Abstract 2081: The CCCTC-binding factor (CTCF) functions to downregulate the transcription of MGMT gene in human glioblastoma cells

Abstract

Abstract The O6-methylguanine-DNA methyltransferase (MGMT) gene located at chromosome 10q26 encodes a DNA repair protein that abrogates the therapeutic effects of alkylating agents by preventing the formation of mutagenic lesions or cytotoxic DNA crosslinks. MGMT is highly expressed in human gliomas and its transcriptional regulation has emerged as a major translational focal point in neuro-oncology, because the epigenetic silencing of the MGMT gene which occurs in a subset of these malignancies confers improved treatment responses to both chemo and radiation therapies. While the promoter methylation and its contribution to glioma therapy has been much studied, the regulation of MGMT transcription per se, proteins accessory to the process and the chromatin structure thereof are not well understood. The CCCTC-binding transcription factor (CTCF) is one such multifunctional protein involved not only in DNA methylation but also with transcriptional activation/repression, and induction of short or long range chromatin looping. CTCF, an 11 zinc-finger containing protein is more frequently known for its insulating effects by binding diverse nucleotide sequences and restricting the function of transcriptional enhancers. Our bioinformatic analysis of the human MGMT gene showed an unusually high (7-10 sites) congregation of CTCF-binding sites distal to the promoter in human cancer cell lines. Given these considerations, we used CTCF siRNA to eliminate the transcription factor in two MGMT-proficient GBM cell lines (SF180 and GBM10). A specific reduction of CTCF protein by individual and pooled siRNAs was consistently accompanied by increased MGMT protein and mRNA levels. Furthermore, the DNA repair activity of MGMT was increased by 50%, which correlated with a proportional reduction of the CTCF protein in siRNA-exposed cells. Currently, we are investigating the effect of CTCF on MGMT reactivation in cells with methylation, MGMT promoter activity in CTCF silenced cells and the effect of enforced CTCF expression on MGMT transcription. Our results for the first time highlight the involvement of multivalent CTCF in MGMT expression. Since CTCF is tightly linked with DNA methylation, further studies should reveal its role in silencing of MGMT in human GBMs (supported by CPRIT RP130266 and NIH RO3CA125872 grants to KSS). Citation Format: Kalkunte S. Srivenugopal, Mohammed Ibrahim Al-Obaide. The CCCTC-binding factor (CTCF) functions to downregulate the transcription of MGMT gene in human glioblastoma cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2081. doi:10.1158/1538-7445.AM2015-2081