FIGURE 7.

Inhibition of USP22 sensitizes melanoma to ferroptosis. (A) Heatmap showing cell viability of USP22‐knockout (sgUSP22) and control (sgCtrl) A375 cells treated with BRAF inhibitors (LGX818, GSK2118436, PLX4032), MEK inhibitors (GDC0973, GSK1120212, MEK162), and BET inhibitor (NHWD‐870), ferroptosis inducer (RSL3), apoptosis inducer (staurosporine), necroptosis inducer (HS‐173), and cuproptosis inducer (Elesclomol‐Cu). (B) GSEA showing enrichment of ferroptosis in RSL3‐treated SK‐Mel‐28 cells after topotecan (TPT) exposure for 24 h. (C) Cell viability of A375 and SK‐Mel‐28 cells treated with RSL3 or topotecan (TPT) either alone or in combination for 24 h. (D) Real‐time PCR and RNA sequencing showing the mRNA expression of ferroptosis marker including PTGS2, CHAC1, and TFRC in SK‐Mel‐28 cells treated with RSL3 alone or in combination with topotecan (TPT). (E and F) The levels of lipid ROS detected by C11‐BODIPY 581/591 in A375 (E) and SK‐Mel‐28 cells (F) exposed to RSL3 (2.5 µM), TPT (0.5 µM) or the combination. (G) Cell viability of USP22‐knockout (sgUSP22) and control (sgCtrl) A375 cells treated with RSL3 (2.5 µM) alone or plus GDC0941 (PI3Ki, 1 µM) or AZD8055 (mTORi, 500 nM). (H) Western blotting indicated the levels of ferroptosis‐associated proteins (SCD, GPX4, DHODH, FSP1, and ACSL4) in A375 cells after treatment with topotecan (TPT, 0.5 µM) or USP22‐knockout (sgUSP22). (I) Schematic view and representative H&E images of lung metastasis of mice after tail vein injection of B16F10 cells in the indicated groups. (J) Graphical summary showing that targeting USP22 by topotecan suppresses melanoma metastasis via inhibiting EMT and sensitizes ferroptosis through SIRT1/PTEN/PI3K axis. One‐way ANOVA analysis was performed in (D–G, I). ns., no significance, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.