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Clinical Cancer Research Vol. 8, 828-835, March 2002
© 2002 American Association for Cancer Research

Molecular Oncology, Markers, Clinical Correlates

High Prevalence of Decreased Expression of KAI1 Metastasis Suppressor in Human Oral Carcinogenesis1

Katsuhiro Uzawa2, Kanae Ono2, Hiroyoshi Suzuki, Chihaya Tanaka, Takashi Yakushiji, Nobuharu Yamamoto, Hidetaka Yokoe and Hideki Tanzawa3

Departments of Clinical Molecular Biology [K. U., K. O., H. Y., H. T.] and Urology [H. S.], Graduate School of Medicine, Chiba University, Chiba 260-8670, Japan, and First Department of Oral and Maxillo-Facial Surgery, Tokyo Dental College, Chiba 261-8502, Japan [C. T., T. Y., N. Y.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Purpose: KAI1 was originally identified in prostate cancer as a metastasis suppressor gene. Recent studies have shown a frequent down-regulation of KAI1 expression in many tumor types, whereas mutation or hypermethylation of the gene is infrequent. The aim of the present study was to examine whether loss of KAI1 expression that might be caused by genetic or epigenetic alterations could contribute to oral carcinogenesis.

Experimental Design: We analyzed mutational and methylation status of the KAI1 gene and both the mRNA and protein level in a series of oral tumors [28 precancerous lesions, 101 primary oral squamous cell carcinomas (OSCCs), and 30 metastatic OSCCs] and OSCC-derived cell lines. We also examined p53 protein expression, which has been reported to be a candidate activator for the KAI1 gene.

Results: With the exception of three microsatellite instabilities in the KAI1 gene, we found no mutations in the coding sequence of the KAI1 gene, no loss of heterozygosity, and no hypermethylation of the KAI1 promoter region in all samples investigated. By immunohistochemistry, however, high frequencies of KAI1 down-regulation were evident not only in the metastatic OSCCs [29 of 30 (97%)] but also in the primary OSCCs [83 of 101 (82%)] and in the precancerous lesions [13 of 28 (46%)]. There was a significant relationship between down-regulation of KAI1 protein expression and primary tumors associated with lymph node metastases (P = 0.0115), whereas there was no statistical correlation between p53 status and KAI1 expression. Taken together, reverse transcription-PCR data were consistent with the protein expression status in 16 patients from whom mRNA was available.

Conclusions: Our data suggest that whereas loss of KAI1 protein expression is associated with primary tumors with lymph node metastases, the down-regulation of KAI1 is an early event in the progression of human oral cancer. The down-regulation of KAI1 is not associated with either mutation, allelic loss, methylation of the promoter, or p53 regulation.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
OSCC,4 which contributes to >90% of all malignant tumors of the oral cavity, is one of the most common human malignancies worldwide (1 , 2) . Most patients with OSCC who have either suspected or proven metastases in regional lymph nodes are candidates for composite resection in which the lesion, surrounding tissues, and lymph nodes of the neck are all removed. This procedure often produces grievous deformities and defects of the jaw and extensive loss of soft tissue, which makes functional and esthetic rehabilitation a long, involved process. Despite its clinical importance, there is no relative tool to identify the progressive metastatic state in oral cancer cells. The carcinogenesis of human OSCC is thought to be a multistep phenomenon in which a variety of genetic alterations can be segregated into early to late stages (3) . During this process, loss of metastasis suppressor gene(s) function should be a crucial step in the progression of cancer cells from a nonmetastatic to a metastatic phenotype. However, the molecular mechanisms of the progression of OSCC to the metastatic state are still unknown.

The KAI1 gene was originally identified as a putative metastasis suppressor gene for prostate cancer (4) , and its protein is a member of the TM4SF (5 , 6) . Additional studies have shown that decreased KAI1 expression could be a useful marker for metastatic/invasive potential in a series of human tumor types, including cancers of the prostate (7 , 8) , bladder (9) , breast (10) , colon (11 , 12) , and pancreas (13 , 14) . In addition, other studies have indicated that the KAI1 mRNA expression level is associated with prognosis in patients with lung (15, 16, 17) , breast (18) , and pancreatic cancers (19) . These observations indicate that down-regulation of KAI1 expression may be an important step in the progression of many types of human malignancies. With regard to SCC, a higher incidence of decreased expression of KAI1 protein was recently reported in esophageal SCCs (20 , 21) . In contrast, a steady level of KAI1 mRNA expression has been found to be similar in both primary and metastatic esophageal SCCs (22) . Thus, whether loss of KAI1 function could contribute to the pathogenesis of human SCCs including OSCC is inconclusive.

To date, considerable evidence has shown that no mutations, no LOH, and no hypermethylation seem to be involved in the molecular basis for down-regulation of KAI1 expression. On the other hand, a recent study showed that the KAI1 gene was directly activated by p53, indicating that down-regulation of the KAI1 gene in tumor cells could be linked to loss of p53 function (23) . At present, no information is available regarding the association between down-regulation of KAI1 and genetic/epigenetic alterations and the state of p53 expression in human oral carcinogenesis.

The purpose of this study was to examine the presence of gene mutation and/or hypermethylation and the state of KAI1 expression during the carcinogenesis of human OSCC. In addition, p53 tumor suppressor protein expression was evaluated and compared with KAI1 expression.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Tissue Samples.
Pairs (101) of tumor and corresponding normal oral mucosa specimens and 30 metastatic lesions were obtained from 101 unrelated Japanese patients with OSCC at the time of surgical resection at the Department of Oral Surgery, Chiba University Hospital, between 1992 and 1999. Informed consent was obtained from all patients and the patients’ families. In addition, 28 precancerous tissues that were pathologically diagnosed as leukoplakia were obtained in the same manner as mentioned above. The resected tissues were divided into two parts, one of which was frozen immediately after careful removal of the surrounding normal tissues and stored at -80°C until use; the other was fixed in 10% buffered formaldehyde solution for pathological diagnosis and immunohistochemical staining. Histopathological diagnosis for each cancerous tissue was performed according to the International Histological Classification of Tumours (24) by the Department of Pathology, Chiba University Hospital. Clinicopathological staging was determined by the TNM (tumor-node-metastasis) classification of the Union Internationale Contre Cancer (25) . All patients had SCC that was histologically confirmed, and the tumor samples were checked to ensure that tumor tissue was present (>80% of specimens).

Immunohistochemistry.
Paraffin-embedded tissue sections (4 µm) from 101 paired primary OSCCs, adjacent normal oral tissue, 30 lymph node metastases, and 28 precancerous lesions (leukoplakias) were used for immunohistochemical detection of KAI1 and p53. After deparaffinization and hydration, the slides were heated in a microwave for 15 min in 0.01 M citrate buffer (pH 6) and rinsed three times in PBS solution. After quenching the endogenous peroxidase activity in 0.3% H2O2 for 30 min, the sections were blocked for 2 h at room temperature with 5% BSA before reacting with mouse antihuman KAI1 monoclonal antibody C33 (kindly provided by Dr. Osamu Yoshie; Shionogi Institute for Medical Science, Osaka, Japan) at a dilution of 1:50 or with antihuman p53 monoclonal antibody BP53-12 (Wako, Osaka, Japan) at a dilution of 1:100. The sections then were incubated with each primary antibody for 1 h at room temperature in a moist chamber. With regard to KAI1, upon incubation with the primary antibody, sections were washed three times in PBS buffer and treated with ENVISION reagent (DAKO JAPAN Inc., Kyoto, Japan) followed by color development in 3,3'-diaminobenzidine tetrahydrochloride (DAKO). As for p53, the incubated sections were treated with biotinylated rabbit antimouse immunoglobulin (DAKO) diluted to a ratio of 1:500 in PBS for 30 min and then incubated with biotin-streptavidin-peroxidase complex (DAKO) for 30 min, followed by color development in 3,3'-diaminobenzidine tetrahydrochloride and hydrogen peroxide mixture (DAKO). The slides were then lightly counterstained with hematoxylin, dehydrated in graded ethanol, cleaned in xylene, and mounted. To quantitate the state of KAI1 protein expression, a scoring method was applied (12) . A mean percentage of positive tumor cells was determined on at least 10 fields at x400 magnification in each section. The intensity of KAI1 immunoreaction was scored as follows: (a) weak, 1+; (b) moderate, 2+; and (c) intense, 3+. The percentage of positive cells and the intensity of the stain were multiplied to produce a KAI1 immunostaining score for each case. Cases with a KAI1 score of <30 were defined as negative. p53 protein expression was evaluated in the same subjects analyzed for KAI1 protein expression according to the scoring system used in KAI1 immunohistochemistry. These judgments were made by two independent pathologists, neither of whom possessed any knowledge or information pertaining to the patients’ clinical status. Statistical significance was evaluated by {chi}2 analysis or Fisher’s exact test.

PCR-SSCP Analysis for the KAI1 Gene Mutation.
To screen the sequence variations of the KAI1 gene, PCR-SSCP analysis was performed as described previously (26) for all OSCC cases examined in the immunohistochemical analysis. Genomic DNA was isolated as described previously (27) . Ten sets of oligonucleotide primers were used to amplify exons 1–10 of the KAI1 gene. The sequence of each primer and each PCR condition were based on those reported previously (8) . After the amplification, the PCR products were electrophoresed under several different conditions (at 4°C, 15°C, and room temperature).

Methylation Assay of the KAI1 Gene Promoter Region.
To determine whether hypermethylation of a CpG island within the KAI1 promoter is responsible for the loss of KAI1 protein expression, 25 DNA samples with negative KAI1 immunoreaction (20 primary and 5 metastatic tumors) were randomly selected for COBRA (28) . The bisulfite modification and DNA purification were carried out on 1 µg of EcoRI (New England Biolabs, Inc., Beverly, MA)-digested DNA using the CpGenome DNA Modification kit (Intergen Discovery Products, Purchase, NY). The modified DNA was amplified with a sense primer (5'-AGGGTAGGGTAGGATTAGGAA-3'; KAI1-F; -494 to -474) and a reverse primer (5'-CTCCTTTTCACCCACCAACTACT-3'; KAI1-RS; +206 to +228) for the first round, and nested primers KAI1–2F [5'-AAGGTTGGTTGGGGTAYGGTTAT-3' (Y = C or T); -179 to -156] and KAI1–2RS [5'-AAAACXAAAACTAAAACTAACTTTACC-3' (X = A or G); +126 to +152] were used to amplify KAI1. The location of each primer was based on the published KAI1 promoter sequence (29) . The PCR reactions were performed in a final volume of 100 µl that contains 200 ng of DNA, 2.5 pmol of each specific primer flanking the CpG island, 50 µM deoxynucleotide triphosphates, 10 mM Tris-HCl buffer (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, and 0.5 unit of Taq polymerase (Perkin-Elmer Corp., Branchburg, NJ). After ethanol precipitation, the PCR products were digested with methylation-sensitive enzymes (BstUI and TaqI) and with HpaII (methylase-sensitive enzyme; New England Biolabs) separated on a 3% agarose gel and visualized by ethidium bromide. Peripheral blood DNA treated in vitro with SssI methylase (New England Biolabs) was used as a positive control for methylated alleles. DNA obtained from leukocytes was used as a negative control for methylated genes.

Microsatellite Analysis at the KAI1 Locus.
To test whether allelic loss on the KAI1 locus could contribute to the pathogenesis of OSCC, we used PCR-based LOH analysis with microsatellite markers (D11S1326, D11S1344, D11S1355, D11S1356, and D11S903) mapped around the KAI1 locus. LOH for tumor DNA samples examined in the PCR-SSCP analysis was assessed by scanning densitometry and analyzed using NIH software (Image version 1.62; Dr. W. Rasband, NIH, Bethesda, MD).

RT-PCR Assay of the KAI1 Gene in OSCCs.
Total RNA was isolated from randomly selected primary OSCC specimens (12 KAI1-positive and 4 KAI1-negative cases) and from paired specimens of noncancerous oral tissue using an SV Total RNA Isolation System (Promega, Madison, WI) according to the manufacturer’s protocol. To create first-strand cDNA, 1.5 µg of total RNA were used for the RT reaction. The reaction was performed using a Ready-To-Go T-Primed First-Strand Kit (Amersham Pharmacia Biotech, Uppsala, Sweden). To obtain reproducible quantitative performance of the RT-PCR assay, we titrated the amount of starting cDNA and the number of amplification cycles. All subsequent assays were carried out using parameters that yielded amplification of both the KAI1 and GAPDH genes within a linear range. cDNA was amplified by PCR using primers specific for cDNA of the KAI1 gene (5'-AGTCCTCCCTGCTGCTGTGTG-3', sense; 5'-TCAGTCAGGGTGGGCAAGAGG-3', antisense). The GAPDH gene (5'-CATCTCTGCCCCCTCTGCTGA-3', sense; 5'-GGATGACCTTGCCACAGCCT-3', antisense) was also amplified as an interior control, resulting in a 305-bp product. cDNA preparations were done in the presence and absence of reverse transcriptase, the latter of which acted as a control for contaminating genomic DNA from which fragments of the pseudogene can be amplified with these primers. PCR reactions were performed in a 9700 Perkin-Elmer Thermal Cycler at 94°C for 1 min, with 29 cycles of 94°C for 40 s, 60°C for 90 s, and 72°C for 90 s, followed by an extension step at 72°C for 5 min. After amplification, an aliquot of the PCR product was separated on a 1.5% agarose gel and stained with ethidium bromide. The density of the ethidium bromide-stained bands was analyzed using the NIH Image software. The results were normalized as a ratio of each specific mRNA signal to the GAPDH gene signal within the same RNA sample. cDNA obtained from normal oral epithelium was used as a positive control. Reproducibility was confirmed by processing all samples at least twice.

Evaluation of KAI1 mRNA Expression in OSCC-derived Cell Lines by 5-AzaC Treatment.
The following OSCC-derived cell lines were analyzed: SAS, HSC-2, HSC-3, HSC-4, Ca9-22, Ho-1-u-1, Ho-1-N-1 (Human Science Research Resources Bank, Osaka, Japan), and OK-92 (established in our department from a carcinoma of the tongue). All OSCC-derived cell lines were grown in RPMI 1640 with 10% fetal bovine serum and 50 units/ml penicillin and streptomycin. To assess restoration of KAI1 expression, the cells were treated with different concentrations (0 and 2 µM) of the DNA methyltransferase inhibitor (5-AzaC) as described previously (30) . On day 5, the cells were washed with PBS and grown for another 10 days without the 5-AzaC. Total RNA isolation and RT-PCR analysis was performed as described above. To confirm whether the demethylating reagent was active, p16 mRNA expression in Ca9-22 cells was examined because this gene has been reported to be down-regulated due to the hypermethylation of the gene (31) .


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Immunohistochemistry.
A total of 101 OSCC patients were identified for whom there was adequate histological material available for immunohistochemical analyses of KAI1 and p53 protein expression. The correlation between the clinicopathological characteristics of patients with OSCC and KAI1 expression is summarized in Table 1Citation . Using the ENVISION reagent (DAKO), a strong KAI1 immunoreaction was successfully detected in the cellular membrane of the normal oral epithelial cells on paraffin-embedded normal oral tissues (Fig. 1A)Citation . To confirm the reactivity of the antibody (C33) in paraffin-embedded sections, we compared the detection of KAI1 antigen in frozen versus paraffin-embedded tissues and found that KAI1 is also expressed at high levels in the normal cellular membrane and down-regulated in the tumor cellular membrane in 10 randomly selected frozen sections (data not shown). The KAI1 immunostaining scores for leukoplakias, primary OSCCs, and metastatic OSCCs range from 0–240, 0–300, and 0–86, respectively. Among the primary tumors examined, 83 cases (82%) showed significant down-regulation of KAI1 protein (Table 1Citation ; Fig. 1, B–DCitation ). Twenty-nine of 30 (97%) metastatic OSCCs within the lymph nodes showed absent or reduced KAI1 immunostaining (Table 1)Citation . Moreover, almost all of the primary OSCCs with regional lymph node metastasis (97%) showed down-regulation of KAI1 protein expression, and a statistically significant difference was observed between the KAI1-reduced primary OSCCs with and without lymph node metastasis (P = 0.0115; Table 1Citation ). On the other hand, there was no statistically significant difference between KAI1 expression and other clinicopathological features (Table 1)Citation . Twenty-eight oral precancerous lesions histologically diagnosed as leukoplakia were also examined for the state of KAI1 protein expression using the same immunohistochemical method. Thirteen (46%) of the leukoplakias (n = 28) were defined as KAI1 negative. Representative results are shown in Fig. 1, E and FCitation .


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  		Table 1 Correlation between the expression of KAI1 and clinical classification in primary OSCCs

 


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  		Fig. 1. Immunohistochemical staining of KAI1 in normal and cancerous oral tissues. A, normal oral tissue exhibited strong KAI1 protein expression that was limited to the cell membrane. Original magnification, x200. B, moderately differentiated primary OSCC. Note that moderate cytoplasmic background staining but almost no specific membrane staining is observed. Original magnification, x100. C, dysplastic oral epithelium adjacent to the tumor showing KAI1-positive staining. Note that invasive carcinoma cells display no membrane staining. Original magnification, x400. D, negative staining of tumor cells of the poorly differentiated primary OSCC. Original magnification, x400. E, KAI1-positive case of leukoplakia. Note that a strong positive immunoreaction for KAI1 was detected on the epithelial cell membrane, especially around the basal cell layers. Original magnification, x100. F, KAI1-negative case of leukoplakia. The border between normal epithelium (left side) and the dysplastic lesion (right side) is indicated. Note that whereas normal epithelial cellular membranes were stained as KAI1 positive, reduced KAI1 protein expression was evident in the lesion. Original magnification, x100. G and H, a metastatic OSCC case showing lack of KAI1 immunostaining with overexpression of p53 protein. Original magnification, x400.

 
p53 protein expression was assessed to determine whether there is a relative correlation between KAI1 expression and p53 expression. Of 101 OSCC cases examined for p53, 31 (31%) had positive staining in the nuclei of the tumor cells (Table 2)Citation . There was no correlation between nuclear p53 staining and any clinical feature except lymph node involvement (P < 0.0001). Nineteen primary OSCCs [19 of 101 (19%)] showed positive p53 immunoreaction with down-regulation of KAI1 protein expression, but no significant correlation between KAI1 and p53 was identified (Tables 3Citation and 4Citation ). A typical result showing KAI1 down-regulation and p53 up-regulation in the same section of metastatic OSCC is indicated in Fig. 1, G and HCitation .


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  		Table 2 Correlation between the expression of p53 and clinical classification in primary OSCCs

 

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  		Table 3 KAI1 and p53 protein expressions in metastatic OSCCs

 

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  		Table 4 Relationship between protein expression of KAI1 and p53 in 101 primary OSCCs

 
Mutation Analyses of the KAI1 Gene.
PCR-SSCP analysis to screen for KAI1 gene mutation was done for all subjects examined in immunohistochemical analysis. However, no mobility shift in SSCP patterns was detected in either KAI1-negative or KAI1-overexpressing cases. Representative results of SSCP analysis for exon 4 of the KAI1 gene are shown in Fig. 2ACitation . To evaluate the state of allelic imbalance, which includes LOH and MSI, at the KAI1 locus, PCR-based microsatellite analysis was performed using five microsatellite markers at the region of the KAI1 locus. No LOH was found in any of these tumors, whereas three primary tumors and one metastasis showed MSI (Fig. 2B)Citation . In addition, these four tumors exhibited reduced or no KAI1 protein.



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  		Fig. 2. A, PCR-SSCP analysis of exon 4 of the KAI1 gene in DNAs from OSCC patients. No mobility shift band was detected compared with each normal control. N, normal tissues; T, primary OSCCs. B, MSI at the microsatellite markers (D11S1355 and D11S903) flanking the KAI1 locus in oral tumors. N, normal tissues; T, primary OSCCs; M, metastatic OSCC. C, COBRA analysis of the KAI1 promoter. In the left panel, positive DNA control treated with SssI methylase shows digested bands resulting in cleavage by methylation-specific restriction enzymes (TaqI and BstUI). On the other hand, control DNA treated with HpaII (methylase-sensitive enzyme) showed no digested band, indicating the complete bisulfite reaction in the experiment. Bisulfite-treated DNA obtained from a stage IV tumor sample showing no KAI1 mRNA expression reveals only an undigested band in each lane (right panel).

 
Methylation Analysis of the KAI1 Gene.
To determine whether methylation in the promoter region was heterogenous, 25 tumors (20 primary and 5 metastatic OSCCs) with absent or reduced KAI1 protein expression were subjected to the COBRA procedure. The bisulfite-modified DNAs were amplified in a 331-bp fragment in the most GC-rich area within the KAI1 5' regulatory region and first exon. The PCR fragment was digested with BstUI, TaqI, and HpaII. No digested fragment was detected in any of the samples examined, whereas control DNA treated with SssI methylase revealed BstUI- or TaqI-digested bands (Fig. 2C)Citation . Typical results of a stage IV OSCC are also shown in Fig. 2CCitation .

KAI1 Gene Expression in OSCC Tissues Analyzed by RT-PCR.
All cases showing down-regulation of KAI1 protein expression (n = 12) by immunohistochemistry revealed significantly reduced mRNA expression, whereas a steady-state level of gene expression was observed in KAI1-positive cases (n = 4). The KAI1 mRNA expression level, normalized to each GAPDH mRNA expression, ranged from 0 to 1.0. Typical examples of the RT-PCR analysis are shown in Fig. 3ACitation .



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  		Fig. 3. RT-PCR analysis of freshly resected tissue samples [A (T, tumors; N, corresponding normal tissues; M, molecular marker)] and OSCC-derived cell lines [B (Lane 1, SAS; Lane 2, HSC-2; Lane 3, HSC-3; Lane 4, HSC-4; Lane 5, Ca9-22; Lane 6, OK-92; Lane 7, Ho-1-u-1; Lane 8, Ho-1-N-1; N, normal oral tissue; M, molecular marker)]. Representative results for the tumor and corresponding normal tissue are shown in A. Absent or significantly reduced KAI1 expressions in mRNA from tumor tissues are evident in three OSCC patients when compared with each corresponding normal tissue. In B, down-regulation of the KAI1 gene is seen in the cell lines, as compared with KAI1 mRNA expression in normal oral tissue. On the other hand, no restoration of KAI1 mRNA expression is detected in any OSCC-derived cell lines showing KAI1 down-regulation after treatment with 5-AzaC (B). p16 mRNA expression in the Ca9-22 cells was used as a positive control in the demethylating study (C). Each panel indicates quantitation of the KAI1 gene RT-PCR products normalized to the level of GAPDH mRNA.

 
Demethylating Study of OSCC-derived Cell Lines.
To analyze mRNA expression of the KAI1 gene in cell lines derived from human OSCC, we performed a KAI1-specific RT-PCR. Three (SAS, Ca9-22, and Ho-1-u-1) of eight (38%) cell lines showed either significantly reduced KAI1 mRNA expression or no KAI1 mRNA expression at all (Fig. 3B)Citation . For these three cell lines, the COBRA assay was done to determine whether each KAI1 promoter might be methylated, and no evidence of methylation was obtained. It is possible that methylation elsewhere in the promoter region might inhibit KAI1 mRNA expression. To address this issue, the cell lines were exposed to demethylating agent 5-AzaC, which could reactivate mRNA expression suppressed by gene methylation. However, no reactivation of KAI1 mRNA expression was found in these cell lines (Fig. 3B)Citation . p16 mRNA expression was restored by 5-AzaC treatment in the Ca9-22 cells (Fig. 3C)Citation , indicating that this chemical agent was active in the demethylating experiment.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
KAI1, a novel member of the TM4SF, is a metastasis suppressor gene located at chromosome 11p11.2 and encodes a transmembrane protein of 267 amino acids (4) . KAI1 has been shown to suppress metastasis in a Dunning rat model of prostatic adenocarcinoma (4) . Furthermore, the down-regulation of KAI1 is associated with tumor progression and/or metastasis in multiple human tumors of the lung (16) , breast (18) , pancreas (19) , bladder (9) , colon (11 , 12) , and esophagus (20 , 21) . Recent studies have reported that this gene inhibits cellular invasion and mortality in colon cancer cell lines in vitro (32) and that overexpression of KAI1 in breast cancer cells results in suppressed experimental invasion in vitro and metastasis in vivo (33) . Therefore, KAI1 will link to the cell surface molecules, such as integrins, E-cadherin, and other TM4SF members, and loss of KAI1 function may play a significant role in the progression of a wide variety of malignant tumors.

Structural abnormalities, including gene mutation, LOH, and MSI, are important factors in the loss of gene expression. In the current study, however, we found that structural abnormalities of the KAI1 gene do not occur in either primary or metastatic OSCCs. Corresponding to our results, no KAI1 gene mutation was recently identified in patients with prostate cancer (8) and esophageal cancer (21) , and no LOH or MSI was detected around the KAI1 locus in lung cancer patients (34) . Thus, it seems that down-regulation of the KAI1 gene does not commonly involve either gene mutation or allelic imbalance. On the other hand, with the accumulating knowledge regarding loss of tumor suppressor genes, abnormal methylation at the promoters of tumor suppressor genes is another mechanism for the suppression of gene activity, which is defined as a third pathway (35) . Indeed, the promoters of several tumor suppressor genes, such as p16, p15, and E-cadherin, are highly methylated with a rare gene mutation in human OSCC. Jackson et al. (36) first examined the DNA methylation status within the promoter region of the KAI1 gene in invasive bladder cancers and bladder cancer cell lines. They showed significant down-regulation of KAI1 mRNA expression in the tumors and cell lines, with no evidence of methylation. In the present study, we did not detect methylation of the KAI1 gene promoter region in malignant oral tumors or in human OSCC-derived cell lines. In addition, whereas three of eight cell lines showed absent or significantly reduced KAI1 mRNA expression, treatment with demethylating agent 5-AzaC was not effective for reexpression of the gene expression. Therefore, these observations suggest that epigenetic change is not the main mechanism of KAI1 down-regulation, although we cannot rule out the possibility that methylation in a more upstream region might be associated with this regulation.

Mashimo et al. (23) demonstrated that the KAI1 gene is directly activated by p53, indicating that loss of p53 function may lead to down-regulation of the KAI1 gene expression. Furthermore, they found a direct relationship between KAI1 and p53 expression in primary prostate cancers. Their findings were of particular interest because genetic alterations of p53 are among the most frequent events in OSCC. Thus, we postulated that there might be a relative correlation between p53 and KAI1 expression in OSCCs. However, we failed to identify any significant correlation between the expression of these proteins, suggesting that p53 does not play an essential role in the process of KAI1 down-regulation in oral carcinogenesis. Similar to our results, no statistically significant difference was reported between p53 and KAI1 expression in SCCs of the esophagus (21) and cervix (37) . An in vitro study by Duriez et al. (38) revealed that KAI1 gene expression is not regulated through a p53-dependent pathway in response to DNA damage in a series of human cell lines. The discrepancies in these above-mentioned results might be due to differences between the methods or organs used. It is possible that transcriptional silencing of the KAI1 gene may be responsible, based on the fact that the 5' KAI1 promoter region contains several binding sites for transcriptional factors such as T-cell-specific transcription factor 1, Myb, and meprin 1. More recent evidence has demonstrated that KAI1 gene expression is mediated by nuclear factor {kappa}B independently of mutant p53 at the transcriptional level (39) . Thus, more detailed functional studies of the above-mentioned transcriptional factors will be required to address this hypothesis.

To date, most studies of the KAI1 gene have suggested that this gene may influence the metastatic ability of human cancers. We observed down-regulation of the KAI1 protein in 97% of metastatic oral tumors. Therefore, our observations for OSCCs agree with the evidence that KAI1 expression is reduced in the majority of metastatic tumors. With regard to the state of KAI1 protein expression reported in human SCCs, the frequencies of KAI1 down-regulation in lung, esophageal, and cervical SCCs are 66% (15) to 76% (16) , 66% (21) to 81% (20) , and 71% (37) , respectively. In invasive SCCs from various tumor sites (lung, head and neck, and cervix), extensive down-regulation of KAI1 (79%) has also been detected (40) . These data are in agreement with our observation that KAI1 was down-regulated in 83 of 101 (82%) primary OSCC tumors. On the other hand, we observed KAI1 down-regulation, for the first time, in almost half (46%) of the precancerous lesions investigated. Therefore, we suggest that the marked down-regulation of KAI1 can be seen as a precipitous event in oral carcinogenesis.

Finally, all of the primary OSCCs pathologically proven to have metastatic lesions were found to have extensive KAI1 down-regulation. At present, it is not known how KAI1 down-regulation is involved in the metastatic process of OSCC. Nevertheless, the frequent loss of KAI1 function in primary OSCC may be an important factor in the progression to the metastatic phenotype.


   ACKNOWLEDGMENTS
 
We thank Dr. Osamu Yoshie for the generous and much appreciated gift of C33 antibody, which was essential to this research. We also thank Lynda C. Charters and Allan Earle for proofreading this manuscript.


   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 research grants from the Ministry of Education, Science and Culture, Japan. Back

2 K. U. and K. O. contributed equally to this study. Back

3 To whom requests for reprints should be addressed, at Department of Clinical Molecular Biology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan. Phone: 81-43-226-2300; Fax: 81-43-226-2151; E-mail: tanzawap{at}med.m.chiba-u.ac.jp Back

4 The abbreviations used are: OSCC, oral squamous cell carcinoma; COBRA, combined bisulfite restriction analysis; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LOH, loss of heterozygosity; MSI, microsatellite instability; SSCP, single-strand conformational polymorphism; RT, reverse transcription; TM4SF, transmembrane-4 superfamily; SCC, squamous cell carcinoma; 5-AzaC, 5-aza-2'-deoxycytidine. Back

Received 7/24/01; revised 12/20/01; accepted 1/ 2/02.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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