COPS5 Triggers Ferroptosis Defense by Stabilizing MK2 in Hepatocellular Carcinoma

Abstract

Sorafenib, which is proven to serve as a potent ferroptosis inducer, is used as a first-line treatment for patients with advanced hepatocellular carcinoma (HCC), but it has limited clinical benefits, mainly due to drug resistance. Herein, using genome-wide CRISPR/Cas9 knockout screening and multiple functional studies, this work identifies COP9 signalosome subunit 5 (COPS5) as a driver of sorafenib resistance and a suppressor of ferroptosis in HCC. Consistently, the amplification and overexpression of COPS5 are frequently observed in clinical HCC samples, which are associated with poor patient prognosis and might predict patient response to sorafenib therapy. Mechanistically, COPS5 stabilized mitogen-activated protein kinase 2 (MK2) through deubiquitination and, in turn, induced the activation of heat shock protein beta-1 (HSPB1), a ferroptosis repressor, thereby protecting HCC cells from ferroptosis and consequently leading to sorafenib resistance and tumor progression, while its own expression could be induced by sorafenib treatment via activating transcription factor 4 (ATF4)-activated transcription. Furthermore, pharmacological inhibition of COPS5/MK2 synergize with sorafenib to induce ferroptosis and suppress HCC progression. This data reveals the crucial role of COPS5 in triggering ferroptosis defense and sorafenib resistance through the activation of the MK2-HSPB1 axis in HCC and highlights the potential of targeting COPS5/MK2 combined with sorafenib as a promising strategy for treating HCC.

Keywords: COPS5; ferroptosis; hepatocellular carcinoma; sorafenib; therapeutic resistance.

Figure 1

CRISPR/Cas9 knockout screening indicates COPS5 as a candidate driver for sorafenib resistance in HCC. A) The viability of HCC cells treated with various concentrations of sorafenib (Sora) for 72 h was tested using the CCK‐8 assay, and the IC50 values were determined. B) Schematic overview of the workflow of the CRISPR/Cas9 knockout screening. This figure was created via BioRende.com. C) Robust ranking aggregation (RRA) scores and volcano plots showing the genes depleted in sorafenib‐treated versus vehicle‐treated mice via CRISPR/Cas9 screening. D) Venn diagram showing the overlap of the depleted genes in the two sorafenib‐treated mouse models. E) COPS5 expression in HCC versus normal liver tissues from the TCGA–LIHC dataset was analyzed in the UALACN database. F) Association between COPS5 expression and overall survival was analyzed using TCGA–LIHC data via the GEPIA website. G) COPS5 expression in HCC tissues from sorafenib non‐responders and responders in the GEO dataset GSE109211.^{[ 13 ]} H) ROC curve depicting the correlation between COPS5 expression and responsiveness to sorafenib in patients with HCC from the GSE109211 dataset.^{[ 13 ]} I, J) IHC analysis of COPS5 expression in 175 HCCs and 76 NATs. Scale bar: 300 (upper) and 100 µm (lower). K) Overall survival curves for patients with HCC based on COPS5 expression as determined by IHC staining. L) Association between overall survival and COPS5 expression in patients with HCC receiving sorafenib therapy according to the Kaplan‐Meier plotter database. M) The protein levels of COPS5 in HCC and normal hepatic cell lines. N) Correlations between COPS5 protein levels and the sorafenib IC50 in HCC cell lines. O) Copy number alterations of COPS5 in HCC samples from cBioPortal using the TCGA dataset. P) Correlation between COPS5 expression and copy number in TCGA–LIHC samples. Data are presented as mean ± SD. Statistical analysis was conducted using a two‐tailed t‐test (E and G), a log‐rank test (F, K, and L), and a two‐tailed Pearson's test (N and P).

Figure 2

COPS5 depletion sensitizes HCC cells to sorafenib in vitro. A) Western blots showing COPS5 expression in COPS5‐KO/KD and control cells. B) Cell viability of COPS5‐KO/KD and control cells upon treatment with sorafenib for 72 h. C,D) Colony formation analysis of control, COPS5‐KO (C), and COPS5‐KD (D) cells treated with 10 µM sorafenib or vehicle. E) Cell death in COPS5‐KO and control cells treated with 10 µM sorafenib or vehicle for 48 h as quantified via Annexin V‐APC/7AA‐D staining. F) Western blots verifying COPS5 re‐expression in COPS5‐KO cells. G,H) Cell viability (G) and colony formation ability (H) of COPS5‐KO cells with or without COPS5 re‐expression and control cells following treatment with sorafenib or vehicle. Data are mean ± SD. *p < 0.05, ** p < 0.01, ***p < 0.001 (two‐tailed t‐test).

Figure 3

COPS5 acts as a ferroptosis repressor. A) The viability of COPS5‐KO and control cells was evaluated after combined treatment with sorafenib and a cell death inhibitor, 1 µM ferrostatin‐1 (Fer), 50 µM deferoxamine (DFO), 5 µM necrostatin‐1 (Nec), 10 µM Z‐VAD‐FMK (Z‐VAD), or 10 µM chloroquine (CQ) for 48 h. B) Viability of COPS5‐KO/KD and control cells after 48 h of incubation with increasing concentrations of RSL3. C) Cell viability in response to 5 µM erastin in control cells, COPS5‐KO cells, and COPS5‐KO cells rescued with COPS5 re‐expression. D–F) Fe²⁺ (D), lipid peroxidation (E), and MDA (F) levels of COPS5‐KO and control cells after treatment as indicated. G) Transmission electron microscopy images displaying morphological changes in the mitochondria of COPS5‐KO HepG2 cells with or without COPS5 re‐expression and control cells after treated as indicated. Scale bar: 500 nm. H–K) COPS5‐KO HepG2 and control cells were subcutaneously implanted into BALB/c nude mice, and the mice were treated with vehicle or sorafenib alone or in combination with ferrostatin‐1 for 3 weeks (5 mice/group). Photographs of tumors (H), tumor volumes (I), tumor weights (J), and MDA levels (K) in the xenograft tumors are shown. Data are mean ± SD. *p < 0.05, ** p < 0.01, ***p < 0.001 (two‐tailed t‐test).

Figure 4

COPS5 stabilizes the MK2 protein and activates the MK2‐HSPB1 axis. A) Heat map depicting the differentially expressed proteins in COPS5‐KO versus control cells (left) and MG132‐treated versus untreated COPS5‐KO cells (right). B) Venn diagram illustrating proteins whose expression is downregulated upon COPS5 knockout but rescued by MG132 treatment. C) Western blots analysis of MK2, p‐MK2, p‐HSPB1, HSPB1, and COPS5 in COPS5‐KO/KD and control cells. D) Western blot analysis of MK2 and COPS5 in COPS5‐KO/KD and control cells treated with or without MG132 (20 µM). E,F) Cycloheximide (CHX) chase assay for MK2 protein stability in COPS5‐KO and control HCC cells (E) and HEK293T cells co‐transfected with the MK2‐His plasmid and COPS5‐Flag or control empty plasmid (F). G) Predicted interaction between COPS5 and MK2 by molecular docking. COPS5, green color; MK2, blue color. H) Reciprocal co‐IP between COPS5 and MK2 in HepG2 cells using anti‐COPS5 or anti‐MK2 antibody. I) Co‐IP of COPS5 and MK2 using anti‐Flag or anti‐His antibody in HEK293T cells co‐transfected with COPS5‐Flag and MK2‐His plasmids. J) Ubiquitination of MK2 was determined using IP/western blotting in COPS5‐KO, COPS5‐reexpressing, and control cells in the presence of MG132 (20 µM). Data are the mean ± SD. Statistical analysis was conducted using a two‐tailed t‐test.

Figure 5

MK2/HSPB1 inhibits ferroptosis and mediates COPS5‐induced sorafenib resistance. A) Assessment of the protein levels of MK2, p‐MK2, p‐HSPB1, HSPB1, and COPS5 in the MK2‐KO and control cells. B) Viability of MK2‐KO and control cells treated with sorafenib for 72 h. C) Effects of MK2 knockout on the colony formation of HCC cells in the presence or absence of sorafenib. D) Viability of the MK2‐KO HepG2 cells cotreated with sorafenib and a ferroptosis inhibitor ferrostatin‐1 (1 µM) or deferoxamine (50 µM) for 48 h. E) Western blotting showing the protein levels of MK2, p‐MK2, HSPB1, p‐HSPB1, and COPS5 in COPS5‐KO cells transfected with the MK2 plasmid alone or in combination with HSPB1 siRNAs. F,G) COPS5‐KO cells were transfected with the MK2 plasmid alone or in combination with HSPB1 siRNAs, followed by exposure to sorafenib or the vehicle. Cell viability (F) and colony formation (G) assays were performed. H,I) Fe²⁺ content (H) and lipid peroxidation levels (I) of the MK2‐KO and control cells exposed to sorafenib or vehicle. J–L) BALB/c nude mice were subcutaneously injected with MK2‐KO or control HepG2 cells and then treated with vehicle or sorafenib alone or in combination with ferrostatin‐1 for 3 weeks (5 mice/group). Photographs of the tumors (J), tumor volumes (K), and tumor weights (L) are shown. Data are mean ± SD. Statistical analysis was conducted using a two‐tailed t‐test. *p < 0.05; ** p < 0.01; ***p < 0.001.

Figure 6

ATF4 transcriptionally upregulates COPS5 in response to sorafenib treatment. A, B) mRNA (A) and protein (B) expression levels of COPS5 in HepG2 and SK‐Hep1 cells following treatment with sorafenib for 24 h. C) Protein levels of COPS5 in HepG2 cells exposed to 5 µM sorafenib for 0, 1, 3, 7, 15, and 30 days. D) Western blots analysis of COPS5, MK2, p‐MK2, p‐HSPB1, and HSPB1 expression in HepG2 and SK‐Hep1 cells exposed to sorafenib. E) mRNA expression levels of ATF2, ATF4, NRF2, HIF1A, FOXO3, TFEB, and TP53 in HepG2 and SK‐Hep1 cells following treatment with sorafenib for 48 h. F) mRNA levels of COPS5, ATF4, FOXO3, TFEB, and TP53 in HepG2 and SK‐Hep1 cells transfected with the corresponding shRNAs in the presence or absence of 10 µM sorafenib. G) Effects of ATF4 knockdown on the protein levels of COPS5, MK2, and p‐HSPB1. H) ChIP‒qPCR assay of ATF4 enrichment on the COPS5 promoter in HepG2 cells treated with sorafenib or the vehicle. The primer pairs for the COPS5 promoter region used in the ChIP‒qPCR assay are shown. IgG and anti‐GAPDH antibodies were used as controls. I) Luciferase reporter assays of the COPS5 promoter region with either wild‐type (WT) or mutated ATF4 binding sites in HEK293T cells with or without sorafenib treatment. J) Correlations between COPS5 and ATF4 expression levels in the TCGA–LIHC and GTEx cohorts were analyzed using the GEPIA website. K) Representative IHC images showing ATF4 and COPS5 staining (left) and the correlation between COPS5 and ATF4 expression levels (right) in human HCC tissues. Scale bar: 150 µm. Data are the mean ± SD. Statistical analysis was conducted using a two‐tailed t‐test (A, E, F, H, and I) and a two‐tailed Pearson's test (J and K). ns, no significance; *p < 0.05; ** p < 0.01; ***p < 0.001.

Figure 7

Combining MK2 inhibitors with sorafenib synergistically suppresses HCC growth. A) Protein levels of p‐MK2, p‐HSPB1, and HSPB1 in HepG2 and SK‐Hep1 cells treated with sorafenib (10 µM) alone or in combination with an MK2 inhibitor, 5 µM MK2 Inhibitor III (MK2I) or 5 µM PF3644022 (PF). B) Lipid peroxidation levels in HCC cells subjected to the indicated treatments. C) Viability of HepG2 and SK‐Hep1 cells cotreated with sorafenib (0, 2, 5, and 10 µM) and MK2 Inhibitor III (0, 2, 4, and 8 µM). D) Colony formation assay of HepG2 and SK‐Hep1 cells treated with a combination of sorafenib and MK2 Inhibitor III. E, F) Representative images (E) and viability (F) of PDOs treated with vehicle, sorafenib, MK2 Inhibitor III, or sorafenib plus MK2 Inhibitor III. Scale bar: 250 µm. G–O) The AKT/MET HCC models were established via hydrodynamic tail vein injection (HDTVi) of plasmids encoding the sleeping beauty transposase and transposons with the myr‐AKT gene and MET gene. 3 weeks after HDTVi of the plasmids, the mice were treated with vehicle, sorafenib, MK2 Inhibitor III, or sorafenib plus MK2 Inhibitor III for another 3 weeks (6 mice per group) (G). Representative bioluminescent images (H) and bioluminescence analysis results (I) of the mice in the four groups were shown. Representative photographs (J) and H&E staining (K) of livers, liver weights (L), liver body/weight ratios (M), and serum levels of ALT and AST (N) in mice from the four groups at 6 weeks after HDTVi of the plasmids were also shown. Scale bar: 150 µm (K). Kaplan‒Meier survival analysis of the survival of mice in the four groups were performed (O). Data are mean ± SD. Statistical analysis was conducted using a two‐tailed t‐test (B–D, F, I, L, M, and N) and a log‐rank test (O). ns, no significance; *p < 0.05; ** p < 0.01; ***p < 0.001.

Figure 8

Curcumin synergizes with sorafenib to inhibit HCC progression. A) Western blots showing COPS5, MK2, p‐MK2, p‐HSPB1, and HSPB1 levels in HepG2 and SK‐Hep1 cells treated with increasing doses of curcumin (CCM). B) Protein levels of COPS5, MK2, p‐MK2, p‐HSPB1, and HSPB1 in HepG2 and SK‐Hep1 cells treated with 10 µM sorafenib alone or in combination with 20 µM curcumin. C) Viability of HepG2 cells cotreated with sorafenib (0, 2, 5, and 10 µM) and curcumin (0, 10, 20, and 50 µM). D) Colony formation assay for HepG2 and SK‐Hep1 cells treated with a combination of sorafenib and curcumin. E, F) Representative images (E) and viability (F) of PDOs treated with sorafenib, curcumin, the combination, or vehicle. Scale bar: 250 µm. G–O) The AKT/MET mice were treated with sorafenib, curcumin, the combination, or vehicle for 3 weeks (7 mice per group) (G). Bioluminescent images (H) and statistical analysis of bioluminescent tracking plots (I) of mice are shown. Representative photographs (J) and H&E staining (K) of livers, liver weights (L), liver body/weight ratios (M), and serum levels of ALT and AST (N) in mice from the four groups at 6 weeks after HDTVi of the plasmids are also shown. Scale bar: 150 µm (K). Kaplan‒Meier survival analysis of the survival of mice in the four groups was performed (O). Data are mean ± SD. Statistical analysis was conducted using a two‐tailed t‐test (C, D, F, I, L, M, and N) and a log‐rank test (O). ns, no significance; *p < 0.05; ** p < 0.01; ***p < 0.001.