Clear cell renal cell carcinoma (ccRCC) is a primary renal subtype that is often asymptomatic initially, leading to late diagnosis. Despite effective resection methods, the complex biology of cancer can lead to postoperative recurrence or distant metastasis. Furthermore, ccRCC exhibits significant resistance to conventional radiotherapy and chemotherapy [11]. Mitochondria are important eukaryotic organelles that play an important role in various types of apoptosis, including necroptosis [12]. Necroptosis is characterized by caspase-independent features, similar to necrosis with features such as cell swelling and membrane rupture [8].

Recent studies explore the role of mitochondrial genes (MTGs) in clear cell renal cell carcinoma (ccRCC). Zhang et al. identified MFN2 as a potential prognostic marker [13]. Yan et al. found that inhibiting NR3C1 restricts ccRCC proliferation and migration via stress-mitophagy [11]. Luo et al. proposed MFN2's role in regulating ccRCC progression through mitochondria-dependent EGFR dephosphorylation [14]. Li et al. developed a risk prediction model for necroptosis-related genes in ccRCC [15]. Despite these findings, the prognostic relevance of necroptosis-related mitochondrial genes (nc-MTGs) in ccRCC remains limited. This study aims to further explore the association between nc-MTGs and ccRCC prognosis.

In this study, we analyzed the TCGA database to extract Differentially Expressed Genes (DEGs). Integrating Mito Carta 3.0 and KEGG Pathway datasets, we identified six necroptosis-related mitochondrial genes (nc-MTGs) linked to prognosis—BID, FKBP10, PRELID2, FDXR, POLG2, and ACADM. Using single-factor Cox, LASSO, and multi-factor Cox regression analyses, we established a prognostic model for ccRCC, categorizing patients into high and low-risk groups. Kaplan–Meier, heatmap visualization, and ROC analysis validated the model's robust predictive capacity. External validation used the E-MTAB-1980 dataset. Analysis of immune-related factors and drug sensitivities highlighted differences between the different groups. Elevated BID, FKBP10, and POLG2 expression in the high-risk group suggests their role as risk factors, associated with a poorer ccRCC prognosis.

Recent studies have found that BID is one of the only proteins in BH45 that controls BAK and BAX and is considered to play a key role in the apoptosis signaling pathway [16]. Hu et al. contend that elevated expression of BID is linked to unfavorable prognosis in ccRCC patients [17]. The FK506-binding protein (FKBP) is a family of intracellular receptors that can bind specifically to the immunosuppressant FK506 and rapamycin [18]. Cai et al. proposed that FKBP10 promotes glioma cell proliferation by activating the AKT-CREB-PCNA axis [19]. High VSX1 expression promotes the aggressiveness of clear cell renal cell carcinoma through transcriptional regulation of FKBP10[20]. Polymorphisms in POLG2 gene thought to be genetically associated with aggressive bladder cancer in Japanese men [21]. Inhibition of ACADM-mediated fatty acid oxidation by Ma et al. promotes hepatocellular carcinoma through the CAV1/SREBP1 signaling pathway [22], but its role in ccRCC requires further study. Wang et al. claimed that PRELID2 is involved in follicle development in polycystic ovarian syndrome (PCOS) [23], but the role of PRELID2 in tumors requires further exploration. Ferredoxin reductase (FDXR) is a target of p53 and regulates p53-dependent apoptosis. Zhang et al. suggest that FDXR and p53 regulate each other and that the FDXR-p53 loop is critical for tumor suppression through iron homeostasis [24].

Through a meticulous examination of pertinent clinical parameters, we identified four autonomous prognostic factors (age, grade, stage, and risk score) pivotal in assessing the predictability of our risk model. The receiver operating characteristic (ROC) curve substantiated the model's robustness in forecasting 1-, 3-, and 5-year survival rates. To enhance the model's practical utility in clinical settings, a nomogram was employed to graphically represent the survival prognosis for ccRCC patients. Its validity was rigorously assessed and confirmed using an external dataset (E-MTAB-1980), establishing the nomogram's prowess in forecasting patient outcomes as evidenced by the calibration curves.

The GO and KEGG analyses delineated the engagement of these necrosis-related mitochondrial genes (nc-MTGs) in fundamental cellular processes. These genes are implicated in apoptosis, redox reactions, dysregulation of cancer transcription, immune checkpoint pathways, and the chemical induction of reactive oxygen species. Mitochondria, recognized as pivotal organelles, play a multifaceted role by generating energy and actively participating in a spectrum of biological processes. Their malfunction can lead to damage in enzymes of the tricarboxylic acid (TCA) cycle, leakage in the electron respiratory chain, and consequent oxidative stress. This results in a significant alteration in cellular metabolism and signal transduction pathways, contributing notably to apoptosis and resistance to therapy, prominently influential in the development of various human cancers [25]. Simultaneously, mitochondria serve as synthetic organelles capable of not only producing adenosine triphosphate (ATP) but also synthesizing macromolecules. This versatility allows them to cater to the diverse metabolic requisites of different immune cells [26]. Singhal et al. identified metformin, an anti-diabetic medication acting on mitochondrial complex I, as an immunomodulator in tuberculosis [27]. This finding suggests that targeting human mitochondrial metabolism can influence the immune response in disease scenarios. In our study focused on clear cell renal cell carcinoma (ccRCC), we observed promising results for immunotherapy and targeted therapy. Up to now, more than ten kinds of targeted drugs and immune drugs have been approved for the treatment of advanced renal cell carcinoma at home and abroad. Tyrosine kinase inhibitors and VEGF antibodies are widely used as first-line or sequential therapy for renal cell carcinoma. To date, nine drugs have been approved by the U.S. Food and Drug Administration (FDA) for first-line or second-line treatment of advanced RCC: sunitinib, sorafenib, pazopanib, axitinib, temsirolimus, everolimus, bevacizumab + interferon, cabozantinib, and Lenvatinib [28]. At present, the effective rate of targeted therapy is about 30%. Most targeted drugs have significant effect on medium- and low-risk patients, but the effect is not obvious in high-risk patients [29]. Immunotherapy drugs used in the treatment of renal cell carcinoma in China include nivolumab, pembrolizumab, interleukin 2 and recombinant human interferon α2b.For medium to high-risk patients, dual immunotherapy is superior to single targeted therapy [30]. Three large Phase III clinical studies of PD1/PD-L1 in combination with TKI for first-line treatment of RCC, Immotion-151, JAVELIN Renal 101, and KEYNOTE-426, have also achieved breakthrough results, which also provide clinicians with more treatment options [29, 31, 32].

Targeting mitochondrial metabolism to influence immune responses in disease situations remains to be further explored, so we analyzed the relationship between necrosis-associated mitochondrial genes (nc-MTGs) and the tumor immune microenvironment. Immunocyte infiltration analysis showed that higher risk scores were positively correlated with T cell CD4 memory activation, Tregs, and macrophage M0, and negatively correlated with mast cell quiescence, lymphocytes, CD4 + T cells, macrophages M1, and dendritic cells. Increased infiltration of Treg cells has been demonstrated in progressive ccRCC, suggesting an association with poor prognosis. [33, 34]. Conversely, stasis in dendritic cells, mast cells, and eosinophils has exhibited a notable correlation with enhanced prognosis [35]. IFNγ and lipopolysaccharide (LPS) have demonstrated the capability to instigate a proinflammatory macrophage phenotype (M1), and several factors associated with M1 have been linked to protracted survival in ccRCC [36]. We also used the TISCH database to probe BID expression in single-cell immune populations associated with tumor microenvironment (TME). Our results showed that BID is mainly present in monocytes/macrophages (Mono/Macro) and dendritic cells (DC), and previous studies have demonstrated the role of the necrosis-apoptosis axis in macrophages. Our results therefore support the conclusion that BID overexpression is positively correlated with risk score and suggests a poor prognosis. With the approval of immune checkpoint inhibitors targeting PD-L/CTLA-4, overall survival has improved for many cancers [37, 38]. When we compared immunotherapy response among different risk groups, we noted that in CTLA-4 positive/PD-L renal cell carcinoma patients, the response rate to immunotherapy was significantly higher in the high-risk group than in the low-risk group, while there was no discrepancy in the efficacy of immunotherapy among different risk groups in CTLA-4 negative/PD-L patients. Examination of immune checkpoint-related genes revealed that the expression levels of CD40LG, CTLA4, LGALS9, PD-1, TDO2, TIGIT, TNFRSF4, TNFRSF18, and TNFRSF14 were significantly increased in the high-risk group. We generally believe that the higher the expression of immunosuppressive molecules, the better the effectiveness of immune checkpoint inhibitors. Therefore, whether we believe that immune checkpoint expression levels are not the only determinant of immunotherapy, the immune microenvironment of the tumor may be related to this, and as mentioned earlier, a high proportion of patients are in an immunosuppressive microenvironment. In addition, our analysis delves into the relationship between different risk groups and drug sensitivity. The study found that different targeted drugs have different efficacy in different risk groups. For example, high-risk populations are more sensitive to NK inhibitors VIII, PD.0325901, Salubrinal, Bosutinib, Dasatinib, PD.0332991, Pazopanib, Sunitinib, Tipifarinib, and Imatinib. In summary, immunization + targeted therapy is also a good option for high-risk patients. This further increases the reliability of our findings and provides insights into clinical treatment.

We started our experiments with several renal carcinoma cell lines. From these cell lines, we isolated RNA and performed a comparative analysis of BH3 interacting domain death agonist (BID) expression between normal and tumor cells. The results of our PCR analysis clearly indicate that BID is up-regulated in tumor tissue. Subsequently, we will explore the effects of BID reduction on tumor cells by functional experiments in CAKI and ACHN cell lines, where we reduced BID expression by small interfering RNA (siRNA). The results showed that the intervention significantly inhibited renal cell migration, but had no effect on cell proliferation. Simultaneously, we are endeavoring to achieve a more comprehensive understanding of the underlying mechanisms involved in this context.

Our study shows that nc-MTGs have important clinical significance in the prognostic assessment of ccRCC patients. The key roles of these molecules in apoptosis, energy metabolism and antioxidant stress make them potential therapeutic targets and prognostic markers. In clinical settings, by detecting the expression levels of these nc-MTGs, high-risk patients can be identified earlier and more accurately for individualized treatment. As biomarkers, nc-MTGs can provide detailed information about the metabolic state and apoptosis mechanism of tumor cells, complementing and improving the existing prognostic assessment system. By incorporating these molecules into the assessment system for ccRCC patients, we can more accurately predict patient progression and treatment response, optimize treatment strategies, and improve outcomes.

Although genes associated with mitochondrial dysfunction and necrotizing apoptosis provide valuable insights into tumor prognosis and the immune microenvironment, it must be acknowledged that our study has certain limitations. Firstly, our observation that BID expression interferes with tumor cell migration without affecting proliferation merits further exploration. Second, our study is based on public transcriptome data supplemented by basic in vitro experiments. Therefore, more complex in vitro studies and animal experiments are necessary to further explore the underlying mechanisms of mitochondrial dysfunction in ccRCC. Finally, since this is a retrospective study, prospective trials involving larger patient cohorts are needed to improve the reliability of the scoring system.