FDX1 is a crucial regulatory protein of cuproptosis since copper-related reductases can be encoded by FDX1, which converts less toxic Cu2+ ions to more toxic Cu1+ ions [5]. Also, FDX1 can modify protein lipoylation which is a highly conserved lysine post-translational regulation. The deletion of FDX1 results in the depletion of succinate and the accumulation of pyruvate and α-ketoglutarate. Thus, the knock-out of FDX1 can lead to compromised protein lipoylation and rescue cells from copper toxicity. Furthermore, FDX1 has a promotional effect on the lipoylated oligomerization of dihydrolipoamide S-acetyltransferase owing to binding to lipoylated proteins of the tricarboxylic acid (TCA) cycle [5].
Taken together, I hypothesized that FDX1 might be an unrecognized biomarker of cancer. Thus, I conducted a pan-cancers analysis (33 cancer types) based on the TCGA database due to these pivotal functions of FDX1 on Cuproptosis. I found that the FDX1 expression level between tumor tissues and adjacent normal tissues was significantly different in 14 cancer types. The FDX1 expression level was lower in tumor tissues of KIRC. Afterward, FDX1 was determined as an independent protective factor of survival in KIRC by Cox and Kaplan-Meier model. Furthermore, in KIRC tumor tissue, immune genes involving the gene of antimicrobials, cytokine, chemokine receptors, NK cell cytotoxicity, antigen processing and presentation, TCR signaling pathway, and immune Checkpoints had a co-expression relationship with FDX1. They can work together with clinical factors including age, gender, stage, and risk scores to generate prognostic models through the Nomo plots. In reviewing three recent papers, the authors stated that FDX1 might be a prognosis impact factor of lung adenocarcinoma, hepatocellular carcinoma, and colon adenocarcinoma due to the mechanism of copper-induced cell death [11, 15, 16]. Another recent pan-cancers analysis also found that the FDX1 expression levels were down-regulated in tumor tissues of KIRC, ACC, HNSC, THCA, and LGG. In their pan-cancers analysis, FDX1 was considered to be a potential prognostic biomarker of immunotherapy due to its significant association with clinical characteristics, tumor mutational burden, microsatellite instability, immune-related signal pathways, immune cell infiltration, and antitumor drug susceptibility [17]. These results were consistent with our findings.
Considering immune infiltrate, tumor tissue is commonly infiltrated by various immune cells that secrete cytokines and chemokines to impact the efficacy of clinical cancer treatment by regulating the tumor microenvironment. Hence, exploring the underlying mechanisms of immune infiltration is a promising strategy for KIRC treatment [18–21]. In the present study, T CD 8+ cells, B memory cells, and NK cells have negative correlations with the FDX1 expression levels. In general, both T CD8+ cells and Memory B cells are adaptive immunity. T CD8+ cells can clear intracellular pathogens to provide long-term protection [22]. Memory B cells can provide protective immunity against infectious agents. They have a stronger affinity for immunoglobulin (Ig) genes, which are determinants for their malignant transformation levels in oncogenic events [23]. NK cells are cytotoxic lymphocytes, which are involved in early defense and tumor immune surveillance. NK cells directly exert anti-tumor effects by interacting with other immune cells to attack tumor cells [20]. Therefore, the FDX1 expression levels had a negative correlation with the level of immune infiltrates. Moreover, I found that macrophage M1s and M2s, and Mast cells have positive correlations with the FDX1 expression levels. In chronic inflammatory conditions, macrophage cells generate cytokines, tissue repair factors, and angiogenic factors to promote tumor angiogenesis [24–26]. Similarly, mast cells can release vascular growth factors such as fibroblast growth factor 2 [27, 28]. Normally, the infiltrated macrophages and mast cells in the tumor are conducive to the proliferation of tumor cells. Thus, these findings supported the previous view that the FDX1 expression levels are negatively correlated with the level of immune infiltrates. The publication by Zilong Bian et al. also presented that the FDX1 expression level was positively related to the abundance of macrophages in KIRC tissue [29]. Likewise, in the study of Zhen Zhang et al., hepatocellular Carcinoma patients with FDX1-related high-risk gene scores showed high levels of protumor immune infiltration [11]. However, FDX1 was negatively associated with T regulatory cells. It was inconsistent with the above view since Treg cells suppress immune function by releasing suppressive cytokines, leading to cell lysis and apoptosis of T cells, and cutting off the ATP supply of effector T cells [30–32]. One likely explanation presented by Winerdal et al. was that the low infiltration levels of Treg cells were associated with a positive prognosis in patients with cancers since Tregs probably inhibited the expression levels of macrophage genes and the invasive factor metalloproteinase 2, which reduced the invasiveness of tumor cells [31]. In light of these considerations, FDX1 probably affects the occurrence, progression, and metastasis of tumors.
The immune cell regulations on cancers are really complex and most of their principles are not discovered yet. While it goes far beyond our understanding, intensive studies have revealed many fundamental mechanisms over the past decades. For example, the Th17 cell is a subset of CD4+ T cells. The differentiation of Th17 cells is regulated by the TCR signal pathway. Pathogenic Th17 cells and dysfunction of the TCR signal pathway result in multiorgan inflammatory diseases. It is clear that cancer progression and the response to immune therapy are impacted by inflammation [33]. In the present study, I found that FDX1 was involved in multiple immune and inflammation functions since FDX1 and its co-expressed immune genes were enriched in TCR and Th17 cell signal pathways, and other biological process responses, including NK cell-mediated cytotoxicity, Leukocyte transendothelial migration, cell-cell adhesion, phagocytic vesicle, cell proliferation of leukocyte and mononuclear, and plasma membrane. Particularly, FDX1 and its co-expressed immune genes had enrichment effects on the immunological synapse. It is a connection point connected by the chimeric antigen receptor (CAR) between a NK cell and a target cell. It enables a CAR-NK complex to recognize potential antigenic ligands, activate the immune response programs, and secrete some cytolytic molecules including secretory granule lumen and specific granules such as platelet alpha granule and tertiary granule membrane. Cell injury and tissue damage were eliminated by increasing cell number and cell life span[34–37].
On the other hand, chronic inflammation can promote tumor cell growth and induce chemotherapy resistance. In our study, FDX1 and its immune genes had enrichment effects on multiple signaling pathways of Cytokine and Chemokine such as NF-kβ, TLR, and TNF pathways. TNF normally exists in immune cells. It is a cytokine that can directly kill tumor cells due to a systemic inflammatory response. TNF and CD40 (one of the molecules of the TNF receptor family) can coactivate many signal pathways including the NF-kβ and Mitogen-Activated Protein Kinase (MAPK) signal pathways [38, 39]. Similarly, TLR can also trigger NF-kβ and MAPK signal pathways through the interactions of the TLR and myeloid differentiation factor 88 structural domains[40]. Morikawa T et al. reported that TLR was overexpressed in tumor tissues. Activating TLR and its ligand could increase interferon β expression and cell growth inhibition [41]. In addition, with the development of tumor immunotherapy, immune checkpoint inhibitors had already achieved encouraging successes over the last 10 years, particularly in the PD-1 signal pathway. PD-1 is an immune receptor in the CD28 series, when PD-1 binds to PDL-1, the cytotoxic T cells are suppressed and Treg cells are activated. However, if the bindings of PD-1 and PDL-1 are broken by immune checkpoint inhibitors, this immune escape mechanism is no long hijacked [42]. The present analysis of KEGG indicated that FDX1 and its co-expressed immune genes were enriched in the PD-1 signal pathway. Another systemic pan-cancer study by Cai et al. also illustrated that PD-1 was significantly upregulated in KIRC tumor tissue[7]. Collectively, FDX1 is likely an immune therapeutic target for KIRC.
Although the findings of this bioinformatic study provided useful clues on the correction between the FDX1 expression levels and the prognostic of KIRC due to the impact of Cuproptosis and immune infiltration, the underlying molecular mechanisms were not revealed clearly. More in vitro experiments, cancer cell-culture studies, animal tumor models, and human clinical trials are warranted to further explore and verify the molecular mechanisms of FDX1 on KIRC in detail. After all, correlations do not prove causations.