Investigation of PTEN promoter methylation in ameloblastoma

Background Phosphatase and tensin homolog (PTEN) acts as a tumor suppressor gene. Inactivation of PTEN has been reported in various types of cancers. PTEN promoter methylation possibly underlies PTEN inactivation, which results in tumorigenesis. The aim of this study was to investigate whether PTEN promoter methylation contributes to PTEN inactivation in ameloblastoma and its associated protein expression. Material and Methods In total, 20 fresh-frozen ameloblastoma samples were evaluated for PTEN promoter methylation using methylation-specific polymerase chain reaction (MS-PCR). A subset of 10 paraffin-embedded ameloblastoma samples was examined for PTEN expression through immunohistochemistry. Four primary cultured ameloblastoma cells were investigated for PTEN promoter methylation and PTEN transcriptional expression via reverse transcription PCR. Results PTEN promoter methylation was detected in 65% (13/20) of the ameloblastoma samples. Of 10 ameloblastoma samples, 4 exhibited reduced PTEN expression. Of 5 samples with methylated PTEN, 3 (60%) were associated with loss of PTEN expression. However, PTEN expression was detected in 4 (80%) of 5 samples with unmethylated PTEN. In addition, 3 (75%) of 4 primary ameloblastoma cell cultures exhibited an inverse correlation between PTEN promoter methylation and PTEN transcription level. Conclusions PTEN promoter methylation is found in a number of ameloblastomas but not significantly correlated with loss of PTEN expression. Genetic or epigenetic mechanisms other than PTEN promoter methylation may contribute to PTEN inactivation in ameloblastoma tumor cells. Key words:PTEN, promoter methylation, ameloblastoma.


Introduction
Ameloblastoma is the most frequently encountered neoplasm arising from the epithelium of the tooth-forming apparatus. Although this tumor is benign, it exhibits locally invasive behavior and has a high risk of recurrence. Its macroscopic features range from completely solid to multicystic appearance. Its histopathological subtypes include follicular, plexiform, acanthomatous, granular cell, basal cell, and desmoplastic ameloblastomas. In rare cases, ameloblastoma may metastasize despite its benign histology; this type of ameloblastoma is termed as metastasizing ameloblastoma (1). Ameloblastic carcinoma, a malignant counterpart of ameloblastoma, is markedly rare, with only 100 cases reported to date; this tumor exhibits cytological features of malignancy and may or may not metastasize (2). Phosphatase and tensin homolog (PTEN) is located on chromosome 10q23.3 and has been implicated in many familial and sporadic cancers (3,4). Deletions or somatic mutations in PTEN have been detected in many types of cancers, including prostate, breast, and brain cancer (3). Apart from genetic mutation, the epigenetic regulation of PTEN via differential methylation may contribute to its inactivation (5). Methylation of the PTEN promoter region has been reported in some types of cancers and

Results
-MS-PCR and immunohistochemistry of ameloblastoma tissues We examined PTEN promoter methylation and whether it affects PTEN expression in ameloblastomas. PTEN promoter methylation was observed in 65% (13/20) of ameloblastoma samples ( Table 1). The exemplified gel electrophoresis is demonstrated in Fig. 1. Ten samples of these ameloblastoma cases were investigated for immunohistochemical staining of PTEN. We found loss of PTEN expression in 3 of 5 (60%) ameloblastoma samples with PTEN promoter methylation while PTEN expression was present in 4 of 5 (80%) ameloblastoma samples with no PTEN promoter methylation (Table 1). Representative samples showing positive and negative immunostaining of PTEN were shown in Fig. 2.
-Association among PTEN promoter methylation, PTEN expression, and clinicopathological parameters Table 1 shows the association between PTEN promoter methylation and PTEN expression in the ameloblastoma samples. No significant correlation was found between PTEN promoter methylation and PTEN expression (P = 0.52). Furthermore, no correlation between PTEN promoter methylation and age (P = 0.49), gender (P = 0.40), location (P = 0.62) and the histological appearance of ameloblastoma (P = 0.41) was demonstrated. Similarly, no correlation was observed between PTEN expression and age (P = 0.25), gender (P = 1.00), location (P = 0.51), and the histological appearance of ameloblastoma (P = 1.00).
-MS-PCR and RT-PCR of primary ameloblastoma cell cultures PTEN promoter methylation and PTEN expression were examined in four primary ameloblastoma cell cultures using RT-PCR (Fig. 3). Only 1 of 4 samples exhibited PTEN promoter methylation and showed no PTEN transcription. PTEN promoter methylation was inversely correlated with PTEN transcription level in the remaining ameloblastoma samples (75%) ( Table 1).

Discussion
Ameloblastoma is considered the most common benign neoplasm of the jaw (1). To prevent local recurrence, patients with ameloblastoma are mostly treated with radical surgery. Understanding the molecular mechanisms that underlie the formation of this tumor may help in developing an alternative and novel treatment for its cure with minimal tissue or bone removal. PTEN, a putative tumor suppressor gene, is commonly mutated in many types of human neoplasms (3). The protein product of PTEN, a lipid phosphatase, negatively regulates the Akt signaling pathway, thereby stimulating cell cycle arrest and apoptosis (13). Kumamoto and Ooya first reported that the PTEN level is significantly lower in ameloblastic tumors than in tooth germs (14). The absence of PTEN in 33.3% of ameloblastoma samples was subsequently reported (10). These results suggest that the inactivation of PTEN may be involved in the molecular pathogenesis of ameloblastoma. In the present study, we investigated the possible role of PTEN promoter methylation and the associated loss of PTEN expression in a subset of ameloblastoma samples.
To the best of our knowledge, there have been no studies on PTEN promoter in ameloblastoma. Careful analysis of the PTEN promoter has been recommended because it shares a strong homology with the PTEN pseudogene (5). The genomic sequence of the highly conserved and processed PTEN pseudogene (GenBank accession number: AF040103, PTEN pseudogene; AF029308, Homo sapiens chromosome 9 duplication of the T-cell receptor β locus and trypsinogen gene families) is 98% identi-cal to that of PTEN, and this identical sequence is composed of an 841-bp region in the promoter region (15).
In the present study, PTEN promoter methylation was performed using methylation-specific primers that do not amplify the highly homologous PTEN pseudogene because these primers lie outside the sequence homology of the PTEN pseudogene.
Promoter methylation is reportedly one of the epigenetic mechanisms underlying the aberrant expression of tumor suppressor genes and contributing to the development of various types of cancers. For example, the methylation of adenomatous polyposis coli promoter is reportedly associated with tumor in the colon and breasts (16). PTEN promoter methylation is also observed in various types of cancers, including gastric, breast, colorectal, and lung cancer (7,8,(17)(18)(19)(20).
In the present study, PTEN promoter methylation was found in 65% (13/20) of the ameloblastoma samples. However, immunohistochemical staining of PTEN expression was performed in only 10 samples. Of these samples, 3 (60%) of 5 samples with PTEN promoter methylation were associated with loss of PTEN expression, whereas 4 (80%) of 5 samples without PTEN promoter methylation showed PTEN expression. PTEN promoter methylation and decreased PTEN expression were not significantly correlated, indicating that other genetic or epigenetic mechanisms possibly regulate PTEN expression, for example, genetic alterations, transcriptional silencing, post-transcriptional regulation, and modification (21). Previously, PTEN exhibited high frequency of allelic losses (62%) in ameloblastic tumors (9). Moreover, Narayan et al. reported that 5 (25%) of 20 samples of solid/multicystic ameloblastoma exhibited gene alterations in exon 5 of PTEN while no PTEN mutation was observed in normal tooth germs. However, associated protein expression was not examined in those samples (22). Based on the two-hit model (23), it may be possible that PTEN promoter methylation and allelic loss play a role in PTEN inactivation since PTEN is a tumor suppressor gene. It is also possible that PTEN promoter methylation, contributing to a decrease in protein expression, depends on the specific tumor type. Previous studies on lung and ovarian cancers did not see a correlation between PTEN promoter methylation and loss of protein expression (18,24).
Notably, in the present study, 2 (40%) of 5 samples with PTEN promoter methylation showed PTEN expression. This may be attributed to the partial methylation of PTEN at the promoter region. It has been proposed that translational inactivation involves a series of events requiring a sufficient DNA methylation level. The silencing process is then maintained by the spread of methylation (25). This is also supported by the presence of unmethylated bands in several samples following MS-PCR. However, these unmethylated bands also represent normal fibrous tissue stroma in the ameloblastoma samples.
Regarding the in vitro experiment, only 1 (25%) of 4 primary ameloblastoma cell cultures showed promoter methylation and loss of PTEN transcription. This result is consistent with a previous study on breast cancer; none of the breast cancer cell lines exhibited PTEN promoter methylation (17). Lastly, the limitation of the present study is the small sample size that may not represent ameloblastoma cases and cell lines in general; thus, further studies with a larger sample size are required to confirm our findings.
In conclusion, PTEN promoter methylation was detected in a subset (58.3%) of ameloblastoma samples; however, it did not significantly contribute to decreased PTEN expression. Other genetic mechanisms possibly underlie the loss of PTEN expression in ameloblastomas.