Abstract 1376: Prx4 depletion enhances ferroptosis susceptibility in colorectal cancer

Abstract

Abstract Ferroptosis is a form of cell death that is characterized by the accumulation of lipid peroxides. Dysregulation of ferroptosis has been linked to the development of cancer. Several important regulators involved in ferroptosis have been discovered, However, the molecular mechanism behind this process has yet to be completely understood. Members of the peroxiredoxin (Prx) family function as leading cellular antioxidants that react with hydrogen peroxide to keep redox homeostasis as well as contribute to oxidative signaling under both physiological and pathological conditions. In this study, we investigated the role of Prx family members in the regulation of ferroptosis in colorectal cancer cells using loss-of-function and gain-of-function experiments. Firstly, immunoblotting was used to examine endogenous levels of Prx family members in different colorectal cancer cells and cells with high or low expression of Prxs were selected for study. Secondly, each of the endogenously expressed 2-Cys containing Prxs including Prx1, Prx2, Prx3 and Prx4 in representative CRC cell lines was depleted by stably expression of ShRNA targeting their coding regions or Knockout using CRISPR-Cas9. The consequences of Prx knockdown in these cells in response to ferroptosis inducers, including erastin and RSL3, were examined by cell viability and colonogenic assays. Cell death due to ferroptosis in these cells was demonstrated by the measurement of lipid peroxidation using the sensor BODIPY 581/591 C11, Levels of MDA and 4-HNE. The mode of cell death was differentiated from other cell death types by the combined application of various inhibitors. In addition, overexpression were used to validate the results observed in Prx-depleted cells. Finally, Prx4 depleted cells were submitted for RNA sequencing and the data were subjected to GSEA analysis to identify enriched pathways. In conclusion, we found that 2-Cys containing Prxs are widely expressed in CRC cell lines, and depletion of Prx1, Prx2 or Prx4 but not Prx3 sensitizes CRC cells to erastin induced cell death. However, only depletion of Prx4 but not other Prxs sensitizes CRC cells to ferroptotic cell death that is characterized by the accumulation of lipid peroxidation and can be rescued by the treatment with inhibitors of ferroptosis. Moreover, Prx4 depleted cells show higher levels of MDA and 4-HNE. In consistence, overexpression of Prx4 also leads to the resistance of CRC cell to ferroptosis. Importantly, according to gene set enrichment analysis (GSEA) of (KEGG) pathway, pathways including P53 pathway and arachidonic acid (AA) metabolism, which have a well-known significant role in ferroptosis regulation, were activated in Prx4 depleted cells. Thus, our findings reveal an essential role of Prx4 in protecting colorectal cancer against ferroptosis and provide a potential target to enhance the antitumor activity of ferroptosis-based treatment. Citation Format: Aziza Alshahrani, Hong Jiang, Pratik Thapa, Na Ding, Yanning Hao, Qiou Wei. Prx4 depletion enhances ferroptosis susceptibility in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1376.