COP1 drives renal cell carcinoma progression by targeting ACSL4 for ubiquitin-mediated degradation and inhibiting ferroptosis

Abstract

Background: Renal cell carcinoma (RCC) progression is closely linked to dysregulation of the ubiquitin-proteasome system, particularly aberrant ubiquitination processes governing protein degradation and cell cycle control. As a pivotal E3 ubiquitin ligase, COP1 mediates substrate-specific ubiquitination to regulate protein stability. However, its functional role in RCC remains poorly characterized. This study investigates how COP1 drives RCC malignancy and explores its underlying molecular mechanisms.

Methods: We analyzed the expression of COP1 in RCC cells and its relationship with patient overall survival (OS) in databases. The CCK-8 assay was used to detect the effect of COP1 on the proliferation of RCC cells, while the Transwell assay was used to assess the impact of COP1 on the migration and invasion of RCC cells. We employed mass spectrometry, co-immunoprecipitation, Western blot, and RT-qPCR to explore the target proteins that interact with COP1 and their interaction modes. After inducing with ferroptosis inducers, we measured the effect of COP1 on lipid ROS levels in RCC cells. Finally, we validated the role of COP1 in RCC using in vivo experiments.

Results: COP1 was significantly correlated with poor patient prognosis. Functional studies demonstrated that COP1 overexpression markedly increased RCC cell proliferation by 65% (786-O) and 58% (ACHN) (p < 0.001) and enhanced migration/invasion (p < 0.01), while COP1 knockdown suppressed these malignant phenotypes by 40-50%. Mechanistically, COP1 directly bound ACSL4 and promoted its K48-linked ubiquitination, reducing ACSL4 protein stability by 70% (p < 0.001) and suppressing ferroptosis, as evidenced by decreased lipid ROS levels (p < 0.01) and reversal of ferroptosis inhibition by ferrostatin-1. In vivo, COP1 overexpression accelerated tumor growth in xenograft models, with a 2.5-fold increase in tumor volume compared to controls (p < 0.001), accompanied by reduced ACSL4 expression and elevated Ki67 proliferation index. These effects were further amplified by the ferroptosis inhibitor ferrostatin-1, underscoring COP1's role in driving tumor progression through ferroptosis suppression.

Conclusion: Our study establishes COP1 as a critical driver of RCC progression by suppressing ferroptosis through ubiquitin-mediated degradation of ACSL4, thereby providing a novel theoretical foundation for targeted therapeutic strategies in RCC.

Keywords: ACSL4; COP1; RCC; ferroptosis; ubiquitination.

Figure 1

COP1 is Highly Expressed in RCC and Promotes Tumor Cell Proliferation, Migration, and Invasion, Correlating with Poor Prognosis. (A) Kaplan-Meier survival curves of RCC patients stratified by COP1 expression (high vs. low; log-rank test, p < 0.001). RFWD2 is another name for COP1. (B) Western blot validation of COP1 stable overexpression efficiency in 786-O and ACHN cells, and significant downregulation of COP1 after transfection with shCOP1#1/2. GAPDH served as a loading control. (C) CCK-8 assay demonstrated significantly enhanced cell proliferation in COP1-overexpressing 786-O and ACHN cells. (D) COP1 knockdown markedly suppressed cell proliferation in both cell lines compared to controls. (E) Transwell experiments revealed increased migratory and invasive capacities in COP1-overexpressing cells. (F) COP1 knockdown significantly attenuated cell migration and invasion. (G) The statistical analysis of the result that COP1-overexpressing cells in 786-O and ACHN RCC cells have stronger migration ability in Transwell experiments. (H) The statistical analysis of the result that COP1-overexpressing cells in 786-O and ACHN RCC cells have stronger invasion ability in Transwell experiments. (I) Statistical analysis of the result that the invasion and migration abilities of 786-O and ACHN RCC cells are downregulated after COP1 knockdown in Transwell experiments. *p < 0.05, **p < 0.01, ****p < 0.0001.

Figure 2

COP1 Interacts with ACSL4 and Downregulates ACSL4 Protein Levels in RCC. (A) CO-IP experiments in 786-O cells demonstrated a physical interaction between ACSL4 and COP1. (B) Western blot analysis in 786-O cells revealed that overexpression of COP1 significantly reduced ACSL4 protein levels. (C) Screening of the ferroptosis database identified 16 ferroptosis-related proteins, including ACSL4, that interact with COP1. (D) RT-PCR experiments showed no significant changes in ACSL4 mRNA levels upon COP1 overexpression in 786-O and ACHN cells. (E) Immunofluorescence co-localization experiments in 786-O cells confirmed the interaction between ACSL4 and COP1. The protein labeled with green fluorescence is COP1, and the protein labeled with red fluorescence is ACSL4. (F) Co-localization analysis of the immunofluorescence image by using ImageJ software. Pearson’s r = 0.518. ns, no significant difference in statistics.

Figure 3

COP1 Promotes Ubiquitination-Dependent Degradation of ACSL4 and Suppresses Ferroptosis in RCC Cells. (A) ACSL4 and COP1 protein levels in 786-O cells (Control vs. OE-COP1) after CHX treatment (50 μg/mL). Tubulin: loading control. (B) Quantified ACSL4 half-life in 786-O cells (Control vs. OE-COP1). (C) ACSL4 and COP1 protein levels in ACHN cells (Control vs. OE-COP1) after CHX treatment. Tubulin: loading control. (D) Quantified ACSL4 half-life in ACHN cells (Control vs. OE-COP1). (E) Co-IP assays confirmed COP1-mediated ubiquitination-dependent degradation of ACSL4 in ACHN cells. (F) Co-IP analysis revealed enhanced K48-linked polyubiquitination of ACSL4 in ACHN cells co-transfected with COP1. (G, H) Cell viability assays using ferroptosis inducers (RSL3 and Erastin) showed that COP1 overexpression increased cell survival and suppressed ferroptosis, whereas COP1 knockdown reduced viability and accelerated ferroptosis. (I, J) Rescue experiments: Ferrostatin-1, Z-VAD-FMK, and Necrostatin-1 were applied to COP1-overexpressing ACHN cells treated with Erastin/RSL3. Cell viability was measured to validate ferroptosis specificity. (K-P) Flow cytometry analysis demonstrated altered lipid peroxidation levels in COP1-overexpressing or knockdown ACHN cells under RSL3/Erastin treatment. (Q, R) Exogenous ubiquitination assay in 786-O cells. Increased ubiquitination of ACSL4 observed in COP1-overexpressing (OE-COP1) cells compared to control (Vector) cells. (S, T) Exogenous ubiquitination assay in ACHN cells. Enhanced ubiquitination of ACSL4 detected in COP1-overexpressing (OE-COP1) cells relative to control (Vector) cells. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4

COP1 Suppresses Ferroptosis and Promotes Tumor Growth In Vivo. (A) Representative images of subcutaneous xenograft tumors in nude mice derived from ACHN cells under different treatment conditions. Six representative tumors are shown for each group. (B, C) Quantitative analysis of tumor weight and volume. (D) Representative immunohistochemical (IHC) staining images of PRMT9, GPX4, and Ki-67 in tumor sections. Scale bar, 50 μm. *p < 0.05, ****p < 0.0001.

Figure 5

Schematic representation. Schematic representation of the proposed mechanisms.