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Expression of an Inhibitor of Apoptosis, Survivin, in Oral Carcinogenesis
C. Tanaka1,
K. Uzawa2,3,*,
T. Shibahara1,
H. Yokoe3,
H. Noma1 and
H. Tanzawa2,3
1 First Department of Oral and Maxillo-Facial Surgery, Tokyo Dental College, 1-2-2 Masago, Mihama-ku, Chiba 261-8502, Japan;
2 Department of Clinical Molecular Biology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan;
3 Division of Oral Surgery, Chiba University Hospital, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan;
Correspondence: * corresponding author, uzawak{at}faculty.chiba-u.jp
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ABSTRACT
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A novel inhibitor of apoptosis, survivin, plays a role in oncogenesis. To determine the potential involvement of survivin in oral carcinogenesis, we investigated the distribution of survivin protein expression in oral squamous cell carcinomas (OSCCs) and oral pre-malignant lesions. The mRNA expression level and methylation status of the gene also were evaluated in OSCCs and OSCC-derived cell lines. In immunohistochemistry, 58% of tumors and 37% of pre-malignant lesions examined were positive for survivin, while no immunoreaction was observed in corresponding normal tissues. The reverse-transcription/polymerase chain-reaction revealed similar changes in survivin gene expression levels. Furthermore, of the 9 normal oral tissues with no survivin gene expression, 4 showed methylation of the gene, while no methylation was detected in the corresponding tumorous tissues. The results suggest that survivin plays an important role during oral carcinogenesis, and that the gene expression may be regulated by an epigenetic mechanism.
Key Words: survivin • oral squamous cell carcinoma • DNA methylation • inhibitor of apoptosis
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INTRODUCTION
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Apoptosis plays a major role during embryonic development and in the maintenance of tissue homeostasis (Vaux et al., 1994). However, when dysregulation of apoptosis occurs, unwanted cells survive or required cells die, resulting in several diseases, including cancer (Thompson, 1995). The inhibitors of apoptosis (IAP) protein family are novel intracellular proteins that suppress the apoptosis induced by a variety of stimuli (Deveraux et al., 1998). Recently, a novel member of the IAP family, termed survivin, was identified (Ambrosini et al., 1997). Survivin in most adult normal tissues is undetectable, whereas its expression in human common tumors has been associated with increased aggressiveness and decreased patient survival (Kawasaki et al., 1998; Lu et al., 1998; Monzo et al., 1999; Swana et al., 1999; Tanaka et al., 2000), suggesting that apoptosis inhibition by survivin is an important predictive/prognostic parameter of poor outcome in human cancers and that survivin will be a diagnostic/therapeutic target in the development of malignant tumors.
Regarding oral squamous cell carcinomas (OSCC), it is unknown whether survivin is associated with oral carcinogenesis. In this study, we therefore examined the state of survivin mRNA and survivin protein expression in OSCCs and oral pre-malignant lesions. In addition, to determine whether expression of survivin could be regulated by an epigenetic mechanism, we evaluated the methylation status of the CpG sites in exon 1 of the survivin gene in DNA samples from primary tumors and adjacent normal tissues.
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MATERIALS & METHODS
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Tissue Specimens
Tumors or pre-malignant lesions (leukoplakias), with patient-matched normal epithelium where available, were obtained at the time of surgical resection at Chiba University Hospital after the patient’s informed consent had been obtained under a protocol reviewed and approved by the institutional review board of the Chiba University, Japan. Resected tissues were immediately frozen and stored in liquid nitrogen. Histopathologic diagnosis of each neoplastic tissue was performed, according to the WHO criteria, by the Department of Pathology, Chiba University Hospital. Clinicopathologic staging was determined by the TNM classification of the International Union against Cancer. All patients had SCC or leukoplakia that was histologically confirmed, and tumor samples were checked to ensure that tumor tissue was present (> 80% of specimens).
DNA/RNA Isolation
Genomic DNA was extracted by proteinase K digestion and the phenol-chloroform extraction procedure. Total RNA was isolated by means of an SV Total RNA Isolation System (Promega, Madison, WI, USA) according to the manufacturer’s protocol. The samples were stored at –80°C until use.
Immunohistochemistry
Immunohistochemical staining was performed on 4-µm sections of paraffin-embedded specimens with the use of goat anti-human Survivin polyclonal antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA). In brief, after deparaffinization and hydration, the slides were treated with endogenous peroxidase in 0.3% H2O2 for 30 min, after which the sections were blocked for 2 hrs at room temperature with 1.5% blocking serum (Santa Cruz) in phosphate-buffered saline (PBS) before reacting with anti-Survivin antibody (1:100 dilution) at room temperature in a moist chamber for 30 min. After being washed with PBS buffer, the slides were treated with biotinylated secondary antibody and avidin-biotin enzyme reagent (Santa Cruz), followed by color development in 3,3'-diaminobenzidine tetrahydrochloride (DAKO JAPAN Inc., Kyoto, Japan). Finally, the slides were lightly counterstained with hematoxylin. As a negative control, duplicate sections were immunostained without exposure to primary antibodies. For quantitation of the state of survivin protein expression, the mean percentage of positive tumor cells was determined in at least five random fields at 400x magnification in each section. The intensity of the survivin immunoreaction then was scored as follows: 1+, weak; 2+, moderate; and 3+, intense. The percentage of positive tumor cells and the staining intensity were then multiplied to produce a survivin-immunohistochemical staining (survivin-IHC) score. Cases with a survivin-IHC score less than 100 were considered negative. These judgments were made by two independent pathologists, neither of whom had any knowledge or information pertaining to the patients’ clinical status. Any discrepancy in the scoring of slides was resolved jointly by the pathologists by discussion, and a consensus observation was recorded after discussion.
Statistical significance was evaluated by  2 analysis, Fisher’s exact test, or Student’s t test.
RT-PCR
To create first-strand cDNA for the survivin gene, we used 500 ng of total RNA for the RT reaction. The reaction was performed with the use of a Ready-To-Go T-primer First-Strand Kit (Amersham Pharmacia Biotech, Uppsala, Sweden). Ten percent of the cDNA was amplified by PCR with a forward primer, 5'-AGAACTGGCCCTTCTTGGA-3', and a reverse primer, 5'-AAGGAAAGCGCAACCGGACG-3' in a final volume of 25 µL containing 12.5 µL of PCR Master Mix (Roche Molecular Biochemicals, Mannheim, Germany), 1 µL of the cDNA obtained from the RT reaction, 0.5 µL of each of the specific primers (20 pM), and 10.5 µL of water. To confirm the identity of the PCR products, we cloned them into a pCR 2.1 vector (Invitrogen, Carlsbad, CA, USA) and sequenced them on an ALF express IITM DNA Sequencer (Amersham Pharmacia Biotech). After PCR amplification, an aliquot of the PCR product was separated on a 3% agarose gel and stained with ethidium bromide. The density of the ethidium-bromide-stained bands was analyzed by NIH image 1.62. The results were normalized as a ratio of each specific mRNA signal to the glyceraldehye-3-phosphate dehydrogenase gene signal within the same RNA sample. cDNA obtained from normal oral epithelium was used as a negative control. We confirmed reproducibility by processing all samples at least twice.
Methylation Assay
To determine if methylation of a CpG island of the survivin exon 1 could contribute to the mRNA expression of survivin, we used a PCR-based methylation assay to analyzed DNA samples obtained from 9 pairs of OSCCs and corresponding normal oral tissues, and 8 OSCC-derived cell lines. In brief, 10 µg of DNA was digested with Hind III (1 µg/unit, Boehringer GmbH, Mannheim, Germany) at 37°C for 16 hrs. The digested DNAs were then precipitated with ethanol, dissolved in distilled water, and further digested with mCpG-sensitive Hpa II (1 µg/unit, Toyobo, Tokyo, Japan) at 37°C for 16 hrs. The Hpa II-digested DNAs then were amplified with specific primers for survivin exon 1 as previously reported by Hattori et al.(2001). The PCR reactions were performed in a final volume of 25 µL containing 1 µL of digested DNA, 2.5 pmol of each specific primer, 50 µM of dNTPs, 10 mM Tris-HCl buffer (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, and 0.5 unit of AmpliTaq Gold (Applied Biosystems, Foster City, CA, USA). The amplified PCR products were separated on 3% agarose gel and visualized by ethidium bromide after the run.
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RESULTS
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Immunohistochemistry
In total, we identified 71 OSCC patients for whom there was adequate histologic material available for immunohistochemical analysis. The correlation between the clinicopathologic characteristics of patients with OSCC and survivin expression status is summarized in the Table . All normal oral mucosa specimens showed absence or significant down-regulation of survivin expression and were considered survivin-negative. Among the tumors examined, 41 cases (58%) revealed a survivin-immunoreaction in the cytoplasm of the tumor cells (Table). However, there was no statistically significant difference between survivin expression and clinicopathologic features (Table). More interestingly, 14 of 38 leukoplakias (37%) were considered survivin-positive. Representative results for survivin protein expression in normal oral tissue, leukoplakia, and primary OSCC are shown in Fig. 1.
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Figure 1. Immunohistochemical staining of survivin in normal, pre-malignant (leukoplakia), and malignant (squamous cell carcinoma) tissue. (A) Normal oral tissue exhibited negative survivin protein expression. Original magnification, X50. (B) Moderately differentiated primary OSCC. Note the strong cytoplasmic immunoreaction for survivin. Original magnification, X100. (C) Survivin-positive case of leukoplakia. Note that strong positive immunoreaction for survivin was detected on the epithelial cell cytoplasm. Original magnification, X25. (D) The border between normal epithelium (right side) and the dysplastic lesion (left side) is indicated. Note that while no survivin expression was detected in normal epithelial cellular cytoplasm, strong survivin protein expression was evident in the lesion. Original magnification, X50. Bar = 100 µm.
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The survivin-IHC scores for normal tissues, leukoplakias, and OSCCs ranged from 0 to 5 (mean, 1.7), from 15 to 234 (mean, 93.9), and from 2 to 282 (mean, 113.4), respectively. The survivin expression levels in both primary OSCCs and leukoplakias were significantly higher than that in normal oral tissues (p < 0.0001, Fig. 2). In contrast, we found no significant difference in survivin-IHC scores between OSCCs and leukoplakias (p = 0.3364, Fig. 2).
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Figure 2. State of survivin protein expression in normal tissues (n = 71), leukoplakias (n = 38), and primary OSCCs (n = 71). The survivin-IHC scores were calculated as follows: survivin-IHC score = (the percentage of positive tumor cells) X the staining intensity. Survivin protein expressions in leukoplakias and OSCCs were significantly higher than in normal oral tissues (p < 0.0001, Student’s t test), whereas no statistical difference in the protein expression was observed between leukoplakias and OSCCs (p = 0.3364, Student’s t test). The results represent the mean ± SD.
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RT-PCR and Methylation Assay
Using RT-PCR analysis, we examined the expression levels of survivin mRNA in 21 OSCC patients from whom RNA was available from primary tumors and matched normal epithelium and in 8 OSCC-derived cell lines. We confirmed the presence of the survivin mRNA by cDNA sequence analysis of a 196-bp RT-PCR product in human OSCC tissue (data not shown). No normal tissue, with the exception of one case, had survivin expression, whereas the expression was detected in 12 OSCCs (57%) tested. Four of 8 cell lines (50%) revealed survivin mRNA expression (Fig. 3). No statistical significance was observed between the presence of the gene expression and any clinicopathologic features studied (data not shown).
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Figure 3. Representative results of RT-PCR and methylation assay in tissue samples obtained from normal mucosa and tumor, and in OSCC-derived cell lines. Each lower panel reveals GAPDH mRNA expression as an internal control for RT-PCR analysis. N, normal tissue; T, primary OSCC; M, molecular marker ( X174-HaeIII). (A) RT-PCR analysis with the use of specific primers for survivin gene showed that survivin was expressed in tumors but not in corresponding normal tissues, except for case No. 8. Methylation assay revealed survivin-methylation in normal tissues of cases 17, 21, 9, and 13, respectively, which showed no survivin gene mRNA expression. (B) Survivin mRNA expression was detected in OSCC-derived cell lines (HSC-3, OK-92, Ca9-22, and SAS). All cell lines, with the exception of Ca9-22 cell line, showed survivin-methylation.
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We investigated the methylation status of the survivin gene in 9 pairs of OSCCs and matched normal oral tissues. Among them, 4 normal tissues (44%) had a survivin-methylated allele, whereas tumorous tissues showed no evidence of survivin-methylation (p = 0.041). All normal tissues showing survivin-methylation had no gene expression, and 3 of 4 survivin-mRNA-positive cell lines had unmethylated survivin. Representative results are shown in Fig. 3 .
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DISCUSSION
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This study has showed that expression of survivin was detected in almost half of the specimens obtained from primary OSCCs and OSCC-derived cell lines, while absence or significant down-regulation of survivin was observed in normal tissues. In addition, 37% of pre-malignant lesions evaluated showed survivin expression. More importantly, survivin was methylated specifically in normal tissues, whereas tumors and the cell lines had unmethylated survivin.
Considerable evidence suggests that elevated expression of survivin may promote tumorigenesis, and in fact survivin is highly expressed in human common cancers (Kawasaki et al., 1998; Lu et al., 1998; Monzo et al., 1999; Tanaka et al., 2000; Gianani et al., 2001). In contrast, several recent studies have reported survivin gene expression in several normal tissues (Grossman et al., 1999; Gianani et al., 2001; Lehner et al., 2002). In our study, we found that the vast majority of normal tissues had no survivin mRNA expression, with the exception of one tissue specimen. At present, it is unclear why normal cells with survivin expression exist. One possible explanation is that the mRNA sample containing survivin mRNA may be obtained from mitotically active cells, because survivin has been shown to regulate the cell cycle in the G2-M phase (Li et al., 1998).
Lo Muzio et al.(2001) reported a significant correlation between survivin protein expression and tumor aggressiveness in OSCCs and skin SCC. The incidence of survivin expression was consistent with the present study that showed protein expression in 50% to 60% of the tumors analyzed. In laryngeal SCCs, survivin gene expression was closely associated with survival rates (Dong et al., 2002). Thus, we postulated that the state of survivin expression may be an important discriminator for the progression of head and neck SCCs. However, the results were not as good as expected, because we could not find a relation between the degree of survivin expression and the grade/stage of OSCC examined in this study. Thus, we hypothesize that survivin protein accumulation might be an early event during oral carcinogenesis, since one-third of oral pre-malignant lesions examined in this study had protein expression. In this context, the expression of survivin has been demonstrated in several types of pre-malignant lesions or early stages of cancer, including colon polyps (Gianani et al., 2001), pancreatic adenoma (Satoh et al., 2001), and Bowen diseases (SCC in situ) (Grossman et al., 1999). Further studies on a large series of patients will provide more accurate information about the involvement of survivin expression in the development of oral carcinogenesis.
It has been accepted that gene expression in cells is regulated by DNA methylation, and hypermethylation in the promoter region of a given tumor suppressor gene may lead to its inactivation. Regarding the survivin gene, the promoter region was reported to be unmethylated in both normal and tumor tissues (Li and Altieri, 1999). On the other hand, 86% of ovarian cancers were positive for survivin, whereas 83% of survivin-negative cases were methylation-positive for the gene (Hattori et al., 2001). More recently, a transfection experiment (Bao et al. 2002) showed that survivin expression in an ovarian cancer cell line could be controlled by transcriptional regulation. We also found similar evidence of survivin-methylation in normal tissues and tumors with no survivin expression. These observations raise the possibility that up-regulation of the survivin gene may be the result of epigenetic regulation. In addition to epigenetic regulation, survivin expression could be controlled by survivin-related protein, because one OSCC patient revealed no survivin-methylation with the absence of gene expression (Fig. 3). Recent in vitro studies have shown that survivin expression is regulated in transcriptional response by wt-p53 (Hoffman et al., 2002) or by wt-adenomatous polyposis coli protein (Zhang et al., 2001). Lu et al.(1998) reported a significant correlation between survivin expression and accumulation of mutant p53 in gastric cancer. It would therefore be interesting to determine if survivin expression could be correlated with these tumor suppressors in OSCCs.
Finally, recent studies have shown that targeting of survivin using survivin antisense cDNA or a survivin mutant adenovirus may be a useful approach for selective cancer gene therapy (Ambrosini et al., 1998; Mesri et al., 2001). These targeting strategies might also be novel therapeutic tools for human OSCCs.
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ACKNOWLEDGMENTS
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This study was financially supported by a Grant-in-Aid for Scientific Research (No. 14571880) from the Ministry of Education, Science, Sports and Culture of Japan.
Received for publication September 12, 2002.
Revision received January 21, 2003.
Accepted for publication March 25, 2003.
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Journal of Dental Research, Vol. 82, No. 8,
607-611 (2003)
DOI: 10.1177/154405910308200807
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ABSTRACT
INTRODUCTION