Understanding the Role of the Peroxiredoxin Family in the Regulation of Ferroptosis in Colorectal Cancer

Abstract

Ferroptosis, a new form of regulated cell death, is typically characterized by excessively iron‐dependent accumulation of lipid peroxidation, which causes the breakage of plasma membrane. Dysregulation of ferroptotic cell death has been linked to the development of cancer, and inducing ferroptosis has been proposed as a potential strategy for cancer treatment. Although several important regulators involved in ferroptosis have been discovered, 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 signalling 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 (CRC) 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. 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 and differentiated with other modes of cell death such as apoptosis, autophagy and necrosis by the combined application of various inhibitors. Finally, overexpression and rescue experiments were used to further validate the results observed in Prx‐depleted cells. 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. In consistence, overexpression of Prx4 also leads to the resistance of CRC cell to ferroptosis. 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.