Early-stage LUAD patients can achieve curative results through surgical intervention. However, due to the lack of obvious clinical symptoms in the early-stage of LUAD patients and limitations in screening methods, most patients are found to have already progressed to the middle and late stages of the disease [22]. Among the factors influencing tumorigenesis, alterations in nucleotide metabolism play a pivotal role, with enhanced synthesis of nucleoside triphosphates being crucial for the development of LUAD [23]. A deeper understanding of metabolism in cancer can assist in the identification of valuable diagnostic biomarkers [24]. In this study, we utilized data from the public datasets and employed a combination of univariate cox regression, Lasso cox regression, and multivariate cox regression analysis to identify four nucleotide metabolism-related genes: RRM2, TXNRD1, NME4, and NT5E, as potential biomarkers. Based on the expression levels of these four prognostic genes and overall survival (OS) data, we established a risk scoring system that categorizes LUAD patients into low and high risk groups. In this context, we have identified key Nucleotide Metabolism-Related Genes (NMRGs) relevant to LUAD progression and prognosis and have analyzed their potential functional mechanisms.
To understand the molecular and immune-related differences between high/low-risk subgroups, we investigated the functions of the four biomarkers. Ribonucleotide reductase M2 subunit (RRM2) is a rate-limiting enzyme in the nucleotide synthesis pathway, and increased RRM2 expression and activity have been associated with various cancer types. It has been overexpressed and linked to tumor progression, invasion, metastasis, and lower patient survival rates in cancers such as gastric, ovarian, colorectal, brain, and breast cancer [25–29]. Consequently, RRM2 has long been considered an important drug target for various proliferative diseases, including cancer [30]. In LUAD cells, RRM2 overexpression enhances tumor cell proliferation and invasion, and it is considered an independent risk factor for the overall survival (OS) of LUAD patients [31]. Our experiments also confirm that RRM2 expression is significantly higher in LUAD tissues compared to adjacent normal tissues. Cytoplasmic selenoprotein thioredoxin reductase 1 (TXNRD1) plays multiple roles associated with malignant tumors, as it can protect normal cells from malignant transformation [32]. However, in liver cancer, upregulated TXNRD1 expression promotes hepatocellular carcinoma progression through the activation of the Akt/mTOR signaling pathway and is associated with lower patient survival rates [33]. There is limited research on TXNRD1 in LUAD, but a study by Jin X suggests that TXNRD1 expression is reduced in LUAD patients who are female, have not received radiotherapy, and have no distant metastases, which aligns with our verification results [34]. Nucleoside Diphosphate Kinase 4 (NME4) is a critical rate-limiting enzyme that regulates nucleotide metabolism and ATP/ITP metabolism [35]. Aberrant overexpression of the NME4 gene in gastric and colon cancer may lead to an imbalance in nucleotide pools in mitochondria, resulting in checkpoint regulation failure and the accumulation of genetic changes, ultimately leading to tumorigenesis [36]. As an oncogenic promoter, NME4 can promote the progression of NSCLC by inhibiting cell cycle arrest and stimulating tumor cell proliferation [37]. NT5E encodes the ecto-5’-nucleotidase (CD73) is a critical rate-limiting enzyme in the extracellular purine metabolism pathway. It plays a crucial role in generating and maintaining adenosine concentrations, thereby influencing tumor cell neovascularization, immune evasion, and immune response [38, 39]. CD73 is highly expressed in most cancers, but its expression levels are lower than adjacent normal tissues in appendiceal adenocarcinoma and ovarian cancer. Moreover, high CD73 expression is closely associated with lower overall survival (OS) but not with recurrence-free survival (RFS) [40]. In vitro experiments, NT5E may promote LUAD proliferation and metastasis through the EGFR/AKT/mTOR pathway [38].
Through GSEA enrichment analysis, in addition to cell cycle, DNA replication, proteasome, and vascular smooth muscle contraction pathways associated with tumor proliferation and invasion, spliceosome-related signaling pathways were also enriched. These findings suggest that differences in survival may be driven by different immune status in LUAD patients [41]. Vascular smooth muscle contraction plays a crucial role in controlling blood flow and the delivery of oxygen and nutrients to tissues. Tumors often possess a higher density of microvasculature than normal tissues. However, these pathological vessels are often less elastic and function differently from normal vessels, often promoting tumor growth through autocrine signaling [42]. The specific mechanisms underlying this phenomenon in LUAD are not yet clear. GSVA analysis indicates a significant upregulation of the glycolysis signaling pathway in the high-risk group. Elevated glycolysis signaling pathways are closely associated with immune therapy resistance and poor prognosis [43]. Studies have shown that chenodeoxycholic acid (CDCA), acting as an integrin α5β1 inhibitor, can inhibit LUAD cell proliferation, migration, and invasion and induce apoptosis through the α5β1/FAK/p53 signaling pathway [44]. Our research suggests that the downregulation of bile acid metabolism pathways is more pronounced in the high-risk group, possibly contributing to the poor prognosis.
Immunotherapy, as an emerging treatment approach, has shown promising results. However, it is not universally effective for all LUAD patients, as its efficacy varies depending on the patient's immune system status and cancer characteristics. Immune evasion is a significant hallmark of cancer progression, and the downregulation of HLA can reduce antigen presentation, thereby promoting immune escape [45]. Our results indicate that 14 HLA family genes are expressed at higher levels in low-risk group patients, suggesting that low-risk patients may benefit from immunotherapy and consequently achieve longer overall survival (OS). We conducted a differential expression analysis of 48 immune checkpoint molecules between high and low-risk groups in LUAD. We found significant differences in the expression of 25 immune checkpoint molecules between the high and low-risk groups. Our study demonstrated that RRM2 has the highest positive correlation with CD276, an immune checkpoint molecule. Aberrant expression of CD276 upregulates the epithelial-mesenchymal transition (EMT) of LUAD cells, promoting LUAD development [46]. However, further studies are needed to understand the interaction between RRM2 and CD276. TIDE, as an algorithm for predicting patient response to immune checkpoint inhibitor (ICI) therapy, suggests that higher scores indicate a higher likelihood of significant immune escape and a lower likelihood of benefiting from ICI treatment [47]. We used the TIDE algorithm, IPS algorithm, and TMB analysis to assess their responses to immunotherapy, and our research suggests that low-risk patients are more likely to benefit from immunotherapy. Furthermore, understanding the composition of immune cells in tumor tissue can help identify new cancer treatment approaches and enhance the efficiency of ICI therapy. Tumor-associated macrophages can promote tumor cell proliferation, migration, and tumor angiogenesis. M0 macrophage high-density infiltration is closely associated with poor clinical outcomes in early-stage LUAD, which is consistent with our research [48, 49]. Many studies suggest that increased infiltration of plasma cells in tumors can significantly prolong the OS of NSCLC patients receiving PD-L1 treatment [50]. Our research suggests a significant downregulation of plasma cell infiltration in the high-risk group, indicating a worse prognosis. High infiltration density of mast cells can extend the survival of early-stage LUAD patients [51] and lead to a higher recurrence-free survival (RFS) for stage I and II postoperative patients [52]. However, another study suggests that mast cells promote LUAD cell metastasis through the release of proteases via exosomes [53]. Therefore, the relationship between mast cells and LUAD remains controversial and requires further investigation.
We assessed the sensitivity of high/low-risk group patients to drugs currently used for the treatment of LUAD. High-risk group patients had obviously higher IC50 values for Doramapimod_1042, BMS-754807_2171, MK-2206_1053, and Nutlin-3a (-)_1047 compared to low-risk group patients, suggesting that the low-risk group may have a better response to chemotherapy and targeted therapy. Doramapimod_1042, BMS-754807_2171, MK-2206_1053 are inhibitors of the p38-MARK, PI3K/AKT, and AKT/PKB pathways, respectively, and they can significantly inhibit the proliferation of lung adenocarcinoma cells and increase apoptosis [54–56]. On the other hand, Nutlin-3a (-)_1047 activates p53 in normal lung epithelial cells and induces apoptosis in lung adenocarcinoma cells [57]. The validation results of gene expression showed consistency with our bioinformatics analysis for RRM2 and NT5E. We speculate that RRM2 inhibitors can activate the cGAS/STING signaling pathway, increase CD8 + T cell infiltration, and have an anti-tumor effect [58]. When given EGFR, AKT, or mTOR inhibitors, the function of NT5E can be significantly inhibited, indicating that NT5E may be involved in the pathogenesis of LUAD through the EGFR/AKT/mTOR axis [38]. The differences in NME4 and TXNRD1 expression may be attributed to sample variations.
We have constructed a novel model based on nucleotide metabolism that underwent comprehensive analysis and validation across multiple databases, showing significant potential for LUAD prognosis prediction and providing new insights into LUAD treatment. However, this model still has its limitations. Firstly, immunotherapy has emerged as a viable treatment option for LUAD, but the selection and specific mechanisms of combination therapy remain to be further explored. Additionally, before the application of NMRGs in predicting LUAD treatment responses in clinical settings, further validation and support from real-world and basic research data are required.