Panoply™ Human CTSS Over-expressing Stable Cell Line

Ferroprelation, a newly discovered iron-dependent programmed cell death characterized by excessive lipid peroxidation, is emerging as a promising target for cancer therapy. Here, researchers first identified cathepsin S (CTSS) as a novel regulator of ferroptosis. CTSS is upregulated in ferroptosis-resistant hepatocellular carcinoma (HCC) cells, and inhibition of CTSS sensitizes HCC cells to ferroptosis. Mechanistically, ferroptosis stress induces CTSS maturation and promotes the autophagy-lysosomal degradation of Kelch-like ECH-associated protein 1 (KEAP1). This process blocks KEAP1-dependent, ubiquitination-mediated degradation of nuclear factor E2-associated factor 2 (NRF). Consequently, accumulated NRF2 translocates from the cytoplasm to the nucleus and drives the transcription of anti-ferroptosis genes. In vivo studies have shown that CTSS depletion induced by shRNA or the specific inhibitor LY3000328, combined with ferroptosis inducers, can inhibit the growth of hepatocellular carcinoma (HCC) tumors in an orthotopic xenograft mouse model. These data suggest that CTSS can enhance ferroptosis in HCC cells and may be a potential therapeutic target for overcoming ferroptosis resistance in HCC patients.

To clarify the role of CTSS in KEAP1 degradation, researchers constructed a catalytically inactive CTSS mutant (CTSS-C25A) by mutating the cysteine residue at amino acid position 25. Compared with control vector cells, wild-type CTSS-overexpressing cells showed increased cell viability, indicating enhanced resistance of CTSS-WT HCC cells to erastin and RSL3 (Figure 1G). Consistent with this, an increased GSH/GSSG ratio and decreased MDA levels were observed in CTSS-WT-overexpressing HCC cells, suggesting that CTSS overexpression confers ferroptosis resistance to HCC cells. Interestingly, ectopic overexpression of CTSS-C25A had no effect on cellular sensitivity to ferroptosis inducers, nor on intracellular GSH/GSSG ratio and MDA levels. These results highlight the crucial role of CTSS protease activity in regulating cellular sensitivity to ferroptosis inducers. To further verify whether CTSS degrades KEAP1 through its protease activity, researchers co-transfected HEK293T cells with the His-KEAP1 plasmid and equal amounts of CTSS-WT or CTSS-C25A plasmids. Notably, in cells transfected with CTSS-WT, the level of exogenous KEAP1 protein was reduced, while CTSS-C25A transfection had no effect on KEAP1 abundance (Figure 1H). This indicates that CTSS-mediated KEAP1 degradation is strictly dependent on its proteolytic activity.

Figure 1. CTSS promotes lysosomal degradation of KEAP1. (Xu R C, et al., 2025)