Homozygous deletions (HD) provide an important resource for identifying the location of candidate tumor suppressor genes. To identify the tumor suppressor gene in oral cancer, we employed high-resolution comparative genomic hybridization (CGH)-array analysis. We identified a homozygous loss of FAT (4q35), a new member of the human cadherin superfamily, from genome-wide screening of copy number alterations in one primary oral cancer. This result was evaluated by genomic polymerase chain reaction in 13 oral cancer cell lines and 20 primary oral cancers and Southern blot in the cell lines. We found frequent exonic HD of FAT in the cell lines (3/13, 23%) and in primary oral cancers (16/20, 80%). FAT expression was absent in these cell lines. Homozygous deletion hot spots were observed in exon 1 (9/20, 45%) and exon 4 (7/20, 35%). Moreover, loss of gene expression was identified in other types of squamous cell carcinoma. The methylation status of the FAT CpG island in squamous cell carcinomas correlated negatively with its expression. Our results identify mutations in FAT as an important factor in the development of oral cancer and indicate the importance of FATs function in some squamous cell carcinomas.
Oral cancer is the most common head and neck cancer, affecting more than 400 000 people worldwide every year, with most histologically designated as squamous cell carcinoma (IARC. GLOBOCAN 2002, 2004). Although easily detected and often cured in its early stage, the advanced stage has a poor prognosis. Most patients, after multidisciplinary treatment, suffer from severe cosmetic and/or functional difficulties. The development of oral cancer is thought to be because of an accumulation of multiple genetic changes. Preventive strategies aimed at inhibition of various stages of early carcinogenesis are becoming a major focus of investigations. To achieve this goal, a comprehensive understanding of the genetic changes associated with oral carcinogenesis is essential.
Concerning gain of fragment, amplification at 11q and 3q clearly participates in oral cancer carcinogenesis and concerns nearly 70% of cases. 3q amplification has been identified in early tumor development. Although there are some reports of overexpression and/or amplification of several proto-oncogenes, such as bcl-2, ras and c-myc (Haughey et al., 1992; Kiaris et al., 1995; Teni et al., 2002), little is known about the molecular changes associated with tumorigenic transformation.
Possible roles of several tumor suppressor genes have also been demonstrated in oral cancer. Genetic alterations of these genes, such as p53, were found in about 44% of primary head and neck squamous cell carcinomas (Siegelmann-Danieli et al., 2005). To date, frequent loss of heterozygosity or recurrent segmental DNA copy number changes have been identified in oral cancer. Loss of chromosome 3p and/or 9p has been found in more than 80% of tumors and loss of 17p involved in more than 50% of cases. These alterations are associated with early tumor development. Loss of 5q, 8p, 4q, 10q or 13q has been identified in nearly 30% of tumors, and loss of 11p in less than 20%. These alterations are preferentially associated with tumor progression. To date, in oral cancer including oral squamous cell carcinoma (OSCC), loss of heterozygosity (LOH) has been identified on chromosomes 3p, 9p, 13q, 15q and 17p (Beder et al., 2003). These findings suggest that some of these regions may contain a tumor suppressor gene. However, heterozygous deletions are usually of a substantial size, and their common region of overlap is large. In contrast, homozygous deletions (HDs) are a rare event, and are detected at a much lower frequency in human cancers. Because the homozygously deleted region is usually very small, mapping HD may be of considerable help for the fine location and identification of tumor suppressor genes. The identification of the RB1, WT1, BRCA2 and PTEN genes is illustrative of this (Dryja et al., 1984; Lewis et al., 1989; Wooster et al., 1995; Li et al., 1997).
Recently, the development of array-based methods for comparative genomic hybridization (CGH) has provided high-resolution detection of copy number changes, limited only by the size and spacing of the large insert clones included in the array (Pinkel et al., 1998; Hodgson et al., 2001; Simon et al., 2002; Veltman et al., 2002). The quantitative data collected should also allow the identification of single-copy changes in highly aneuploid tumor cells. In the present study, we identified a homozygous loss of the tumor suppressor gene FAT (4q35) by screening a panel of OSCC cell lines for copy-number aberrations using array-based CGH analysis. We observed loss of FAT expression in OSCC cell lines and homozygous exonic deletions in primary oral cancers.
Array-based CGH analysis of primary oral cancer
After achieving reproducible results in CGH-array analysis in some preliminary experiments in OSCC cell lines (data not shown), we prepared a set of cancer/normal genomic DNA collected by laser-capture microdissection (LCM) and whole genome amplification. We analysed one primary oral cancer T1 by CGH array, and the chromosome alteration profiles of 4044 loci are shown in Figure 1a. We paid attention to more remarkable patterns of chromosome abnormalities, such as HDs, which are likely to be landmarks of tumor suppressor genes. We found an HD of one locus including 4q35.2 (Figure 1b). Bacterial artificial chromosomes (BAC) clone: chr 4: 187895547–188007613 (112 kb) at the 4q35.2 locus contained the FAT gene, a new member of the human cadherin superfamily.
Homozygous deletion of FAT in oral cancer cell lines
To evaluate the integrity of the genomic FAT region within the common break region, exon-specific polymerase chain reaction (PCR) analysis was performed using 13 oral cancer cell lines and primer pairs spanning the FAT region from exon 1 to exon 27 (Figure 2). We detected an HD of exons 1–2 in two OSCC cell lines (Ca9–22, SAS), exon 3 in one OSCC cell line (Ca9–22), and exon 4 in three OSCC cell lines (Ca9–22, SAS and HO1-U1) (Figure 3, Table 3). The overall frequency of HD was 23% (3/13). HD hot spots were observed in exon 4 (3/13, 23%). No HD was found in exon 5–27 (data not shown). Concerning other squamous cell carcinoma cell lines, no HD was observed (Table 3). The HDs were further confirmed by Southern blot hybridization using a probe spanning FAT exon 2 (Figure 6). Ca9–22 and SAS were not detected in exon 2. HO1-U1 was weakly detected, suggesting LOH in exon 2.
Homozygous deletion of FAT in LCM-treated primary tumors
To confirm that homozygous loss of FAT was not an artifact that arose during establishment of the cell lines, we performed genomic PCR of FAT exons using 20 sets of normal and tumor cells from laser-capture microdissected areas from frozen tissue. We used primer pairs spanning the exon 1–4 region of FAT, and detected HDs in exon 1 (9/20, 45%), exon 2 (4/20, 20%), exon 3 (2/20, 10%) and exon 4 (7/20, 35%), at an overall frequency of 80% (16/20) (Figure 5, Table 3). HD hot spots were observed in exon 1 (45%) and exon 4 (35%). The HD frequency in primary oral cancers was much higher than that expected from cell line data. The age, sex, tumor location, and tumor-node-metastasis (TNM) stage showed no significant correlation with the HD frequency.
Loss of FAT expression in oral cancer cell lines
Next we determined expression levels of FAT by means of reverse transcriptase (RT)-PCR in all 13 oral cancer cell lines. HSC2 as well as two cell lines (Ca9–22, SAS) with HD showed no expression of FAT mRNA (Figures 4, 5 and 6, Table 3).
Methylation status of FAT CpG island in OSCC cell lines
Because hypermethylation around the CpG island is likely to be associated with silencing of FAT expression, we performed methylation-specific PCR (MSP). Each amplified DNA sample with nested primers was applied to an Applied Biosystems 3700 DNA analyzer (Applied Biosystems, Foster City, CA, USA) and the expected sequences confirmed. Three representative OSCC cell lines (HSC2, SAS, HO1-U1) with HD and without FAT expression were methylated, whereas one OSCC cell line (Ca9–22) was unmethylated. The methylation status of the FAT CpG island in the OSCC cell lines correlated negatively with its expression (Figure 8, Table 3).
Evaluation of FAT gene expression in other squamous cell carcinoma cell lines
To confirm genetic and epigenetic changes in the FAT gene, we performed genomic PCR, RT-PCR and MSP analysis around the CpG island in four other squamous cancer cell lines (from the esophagus, lung and cervix). HD was not found in any cell lines, but one cell line (TE4) showed no expression of FAT mRNA (1/4, 25%) (Figure 7). In addition, TE4 revealed aberrant hypermethylation in the FAT CpG island region (Figure 8, Table 3).
In this study, we carried out genome-wide screening to identify a putative tumor suppressor gene locus in oral cancer using an effective combination of LCM, whole genome amplification, and CGH array. Homozygous loss at 4q35.2, the location of FAT, had never been documented in oral cancer before, prompting us to examine whether a tumor suppressor gene involved in oral tumorigenesis might lie within this region. Consequently, we found a homozygous loss of the FAT gene (4q35), a putative tumor suppressor gene and new member of the human cadherin superfamily. The most frequent loss was exon 1 (45%) and the second most frequent was exon 4 (35%) in OSCC primary tumors. We found frequent exonic HDs of FAT in cell lines (3/13, 23%) and in primary oral cancers (9/20, 45%). FAT expression was absent in these cell lines. Moreover, exonic HD and loss of gene expression were identified in other types of squamous cell carcinomas. Our results identify mutations in FAT as an important factor in the development of oral cancer and indicate the importance of FAT's function in some squamous cell carcinomas.
The FAT gene (also called CDHF7, FAT1, ME5 and hFAT1) was originally discovered in Drosophila, where it is thought to function as a tumor suppressor (Watson et al., 1994). Its deletion causes hyperplastic overgrowth of larval imaginal discs, and defects in differentiation and morphogenesis (Bryant et al., 1988; Mahoney et al., 1991). Of particular interest is the fact that FAT interacts with components of the EGFR pathway in Drosophila (Garoia et al., 2000). Sequencing analysis of full-length cDNA revealed that the FAT gene encodes a huge protein (27 exons, 4590 amino acids, 506 kDa) with 34 cadherin-like domains, five EGF-like repeats interspersed with two laminin A-G chain motifs, a transmembrane domain and a novel cytoplasmic domain (Mahoney et al., 1991; Ponassi et al., 1999). FAT shows similarity to a single-pass type I membrane protein, and is expressed at high levels in a number of fetal epithelia (Tanoue and Takeichi, 2005). It probably functions as an adhesion molecule and/or signaling receptor, and is likely to be important in developmental processes and cell communication. Transcript variants derived from alternative splicing and/or alternative promoter usage exist, but they have not been fully described. Furthermore, its mechanisms of action have not been elucidated.
Among the hallmarks of cancer are defective cell–cell and cell–matrix adhesion. Cadherins have been reported to be associated with human cancer. For example, a reduction or loss of E-cadherin expression is often found in gastric cancers. Mutations of the E-cadherin gene occur in approximately 50% of diffuse-type gastric cancers (Keller et al., 1999). Furthermore, a loss or reduction of E-cadherin expression has also been shown to result from hypermethylation of the E-cadherin promoter in gastric cancers (Tamura et al., 2000). From this point of view, we confirmed similar aspects of FAT in oral cancer. Reduction of cell–cell and cell–matrix adhesion is early tumorigenesis events implicated in the invasive and metastatic process. The fact that abnormal adhesive marker expression is a feature commonly shared by most human cancers makes the cadherins potential candidates for anticancer targeted therapy.
Our data suggest that the human protocadherin FAT gene acts as a tumor suppressor in oral cancer and might play a role in other types of squamous cell carcinomas. This should be examined further by functional analysis of this gene mutation. In addition, extensive investigations using different populations and a large sample size are required to clarify the role of these homozygous deletions. Because of the huge size of mRNA, the full-length cDNA of FAT is not available. However, we are now preparing the full-length cDNA and planning a rescue experiment in homozygously deleted OSCC cell lines to understand the tumorigenesis and progression.
Materials and methods
Cell lines and primary tumors
A total of 13 OSCC cell lines (HSC2, HSC3, HSC4, OSC4, OSC5, OSC6, SCC9, SCC25, Ca9–22, SAS, KN, KB and HO1U1) and four other SCC cell lines (TE4, TE8, EBC1 and ME180) were used. TE4 is highly differentiated squamous cell carcinoma of esophagus and TE8 is moderately differentiated squamous cell carcinoma of esophagus. EBC1 is squamous cell carcinoma of lung. ME180 is highly invasive squamous cell carcinoma of the cervix. HSC3, HSC4, Ca9–22, SAS, KB, HO1U1, TE4, TE8, EBC1 and ME180 were obtained from the Cell Resource Center for Biomedical Research (Institute of Development, Aging and Cancer, Tohoku University) and the others were isolated from a series of oral tumors, the majority of which have been described elsewhere (Prime et al., 1990; Ishwad et al., 1999; Loughran et al., 1997). All cell lines were cultured in Dulbecco’s modified Eagles’ medium, containing 1% penicillin/streptomycin and 10% fetal bovine serum. Cell lines were incubated at 37°C in an atmosphere of 5% CO2.
Primary tumor samples were obtained from 20 patients who were undergoing surgery at Ehime University Hospital, with prior written consent from each patient and after approval by the local ethics committee. The resected tumor tissues were snap-frozen at −80°C until use. The clinicopathological features of the 20 cases are shown in Table 1.
DNA and RNA extraction from cell lines and reverse transcription
Genomic DNA was extracted by the phenol–chloroform extraction method from all cultured cells. Total RNA was extracted from all cell lines using an ISOGENE kit (Nippon Gene, Toyama, Japan). A total of 2 μg of mRNA was reverse-transcribed using a QuantiTect Reverse Transcription kit (Qiagen, Tokyo, Japan) according to the manufacturer’s protocol. Then, 1 μl cDNA was used for FAT RT-PCR amplification in a total volume of 25 μl containing 10 pmol of each forward and reverse primer, 200 μ M of dNTP (Takara, Ohtsu, Japan), 1 × PCR buffer and 0.5 U Taq DNA polymerase (Takara). Amplification was performed in a thermal cycler (Takara Dice) for 35 cycles of 94°C for 15 s, 52°C for 30 s and 72°C for 30 s. The PCR product was separated on 2% agarose gel and visualized by ethidium bromide staining. Primers designed from FAT exons 2–4 are shown in Table 2. Other primer sequences are available upon request. β-Actin primers were used as an external control.
LCM and DNA extraction of primary tumors
Frozen tissues were prepared, and following the staining of tissue sections with Mayer’s hematoxilin and eosin Y solutions, 1000 cancer cells and 1000 normal cells were separately microdissected and pressure catapulted using the PALM Microbeam microscope system (PALM Microlaser Technologies, Bernried, Germany). One set of slides were examined under a microscope by two pathologists to provide a detailed confirmation of the identification of cancer and normal cells. All normal cells adjacent to cancers were microdissected from regions at least 2 mm away from cancer margins. Genomic DNA was extracted using a protocol described elsewhere (Rook et al., 2004).
Whole genome amplification and purification
A 1 μl dilution from all microdissected samples was used as the starting material for amplification. Whole genome amplification was carried out according to the GenomiPhi kit manufacturer’s instructions (Amersham Biosciences), using an incubation time of 16 h. GenomiPhi reactions were checked on 0.6% agarose gel. Amplification was considered successful when a smear of DNA fragments, ranging from 1 to 20 kb, was visible.
Construction of BAC library
The array used in this study consists of 4044 human BACs, which were spaced approximately 800 kb on average across the entire genome (MacArray Karyo 4000 from Macrogen Inc., Seoul, Korea). BAC clones were selected from the proprietary BAC library of Macrogen, Inc. Briefly, the pECBAC1 vector (Frijters et al., 1997) was digested with HindIII, and size-selected HindIII-digested pooled male DNAs were used to generate a BAC library. These vectors were then transformed into and grown in Escherichia coli DH10B strain.
Construction of BAC-mediated array CGH microarray
Clones were first selected to yield an average genomic coverage of 800 kb resolution. All clones were two-end sequenced using an ABI PRISM 3700 DNA Analyzer (Applied Biosystems, Foster City, CA, USA), and their sequences were blasted and mapped according to their positions described in the University of California, Santa Cruz (UCSC) human genome database (http://www.genome.ucsc.edu). Confirmation of locus specificity of the chosen clones was performed by removing multiple loci-binding clones by individual examination under standard fluorescence in situ hybridization (FISH) as described previously (Pinkel et al., 1986). These clones were prepared by the conventional alkaline lysis method to obtain BAC DNA. The DNA was sonicated to generate ∼3-kb fragments before mixing with 50% dimethyl sulfoxide (DMSO) spotting buffer. The arrays were manufactured by an OmniGrid arrayer (GeneMachine, San Carlos, CA, USA) using a 24-pin format. Each BAC clone was represented on an array as triplicate spots, and each array was prescanned using a GenePix 4200A scanner (Axon Instruments Inc., Foster City, CA, USA) for proper spot morphology.
DNA labeling for array CGH
The labeling and hybridization protocols described by Pinkel et al. (1998) were used with some modification of the labeling procedure. Briefly, 21 μl solution containing 500 ng normal DNA (reference DNA) or tumor DNA (test DNA), 20 μl BioPrime DNA Labeling System random primers solution (Invitrogen, Carlsbad, CA, USA), and water were combined and incubated for 5 min at 95°C, and subsequently cooled on ice. After the addition of 5 μl 10 × dNTPs labeling mix (1 mM dCTP, 2 mM dATP, 2 mM dGTP and 2 mM dTTP), 3 μl 1 mM Cy-3 or Cy-5 dCTP (GeneChem Inc., Daejeon, Korea), and 40 U BioPrime DNA Labeling System Klenow fragment (Invitrogen), the mixture was gently mixed and incubated overnight at 37°C. The addition of 5 μl BioPrime DNA Labeling System Stop Buffer (Invitrogen) ended the reaction. After labeling, unincorporated fluorescent nucleotides were removed using QIAquick Spin columns (Qiagen, Germany). In one tube, the Cy3-labeled sample and Cy5-labeled reference DNAs were mixed together, and 50 μg human Cot I DNA (Invitrogen), 20 μl 3 M sodium acetate and 600 μl cold 100% ethanol were added for DNA precipitation.
Array hybridization, imaging and data preprocessing
The pellet was resuspended in 40 μl hybridization solution containing 50% formamide, 10% dextran sulfate, 2 × SSC, 4% SDS and 200 μg yeast tRNA. The hybridization solution was denatured for 10 min at 72°C and was subsequently incubated for 1 h at 37°C to allow blocking of repetitive sequences. Hybridization was performed in slide chambers for 48 h at 37°C. After post-hybridization washes, arrays were rinsed, spin-dried and scanned into two 16-bit TIFF image files using a GenePix 4200 A two-color fluorescence scanner (Axon Instruments Inc., Foster City, CA, USA), and individual spots were analysed with GenePix Pro 3.0 imaging software (Axon Instruments). Clones on the X and Y chromosomes were not used for further analysis because their intensity may distort the entire data set. The log2-transformed fluorescence ratios were calculated from background-subtracted median intensity values, and these ratios were used to perform normalization according to intensity normalization methods. Regions of gain were defined as log2 ratio>+0.25, and those suggested to contain loss as log2 ratio<−0.25.
Genomic PCR analysis of FAT gene
The extent of homozygous deletions within the common break region of the FAT gene was studied by PCR. Exon-specific PCR amplification was carried out using genomic DNA samples prepared from 13 oral cancer cell lines and 20 primary tumors and primer pairs spanning the FAT region from exon 1 to exon 27. Primer sequences were obtained from the genomic database (http://www.gdb.org) and are available upon request.
One microliter of genomic DNA was used for the first PCR amplification, in a total volume of 25 μl containing 10 pmol of each forward and reverse primer, 200 μ M of dNTP (Takara Inc.), 1 × PCR buffer and 0.5 U Taq DNA polymerase (Takara Inc.). Amplification was performed in a thermal cycler (Takara Dice) for 35 cycles of 94°C for 15 s, 60°C for 30 s and 72°C for 30 s. The PCR product was separated on 2% agarose gel and visualized by ethidium bromide staining. Only completely negative signals were determined as homozygous deletions. Blood lymphocyte DNA (from a healthy individual) with known FAT integrity was used as a positive control. Another PCR assay for β-globin DNA detection was performed with each processed specimen to control for DNA integrity and for the presence of an adequate quantity of DNA. Positive amplification signals were seen in all tested samples, thus confirming the quality of the DNA samples.
Southern blot analysis
High-molecular weight DNA from OSCC cell lines was isolated by phenol–chloroform treatment (Sambrook et al., 1989). Three micrograms of DNA was digested with the restriction enzyme, AvaII or AlwI, run on 0.7% agarose gel and blotted on a nylon membrane. The filter was probed with a 32P-radiolabelled fragment of exon 2 (made by long PCR amplification of FAT exon 2 with LA Taq polymerase (Takara Inc.). Southern blotting and hybridization were performed according to standard procedures.
Bisulfite modification of genomic DNA and methylation-specific PCR analysis of FAT gene promoter region
Methylation-specific PCR (MSP) was applied to investigate the methylation status of the promoter regions of the FAT gene. Genomic DNA were treated with sodium bisulfite using a MethylEasy DNA Bisulphite Modification Kit (Human Genetic Signatures, NSW, Australia), and PCR was performed to distinguish methylated from unmethylated DNA as described by Herman et al. (1996). We used the following PCR primers: (1) for unmethylated FAT sequence, 5′-IndexTermTTGTTTTTTTGTTTTTAGGAAATGG-3′ (sense) and 5′-IndexTermCATCCAAAAACATATTTATCCCAAC-3′ (antisense); (2) for methylated FAT sequence, 5′-IndexTermTTCGTTTTTTCGTTTTTAGGAAAC-3′ (sense) and 5′-IndexTermCCAAAAACGTATTTATCCCGAC-3′ (antisense). Primers for MSP analysis were designed using Methprimer software (http://www.urogene.org/methprimer/index1.html). PCR products were visualized on 3% agarose gels stained with ethidium bromide and sequenced (Table 3).
oral squamous cell carcinoma
loss of heterozygosity
fluorescence in situ hybridization
comparative genomic hybridization
bacterial artificial chromosome
reverse transcriptase-polymerase chain reaction
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We thank the patients and their families for helping and participating in this work. We are grateful to Dr Norio Takahashi for performing preliminary experiments and helpful discussion about CGH array. This study was supported in part by grant-in-aids from the Ministry of Education, Culture, Sports, Science and Technology, Japan and from the Ministry of Health, Labor and Welfare of Japan.
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Cite this article
Nakaya, K., Yamagata, H., Arita, N. et al. Identification of homozygous deletions of tumor suppressor gene FAT in oral cancer using CGH-array. Oncogene 26, 5300–5308 (2007). https://doi.org/10.1038/sj.onc.1210330
- oral cancer
- tumor suppressor gene
- homozygous deletion
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