PO-250 Investigating BRF2-dependent RNA polymerase III transcription deregulation in cancer

Abstract

IntroductionSmall subsets of RNA Polymerase (Pol) III genes, including the selenocysteine tRNA gene, rely on the presence of the BRF2 transcription factor. Previously, we identified a redox-sensitive switch (Cysteine 361) which, when oxidised, can downregulate Pol III transcription at all BRF2-dependent promoters (Gouge et al. 2015).In cancer cells, BRF2 is frequently amplified and/or overexpressed. We hypothesised that activation of BRF2 is required for maintaining high levels of selenocysteine tRNA, as well as selenoproteins, during prolonged oxidative stress. Selenoproteins are required for redox homeostasis and ROS detoxification and we were able to demonstrate that cancer cells with high levels of expression of BRF2 are more resistant to oxidative stress-induced apoptosis (Gouge et al. 2015).We therefore set out to comprehensively understand the role of BRF2 activation in oncogenesis and its link to oxidative stress response pathways, which are often deregulated in cancer cells.Material and methodsHAP1 cells (fully haploid human CML-based cell line) were generated that carried a C361A mutation, which is a redox-insensitive mutant of the molecular pin. These were tested for their proliferative capacity and ability to withstand exogenous oxidative stress. Full length human BRF2 was overexpressed using lentiviral vectors in malignant and non-malignant cell lines; proliferative capacity and BRF2-dependent transcripts were monitored.Results and discussionsWe found that C361A HAP1 cells were more tolerant to exposure to exogenous oxidative stress using tBHP. However, the proliferative capacity of HAP1 3D spheroids without exogenous stress was not significantly altered by the presence of this mutation. We are investigating the effects of exogenous stress exposure in these cells to identify which BRF2-dependent transcripts are altered following exposure to tBHP.Overexpression of BRF2 in non-malignant cells is not sufficient to induce increased proliferative potential. Experiments to elucidate the effects of overexpression in malignant cell lines are still ongoing.ConclusionWe are continuing to investigate the effects of BRF2 amplification and overexpression on the transcriptional programme of cancer cells under oxidative stress conditions. Since selenocysteine-containing proteins have been implicated in cellular response to ferroptosis, we would like to investigate whether this is mediated by BRF2 overexpression and whether this can be exploited in a therapeutic setting.