Genetic Ablation of the Cystine Transporter xCT in PDAC Cells Inhibits mTORC1, Growth, Survival, and Tumor Formation via Nutrient and Oxidative Stresses

Abstract

Although chemoresistance remains a primary challenge in the treatment of pancreatic ductal adenocarcinoma (PDAC), exploiting oxidative stress might offer novel therapeutic clues. Here we explored the potential of targeting cystine/glutamate exchanger (SLC7A11/xCT), which contributes to the maintenance of intracellular glutathione (GSH). Genomic disruption of xCT via CRISPR-Cas9 was achieved in two PDAC cell lines, MiaPaCa-2 and Capan-2, and xCT-KO clones were cultivated in the presence of N-acetylcysteine. Although several cystine/ cysteine transporters have been identified, our findings demonstrate that, in vitro, xCT plays the major role in intracellular cysteine balance and GSH biosynthesis. As a consequence, both xCT-KO cell lines exhibited amino acid stress with activation of GCN2 and subsequent induction of ATF4, inhibition of mTORC1, proliferation arrest, and cell death. Tumor xenograft growth was delayed but not suppressed in xCT-KO cells, which indicated both the key role of xCT and also the presence of additional mechanisms for cysteine homeostasis in vivo. Moreover, rapid depletion of intracellular GSH in xCT-KO cells led to accumulation of lipid peroxides and cell swelling. These two hallmarks of ferroptotic cell death were prevented by vitamin E or iron chelation. Finally, in vitro pharmacologic inhibition of xCT by low concentrations of erastin phenocopied xCT-KO and potentiated the cytotoxic effects of both gemcitabine and cisplatin in PDAC cell lines. In conclusion, our findings strongly support that inhibition of xCT, by its dual induction of nutritional and oxidative cellular stresses, has great potential as an anticancer strategy. Significance: The cystine/glutamate exchanger xCT is essential for amino acid and redox homeostasis and its inhibition has potential for anticancer therapy by inducing ferroptosis.

Amino Acid Transporters as Targets for Cancer Therapy: Why, Where, When, and How

“…Depletion of glutathione by APR-246, which binds thiol-groups, in combination with inhibition of xCT could make cancer cells more vulnerable to oxidative stress. Accordingly, it may be necessary to increase oxidative stress to facilitate cell death in xCT ko cells [80,81]. This is consistent with the absence of xCT in genome-wide ko screens.…”

Reprogramming of Amino Acid Metabolism in Pancreatic Cancer: Recent Advances and Therapeutic Strategies

“…Moreover, the expression of SLC7A11 is upregulated in the pancreatic tumor tissues and pancreatic cancer cells can increase SLC7A11 expression in response to oxidative stress, which results in the increase in GSH synthesis and enables tumor cells to survive in the presence of elevated ROS (91). Recent studies demonstrated that genetic deletion of SLC7A11 induces PDAC cell and tumor ferroptosis, and PDAC cells can use cysteine to synthesize GSH and coenzyme A to down-regulate ferroptosis (92,93).…”

“Induction of pancreatic tumor-selective ferroptosis through modulation of cystine import”

“…To date, in vivo evidence for the role of system x c in cancer has largely relied on ex vivo assays, direct injection of chemical probes (19), or implantation of cells with existing alterations that could impact engraftment (20). In order to determine the reliance of established pancreatic tumors on system x c in a physiological context, we employed a dual recombinase genetic engineering strategy based on the wellvalidated "KPC" mouse (21 Figure 5 for more information) developed tumors histopathologically identical to those from KPC mice: typically a single, focal mass with moderate differentiation and robust stromal desmoplasia (Supplementary Figure 6A), in the context of surrounding premalignant histopathology (Supplementary Figure 6B).…”