Genetic alterations play an important role in the development of CC [4, 7, 8]. Our extensive molecular analyses identified a number of specific genetic changes in invasive CC and its precursor lesions [6, 14–19]. However, epigenetic alteration in cervical tumorigenesis is not well characterized [12, 13]. In the present study, we identified an overall methylation in 87.8% CC cases. Previous studies by Dong et al (79%) and Virmani et al (72.4%) have also reported a similar overall high frequency of promoter methylation [12, 13]. We identified promoter methylation in >25% cases in CDH1, DAPK, and RARB genes, while it was less frequent (10–25% cases) in HIC1, FHIT, RASSF1A, and APC genes in CC patients. The promoters of CDKN2A, MGMT, BRCA1, TP73, TIMP3, GSTP1, and MLH1 genes were rarely (<10% cases) methylated.
CDH1 is the most commonly methylated gene in the present study showing promoter methylation in 51.1% of CC cases. Dong and co-workers have shown earlier that the CDH1 promoter is methylated in 28% of invasive CC . E-cadherin, CDH1, is a major adhesion component of epithelial cells, which plays an important role as an invasion suppressor gene. Loss of function of CDH1 gene has been shown to occur in widespread of epithelial tumor types by mutational or promoter hypermethylation mechanisms [11, 20]. We have also identified loss of expression of CDH1 gene in 37.5% of CC cell lines analyzed. Thus, these data suggest that inactivation of the CDH1 gene by promoter methylation plays a major role in CC tumorigenesis. However, our immunohistochemical analysis of E-cadherin protein showed no evidence of decreased expression in precursor CIN lesions. Therefore, these data indicate that the methylation of CDH1 gene is a late event in the development of CC.
DAPK gene is methylated in 43.3% cases in the present study. Dong and co-workers also previously showed promoter methylation in 51% of CC . DAPK is a positive mediator of the programmed cell death induced by gamma-interferon . Loss of DAPK expression has been shown to occur in a number of human malignancies, primarily by promoter hypermethylation . It has also been shown that loss of DAPK gene expression was associated with aggressive and metastatic phenotype in many tumor types . In the present study, in addition to showing high frequency of promoter methylation of DAPK gene, we found a complete lack of expression in two of the methylated cell lines and four of 6 unmethylated cell lines. Thus, our results suggest that loss of expression occurs in CC cell lines by mechanism other than complete promoter methylation. Therefore, our data further suggest a role for DAPK in CC tumorigenesis.
The third most commonly methylated gene in the present study is RARB. A previous study also has reported promoter methylation in 33.3% of invasive CC, and 11% and 29% of low- and high-grade CIN lesions, respectively . These authors suggest that RARB methylation is an early event in multistage cervical carcinogenesis. Our semi-quantitative analysis of RARB gene expression by RT-PCR showed down-regulated expression in all the eight cell lines analyzed whether or not the promoter methylated (Fig. 3). In addition, our immunohistochemical analysis of RARB showed lack of expression in 60% of high-grade CINs. It has been reported that RARB mRNA is down regulated in a number of tumor types , including cervical cancer . Taken together, these data support the concept that RARB play an important role early in cervical cancer progression. The direct role of RARB in regulating gene expression and its retinoid-mediated antiproliferative, differentiative, immuno-modulatory, and apoptotic-inducing properties may offer a therapeutic target in the future for CC treatment .
As reported in a previous study, we also found promoter methylation of HIC1 in 22.2% of CC . HIC1 is a zinc finger transcription factor that is transcriptionally silenced by promoter methylation in several types of human cancer . Heterozygous Hic1 mouse with promoter methylation of wild type allele develop carcinomas, sarcomas and lymphomas, suggesting that this gene acts as a tumor suppressor gene . HIC1 gene was down regulated in majority of the CC cell lines examined by us and this down-regulated expression was reactivated upon treatment with demethylating and HDAC-inhibiting agents. These results support the tumor suppressor role of HIC1 and its inactivation by promoter methylation in CC may be a critical epigenetic change in tumor development.
The present methylation profile of CC identified significant differences in the methylated genes between SCC and AC. We found that methylation of HIC1, APC, and BRACA1 genes was more common in AC while CDH1 promoter methylation was seen only in SCC. Such differences in the two major histologies have also been reported previously for DAPK, HIC1, and APC genes . However, we found CDH1 promoter methylation restricted to SCC in the present study, while Dong et al found methylation in both histological types . This difference may be due to small number of AC patients we studied. Distinct patterns of promoter methylation between SCC and AC, however, suggest that epigenetic pathways of tumor suppression may be different in these histologic subtypes. Contrary to the previous reports, we found a low frequency of p16/CDKN2A gene promoter methylation [12, 13, 28, 29]. The reason for these differences is currently unclear.
Our statistical correlative analysis of promoter methylation with clinicopathologic parameters, HPV type, and microsatellite instability identified significant associations. A most significant finding was that overall promoter hypermethylation is associated with the progression and predicts significantly poor disease-free survival. This was most evident in BRCA1 and MGMT genes, where all the patients with promoter methylation were dead (Table 2). The multivariate Cox regression analysis showed that promoter methylation is an independent predictor of outcome for BRCA1 (3-fold risk) and MGMT (2.3 fold risk) genes. Promoter methylation of cancer-related genes has been reported to predict patient prognosis for a number of genes [30, 31]. BRCA1 plays a critical role in DNA repair and recombination, cell cycle checkpoint control, and transcription. This gene has been shown to be hypermethylated in tumors arising from breast-ovarian cancer syndrome . Although we detected complete promoter methylation of BRCA1 gene in only 6% of primary tumors, other mechanisms that inactivate this gene might exist. The present MSP data and its relation to poor clinical outcome warrants a further study of BRCA1 role in CC. MGMT gene encodes O(6)-methylguanine-DNA methyltransferase and this enzyme effectively removes DNA adducts formed by alkylating agents . Epigenetic gene silencing of MGMT confers enhanced sensitivity to alkylating-based chemotherapy, and lack of methylation associates with failure to respond to chemotherapy [34, 35]. On the contrary, all patients that had MGMT promoter methylation were dead within 21 months of initial diagnosis, which predicts a poor prognosis. A possible explanation for this failure to respond may be that all the four patients in the present study were treated only with radiotherapy with or without combination of surgery. The standard treatment for advanced-stage cervix cancer has been the combination of radical surgery and radiotherapy . The utility of cytotoxic chemotherapy in patients with advanced CC has been recognized recently . Thus, the epigenetic gene silencing of MGMT, although in a small proportion, may serve as indicator of responsiveness to treatment with chemotherapeutic drugs that cause DNA adducts in CC.
Most patients in the present study population were treated with traditional radiation and surgery treatment protocols. To assess the impact of promoter methylation on treatment, we compared the patients followed for more than 5 months after treatment. This analysis suggests that RARB methylation predict a worse prognosis (P = 0.02). Twelve of the 15 (80%) patients with RARB methylation were DOC or did not respond to the current treatment, while only 3 of the 15 (20%) patients with RARB methylation exhibited complete response. Since RARB functions as a tumor suppressor gene and its chemopreventive effects are purely based on retinoid induced reactivation, the patients that received radiation therapy will have no effect on these tumors. Our data, therefore, suggest that the CC patients showing RARB promoter methylation may benefit from targeted chemopreventive treatment of combination of retinoic acid, demethylating, and chromatin modifying agents that reactivate the gene expression. An understanding of the exact mechanisms of RARB loss of function in CC is essential for such a strategy.
Microsatellite instability (MSI) is hallmark of mismatch repair-deficient cancers, which has been observed in all cancers arising from Hereditary Non-polyposis Colorectal Cancer (HNPCC) syndrome, some sporadic colorectal and other cancers . MSI is generally associated with germ-line mutations in one of two MMR genes, hMLH1 and hMSH2. Epigenetic inactivation of promoter hypermethylation of APC and HIC1 also results in MSI phenotype in sporadic colorectal cancers [39, 40]. Accumulating evidence supports the view that promoter methylation of mismatch repair genes may be related to MSI phenotype [41, 42]. A number of previous studies have shown the existence of MSI phenotype in CC [43, 44]. In the present study, we found that MSI-H occurs at an increased frequency in tumors with HIC1 and APC promoter hypermethylation. Association of APC promoter methylation with MSI phonotype has been previously reported in endometrial cancer . Although the significance of these results is currently unclear, we believe that inactivation of these genes by promoter methylation causes microsatellite instability and may contribute to the development of CC.
Aberrant promoter methylation changes that occur in cancer are associated with transcriptional repression and loss of function of the methylated gene . Since we found evidence of down-regulated expression of several methylated genes in CC cell lines, we tested to see if this down-regulation could be reactivated by methylation and HDAC-inhibiting agents. Transcriptional silencing of genes resulting from DNA hypermethylation of CpG islands is reversed by treatment with the hypo-methylating agent 5-aza-2'-deoxycytidine and HDAC-inhibiting agent n-butyrate in a dose and duration-dependent manner. We found that 5-Aza-2'-deoxycytidine treatment alone or in combination with n-butyrate resulted in reactivation of gene expression in most cell lines that showed promoter methylation. In some cell lines, exposure to n-butyrate alone has resulted in reactivation of the gene expression, for example in HIC1 gene (Fig. 4). These data, therefore, suggest that other epigenetic mechanisms involving defects in chromatin modification factors such as acetylation and methylation of histone proteins may also play role in gene silencing. Thus, our data also suggest that epigenetic changes that accompany methylation are common in CC and play critical role in CC development.