Demethylation of the MCJ gene in stage III/IV epithelial ovarian cancer and response to chemotherapy
Introduction
In recent years, it has become clear that epigenetic changes, particularly alterations in DNA methylation and histone acetylation, play a key role in the genesis and progression of human cancer and such epigenetic changes are now important targets for the development of novel therapeutic approaches [1]. DNA methylation occurs almost exclusively at CpG dinucleotides (cytosine residues immediately followed by guanines) and about 70% of CpG sites in the human genome are methylated [2]. CpG dinucleotides are under-represented throughout genome, with the exception of short stretches of DNA known as CpG islands. These CpG islands are GC-rich stretches of DNA of up to a few kilobases in length with close to the expected number of CpG dinucleotides and are frequently associated with human genes, often mapping to the promoter/first exon of the gene. In contrast to the bulk of DNA, the CpG sites within CpG islands are almost always methylation free [2]. Essentially all tumor types exhibit gross alterations in the pattern of DNA methylation, with aberrant methylation of CpG islands being observed at up to several thousand different loci in a single tumor [3]. Hypermethylation of CpG islands located within the 5′ region of genes is known to lead to transcriptional repression [4] and genes known to be critical both in tumor development and also in the response of tumors to therapy are known to be inactivated by this mechanism [3].
The methylation controlled DNA J (MCJ) gene was originally identified by Shridhar and colleagues [5]. Transfection of the gene back into MCJ-deficient cell lines had no apparent effect on cell growth but rendered the cells more sensitive to a number of important chemotherapeutic agents, including cisplatin and paclitaxel, which are the mainstays of chemotherapeutic treatment for ovarian cancer patients [6]. Consistent with this observation, we have found loss of expression of MCJ in eight out of ten independently derived cisplatin-resistant derivatives of the ovarian carcinoma cell line A2780 [7].
We have recently identified a CpG island within the MCJ gene, which begins within the 1st exon of MCJ and extends into the first intron [7]. Even though the CpG island does not overlap the gene promoter, hypermethylation of the island results in transcriptional repression of the gene and reversal of this DNA methylation results in gene re-expression. Although there are also a limited number of CpG sites within the MCJ promoter, expression of MCJ is independent of the methylation status of these promoter CpG sites [7], indicating that methylation within the CpG island, and not the promoter region, is critical in silencing of the MCJ gene. In addition, methylation of the CpG island is associated with reduced levels of histone acetylation [7], consistent with current models of DNA methylation conferring transcriptional silencing through binding of MBD (methyl binding domain) protein containing complexes and subsequent chromatin remodeling [4]. These results suggest that the DNA methylation status of the MCJ CpG island could influence gene expression in ovarian tumor cells and lead to alterations in response to chemotherapy. To begin to address this question, we have examined the methylation status of the MCJ CpG island (at a total of 35 CpG sites) in a study of 41 retrospectively collected stage III/IV ovarian tumors by sequencing of bisulfite modified DNA. This analysis demonstrated widely variable levels of CpG island methylation in the ovarian tumors and identified a correlation between patients whose tumors maintained high levels of MCJ methylation, which would be predicted to result in very low levels of MCJ gene expression, with poor response to chemotherapy and reduced overall survival. These results are consistent with a role for MCJ in determining chemosensitivity in vivo.
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Tissue samples
Ovarian tumor samples were obtained from the Western Infirmary and Stobhill General hospitals, Glasgow, UK and Pembury Hospital, Kent, UK. Ethical approval for all samples collected had been obtained and samples were collected according to MRC operational and ethical guidelines on “Human tissue and biological samples for use in research”. The tumor specimens underwent gross examination, sampling and microscopy by an experienced histopathologist. The tissue submitted for research purposes was
Results
Unlike the vast majority of genes with CpG islands, cell type specific DNA methylation of MCJ is observed in normal cells [7]. Thus, the gene is methylated and not expressed in epithelial cells, including ovarian surface epithelial cells from which epithelial ovarian tumors are derived. Therefore, to assess the potential role of MCJ demethylation in ovarian cancer, it was necessary to quantify the levels of CpG methylation across the MCJ CpG island. Quantitation of the level of methylation of
Discussion
Expression of the MCJ gene is controlled, at least in part, by methylation of its CpG island in both normal and neoplastic cells [7]. Analysis of MCJ CpG island methylation in stage III/IV epithelial ovarian tumors identified widespread methylation, with 93% of tumors exhibiting the presence of methylated clones. This high level of methylation was not surprising as the gene is also methylated in normal ovarian surface epithelial cells from which this tumor type is derived [7]. However, the
Acknowledgments
The authors would like to thank Andrea Hay and Jan Gorman for retrieval of clinical information. This work was supported by a Cancer Research UK programme grant awarded to R.B. and by a clinician scientist fellowship awarded to K.O.
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