro-fluorescein phosphoramidite- associated strongly with prostate cancer progression (18). labeled primers for microsatellite loci (Research Genetics, These observations suggest that chromosome region 8p21–22 Huntsville, AL) with tumor and normal template DNAs were plays an important role in the development of various tumors, performed as reported (35), with minor modifications. Briefly, m including prostate and breast cancers. PCRs were done in a 20 l of buffer containing 2.5 mM MgCl2, Functional evidence of the presence of tumor suppressor 1.5 mM dNTP, and 0.5 unit of Ampli Taq Gold (Perkin– gene(s) at 8p was shown experimentally by chromosome Elmer). PCR conditions were as follows; after 95°C for 12 min, transfer into tumor cells (19). Somatic cell hybrids of cancer a total of 30 cycles consisting of 10 cycles at 94°C for 15 sec, cells with transferred normal human chromosome 8p22–23 55–58°C annealing for 15 sec, and 72°C for 30 sec, and 20 cycles fragments lost their ability to produce tumors (19). Additional at 89°C for 15 sec, 55–58°C annealing for 15 sec, and 72°C for microcell fusion experiments suggested the possible location of 30 sec, followed by 72°C for 30 min. After heat denaturation, metastasis suppressor gene(s) at 8p (20–22). It is possible that the samples were loaded on a 6% denaturing gel on the two or more genes at 8p may be involved in suppression of Applied Biosystems 373 DNA sequencer. Data collection and cancer development. fragment analysis were done with the Applied Biosystems Efforts toward positional cloning of the suppressor gene(s) Prism Genescan and the Applied Biosystems PRISM GENO- allowed the isolation of a candidate tumor suppressor gene, TYPER ANALYSIS software (Perkin–ElmeryApplied Biosys- N33, at 8p22 near the macrophage-scavenger-receptor (MSR) tems). Cases were judged as LOH when an allele peak signal gene locus (23–26). The N33 gene was silenced in several from tumor DNA was reduced by 50% compared with the cancer cells, although no point mutations were found. Another candidate gene, PRLTS, at 8p21.3–22 showed point mutations in four cancer cases (27, 28). The frequency of alterations in Abbreviations: LOH, loss of heterozygosity; YAC, yeast artificial chromosome; BAC, bacterial artificial chromosome; RT, reverse this gene was, however, very low (28). Alterations of N- transcription. acetyltransferase (NAT)1 and NAT2 genes at 8p22 have also Data deposition: The sequences reported in this paper have been deposited in the GenBank database (accession nos. AF123652– The publication costs of this article were defrayed in part by page charge AF123659). ‡To whom reprint requests should be addressed at: Kimmel Cancer payment. This article must therefore be hereby marked ‘‘advertisement’’ in Institute, Jefferson Medical College, Thomas Jefferson University, accordance with 18 U.S.C. §1734 solely to indicate this fact. BLSB Room 1050, 233 South 10th Street, Philadelphia, PA 19107- PNAS is available online at www.pnas.org. 5799. e-mail: [email protected]. 3928 Downloaded by guest on September 29, 2021 Medical Sciences: Ishii et al. Proc. Natl. Acad. Sci. USA 96 (1999) 3929 normal counterpart. When tumors showed 40–60% reduction FEZ1 exons 1–3 were used for PCR with genomic DNA. The of a normal counterpart, the analyses were repeated two more PCRs were done with the same conditions as those described times, and average reductions were used as final data. for the LOH studies, except that 4% (wtywt) dimethyl sulfox- Yeast Artificial Chromosome (YAC) and Bacterial Artificial ide was added, and PCR amplifications were a total of 35 Chromosome (BAC) DNAs. PCR amplifications were done to cycles. The PCR products were cleaned with the Qiagen PCR screen human YAC and BAC libraries (Research Genetics). purification column and sequenced. Sequencing reactions and YAC clones were embedded into agarose and separated by analysis were performed by using the Applied Biosystems pulse-field gel electrophoresis (36, 37). After the gel electro- Prism BigDye terminator reaction chemistry on a Perkin– phoresis, the YAC DNAs were cut out from the gels. BAC Elmer Gene Amp PCR system 9600 and the Applied Biosys- DNAs were purified by using the Qiagen (Chatsworth, CA) tems Prism 377 DNA sequencing system. purification kit. BAC DNAs were sequenced with T7 and SP6 primers. Southern blot hybridization and PCR analysis re- RESULTS AND DISCUSSION sulted in overlapping clones. From these results, contigs were constructed. Deletion Analysis of Chromosome 8p in Cancer. DNAs from cDNA Selection. Gels containing YAC DNAs were digested 53 primary esophageal cancers and matched normal tissues with MboI and were extracted from the gel with Gene Clean were analyzed for allele loss at 22 microsatellite loci on III (Bio 101). Two deoxyoligonucleotides, 59-GATCTCGAC- chromosome 8p. Representative data are shown in Fig. 1A. GAATTCGTGAGACCT-39 and 59-TGGTCTCACGAAT- Allelic loss was assessed by the reduction of the signal intensity TCGTCGA-39, were annealed to make an adapter-linker, of an allele, compared with its normal counterpart (35). The which was ligated to the digested genomic DNA. PCR ampli- variability of relative intensity of tumor alleles is caused by fications with 15 cycles were performed with 59-biotinylated either intratumoral heterogeneity or normal tissue contami- primer corresponding with the adapter-linker. cDNAs were nation. Histological examination showed that different tumors reverse-transcribed from prostate poly(A)1 RNA (CLON- had different ratios of neoplastic to stromal cells and to TECH) with NotI-primer adaptoryoligo(dT)-primers (Super- infiltrating lymphocytes. The apparent incomplete allele loss script Plasmid system; GIBCOyBRL). After the first strand seen in some samples, such as E46 at D8S136 and D8S264 (Fig. cDNA end was blunted, a SalI adaptor (GIBCOyBRL) was 1A) likely is caused by the contamination of normal cells. The ligated to cDNAs. cDNAs were amplified by PCR with the SalI allelic losses for each matched DNA pair are summarized in adapter primers, and the products were cleaned with the Fig. 1B. Twenty-three patients (43%) showed loss of an allele Qiagen PCR purification column. at one or more loci on 8p. Sixteen of 23 tumors (70%) showed The methods of blocking, hybridization, and washing (26, a commonly lost 1.5-Mb region near the D8S261 loci, and 14 38) were adapted with minor modifications. Repetitive se- patients (61%) had potential common LOH regions near quences were blocked with equal amounts (wtywt) of Cot-1 D8S254 (Fig.
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