Antioxidants have historically been thought of as cancer preventative agents; however, increasing evidence in recent years indicate that antioxidants promote cancer cell survival. Using 3D spheroid models, our lab previously demonstrated that normal cells die in the inner layers of spheroids, whereas cancer cells survive. Multiple lines of evidence indicate that survival of inner cells within tumor spheroids is dependent on escape from apoptosis and reactive oxygen species (ROS)-induced cell death through upregulation of anti-apoptotic and antioxidant programs. While the mechanisms associated with apoptosis are well-established, there is relatively little understood about the cellular processes involved in ROS-induced cell death (origins of ROS, types of oxidative stress experienced, mechanisms of cell death, antioxidants involved etc.). Tumor cells with mutations in the nuclear factor erythroid 2-related factor 2 (NRF2) pathway that lead to hyperactivation of this transcription factor serve as an excellent model to study the role of antioxidant programs in the regulation of survival processes. NRF2 is the master regulator of the antioxidant response directly regulating expression of antioxidant enzymes as well as proteins required for the production of non-enzymatic antioxidants. Furthermore, mutations in the NRF2 pathway are frequent in tumor types that are associated with oxidative stress such as lung cancer. We found that knock-down of NRF2 affected cell survival and growth in 3D by regulating (i) cell proliferation and (ii) inner cell survival. To better understand the specific processes responsible for ROS-induced cell death and the antioxidant program(s) utilized by cancer cells to bypass these death programs, we performed CRISPR screens in 3D using lung cancer cell lines with NRF2 hyperactivation to identify downstream targets of NRF2 and/or antioxidants that collaborate with NRF2 to regulate cell survival and growth in 3D. As predicted, we observed drop-out of sgRNAs targeting NRF2. Notably, a top drop-out hit was glutathione peroxidase 4 (GPX4), an essential antioxidant enzyme that reduces membrane phospholipid hydroperoxides to prevent a form of non-apoptotic cell death known as ferroptosis. We found that inhibition of GPX4 resulted in elevated levels of lipid peroxidation and death in the inner cells of 3D spheroids which were rescuable by the ferroptosis inhibitor Ferrostatin-1, implicating ferroptotic cell death in 3D biology. Surprisingly, knock-down of NRF2 resulted in stabilization of GPX4 at the post-transcriptional level. Finally, knock-down of NRF2 combined with GPX4 inhibition induced cell death in both inner and outer cells of 3D spheroids. These data suggest that cancer cells exploit the functional interplay between NRF2 and GPX4 to survive in high oxidative stress environments. This work was supported by funding from the ROADS Program funded by F. Hoffmann-La Roche Ltd..

Note: This abstract was not presented at the meeting.

Citation Format: Patricia Cho, Nobuaki Takahashi, Laura M. Selfors, Hendrik J. Kuiken, Joan S. Brugge. Interplay between NRF2 and GPX4 is critical for cancer cell survival in 3D spheroids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 884.