SUMO2 inhibits ACSL3 protein degradation to antagonize erastin-induced ferroptosis in hepatocellular carcinoma

Abstract

Hepatocellular carcinoma (HCC) is the prevalent form of primary liver cancer with a high rate of morbidity and death. Ferroptosis is a kind of regulatory cell death mode that depends on iron. Small ubiquitin-like modifier 2 (SUMO2) is linked to HCC progression; however, its role in ferroptosis within HCC remains unclear. Our goal was to evaluate the regulatory effects and molecular mechanisms of SUMO2 in HCC ferroptosis. SUMO2 was screened by bioinformatics analysis, and its expression was verified in HCC tissues. Stable SUMO2 knockdown and overexpression cell lines were created. The downstream target protein acyl-CoA synthetase long-chain family member 3 (ACSL3) of SUMO2 was screened to assessed the mechanism of SUMO2 regulating ferroptosis in HCC cells. In HCC tissues, SUMO2 expression was higher and linked to a worse prognosis for patients. SUMO2 overexpression reduced malondialdehyde content, prevented mitochondrial crest loss, and increased glutathione level under ferroptotic stimuli. Meanwhile, overexpression of SUMO2 lowered the expression of molecules that promote ferroptosis and raised the expression of molecules that prevent it. SUMO2 knockdown produced the opposite effects. Mechanistically, SUMO2 elevated ACSL3 protein level by inhibiting its entry into the ubiquitin-proteasome degradation pathway and enhanced its protein stability. The inhibitory effects of SUMO2 on ferroptosis in HCC cells were reversed by ACSL3 knockdown in SUMO2-overexpressing cells. In summary, SUMO2 binds to ACSL3, preventing its protein degradation, thereby increasing its protein stability and level, which in turn negatively regulates ferroptosis in HCC cells. These results point to interesting targets and therapeutic approaches for HCC.

Keywords: Erastin; ACSL3; Ferroptosis; Hepatocellular carcinoma; Protein degradation; SUMO2.

Fig. 1

SUMO2 is highly expressed in hepatocellular carcinoma (HCC) tissues and is associated with poor prognosis. A mRNA expression of SUMO family members in HCC tissues. B, C mRNA expression of SUMO family members based on different grades and stages of HCC. D Association between mRNA expression of SUMO family members and vascular infiltration in HCC. E Relationship between SUMO1 and SUMO2 mRNA expression and different survival in patients with HCC. F, G SUMO2 mRNA and protein expression in 10 patients’ paired HCC and adjacent tissues, as detected via qRT-PCR and Western blotting. H Immunohistochemistry detection of SUMO2 protein expression in the tissue microarrays of paired HCC and adjacent tissues of 86 patients. I Differences in SUMO2 protein expression based on different clinical indicators in 86 patients with HCC. J Analysis of SUMO2 protein expression in normal hepatocytes (HL7702) and six HCC cell lines (SK-HEP-1, HCCLM3, SMMC-7721, MHCC97H, PLC/PRF/5, and Huh7 cells) by Western blot. *P < 0.05, **P < 0.01, ***P < 0.001; ns not significant

Fig. 2

SUMO2 negatively regulates erastin-induced ferroptosis in HCC cells. A, B qRT-PCR and Western blotting were employed to verify the efficiency of SUMO2-knockdown using three independent short hairpin RNA (shRNA) sequences. C, D We used Western blotting and qRT-PCR to assess the effectiveness of SUMO2 overexpression. Cells with stable SUMO2 knockdown or overexpression were subjected to treatment with erastin (10 µM) for 24 h. Intracellular malondialdehyde (MDA) (E) and glutathione (GSH) (F) levels were measured using a kit. Additionally, the structure of the cell mitochondria was visualized using a transmission electron microscopy (G). *P < 0.05, ***P < 0.001; ns not significant

Fig. 3

Ferroptosis-related molecule expression in cells with stable SUMO2 knockdown or overexpression. The mRNA and protein expression of molecules linked to iron metabolism (TF, TFRC, DMT1, FTH1, and NRF2) (A, B), lipid peroxidation (ACSL4 and ALOX15) (C, D), and redox pathways (SLC7A11, GPX4, and TP53) (E, F) in cells with stable SUMO2 knockdown or overexpression were assessed via qRT-PCR and Western blotting. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 4

SUMO2 affects the protein level of ACSL3 via the ubiquitin-proteasome pathway. A Workflow for the identification of proteins that interact with SUMO2 using immunoprecipitation combined with liquid chromatography and mass spectrometry. B Western blotting was used to confirm that SUMO2 bound to ACSL3. C, D Western blotting and qRT-PCR were used to evaluate the protein and mRNA level of ACSL3 in cells with stable SUMO2 knockdown or overexpression. E, F Cells with stable SUMO2 knockdown or overexpression were treated with MG-132 (20 µM) for 6 h, and Western blotting was employed to assess the level of ACSL3 protein. G, H Cells with stable SUMO2 knockdown or overexpression were treated with cycloheximide (50 µg/mL) for 0, 2, 4, and 6 h, and Western blotting was performed to detect ACSL3 protein level. ns not significant

Fig. 5

ACSL3 knockdown reverses the negative regulatory effect of SUMO2 overexpression on ferroptosis in HCC cells. After ACSL3 knockdown in cells overexpressing SUMO2, erastin (10 µM) treatment was performed for 24 h, and intracellular MDA (A) and GSH (B) contents were detected using a kit. C–E Analysis of ferroptosis-related molecules’ protein level using Western blot. *P < 0.05, **P < 0.01, ***P < 0.001