Ferroptosis was first described in 2012 when Dixon et al performed research on a small molecule termed erastin . Ferroptosis is a new form of cell death dependent on iron and is associated with oxidative damage . Features of ferroptosis include cytoplasmic and lipidic ROS accumulation, a reduction in mitochondrial volume, an increase in mitochondrial membrane density, rupture or loss of mitochondrial cristae and rupture of the mitochondrial outer membrane . Recently, ACSL4 was demonstrated to serve as an essential component of ferroptosis through gene screening of Genome-Wide CRISPR and microarray analysis of anti-ferroptosis cell lines . In the present study, the expression levels of ACSL4 and clinicopathological features in ccRCC were assessed. Additionally, markers of ferroptosis, including lipid ROS and Fe2+ was evaluated when ACSL4 was dysregulated. Therefore, it was concluded that ACSL4 is reduced in ccRCC and it was speculated that ferroptosis reduction may be associated with the pathogenesis of ccRCC.
It has been reported that ferroptosis is closely correlated with human diseases . Studies on ferroptosis on ccRCC are limited. Heike and his colleagues suggested that reduced fatty acid metabolism due to inhibition of β-oxidation renders renal cancer cells highly dependent on the GSH/GPX pathway to prevent lipid peroxidation and ferroptotic cell death . As a pivotal indicator and regulator of ferroptosis, ACSL4 functions as a critical determinant of ferroptosis sensitivity by modulating the cellular lipid composition. ACSL4-knockout cells were resistant to lipid peroxidation and ferroptosis . ACSL4-mediated production of 5-hydroxyeicosatetraenoic acid (5-HETE) contributed to ferroptosis. Pharmacological inhibition of 5-HETE production by zileuton limited ACSL4 overexpression-induced ferroptosis . ACSL4 enriches cellular membranes with long polyunsaturated ω6 fatty acids. Moreover, ACSL4 is preferentially expressed in a panel of basal-like breast cancer cell lines and predicts their sensitivity to ferroptosis . Ectopic expression of ACSL4 in ACSL4-negative prostate cancer (PCa) cells increases proliferation, migration and invasion, while ablation of ACSL4 in PCa cells expressing endogenous ACSL4 reduces cell proliferation, migration and invasion. The study further indicate that ACSL4 upregulates distinct pathway proteins including p-AKT, LSD1 and β-catenin . As such, ACSL4 is regarded as a sensitive regulator of ferroptosis. However, ACSL4 has not been investigated in ccRCC associated with ferroptosis. In our study, we found that ACSL4 expression was down-regulated in tumor and related to poor prognosis, which suggest ACSL4 could serve as a biomarker and potential therapeutic target for ccRCC.
Similar to other cell death patterns, ferroptosis is closely associated with certain signaling pathways. Iron accumulation and lipid peroxidation are the key links associated with the occurrence of ferroptosis . To validate the role of ACSL4 in ferroptosis in ccRCC, an ACSL4 overexpression plasmid or si-ACSL4 were transfected into four different ccRCC cell lines. The results indicated that the inhibition of ACSL4 can rescue cells from erastin-induced cell death and lipid ROS production. In contrast, erastin-induced Fe2+ accumulation was not affected by ACSL4 expression. As a fatty acid activating enzyme, the preferred substrates of ACSL4 are long-chain polyunsaturated fatty acids, such as AA and eicosapentaenoic acid. ACSL4 catalyzes these fatty acids and synthesizes the corresponding coenzyme A , which in turn affects the lipid peroxidation process.
In addition to the crosstalk between ferroptosis and other forms of cell death, the function and assay of ferroptosis related molecular and pathway need to be investigated. Some newly reported proteins such as PEBP1 , NCOA4 , and metallothionein-1G  are correlated with ferroptosis via iron metabolism and lipid peroxidation. In our GO and KEGG analysis, we found that the gene co-expressed with ACSL4 is enriched in the pathway of protein ubiquitination. BAP1 encodes a nuclear deubiquitinating enzyme to reduce histone 2A ubiquitination on chromatin. Recent studies reveal that BAP1 decreases H2Aub occupancy on the SLC7A11 (a ferroptosis inhibitor) promoter and represses SLC7A11 expression in a deubiquitinating-dependent manner and that BAP1 inhibits cystine uptake by repressing SLC7A11 expression, leading to elevated lipid peroxidation and ferroptosis. Furthermore, BAP1 inhibits tumour development partly through SLC7A11 and ferroptosis, and that cancer-associated BAP1 mutants lose their abilities to repress SLC7A11 and to promote ferroptosis . This study reveals that, same as SLC7A11, ACSL4 ubiquitination may also be involved in the process of ferroptosis, which requires in-depth research.
Further studies on ferroptosis are needed not only to clarify the molecular mechanism but also to provide an opportunity for designing new therapeutic interventions. For example, in the treatment of advanced hepatocellular carcinoma, sorafenib resistance has been shown to result from the metallothionein-1Ginduced inhibition of ferroptosis . Lachaier et al compared the levels of ferroptosis induced by sorafenib with those induced by the reference compound erastin in a panel of human cell lines originating from various cancer tissues. They found sorafenib induced ferroptosis in Caki-1 and ACHN (kidney tumors) cell lines. Also, they found a positive correlation between the ferroptotic potency of sorafenib and erastin. Compared to other kinase inhibitors, sorafenib is the only drug that displays ferroptotic efficacy, which establishes sorafenib as the first clinically-approved anticancer drug that can induce ferroptosis . Doll and his colleagues further demonstrate that pharmacological targeting of ACSL4 with the antidiabetic compound class, thiazolidinediones, ameliorates tissue demise in a murine model of ferroptosis, suggesting that ACSL4 inhibition is a viable therapeutic approach to prevent ferroptosis-related diseases .