In the study, we downloaded RNA sequencing, clinical information from TCGA and ICGC dataset. Next, we identified DEGs between patients with > 3 year OS and < 1 year OS by integrated bioinformatics analysis of iron metabolic genes in TCGA dataset. Then, we constructed and validated an iron metabolic signature in TCGA and ICGC dataset, which showed a strong prognostic performance for predicting the survival of HCC patients. In addition, the gene signature was an independent prognostic factor by conducting Multivariate Cox regression analysis. Meantime, WGCNA analysis, GSEA analysis, Go and KEGG enrichment analysis were performed to reveal molecular mechanisms, and cell cycle therapy which may be benefit to high-risk patients. And immunotherapy response prediction was analyzed and revealed immunotherapy may be significant for high risk group. Furthermore, two independent prognostic factors were combined to build Nomogram for predicting HCC prognosis. Finally, we analyzed the expression of the six genes using qRT-PCR in twelve HCC samples.
Iron plays a crucial role in biological processes in mammalian cells by functioning as iron-and haem-containing enzymes, including the cellular stress response, mitochondrial metabolism, chromatin remodeling, DNA repair, mitochondrial metabolism, DNA replication.
thus iron is an essential nutrient that facilitates cell proliferation and growth.3 Besides, iron is reported to be associated with tumor biological process, such as tumorigenesis, angiogenesis, invasion, and metastasis.4 Iron is unraveled to participate the formation of enzymes involved in DNA synthesis and the cell cycle, and it can regulate proteins that control cell cycle, Turner J et al 1 reported that a kind of iron chelator, tachpyridine, can induce G2 arrest and promote cancer cells to be sensitive to ionizing radiation, indicating iron chelators can be considered as radioenhancing agents for cancer treatment.
Meantime, iron may promote tumour initiation via free radicals. Recently, proteins involved in iron metabolism are reported to contribute to cancer development, including iron efflux pumps, oxidases and reductases, iron regulators as well as siderophore-binding proteins.2
Ferroptosis is iron-dependent form of regulating cell death, which present different characteristics with apoptosis, necrosis.[9] Reactive oxygen species (ROS) may appear in mitochondria during iron-involving oxidative phosphorylation, and excessive ROS could induce oxidative stress response, which could damage large molecular substances such as nucleic acids, lipids and proteins.[21] Recently, an increasing number of studies has indicated that various kinds of diseases are associated with ferroptosis, including periventricular leukomalacia, Huntington's disease and renal functional damage.[22–24] In addition, many kinds of cancers have revealed the close relationships with ferroptosis, including liver cancer, osteosarcoma, prostate adenocarcinoma, cervical carcinoma and diffuse large B‐cell lymphoma.[25, 26]Ferroptosis is also confirmed to be associated with anti-tumor efficacy of chemotherapeutic drugs and immunotherapy. For advanced liver cancer, Sorafenib, a multi‐kinase inhibitor, is the first choice for treatment for 40% of newly diagnosed HCC patients.[27] which could induce and activate ferroptosis to play an anti-cancer role in HCC patients.
The prognostic model contained 13 genes (ABCB6, CCNB1, FLVCR1, G6PD, IGSF3, OSBP2, PLOD2, PPAT, RAP1GAP, RRM2, SLC7A11, CYP3A5 and CYP2C9), Six genes were significantly different between normal and cancer tissues by analyzing TCGA and ICGC dataset, qRT-PCR demonstrated the same trend, including G6PD, CCNB1, FLVCR1, ABCB6, RRM2 and CYP2C9. And survival analysis revealed the six genes were significantly associated OS,DFS, DSS, PFS using Kaplan-Meier. Thus we focused on the six genes. G6PD is a housekeeping enzyme expressed in most cells. Various studies suggested that G6PD deficiency may be protective against HCC in vitro and in vivo and patients with G6PD deficiency showed a 55% risk reduction for HCC compared with patients with normal G6PD activity.[28] Aberrant activation of G6PD is linked to tumorigenesis and malignancy in rapidly growing cancer cells. Previous studies reported that silencing of PPP enzyme G6PD prevents erastin-induced ferroptosis, which reveals G6PD act a protective role by inducing ferroptosis.[9] Now it’s known, RRM2 is one of the genes with high expression and poor prognosis in malignant tumors. During cell division, RR is essential for DNA synthesis. It encodes enzymes that catalyze the conversion of ribonucleotides to deoxyribonucleic acids. The activity of RRM2 subunit reductase requires two Fe3 + ions to form tyrosil radical on tyr122, and tyrosil radical plays a key role in the enzyme activity. [29]Animal experiments revealed that iron deficiency decreased the expression of RRM2 and reduced the protein abundance of RRM2.[30]And RRM2 may inhibit ferroptosis.[31] The expression level of CCNB1 was abnormally elevated in HCC and high expression is associated with better prognosis, as an oncogene, CCNB could inhibit the apoptosis of HCC cells and promote cell invasion.[32] FLVCR1 is a cell surface heme exporter, essential for erythropoiesis and systemic iron homeostasis.[33] The defect of FLVCR1 gene may lead to porphyrin gene overload, excess porphyrins could cause oxidative damage to the cell membrane components and eventually produce cell death.[34]Francesca Vinchi et al investigated the role of Flvcr1a in liver function in mice, the result revealed that FLVCR1 regulates heme synthesis and degradation as well as cytochromes P450 expression and activity.[35] Further study demonstrated that FLVCR1 mediates heme export from macrophages that ingest senescent red cells and regulates hepatic iron. Iron metabolism is disordered in tumors compared with normal tissues and the iron intake gene, FLVCR1, were the most relevant iron metabolism genes in Hepatocellular Carcinoma.[36] ABCB6 encode a mitochondrial transporter. It localizes to both the outer mitochondrial membrane and the plasma membrane. Expression of ABCB6 has been associated with iron homeostasis, [37] and ABCB6 may play a role in heme metabolism. Previous study has found ABCB6 can rescue the rapid growth phenotype of ATM1 deleted yeast cells, which means that ABCB6 acts as a backup system.[38] In HCC, prognostic analysis has indicated that high expression of ABCB6 was associated with worse prognosis. Because ABCB6 may inhibit the ferroptosis of liver cancer cell, and ferroptosis is considered to be a potential anticancer therapy.[39] Increased expression of cytochrome P450 CYP2C9, together with elevated level of its products epoxyeicosatrienoic acids, is associated with aggressiveness in cancer, previous study revealed that high activity CYP2C9 genotype is associated with increased risk of colorectal cancer, [40] while a low activity CYP2C9 genotype (CYP2C9*2) is associated with increased risk of colorectal adenoma [41] and lung cancer[42] and low expression is associated with poor prognosis in HCC.[43] which is consistent with our study.
As a notable immunotherapy, immune checkpoint blockade have emerged as powerful tools in the management of multiple cancer types, and immune checkpoint drugs have already been FDA approved in renal cell carcinoma, melanoma, non-small cell lung cancer, Hodgkin lymphoma and urothelial bladder cancer, including pembrolizumab, ipilimumab and nivolumab.[44] Several trials investigating the efficacy of immune checkpoint blockades in HCC are in progress (NCT02837029, NCT02947165, NCT02572687, NCT02740985, NCT01853618, NCT02668770, NCT02239900, NCT02423343, NCT01658878). The treatment of anti-PD-1 antibody nivolumab in HCC demonstrated a significant and stable response.[45] Thus it is promising for HCC by applying immunotherapy. In our study, we found risk score has a strongly positive correlation with immune checkpoints, and the high-risk subtype had significantly higher immune checkpoints expression, including PD-L1,PD-L2, CTLA-4, and TIM-3. Moreover, ImmuneCellAI indicated that high-risk group might present with a better response for immunotherapy. Thus high-risk patients may have a better response for immunotherapy.
Cell cycle relevant pathways were significantly upregulated in high risk group, which suggested cell cycle inhibitors may be more suitable for high-risk patients. CDK4 and CDK6 inhibitors have been reported to enhance the response of immune checkpoint blockade therapies in vitro.[46, 47] and CDK4 and CDK6 inhibitors could T cell exclusion signature status to get a better response in vitro.[47] Thus combination of cell cycle inhibitors and immunotherapy may be synergetic to enhance antitumous effect for patients in high risk group for HCC.
Several limitations should be acknowledged in our study. Firstly, a multicenter and prospective cohorts are needed to validate our results. Secondly, effect of combination of cell cycle inhibitors and immunotherapy is also needed to elucidate in vivo and in vitro experiment.