Abstract 2917: Therapeutic targeting of the mitochondria: An evaluation of the transcriptional link between the antioxidant response and autophagy

Mitoquinone (MitoQ), a mitochondrially targeted chemotherapy induces autophagy via excess reactive oxygen species (ROS) production. Elevations in ROS stimulate nuclear factor (erythroid-derived 2)-like 2 (Nrf2) in breast cancer cell lines. Nrf2 is a nuclear transcription factor that activates the antioxidant response element when under oxidative stress. The aim of this study was to evaluate transcriptional and protein changes associated with Nrf2 activation to identify potential links between oxidative stress, autophagy and cell death. By suppressing Nrf2 and its targeted antioxidant enzymes we should induce elevated ROS levels, which can thereby alter cellular homeostasis and impact the ultimate fate of cancer cells. In the breast cancer cell line MDA-MB-231 RNA was extracted after 24 hours of Nrf2 or non-target pool (NTP) silencing and 18 hours of MitoQ treatment. RNA was sequenced using an Illumina HiSeq and differential expression was analyzed using DESeq in the statistical program R. To avoid bias, raw read counts were filtered to remove low counts possibly due to constraints of the instrumentation. To validate efficient silencing of Nrf2 we confirmed that Nrf2 transcript levels decreased and that there were observed reductions in transcripts containing the Nrf2 transcriptional promoter, the antioxidant response element (ARE) including FTH1, FTL, HMOX1, SRXN1, TXNRD1, NQO1, GCLC, GCLM, GSR and SQSTM1. From there we validated RNAseq data with qPCR using 25 random transcripts and found significant positive correlation between the two methods. Comparisons were first made between the MitoQ and DMSO treatment by silencing (NTP or Nrf2). 2775 genes were differentially expressed (DE) for MitoQ vs DMSO NTP siRNA and 1869 genes were DE for MitoQ vs DMSO Nrf2 siRNA both at an α |1|. We then compared the degree of overlap in DE gene lists between the NTP and Nrf2 siRNA comparisons to identify genes affected only by Nrf2 silencing in combination with MitoQ treatment. 1405 mRNAs were common between silenced and non-silenced sample sets, only 464 mRNAs were due to Nrf2 silencing alone. We are currently performing pathway analysis on these transcripts to determine the role of Nrf2 silencing with redox-active therapies. We are also evaluating autophagic protein levels at later time points, 24 to 48 hours, to further evaluate the potential signaling between oxidative stress and the autophagic response driven by Nrf2. Citation Format: Kaytee L. Pokrzywinski, Francesca Mascia, V. Ashutosh Rao. Therapeutic targeting of the mitochondria: An evaluation of the transcriptional link between the antioxidant response and autophagy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2917.