Cell death is a vital necessity for maintaining homeostasis, development and the prevention of excessively proliferative malignancy, such as cancer. To sustain the infinite self-renewal capacity, cancer cells exhibit the overwhelming demands for supply, including the energy metabolism, (anti)oxidant modification and iron intake . The iron-dependent mechanism makes cancer cell more susceptible to iron-catalyzed necrosis, namely ferroptosis . The proposal of ferroptosis has challenged the previous dogma that almost cell deaths were subject to the caspase-dependent apoptosis. Ferroptosis has highlighted the significance of iron metabolism and created a new promising area combining with caner management. This unique type of death has attracted considerable studies exploring the ferroptotic potential mechanisms and pathways in various cancers [19–21]. However, the specific role of ferroptosis in EAC is unclear. In the current study, we systematically investigated the ferroptosis-related genes expression profiles in EAC. We found that almost half of genes (46.67%, 28/60) were expressed obviously different between the EAC and normal tissues. Functional enrichment analyses results showed these genes were mainly associated with iron-related pathways, such as metabolic and oxidative process. Survival analysis showed four genes (CARS1, GCLM, GLS2 and EMC2) had prognostic values. In addition, the immune cell enrichment analysis revealed the ferroptosis had a close connection with tumor immunity. These findings strongly implied the great potential roles of ferroptosis in EAC.
The essence of ferroptosis is a metabolic necrosis triggered by an iron-catalyzed excessive peroxidation of polyunsaturated fatty acids (PUFAs) . Non-enzymatic lipid peroxidation or auto-oxidation of lipids is indispensable for the initiation of the PUFAs oxidation . Beyond these, enzymatic lipid peroxidation is another chain reaction to catalyze the PUFAs mediated by lipoxygenase (LOX) family [15, 23]. The toxic consequence of continuous oxidation is the loss of the membrane integrity, leading to the occurrence of ferroptosis ultimately. Consistent with our results, functional enrichment analysis of this study demonstrated these different expressed FRG were mainly associated with the oxidative and iron-related reactions. In addition, the GO and KEGG results also showed there was a close link with GSH metabolism and biosynthesis. GSH, an anti-oxidant, could regulate the sensitivity and resistance of ferroptosis by serving as a cofactor for GPX4 (a member of enzymes) to reduce the lipid hydroperoxides [24–25]. GSH could be able to reduce the accumulation of phospholipid hydroperoxides and be responsible for the detoxification. Direct or indirect inhibition of GSH can induce the initiation of ferroptosis . Therefore, it’s conceivable that, anti-cancer therapy that targeting GPX4 and /or GSH may bring satisfactory effect.
The survival analysis and prognostic ROC models were developed based on four genes (CARS1, GCLM, GLS2 and EMC2) in this study. The FRG can be roughly classified into four categories according to their functions in ferroptosis: iron metabolism, lipid metabolism, (anti)oxidant metabolism (CARS1, GCLM) and energy metabolism (GLS2, EMC2) [14–15]. Among these, GLS2 (Glutaminase) is an independent risk factor for OS in patients with EAC in our study. The human GLS2 gene is located in chromosome 12, consisting of 18 kb and 18 exons . The GLS2 could regulate the biosynthesis of GSH during the process of ferroptosis and serve as a target of p53 gene . It’s been confirmed that GLS2 has complex connections with cancers, and the up- or down-regulated expression level is significantly associated with patients’ survival in different cancers [28–30]. Hence, it’s easy to understand that the roles of GLS2 are likely to be tumor type-specific, and also enhance the notions that the implication of GLS2 in ferroptosis needs to be carefully interpreted in a context-dependent manner. These studies are in line with our results, pointing out GLS2 has positive correlation with patients’ prognosis. It’s reasonable to hypothesize the abnormal of GLS2 could promote the development of tumor through regulating the ferroptotic pathway. Although the mechanisms by which the GLS2 mediate the ferroptosis and leads to cancer remain elusive, Niu Y et al gave us a hint that the occurrence of cancer may be a consequence of ferroptosis mediated by the miRNA/GLS2 axis .
Glutathione (GSH) is the most abundant antioxidant in the cell, which synthetizes from glutamate, cysteine and glycine [14, 32]. GCLM, also known as glutamate-cysteine ligase modifier, is the first-rate limiting enzyme of GSH synthesis. The inhibition of GCLM will induce the ferroptosis. The gene expression profiles show the GCLM level is up-regulated in many tumors [33–34]. Moreover, the GCLM expression level is negatively associated with patients’ relapse free survival (RFS) and OS . Our analysis is consistent with these findings, suggesting the GCLM is an oncogene (HR༞1) in EAC. The existence of GLCM that drives ferroptosis has important implications for cancer therapy. A recent study by Sharma P identified the Andrographis, a medicine herb, could overcome the colorectal cancer chemoresistance by regulating the ferroptosis genes, such as GCLM . Therefore, drugs that target ferroptosis can be exploited and provide an efficient strategy for clinical application. CARS1 alias: CARS (cysteinyl-tRNA synthetase), is still at infancy in the area of ferroptosis.
Study has shown that knockdown of CARS1 could cause increased transsulfuration pathway activity, and resistance to ferroptosis [14–15]. EMC2, also refer to TTC35, is ER (endoplasmic reticulum) membrane protein complex subunit 2. This gene share similarities with other ferroptosis-related gene, and its knockdown suppresses erastin-induced ferroptosis .
The notion that immunity promotes or suppresses the tumor is well accepted, and one of the most impactful anti-cancer therapies developed in recent years is the immune checkpoint therapy. Our results demonstrated that the immune statue was significantly different between the low-risk and high-risk EAC patients, including the DCs, CD8 + T cells, type I IFN response, type II IFN response et.al. The enigmatic and sophisticated relations linking immunity with ferroptosis are being gradually revealed with the progress of the experimental trials in vivo and vitro. Researchers found DCs in tumor-bearing hosts accumulate plenty of lipids and PUFAs, causing the impaired ability to present the antigen and stimulate the inadequate CD8 + T cells responses, lending support to the idea that DCs and CD8 + T cells contribute to the ferroptosis through regulating the lipids and PUFAs [35–36]. As expected, experiment ex vivo demonstrated that T cell lipid peroxidation could induce ferroptosis and prevent immunity to infection in the study by Matsushita M et al . Consistent with the results of Matsushita M et al, preclinical models have confirmed CD8 + T cells could enhance ferroptosis-specific lipid peroxidation and increase ferroptosis by releasing the IFN-γ (II IFN), thus increasing the efficacy of immunotherapy . In this study, we found the contents of DCs and CD8 + T cells were significantly higher in the low-risk group than those in high-risk group. One plausible explanation is DCs and CD8 + T cells activate the ferroptosis process by releasing signals, such as IFN-γ. Additionally, we also found the type I IFN (IFN-α, β) was also higher in the low-risk group, indicating the type I IFN may be necessary to initiate the ferroptosis. Undeniably, more work is warranted to confirm the above results.
The strength of this study is that we performed a systematically analysis based on the national database for the first time, and summarized the current knowledge about the ferroptosis genes in the EAC. Notably, it should be aware that the methods provided in this study did not meet all the requirements for the gene expression levels. Meanwhile, there are some limitations in our study. Firstly, the clinical information downloaded from the TCGA is incomplete, especially the therapy, which may be helpful to understand whether FRG are biomarkers of treatment. Secondly, the mechanism how ferroptosis modulate the precise process of EAC are unclear. Lastly, the prognostic model needs to be verified in a large-scale and multicenter clinical cohort. Notwithstanding its limitations, this study does provide a comprehensive overview of FRG profiles in EAC and these limitations can be solved if there are enough data in the future.