Bladder cancer is one of the leading causes of cancer-related death worldwide. As the second most frequent genitourinary malignancy, BC is the 10th most common cancer globally according to global cancer data, with 573,278 new cases diagnosed and 212,526 deaths in 2020 according to Globocan prediction (1). The incidence and mortality of BC have been continuing to increase. Urothelial carcinoma is the most common histologic type, accounting for approximately 90% of primary BC (2). Among the newly diagnosed BC, non-muscular invasive bladder cancer (NMIBC) accounts for approximately 70% and transurethral resection of bladder tumor (TURBT) is the main treatment (3, 4). About 63.24% and 11.76% of the NMIBC patients after TURBT have tumor recurrence and progression (5). Likewise, nearly 50% of muscular invasive bladder cancer (MIBC) patients undergoing radical cystectomy still have local recurrence or distant metastasis, with a 5-year survival rate of only 66% (6). Furthermore, for 30 years, clinicians were stuck with the same, limited range of therapeutics to offer patients with BC, and 5-year survival rates were flat (7). Onset of BC is a complex process, which a multi-factor, multi-step, and multi-gene participation in (8). Therefore, a better understanding of the molecular characterization involved in tumorigenesis and the identification of novel prognostic biomarkers are essential for improving the clinical outcome of patients. The complex etiologic factors, along with the high-level heterogeneity of BC (9), make the prognosis significantly different and prognostic prediction challenging. This calls for the development of novel prognostic models.

Ferroptosis is a newly discovered iron-dependent form of regulated cell death (RCD) that is driven by the lethal accumulation of lipid peroxidation (10, 11). In 2012, it was firstly described that ferroptosis differs from apoptosis, necrosis, and autophagy in terms of morphology, biochemistry, and genetics. Ferroptosis is characterized by the rupture and blistering of cell membranes, mitochondrial shrinkage, increased membrane density, decreased or disappearance of mitochondrial ridges, rupture of outer mitochondrial membranes, and normal-sized nuclei without condensed chromatin (10). Studies have demonstrated strong association of ferroptosis with mammalian neurodegenerative diseases, cancer, and stroke (12). Since the first demonstration in 2012, ferroptosis has received widespread attention as a potential therapeutic pathway for cancer treatment. In recent years, the induction of ferroptosis has emerged as a promising therapeutic alternative to trigger cancer cell death, especially for malignancies that are resistant to traditional treatments (13, 14). Various studies have determined the key role of ferroptosis in killing tumor cells and inhibiting tumor growth (15, 16). A large number of studies demonstrated the potential clinical value of utilizing these deregulated proteins as prognostic biomarkers of malignancy (17–19). Some previous studies have also confirmed the important significance of ferroptosis for the treatment of bladder cancer (20, 21), However, whether these ferroptosis-related genes (FRGs) are correlated with BC patient prognosis remains unclear.

The objective of this study was to determine the prognostic value of FRGs in BC. mRNA expression profiles and corresponding clinical data of bladder urothelial carcinoma (BLCA) were extracted from the public databases. Ferroptosis-related differentially expressed genes (DEGs) closely associated with the prognosis were identified to construct predictive models for the prognosis of BLCA in the TCGA cohort. Then, we validated it in the GEO cohort. Besides this, functional enrichment analysis was performed to explore the underlying mechanisms.

To systematically describe our study, the flow chart of the study is shown in Figure 1 . From the TCGA RNA-seq dataset, we obtained expression data of 430 BLCA patients from the TCGA cohort and 165 patients from GEO (GSE13507). A total of 372 BLCA patients with complete clinical information were finally enrolled in the TCGA cohort. Baseline demographic and clinical characteristics of the included patents are shown in Table 1 .

Bladder cancer is a molecular heterogeneous malignant tumor; the treatment strategies for advanced-stage patients are limited. The molecular characteristics are closely related to prognosis of bladder cancer. Therefore, it is vital for improving the clinical outcome of BC patients to identify key biomarkers and targets affecting prognosis. The development of high-throughput array technology provides an opportunity to explore novel genes involved in the occurrence and development of BC. Increasing evidence has shown that ferroptosis plays a crucial role in tumorigenesis and cancer therapeutics (22). In this study, the differential expression of FRGs between BLCA tumor tissues and adjacent normal tissues were systematically investigated. FRGs associated with the prognosis of BLCA were determined by Cox proportional hazards regression analysis. Results significantly indicated the feasibility of building a prognostic model with these FRGs.

A novel prognostic model integrating nine ferroptosis-related DEGs was, for the first time, constructed and externally validated. These genes that make up the prognostic model were ALB, BID, FADS2, FANCD2, IFNG, MIOX, PLIN4, SCD, and SLC2A3. It was reported that ALB (albumin) may act synergistically with transferrin to limit iron supply, which may lead to the promotion of ferroptosis (23). The expression level of ALB was upregulated in BLCA tumor tissue compared with normal tissues ( Figure 5A ). The OS of the high-expression group was better than that of the low-expression group, which was consistent with the expression in the different risk groups based on the prognostic signature ( Figure 3C ). Ferroptosis is defined as an oxidative and iron-dependent pathway of regulated cell death, which is different from caspase-dependent apoptosis. Mitochondrial transactivation of BID links ferroptosis to mitochondrial damage as the last execution step of oxidative cell death (24). Overexpression of BID may promote the suppression of ferroptosis, indicating a worse prognosis ( Figure 5B ). FADS2 is abnormally expressed in many malignant tumors, and its expression is significantly correlated with tumor proliferation, cell migration invasion, and ferroptosis (25). Activation of FADS2 involved in the Warburg effect inhibits ferroptosis (26). Upregulation of FADS2 was associated with poor prognosis in BLCA ( Figure 5C ). SCD, like FADS2, was involved in Warburg effect (26). A study (27) confirmed that SCD was an enzyme that catalyzes the rate-limiting step in monounsaturated fatty acid synthesis in ovarian cancer cells; inhibition of SCD1 could induce both ferroptosis and apoptosis. SCD was highly expressed in ovarian cancer tissue. The expression levels of SCD in BLCA was also high ( Figure 5H ). FANCD2, a protein that mediates DNA repair, suppresses ferroptosis by transcription and transcription-independent mechanisms (28). FANCD2 is closely correlated to tumorigenesis and progression (29). A study indicated that FANCD2 expression correlated with the activation of apoptotic, cell cycle, and EMT pathways in clear cell renal cell carcinoma (30). The high expression level of FANCD2 was related to better prognosis in BLCA ( Figure 4C , 5D ), which suggests that the role of FANCD2 in BLCA may be consistent with other studies. IFNG (interferon gamma, INFγ)released from CD8+ T cells downregulates the expression of SLC3A2 and SLC7A11, two subunits of the glutamate-cystine antiporter system xc-, inhibits the uptake of cystine by tumor cells, and consequently promotes tumor cell lipid peroxidation and ferroptosis (31). Expression of IFNG was negatively associated, in BCLA patients, with patient outcome ( Figure 5E ). Overexpression of myo-inositol oxygenase (MIOX) exacerbates cellular redox injury in cisplatin-induced acute kidney injury (AKI) by accelerating ferroptosis (32). It is reasonable to assume that MIOX may play an anti-cancer role by promoting ferroptosis in BLCA. This could explain why MIOX is highly expressed in the low-risk group ( Figure 3C ). PLIN4 (Perilipins4) is one of the families of lipid droplet-associated proteins that participate in lipid metabolism regulation. It can be used as diagnostic markers of liposarcoma and to differentiate liposarcoma subtypes (33). Compared with the corresponding normal tissues, the expression of PLIN4 in BLCA tumor tissues was downregulated ( Figure 5G ), and higher expression of PLIN4 was associated with better prognosis ( Figure 5G ). PLIN4 could also be used as prognostic markers. Upregulation of the SLC2A gene that encodes the glucose transporter (GLUT) protein is associated with poor prognosis in many cancers. It was observed that upregulation of the SLC2A3 genes is associated with decreased OS and DFS in colorectal cancer patients (34). Likewise, we found that SLC2A3 expression was high in the high-risk group ( Figure 4C ). The nine FRGs were either positively or negatively correlated with ferroptosis. They were differentially expressed in different risk groups, which was consistent with their gene functions in cancers. However, not all nine genes had expression levels consistent with their functions in BLCA ( Figure 5 ). Therefore, the specific role of these genes in BLCA has to be further investigated.

We further demonstrated that the risk score of the nine-gene signature was an independent prognostic indicator of OS for patients with BLCA. The high-risk group was found to have a significantly higher percentage of BLCA patients with worse clinicopathological features, such as an advanced T stage, lymph node metastasis, and histologic grade ( Table 2 ). In addition, micropapillary carcinoma of the bladder (MPBC) is a variant type of infiltrating urothelial carcinoma, which portends a poor biological behavior in terms of disease stage at first diagnosis and clinical outcome (35). We tried to assess the risk of MPBC patients by our risk score, but unfortunately, the correlation between risk score and diagnosis subtype was not statistically significant. Thereafter, an individualized prognostic prediction model was constructed with nomograms, where the risks of individuals in the clinical context were quantified by integrating multiple risk factors including risk score. Calibration curves suggested high consistency between the actual and predicted OS rates. According to the aforementioned results, it was suggestive that the prognostic risk score model based on the nine-gene signature was a powerful prognostic indicator in BLCA patients.

To determine the role of ferroptosis-related classical signaling pathways in different risk groups, GO and KEGG enrichment analysis of DEGs in the high-risk and low-risk groups. Expectedly, the results indicated that DEGs were significantly enriched in biological oxidation, fatty acid metabolism, and iron metabolism ( Figure 5C ). These biological processes are all critical for the execution of ferroptosis (13, 17). Interestingly, many immunity-related functions and pathways were significantly enriched, such as chemokine receptor binding, humoral immune response, IL-17 signaling pathway, protein interaction with cytokine–cytokine receptor, and toil-like receptor signaling pathway ( Figures 8C, D ). ssGSEA revealed that the infiltrating scores of immune-cell and immunity-related pathways were significantly different in different risk groups. At present, many researchers have proven that ferroptosis is related to immunity. We found that NK cells, CD8+ T cells, and MHC class I molecules were significantly higher in the low-risk group ( Figures 9A, B ). A study indicated that ferritin heavy chain in tumor cells may modulate the expression of MHC class I molecules and influence NK cells (36). MHC class I molecules enable CD8+ T cells to recognize and kill tumor cells (37). CD8+ T cells release interferon (IFN)γ, and (IFN)γ can regulate the lipid peroxidation and ferroptosis pathways in tumors (31). In addition, studies have demonstrated that increased tumor-associated macrophages (38, 39) or Treg cells (39, 40) are related to poor prognosis in HCC patients due to their role in immune invasion. The fractions of macrophages and Treg cells were higher in high-risk group BLCA patients in the GEO cohort ( Figure 9C ), which were consistent with the abovementioned research results.

In recent years, immune checkpoint inhibitor treatment has become a new and promising therapy for BC. The recent IMvigor010 study (41) was designed to evaluate the role of a checkpoint inhibitor in muscle-invasive urothelial carcinoma (MIUC). Although the trial did not meet its primary endpoint of improved disease-free survival (DFS) in the atezolizumab group over observation because of higher frequencies of adverse events, we also could find that adjuvant checkpoint inhibitor therapy may have some advantages in muscle-invasive urothelial carcinoma. The stirring CheckMate274 study presented by Dean Bajorin at the 2021 ASCO Genitourinary Cancers Symposium indicated that the adjuvant nivolumab, a PD-1 immune checkpoint inhibitor, significantly improved DFS in patients with high-risk MIUC after radical surgery, especially in PD-L1≥1% patients. There was significant difference in checkpoint between the high-risk and low-risk patients in our study ( Figure 9D ). The expression of immune checkpoint molecules including PD1, PDL1, and CTLA4 was significantly higher in the high-risk group in GEO cohorts ( Figure 10B ). This indicates that patients in the high-risk group may benefit more from immune checkpoint inhibitor therapy than patients in the low-risk group and provides new insight for BC immunotherapy. Considered together, these findings suggest that poor prognosis of patients with high risk might be correlated with immunosuppression, and ferroptosis could play a role in the immunotherapy of BC.

Despite the confirmation of our prognostic model in various datasets, this study was limited because it was a retrospective study. A further well-designed prospective study is necessary to validate the clinical value of the developed model. Besides, it was inevitable that by merely considering a single hallmark to build a prognostic model, many prominent prognostic genes in BC might have been excluded.

In conclusion, a novel prognostic model based on the nine-FRG signature in BLCA was the first established and validated. The prognostic models exhibited superior predictive performance and could independently predict the prognosis of BC patients. Understanding the roles of the signature and the relationship between ferroptosis and tumor immunity can provide insights into prognostic and therapeutic implications for BC patients.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/ Supplementary Material .

Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.

YY and YH conceived and designed the study. LY and CL provided equal contributions to research design, data analysis and article writing. YQ, GZ, BZ, and ZW revised the manuscript. All authors contributed to the article and approved the submitted version.

This study was supported by Joint Special Fund for Applied Basic Research of Yunnan Provincial Science and Technology Department-Kunming Medical University (2019FE001(-087) and Innovation Fund for Postgraduates of Kunming Medical University (2021D11).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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