Genetic Disruption of the γ-Glutamylcysteine Ligase in PDAC Cells Induces Ferroptosis-Independent Cell Death In Vitro without Affecting In Vivo Tumor Growth

SIMPLE SUMMARY: The newly described form of iron-dependent cell death, called ferroptosis, has emerged as a powerful strategy for eradicating cancer cells. This is of particular importance for pancreatic ductal adenocarcinoma (PDAC), which has been shown to be one of the most aggressive tumors, with a five-year overall survival of less than 8%. The aim of the present study is to identify the most potent and selective target for the induction of ferroptosis in PDAC cells. The results presented here are of great importance not only for the development of novel and more effective anti-cancer therapeutics, but also anticipate potential resistant mechanisms that cancer cells might deploy. This way, ferroptosis-based therapeutics may be a step ahead of highly adaptable cancer cells. ABSTRACT: The conceptualization of a novel type of cell death, called ferroptosis, opens new avenues for the development of more efficient anti-cancer therapeutics. In this context, a full understanding of the ferroptotic pathways, the players involved, their precise role, and dispensability is prerequisite. Here, we focused on the importance of glutathione (GSH) for ferroptosis prevention in pancreatic ductal adenocarcinoma (PDAC) cells. We genetically deleted a unique, rate-limiting enzyme for GSH biosynthesis, γ-glutamylcysteine ligase (GCL), which plays a key role in tumor cell proliferation and survival. Surprisingly, although glutathione peroxidase 4 (GPx4) has been described as a guardian of ferroptosis, depletion of its substrate (GSH) led preferentially to apoptotic cell death, while classical ferroptotic markers (lipid hydroperoxides) have not been observed. Furthermore, the sensitivity of PDAC cells to the pharmacological/genetic inhibition of GPx4 revealed GSH dispensability in this context. To the best of our knowledge, this is the first time that the complete dissection of the xCT-GSH-GPx4 axis in PDAC cells has been investigated in great detail. Collectively, our results revealed the necessary role of GSH in the overall redox homeostasis of PDAC cells, as well as the dispensability of this redox-active molecule for a specific, antioxidant branch dedicated to ferroptosis prevention.