AHR promoter hypermethylation and gene expression in human gliomas

In mammals, CpG islands are found in the proximal promoter regions of almost half of the genes and are generally unmethylated in normal cells, while in cancer cells, the hypermethylation of these promoter regions is the most well categorized epigenetic change to occur (Larsen, 1992; Antequera and Bird, 1993; Costello and Plass, 2001). Promoter CpGs hypermethylation is found in virtually every type of human tumors and result in transcriptional silencing of the gene and subsequent loss of protein expression (Jones and Baylin, 2002; Esteller, 2002).

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The correlation between DNA methylation and gene inactivation is a prerequisite for the identification and functional validation of novel tumor suppressor genes (How Kit et al., 2012).

Inactivation of tumor suppressor genes plays an important role in malignant brain tumor formation and progression. Genetic mechanisms such as mutation, deletion, and structural chromosome rearrangement are known to inactivate tumor suppressor genes. Today, it is well known that also aberrant methylation of CpG islands is one of the major modes of inactivation of tumor suppressor genes in cancer and a growing list of genes are being identified as abnormal methylation of promoter having CpG islands.

The abnormal function of AHR gene has been associated with tumorigenesis pointing to a possible role of AHR as a tumor suppressor gene which is silenced during tumor development. In fact, AHR has been implied to act as a tumor suppressor in prostate and liver tumors, however the underlying molecular mechanism is still unknown (Fan et al., 2010;

Fritz et al., 2007; Peng et al., 2008).

AHR is expressed in normal brain tissue, however in gliomas the expression is frequently lost. In human gliomas, the median AHR expression levels showed a decrease from WHO grade II astrocytomas, to lower levels in Grade III anaplastic astrocytomas and the lowest levels in grade IV glioblastoma (Gramatzki et al., 2009). The mechanisms that alter/silence the expression levels of the AHR gene in cancer, and in the specific case of gliomas, nevertheless are still unknown.

Mutations causing alterations in AHR gene expression are described as be quite rare or even incompatible with survival when leading to a marked loss of expression, suggesting that other mechanisms are at the origin of the loss of expression of AHR (Puga et al., 2002).

Based on these issues and trying to identify the mechanisms that regulate the activity of the AHR in tumor cells, in 2006, Mulero-Navarro and co-workers found that AHR was epigenetically silenced by promoter hypermethylation in 33% of patients with acute lymphoblastic leukemia (ALL). Also, and in order to address whether the low and lost expression of AHR was even due to promoter hypermethylation, treatments with AZA were performed and the AHR mRNA expression was significantly restored. The authors have also evaluated the AHR promoter status in human tumor cell lines from breast, colon, lung, melanoma, osteosarcoma, rhabdomyosarcoma, testis carcinoma, choriocarcinoma, neuroblastoma and embryonic kidney, but minimal AHR promoter hypermethylation was observed (Mulero-Navarro et al., 2006).

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In a more recent study, the methylation status of the AHR gene was investigated in mantle cell lymphoma (MCL). AHR was promoter hypermethylated in 32% of samples. Also, there was a significant inverse correlation between methylation and mRNA levels of AHR, and normal lymph node samples showed significant higher expression levels of AHR than primary MCL (Enjuanes et al., 2011).

Both studies mentioned above concluded that promoter hypermethylation of the AHR gene is frequent and is the source of gene silencing in the tumors under study.

In the present study, and hypothesized that the AHR gene could also be epigenetically regulated by its promoter methylation in human glioma, a series of 188 gliomas (WHO grades I, II and IV) was analyzed by MSP. The methylation specific primers used were the same used by Mulero-Navarro in their study. Within a CpG island, a sequence with 327 nucleotides (-133 to 194), containing 34 CpG dinucleotides susceptible to be methylated, was analyzed (Mulero-Navarro et al., 2006).

AHR gene was found to be promoter hypermethylated in 46.8% of human gliomas and was frequently hypermethylated in 36%, 35.1% and 55.5% of diffuse astrocytomas, oligodendrogliomas and in glioblastomas, respectively. In grade I pilocytic astrocytomas no AHR promoter hypermethylation was found. Another result was that there is a statistical association between the methylation status of AHR promoter and the histological grade of gliomas, with a significant trend to the more malignant glioma likely to be more methylated.

The facts that previous studies show, that there is loss of expression of AHR in gliomas which increases with the degree of malignancy, in conjunction with the results obtained in this study, that there is a frequent promoter hypermethylation of AHR and that hypermethylation also is more frequent as the degree malignancy of gliomas increases, suggests that the AHR gene is epigenetically silenced in human gliomas due to hypermethylation of its promoter, like as seen in ALL and MCL.

However, the major limitation of this study is that analysis of AHR protein expression was not performed, such as the correlation between expression of AHR protein and the promoter methylation status of its gene, thus it can only be hypothesized that the AHR is silenced due to methylation of its promoter.

Although is very tempting to hypothesize that frequent AHR promoter methylation may induce down-regulation of AHR protein expression, however, the frequent methylation of AHR in glioma should be regarded as an epigenetic biomarker but not a therapeutic target until the association between the down-regulation of AHR and its promoter methylation is

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confirmed.The knowledge of the exact role of AHR gene in the development and progression of gliomas can open doors in the diagnosis and clinical management of these tumors.