Human skin cutaneous melanoma (SKCM) is a highly malignant tumor derived from melanocytes, that is prone to occur in adults. The mortality rate of melanoma is up to 75% (Davis et al., 2019). Early-stage, localized melanoma can be curable if appropriate and sufficient treatment is administered (Coit et al., 2013) ; however, the tumor tends to metastasize and spread to other parts of the body (Aris and Barrio, 2015), once metastasized, the 5-years survival rate decreases to only 15% (Coit et al., 2013). SKCM is now the third most commonly diagnosed cancer in the United States, with an estimated 192,000 new cases in 2019. The incidence is six times higher than 40 years ago (Welch et al., 2021). Surgical resection is associated with a satisfactory prognosis for melanoma in the early stages, while treatment of metastatic melanoma mainly relies on immunotherapy (Leonardi et al., 2020). Many studies have explored the relationship between cancer cells, the tumor microenvironment (TME), and the immune system. However, not all treatments that block immune suppression control points are effective for all patients (Rodríguez-Cerdeira et al., 2017). Therefore, identifying robust predictive biomarkers for both clinical prognosis and treatment response is essential.

In recent years, tumor ferroptosis has gathered much interest. Iron is involved in many biochemical processes in the human body, including oxygen transport, various biosyntheses, and the electron transport chain (as a cofactor), playing crucial roles in cell survival (Bogdan et al., 2016). In mitochondrial oxidative phosphorylation, cells produce reactive oxygen species (ROS) while generating ATP. Excessive ROS levels will lead to oxidative stress, directly or indirectly damaging macromolecules, such as proteins, nucleic acids, and lipids, leading to cell damage or death (Yu et al., 2017). Ferroptosis is an iron-dependent form of programmed cell death, which differs from apoptosis, cell necrosis, and autophagy.

The mechanism of ferroptosis is based on influencing iron metabolism in cells, resulting in intracellular ROS production and excessive oxidation of polyunsaturated fatty acids (Dixon et al., 2012; Battaglia et al., 2020). Ferroptosis is mainly regulated by system X_C ⁻ and glutathione peroxidase 4(GPX4). System X_C ⁻ is a Na⁺-dependent cysteine—glutamic acid exchange transporter in the membrane, which completes the intracellular and extracellular glutamate—cysteine exchange (Sato et al., 1999; Bridges et al., 2012). Cell uptake of cysteine is a crucial step in glutathione (GSH) synthesis, and the generation and maintenance of GSH is the key to protecting cells from ROS damage (Yu et al., 2017). GPX4 is an enzyme that decomposes H₂O₂ and organic peroxides into water or the corresponding alcohols, and GSH is an indispensable cofactor in its activation (Ursini et al., 1995). The ferroptosis inducers, erastin and buthionine sulfoximine, reduce the activity of GPX4 and increase the level of ROS in the cytoplasm and lipid (Yang et al., 2014), leading to cell ferroptosis. Ferroptosis is considered as an adaptive process to eliminate malignant cells damaged by nutrient deficiency, infection, or other stress from the body (Mou et al., 2019). A number of studies have demonstrated that ferroptosis plays a role in ischemia-reperfusion injury, cancer, and other diseases. At present, sorafenib and other ferroptosis inducers are used for the treatment of cancer in clinical practice (Fearnhead et al., 2017).

Non-cellular components in the TME may reprogram tumor initiation and invasiveness, but the relationship between iron metabolism and TME is still unclear (Sacco et al., 2021). Tumor-associated macrophages loaded with iron can promote the production of ROS and pro-inflammatory cytokines (tumor necrosis factor-α and interleukin-6), thereby inducing tumor cell death in lung cancer (Costa da Silva et al., 2017). Moreover, new evidence suggests that immune checkpoint blockade decreases tumor growth in a ferroptosis-dependent manner in animal models (Tang et al., 2020), which is considered to be related to antitumor immunity. In addition, the immunotherapy-activated CD8⁺ T lymphocytes induce ferroptosis in cancer cells by downregulating genes (SLC7A11 and SLC3A2) that encode two subunits of the X_C ⁻ system, and the molecular basis behind this phenomenon may be related to interferon (IFN)-γ-mediated transcriptional repression of SLC7A11 and SLC3A2 (Wang et al., 2019). Consequently, identifying biomarkers of iron metabolism within the TME may aid the development of effective cancer treatment strategies.

Focusing on melanoma, many studies have found ferroptosis regulators, as well as traditional ferroptosis-inducing agents. It was found that miR-9 reduced erastin- and RSL3-induced ferroptosis in melanoma cells, and knockout of miR-9 could cause ferroptosis in melanoma cells (Zhang et al., 2018). In addition, the inactivation of miR-137 enhances the anti-melanoma activity of erastin by increasing ferroptosis (Luo et al., 2018). However, it is still unknown whether ferroptosis-related genes are related to the prognosis of SKCM patients.

In the present study, we conducted a comprehensive analysis based on transcript and clinical data obtained from The Cancer Genome Atlas (TCGA) and the FerrD databases. We constructed a predictive model on account of eight ferroptosis-related genes. The model can be regarded as an independent predictor of overall survival (OS) of SKCM. A nomogram was established to further explore the prognosis of SKCM based on risk score and tumor stage. Furthermore, we analyzed the mutational differences of ferroptosis-related genes in high-risk and low-risk groups and the associations between the ferroptosis-related risk score and immune cell infiltration patterns and immunotherapy. External validation cohorts were used to verify the ferroptosis-related risk score predicting the response to immunotherapy of the two subgroups.

Cell death depends on special regulated molecular mechanisms regulation called regulated cell death (RCD) (Galluzzi et al., 2018). Modern medical researches uses these unique biological processes to treat cancer by allowing cancer cells to die, relying on dedicated molecular machinery pharmacologically or genetically. Caspase-dependent apoptosis is a generally recognized RCD process against cancer (Liang et al., 2019). However, tumor cells have the characteristics of resistance to apoptosis, and drug resistance will also occur in the process of chemotherapy-induced apoptosis of cancer (Gottesman et al., 2002). Therefore, it is necessary to find some new forms of RCD to develop anticancer drugs.

Ferroptosis is an iron-dependent form of RCD characterized by the overwhelming accumulation of lethal lipid peroxidation; it is different from apoptosis, necrosis, and autophagy (Dixon et al., 2012). Ferroptosis can be triggered by exogenous small molecules (such as erastin, sorafenib, or sulfasalazine) or regulating physiological conditions (such as the high extracellular glutamate concentration) to block the X_C ⁻ system. Other ferroptosis inducers can directly inhibit GPX4, ultimately leading to lipid peroxide accumulation (Liang et al., 2019). Increasing oxidizable polyunsaturated phospholipids or interfering with the iron balance to destroy the balance of lipid metabolism balance can also sensitize cells to ferroptosis, Strong iron dependence can make cancer cells more susceptible to iron overload and ROS accumulation, enabling tumor microenvironment-targeted, ferroptosis-mediated cancer therapy (Vigil et al., 2010; Hassannia et al., 2019). Studies have found that clear cell carcinomas, highly aggressive malignancies, are usually not susceptible to conventional anticancer treatment. However, their unique metabolic state has been identified to be susceptible to ferroptosis (Zou et al., 2019). In addition, a large number of studies have confirmed the crucial role of ferroptosis in inducing cancer cells death and inhibiting tumor growth. The metabolic status of breast cancer, liver cancer, colorectal cancer, and other malignant tumors is closely related to ferroptosis (Sun et al., 2015). However, most current studies focus on the role of iron metabolism in cancer occurrence and treatment, and the relationship between genes related to ferroptosis and cancer prognosis remains to be explored.

Melanoma cells have a highly mutagenic nature and an immune escape mechanism (Davis et al., 2019), including downregulation of the expression of tumor-associated antigens and melanoma differentiation antigens to inhibit cytotoxic T cell recognition and clearance of tumor cells and secretion of immune inhibitory molecules such as transforming growth factor-beta (TGF-β) and prostaglandin E2 to escape immunity (Palmer et al., 2011; Pitcovski et al., 2017). In addition, melanoma can close the immune response by expressing programmed cell death protein 1/2 (PD-1/2) to avoid immune destruction (Passarelli et al., 2017). So far, the most effective treatment for metastatic melanoma is immune checkpoint inhibitors, such as anti-PD1, PD-L1/2, and CTLA4 antibodies. Still, the complications of these treatments are serious, and many of patients have inadequate treatment responses. So, the combination of ferroptosis and immunotherapy may have good prospects for melanoma therapy. Studies have found that immunotherapy activated CD8⁺ T cells enhance iron-specific lipid peroxidation in tumor cells, and increased ferroptosis contributes to the antitumor effect of immunotherapy (Wang et al., 2019, 8). At present, the relationship between ferroptosis and tumor immunotherapy is still not very clear. Nevertheless, the establishment of some prognostic signatures can be established that use the ferroptosis-related genes to evaluate different tumor immune characteristics to guide individualized immunotherapy (Zhuo et al., 2020; Jiang et al., 2021).

This study found that the ferroptosis-related genes can classify melanoma patients into two classes that exhibit significant differences in clinical and molecular features. Patients were classified into high-risk and low-risk groups by LASSO regression analysis. We established a prognostic model based on eight genes, which were composed of the risk-related genes (CHAC1, CS, GPX2, and ALOX12B) and the protective genes (PML, GCH1, TFAP2C, and NOX4). The GCH1-BH4 pathway is a new pathway that is independent of the GPX4/glutathione system and regulates ferroptosis. GCH1 is a rate-limiting enzyme for the synthesis of tetrahydrobiopterin. Overexpression of GCH1 can eliminate lipid peroxidation and almost completely inhibit ferroptosis (Wei et al., 2020). In high-grade serous ovarian cancers, due to the silencing of PML, ROS content, lipid peroxidation, and lysosomes, and the lysosomal Fe2⁺levels are reduced, which can result in potential ferroptosis and improved sensitivity to immunotherapy (Gentric et al., 2019). NOX4 encodes ROS-producing enzymes enriched in the kidney, where high expression is an essential source of renal ROS. Meanwhile, inhibition of NOX4 reduces the cystine deprivation-induced cell death and lipid ROS, suggesting its vital role in ferroptosis (Yang et al., 2019). TFAP2C plays an essential role in cell differentiation, tissue development, and tumor biology. TFAP2C upregulates the GPX4 gene in tumor cells and regulates some ferroptosis regulators, such as epidermal growth factor receptor (EGFR), CDKN1A, and YAP1, thereby negatively regulating ferroptosis (Dai et al., 2020). Mitochondria play a central role in fatty acid metabolism and provide specific lipid precursors for lipid oxidation. CS participates in mitochondrial lipid metabolism and regulates the activation and synthesis of fatty acids. Silencing CS can rescue cell viability from erastin-induced ferroptosis (Wang et al., 2020). ALOX12B encodes lipoxygenase, which is associated with autosomal dominant fish scale disease and proliferation of epidermoid carcinoma cells (Jiang et al., 2020). Glutathione-specific g-glutamyl cyclotransferase (CHAC1) is a downstream target of the eIF2alpha-ATF4 pathway, and CHAC1 upregulation may be useful as a Pharmacodynamic marker for cystine or cysteine-starved cells (Dixon et al., 2014). Overexpression of CHAC1 led to a robust depletion of glutathione, which was alleviated in a CHAC1 catalytic mutant. On activating the expression of ATF4 and CHAC1, the initial glutathione depletion by inhibiting cystine transport leads to ferroptosis (Ratan, 2020). In summary, numerous studies have shown that the above genes are related to ferroptosis, providing theoretical support for our risk model.

After establishing the ferroptosis-related risk model, our samples were divided into high-risk and low-risk groups according to the median risk score. We used statistical methods such as univariate and multivariate Cox regression analysis to show that the risk score model is an excellent independent prognostic indicator for evaluating the overall survival of melanoma patients. Subsequently, we developed a nomogram, combined with multiple clinical indicators and biological attributes, set up a personalized prognostic prediction scale, and quantified the risk of different individuals with numerous risk factors. In addition, we also analyzed the immune correlation between high- and low-risk groups and found that the risk score could indicate the relationship between prognosis difference and immune tissue infiltration, which could better guide the immunotherapy of SKCM.

Finally, our research also has certain limitations. As a retrospective study, it cannot fully contain all clinical data, so there will be some limitations in variable selection, which must be verified by selecting as many cohorts as possible. In addition, there should be prospective studies to further evaluate the clinical value of our model. Finally, a series of experiments should be carried out to explore the further evaluate the prognostic value of the eight ferroptosis-related gene signature.