According to the cancer statistics report, more than 65,000 patients were diagnosed with RCC, and nearly 15,000 deaths happened in RCC patients in USA every year[33]. About one third of patients with localized RCC recurred or had metastases after surgical treatment[34]. Currently, the prognosis of KIRC is unsatisfactory, with tumor recurrence in about 30% of patients who are considered disease-free. Since sensitive biomarkers for KIRC have not been identified, we hope to explore and identify effective therapeutic targets and useful prognostic biomarkers through the molecular mechanisms of KIRC occurrence and progression.
The present studies showed that selective induction of cancer cell death had become the most effective method of tumor treatment[35~36], and ferroptosis, as a programmed cell death, can regulate the occurrence and progression of tumor cells. Wang et al[37] found that CD8 + T cells can regulate cell ferroptosis to affect the therapeutic effect of tumors. Therefore, it is important to study the relationship between ferroptosis and the immune micro-environment of KIRC, which may change the current pattern of surgical resection, chemotherapy, and radiotherapy in KIRC patients[34], and effectively improve the OS of advanced KIRC patients. The purpose of this study is to investigate the relationship between ferroptosis and the immune micro-environment in KIRC by comprehensive bioinformatics analysis, and to identify effective prognostic biomarkers associated with ferroptosis in KIRC. Finally, therapeutic targets related to ferroptosis were explored and potential drugs for the KIRC treatment were predicted.
In this study, we firstly cross-analyzed the DEGs, which from GSE168845, GSE105261 and GSE11151, with the ferroptosis genes from the FerrDb database, and obtained 17 ferropsis-related DEGs. In this process, it was important to note that since there was only 1 fully overlapped gene, we did not select the intersection of the four datasets, but the overlapped gene of the FerrDb database and each GSE dataset. Then, GO/KEGG analysis of 17 ferroptosis-related DEGs revealed that these genes were associated with iron ion binding, microvillus membrane and regulation of transcription from RNA polymerase II promoter in response to stress. Next, univariate and multivariate Cox regression analyses were used to identify ferroptosis genes associated with poor prognosis. MT1G, LAMP2 and MIOX were identified as independent genes with prognostic value, and a prognostic model was constructed based on this. Subsequently, we explored the immune cell infiltration of the TME by TIMER. We found the proportions of B cells, CD4 + T cells, CD8 + T cells, macrophages, neutrophil and dendritic cells were statistically significant. we also studied the correlation of MT1G, LAMP2 and MIOX expression levels and their methylation status. The results suggested that methylation of m6A had an influence on the MT1G, LAMP2 and MIOX in KIRC. Meanwhile, the analysis of the TF-gene interactions and TF-miRNA coregulatory network delivered miRNAs and TFs interaction with MT1G, LAMP2 and MIOX. Finally, Cmap and PubChem22 database were used to predict potential drugs for treating KIRC by ferroptosis mechanism against MT1G, LAMP2 and MIOX, such as emetine, cephaeline, scoulerline, sanguinarine, cicloheximide, tolfenamic acid, phenoxybenzamine and calmidazolium.
In 2016, sun et al published an article in the authoritative journal "pathology", which found that MT1G promoted the drug resistance of hepatocellular carcinoma to sorafenib by inhibiting ferroptosis[38]. MT1G is a kind of small molecular protein with highly conserved structure, which can induce metal responsive elements and regulate the expression of related genes by combining with metal ions[38]. Lysosomal granule membrane protein (LAMP2) is chiefly expressed in placenta, lung and liver. In the past, it was considered to be a structural protein. Recent studies suggest that LAMP2 not only performs the function of structural protein, but also participates in autoimmune diseases, autophagy formation, endosome fusion, cholesterol transport, liver fibrosis and other physiological and pathological processes[39]. Myo-inositol oxygenase (MIOX) is a proximal tubule specific enzyme that breaks myo-inositol into D-glucuronate[40] and damages the renal tubules through ferroptosis[41]. Because the MIOX promoter has osmotic response elements, carbohydrate response elements, oxidation response elements and sterol response elements, its transcription is regulated by high sugar, oxidative stress, and free fatty acids[42].
Interestingly, with MT1G, LAMP2 and MIOX probes, we had identified 8 potential drugs for the treatment of KIRC through ferroptosis mechanisms. Four of them were alkaloids, such as emetine, cephaeline, scoulerline and sanguinarine. At present, lots of studies had proved that alkaloids screened from medicinal plants and Chinese herbal medicines have anti-proliferation, anti-metastasis and other anti-cancer effects on a variety of tumors in vivo and in vitro[43~44], including RCC and KIRC[45]. Atma, B, et al found that soyauxinium chloride could activate caspases3/7/8/9, change metalloproteinase and increase ROS to cause ferroptosis in CCRF-CEM cells[46]. Lu, S. et al also found that brucine could induce upregulation of ATF3 by endoplasmic reticulum stress to cause the accumulation of H2O2, which result in iron increase, transferrin receptor up-regulation and lipid peroxidation, eventually leading to ferroptosis in glioma cells[47]. However, no studies have shown whether alkaloids can affect the development of KIRC through ferroptosis mechanisms. The exploration of potential drugs is expected to provide a new research direction and theoretical basis for the treatment of KIRC.
In addition to this study, we noted that Lu et al[48], Liang et al[49] and Liu et al[50] respectively explored ferroptosis-related genes in different tumors and established a prediction model for OS. But we're using a different approach, and we're doing a more comprehensive search for ferroptosis-related genes and predicting potential drugs. We believe that this study will provide new insights into the immunotherapy of KIRC.