NCOA4: An Immunomodulation-Related Prognostic Biomarker in Colon Adenocarcinoma and Pan-Cancer

Treatment of cancer in humans requires a thorough understanding of the multiple pathways by which it develops. Recent studies suggest that nuclear receptor coactivator 4 (NCOA4) may be a predictive biomarker for renal cancer. In the present work, TCGA, GEPIA, and several bioinformatics approaches were used to analyze the NCOA4 expression patterns, prognostic relevance, and association between NCOA4 and clinicopathological features and immune cell infiltration. We investigated NCOA4 expression in malignancies. Low NCOA4 expression was associated with poor overall survival in individuals with malignancies such as cholangiocarcinoma, colon adenocarcinoma, and clear cell renal carcinoma. We also analyzed NCOA4 DNA methylation in normal and primary tumor tissues and investigated possible functional pathways underlying NCOA4-mediated oncogenesis. In conclusion, downregulation of NCOA4 is associated with poor prognosis, and NCOA4 may be a predictive biomarker for COAD.


Introduction
Worldwide, cancer is the second leading cause of death, with mortality rates far exceeding those of human immunodeficiency virus/acquired immunodeficiency syndrome, tuberculosis, and malaria combined [1]. Cancer incidence and mortality are increasing at an alarming rate worldwide. It is difficult to pinpoint the exact reasons for this, although they are related both to population aging and growth and to changes in the incidence and distribution of the major risk factors for cancer, some of which are associated with social and economic development. Treatment options for cancer available today include surgical procedures as well as chemotherapy, radiation, targeted therapy, and immunotherapy. Due to the drawbacks of standard treatment, such as intolerance and toxicity, clinical outcomes have remained inadequate in most cases. To better tailor therapy, susceptibility genes need to be incorporated into future targeted and tailored cancer therapies [2]. It is critical to advance our knowledge of carcinogenesis through the discovery and characterization of novel cancer-spanning genes to deepen our understanding of the disease. e availability of freely accessible cancer genome resources such as TCGA ( e Cancer Genome Atlas) [3], GEPIA (Gene Expression Profiling Interactive Research) [4], and TIMER (Tumor Immune Estimation Resource) [5] may now enable a more comprehensive and systematic analysis of cancer genes for a variety of tumors.
Nuclear receptor coactivator 4 (NCOA4), also known as androgen receptor-associated protein 70 (ARA70), was originally discovered to be a component of the RET-fused gene expressed in a subset of papillary thyroid carcinomas [6]. Autophagy is a type of selective autophagy regulated by NCOA4, an autophagy cargo receptor that binds to FTH1 (ferritin heavy chain 1) on the phagophore and transports it to the lysosome for degradation, releasing iron for systemic physiological needs [7]. Arginine on the surface of FTH1 has been shown to specifically bind NCOA4, which can then fuse with a lysosome via nascent autophagosomes, leading to acceleration of ferroptosis cell death. In cancer cells, NCOA4 is a critical regulator of ferroptosis, and mutations in NCOA4 can lead to decreased ferroptosis of tumor cells by reducing intracellular free iron accumulation, glutathione synthesis, and reactive oxygen species, among other effects (ROS). It has been postulated that ferroptosis may be a potential therapeutic target for cancer because cancer cells have higher iron levels and are more susceptible to ferroptosis activation. In the future, it may also be possible to treat a number of ferroptosis-associated diseases by modulating ferroptosis.
us, a more comprehensive understanding of the genes associated with ferroptosis could be much more helpful in developing personalized treatments. Given the already known role of NCOA4 in ferritin degradation, targeting the COPZ1/NCOA4/FTH1 axis may be a promising therapeutic strategy for the treatment of human glioblastoma (GBM) [8]. NCOA4 has the potential to play a critical role in cancer development and progression, as it is thought to be involved in tumor growth and metastasis spread. NCOA4 has only been studied in a small number of cancers. It is therefore critical to better understand the regulatory functions and molecular processes of NCOA4 in the cancer database in order to develop innovative therapeutic approaches for cancer. e role of NCOA4 in prognosis and the immune response was investigated using a number of different datasets, including the TCGA, UALCAN, Kaplan-Meier Plotter, TIMER, and cBioPortal. Additionally, we included factors such as survival status, genetic alterations such as DNA methylation, and critical cellular downstream pathways in our analysis of NCOA4 expression levels among tumor types. A molecular mechanism through which NCOA4 may contribute to the oncogenesis of a variety of human malignancies has been elucidated by our extensive and systematic pan-cancer research efforts.

NCOA4 Expression Pattern in Human Pan-Cancer.
e data from TCGA (https://genomecancer.ucsc.edu/) were used to explore the dysregulation of NCOA4 expression between various kinds of cancer and normal tissues. e TCGA database was merged with GTEx (Genotype-Tissue Expression) [9] to gather RNA sequencing data and clinical features of patients with 33 different kinds of cancer [10]. Because the TCGA data for ACC, DLBC, LAML, LGG, OV, and UCS were insufficient, we used GEPIA2 (https://gepia2. cancer-pku.cn/#analysis) to generate box plots of the data using the usual log2-fold change cut-off of 1, p value cut-off of 0.01, and combined the TCGA and GTEx databases for a comprehensive analysis. e UALCAN tool (https://ualcan. path.uab.edu/analysisprot.html) [11] was used to determine the NCOA4 protein expression levels in distinct cancer types. e level of expression of NCOA4 total protein was determined in tumor and normal tissues.

Immunohistochemistry (IHC).
To assess changes in NCOA4 protein expression levels, we used the online HPA (Human Protein Atlas) (https://www.proteinatlas.org/) [12]. NCOA4 IHC pictures were retrieved in a variety of tissues,  including BRCA, COAD, KIRC, LIHC, LUAD, and THCA. e method of IHC referred to the previous research [13,14]. Fresh tissues from Tongji Hospital's gastrointestinal surgery were sliced into 4 um thick slices, and then immunostaining was performed. Dewaxing of paraffin slices was followed by antigen retrieval using a 10 mmol/L citrate buffer at a pH of 6.0. For 15 minutes, sections were treated with methanol containing 3% hydrogen peroxide. After washing with PBS, the sections were incubated for 30 minutes with blocking serum. e sections were then treated overnight at 4°C with an anti-NCOA4 antibody (Abmart, Shanghai, China). Secondary antibody incubation and staining were conducted according to the manufacturer's instructions using the EnVision ® + System-HRP (DAB) kit (Dako, Glostrup, Denmark). e nuclei were counterstained with hematoxylin. NCOA4 was discovered to be highly expressed in normal tissue, as shown by brown staining under a microscope.

Prognostic Analysis.
Forest plots and Kaplan-Meier curves were used to illustrate the relationship between NCOA4 expression and patient prognosis in 33 cancer types, including OS (overall survival) and DSS (disease-specific survival). Univariate survival analysis was used to compute the HR (hazard ratios) and 95% CI (confidence intervals). We analyzed survival data, including OS and DFS, for all TCGA cancer types (Supplementary Table S1), with or without NCOA4 genetic mutation, using the cBioPortal program (https://www.cbioportal.org/).

Genetic Alteration Analysis.
We collected data on NCOA4 alteration frequency, mutation type, mutated site information, and CNA (copy number alteration) across all TCGA cancers using cBioPortal (https://www.cbioportal. org/) [15]. e UALCAN database was used to compare the methylation status of NCOA4 in various malignancies to that in surrounding normal tissues. e Student's t-test was used to analyze the data, and a p value of 0.05 was considered statistically significant.

Correlation of the NCOA4 Expression with Immune
Infiltration. TIMER tool was used to examine the connection between NCOA4 expression and immune infiltration in all TCGA tumors. Cancer-associated fibroblasts, neutrophils, and macrophages, including M0, M1, and M2, were chosen for comprehensive examination. Estimates were made using the TIMER, TIDE, CIBERSORT, CIBERSORT-ABS, QUANTISEQ, XCELL, MCPcounter, and EPIC algorithms.

NCOA4-Related Gene Enrichment Analysis.
e STRING website (https://string-db.org/) [16] was utilized to investigate the protein-protein interaction (PPI) of NCOA4. Additionally, we establish significant criteria such as the meaning of the network's edge ("evidence"), the sources of active interaction ("experiments"), the minimum required interaction score ("low confidence (0.150)"), and the maximum number of interactors to export ("no more than 50 interactors"). en, we present the experimental evidence for the interaction network of ten NCOA4-binding proteins. GEPIA2 was used to identify the top 100 NCOA4-related genes from all TCGA tumor and normal tissue datasets. NCOA4 expression was compared to the common genes identified by interaction analysis using Spearman's correlation. Calculated p values and correlation coefficients (R) are shown in the respective figure panels. e heatmap representation of the selected genes' expression profile includes the partial correlation coefficient (cor) across all cancer types and the p value for the parity-adjusted Spearman's rank correlation test. To conduct KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis, we combined and filtered the NCOA4-related genes. e enhanced routes were shown using the R packages "tidyr" and "ggplot2." Statistical significance was defined as a p value of 0.05.

Statistical Analysis.
It was utilized to determine the expression of NCOA4 in various tissues. We compared NCOA4 expression levels in tumor and normal tissues using the t-test. Kaplan-Meier curves were used to analyze the survival of patients with varying levels of NCOA4 expression. We employed univariate Cox regression analysis to compute the HR and 95% CI in survival analysis. All R packages were run using version 4.0.3 of the R programming language (https://www.r-project.org/), and statistical significance was considered as a p value of 0.05.

Pan-Cancer Analysis of the Correlation between NCOA4
Expression and Clinicopathology. We examined NCOA4 expression in stage I, II, III, and IV pan-cancer patients to further study the association between NCOA4 expression and clinical features.
e TCGA database analysis found that NCOA4 expression was significantly greater in the early stages of BLCA and KIRC ( Figure 3). Additionally, we reported the relationship between the degree of NCOA4 expression and various clinicopathological characteristics, such as age, gender, T stage, N stage, M stage, and overall survival, in pan-cancer types (Supplementary Tables S2-S14).

Pan-Cancer Analysis of the Prognostic Value of NCOA4.
e purpose of this study was to determine the relationship between NCOA4 expression and prognosis and patient survival. We classified cancer patients according to their level of expression. We examined the connection between the NCOA4 expression and the prognosis of patients with pan-cancer using the TCGA database. OS and DSS were used as survival measures. In terms of OS, the Cox regression analysis of the 33 forms of cancer demonstrated a significant correlation between the NCOA4 expression level and seven types of cancer, namely, ACC, CHOL, COAD, KIRC, LGG, LUAD, and SARC. With the exception of ACC, decreased NCOA4 expression correlates with shorter overall survival in six kinds of cancer (Figure 4(a)). Additionally, we examined the distribution of NCOA4 expression levels and their connection to patient overall survival using the online database GEPIA2 (Figure 4(b)). e survival map and KM curves revealed that less NCOA4 expression in CHOL (p � 0.0061), KIRC (p < 0.001), KIRP (p � 0.575), LGG (p � 0.00018), and SARC (p � 0.03) led to unsatisfactory outcome. On the other hand, elevated NCOA4 expression in OV (p � 0.041) resulted in a negative effect. Interestingly, the TCGA results for CHOL, KIRC, LGG, and SARC were congruent with the GEPIA2 database results. In addition to the TCGA data analysis, we examined the level of NCOA4 expression of alive and deceased patients with ACC, CHOL, COAD, KIRC, LGG, LUAD, and SARC. COAD had statistically significant differences (p < 0.05) and KIRC had a significant difference (p < 0.001) (Figure 4(c)). On the other hand, we used the same approaches to examine the link between NCOA4 expression and disease-free survival. e association between NCOA4 expression and DSS was discovered using Cox regression analysis of 33 different forms of cancer. Reduced NCOA4 expression correlates with shorter disease-free survival in four forms of cancer, namely, CHOL, KIRC, LGG, and SARC ( Figure 5     Journal of Oncology less favorable result ( Figure 5(b)). Additionally, we examined the expression of NCOA4 in alive and deceased individuals with the aforementioned cancer types. However, because the data for the DSS incident was insufficient, we gathered data from CHOL, KIRC, and SARC. In KIRC, there was a statistically significant difference (p < 0.01) ( Figure 5(c)).

Validation of NCOA4 Expression and Prognostic Value in COAD.
From the pan-cancer study results above, we find that NCOA4 gene expression in COAD and KIRC was consistent with protein expression as determined by IHC staining. Additionally, there were statistically significant associations between NCOA4 expression and OS in both cancer types. As a consequence, we chose the COAD for validation. Tongji Hospital obtained fresh tumor and peritumoral surrounding tissues from patients undergoing excision for localized COAD. e expression of the NCOA4 protein is decreased in COAD tissues ( Figure 6). Additionally, we expanded the size of the tissue samples to verify the accuracy of the TCGA bioinformatics data. Shanghai Zhuoli Biotechnology Co. Ltd. (China) produced tissue microarrays with 40 sets of samples. Notably, the expression of NCOA4 protein was much greater in peritumoral neighboring tissues than in tumor tissues (Figures 7(a)and 7(b)). We defined the microarray landscape in terms of blue dots representing tumor tissues and green dots representing normal tissues. In these samples, the level of NCOA4 expression was highly associated with overall survival, and patients with low protein expression had a worse prognosis (p � 0.002). (Figure 7(c)). is discovery established that low NCOA4 levels are related to worse clinical and prognostic outcomes.

Pan-Cancer Analysis of Genetic Alteration and Methylation of NCOA4.
Disruption of the epigenetic landscape occurs often in cancer, frequently as a result of genetic abnormalities in epigenetic regulatory genes [17]. Cancer develops as a result of an accumulation of epigenetic and genetic changes [18]. As a result, we sought to investigate the NCOA4 genetic variants using the cBioPortal (TCGA, Pan-Cancer Atlas). According to our findings, the modification frequency (>5%) with "mutation" of NCOA4 is higher in primary uterine cancers. Furthermore, the frequency of modifications (>5%) in NCOA4 "structural variation" and "amplification" in cholangiocarcinoma was the greatest kind ( Figure 8(a)). e following step was to investigate the new mutations and their placement within NCOA4 (Figure 8(b)). e dominant   Journal of Oncology mutation type for NCOA4 was identified as "Missense." However, the online database does not provide information about the three-dimensional structure of the NCOA4 protein.
We conducted a comprehensive review of these variants in diverse kinds of malignancies to determine whether there is an association between certain NCOA4 genetic variants and patient clinical survival prognosis. Patients with UCEC who had a genetic variant of NCOA4 had a better prognosis in terms of progression-free survival (p � 0.0102), diseasespecific survival (p � 0.0299), but not overall survival (p � 0.0511), or disease-free survival (p � 0.575), when compared to patients who did not have a mutation of NCOA4 (Figure 8(c)). Genome-wide analyses of DNA methylation in a range of cancer types have revealed a high prevalence of cancer-associated methylation alterations. DNA methylation changes occur in every kind of cancer, and more significantly, DNA methylation levels correlate with tumor aggressiveness in the majority of cancers [19]. As a result, we investigated the DNA methylation of NCOA4 using the UALCAN and TCGA datasets (Figure 8(d)). e significant increase in methylation of NCOA4 was shown in BRCA (p < 0.01), CHOL (p < 0.001), HNSC (p < 0.001), LUAD (p < 0.01), and LUSC (p < 0.001). On the contrary, we found a significant decrease in methylation of NCOA4 in KIRC (p < 0.001), KIRP (p < 0.001), READ (p < 0.001), TGCT (p < 0.001), THCA (p < 0.01), and UCEC (p < 0.001).
3.6. Pan-Cancer Analysis of the NCOA4 Expression and Immune Cell Infiltration. Lymphocytes invading tumors affect cancer patient survival. As a result, we examined the expression of NCOA4 in invading immune cells. us, we used the TIMER, CIBERSORT, CIBERSORT-ABS, TIDE, XCELL, MCPcounter, QUANTISEQ, and EPIC algorithms to investigate the relationship between immune cells infiltration and NCOA4 expression in several TCGA tumor types. Interestingly, we observed a negative association between the NCOA4 expression and the estimated macrophage M0 infiltration value for THYM and a positive correlation for TGCT. Additionally, we identified a strong connection between NCOA4 expression and predicted macrophage infiltration values for COAD, LUAD, LUSC, PAAD, and STAD (Figure 9(a)). NCOA4 expression and cancer-associated fibroblast infiltration for the PAAD also exhibited a positive link, whereas neutrophils and NCOA4 expression were shown to be positively correlated with BLCA tumors (Figures 9(b) and 9(c)).

Discussion
Cancer accounts for a significant amount of the world's sickness burden, continuing to be a dangerous disease, with the number of cancer-related deaths increasing [20]. Patient survival has been significantly improved as a result of advancements in cancer diagnosis and treatment technology. It is possible to undertake a detailed study of prognosis at the pan-cancer level using high-throughput RNA expression data and clinical information from the TCGA Pan-Cancer project, which is supported by the National Cancer Institute. With the use of a pan-cancer investigation of approximately 10,000 original tumors spanning 33 various cancer types, we observed that low levels of NCOA4 in malignancies were linked with poor overall survival. e current work conducted a comprehensive analysis of NCOA4 expression in a pan-cancer dataset for the first time. e results from the analysis of 33 cancers' data from the TCGA revealed that NCOA4 was significantly downregulated in BLCA, BRCA, COAD, HNSC, KICH, KIRC, KIRP, LUAD, LUSC, PRAD, READ, THCA, and UCEC but upregulated in LGG, compared with the paracancerous and normal tissues (Figures 1(a) and 1(b)). NCOA4 expression levels may vary among tumors due to their diverse underlying functions and processes. Additionally, we discovered that decreased NCOA4 expression was associated with worse OS results in a variety of malignancies, including CHOL, COAD, KIRC, LGG, LUAD, and SARC (Figure 4(a)). Furthermore, we examined the distribution of NCOA4 expression levels and their connection to patient overall survival using the online   NCOA4   ACC  BLCA  BRCA  CESC  CHOL  COAD  DLBC  ESCA  GBM  HNSC  KICH  KIRC  KIRP  LAML  LGG  LIHC  LUAD  LUSC  MESO  OV  PAAD  PCPG  PRAD  READ  SARC  SKCM  STAD  TGCT  THCA  THYM  UCEC  database GEPIA2. e survival map and KM curves demonstrated that decreased NCOA4 expression in CHOL, KIRC, KIRP, LGG, and SARC results in poorer than desirable outcomes (Figure 4(b)). In addition to the TCGA data analysis, we examined the level of NCOA4 expression in alive and deceased patients with ACC, CHOL, COAD, KIRC, LGG, LUAD, and SARC. COAD and KIRC had statistically significant differences (Figure 4(c)). e TCGA results for CHOL, KIRC, LGG, and SARC were consistent with the GEPIA2 database results. We used the same methodology to examine the relationship between NCOA4 expression and DSS. Cox regression investigation of 33 forms of cancer found that decreased NCOA4 expression in four types of cancer, namely, CHOL, KIRC, LGG, and SARC, results in a shorter disease-free life ( Figure 5(a)). e survival map and KM curves demonstrated that decreased NCOA4 expression results in an unfavorable outcome in CHOL, KIRC, LGG, and SARC ( Figure 5(b)). ese findings show that NCOA4 may be used as a biomarker for predicting tumor patient prognosis. Our study revealed numerous noteworthy findings, particularly at COAD and KIRC. en, we used COAD as the validation cancer in order to investigate the relationship between the level of NCOA4 expression and the result of COAD (Figures 6 and 7). We discovered that a lower level of NCOA4 resulted in a poorer prognosis for COAD patients.
is outcome was consistent with the TCGA dataset analysis. ese findings provide a potential clinical biomarker for the prediction of COAD overall survival.
Cancer is a hazardous disease accounting for a major portion of the world's illness burden, with the number of cancer-related fatalities growing. It has been found that low expression of NCOA4, a ferritinophagy-related gene, correlates with decreased immune cell infiltration and impaired IFN-c receptor signaling in clear cell renal carcinoma (ccRCC) [21]. Vandetanib may be a useful treatment strategy for CRC patients with NCOA4-RET fusion, according to the study in [22]. Hence, NCOA4 has potential as a novel marker to identify patients who may be eligible for ferroptosis-induction therapy or immunotherapy combined with it. As a consequence of breakthroughs in cancer diagnostic and treatment technologies, patient survival has increased dramatically in recent years. In addition, considering the clinical information from the TCGA Pan-Cancer project, which is financed by the National Cancer Institute, it is feasible to conduct a thorough assessment of prognosis at the pan-cancer level. We discovered that low levels of NCOA4 in malignancies were associated with poor overall survival after conducting a pan-cancer examination including almost 10,000 original tumors spanning 33 different cancer types in a single study.
As a result, we sought to investigate the NCOA4 genetic variants using the cBioPortal (TCGA, Pan-Cancer Atlas). According to our findings, the modification frequency (>5%) with "mutation" of NCOA4 is higher in primary uterine cancers. Moreover, the frequency of modifications (>5%) in NCOA4 "structural variation" and "amplification" in cholangiocarcinoma was the greatest kind (Figure 8(a)). e following step was to investigate the new mutations and Journal of Oncology 13   NCOA4   ACC  BLCA  BRCA  CESC  CHOL  COAD  DLBC  ESCA  GBM  HNSC  KICH  KIRC  KIRP  LAML  LGG  LIHC  LUAD  LUSC  MESO  OV  PAAD  PCPG  PRAD  READ  SARC  SKCM  STAD  TGCT  THCA  THYM  UCEC  UCS  14 Journal of Oncology their placement within NCOA4 (Figure 8(b)). e dominant mutation type for NCOA4 was identified as "Missense." Because there is no evidence of NCOA4 being phosphorylated, we examined NCOA4's DNA methylation using the UALCAN and TCGA datasets (Figure 8(d)). DNA methylation, or the methylation of the carbon 5 atom of cytosines, occurs mostly in the CpG context in eukaryotes, but also in the CpHpG and CpHpH contexts in some cell types [23].
DNA methylation abnormalities have been linked to a range of human disorders, including cancer [24], and the most well-studied epigenetic mechanism of cancer is aberrant DNA methylation. Our research revealed a substantial increase in NCOA4 methylation in BRCA, CHOL, HNSC, LUAD, and LUSC. On the other hand, we observed a substantial reduction in NCOA4 methylation in KIRC, KIRP, READ, TGCT, THCA, and UCEC. Additionally,    Journal of Oncology abnormal DNA methylation (hyper-or hypomethylation) may affect NCOA4 gene expression and consequently cancer processes. DNA hypermethylation occurs early and often in a variety of malignancies, and methylation of viral DNA has been proposed as a potential biomarker for cervical illness [25].
Since DNA methylation-mediated gene silence has been implicated in the pathogenesis of a variety of disorders, including cancer, targeting aberrant DNA methylation may one day be deemed a therapeutically useful technique for cancer therapy. Particularly noteworthy is the observation that ferritinophagy increases ferroptosis [26][27][28]. Multiple studies have shown that autophagy contributes to ferroptosis during the early stages of the process by supplying accessible ferric iron through NCOA4-mediated ferritinophagy during the early stages of the process [29]. Additionally, since some oncogenic mutations render tumor cells very vulnerable to ferroptosis, triggering ferroptosis in tumor cells that are susceptible to ferroptosis may hold considerable therapeutic potential for ferroptosis-sensitive tumor cells [30]. However, there is still much that is unknown about the molecular pathways that support the therapeutic potential of ferroptosis regulation. It is impossible to understand tumor progression without understanding the tumor microenvironment, which is comprised of nontumor cells such as  tumor-infiltrating immune cells (TILs), cancer-associated fibroblasts (CAFs), and endothelial cells. e absence of extracellular matrix (ECM) components results in the establishment of a highly dynamic tumor microenvironment (TME) with intricate interactions between multiple internal components that promote tumor growth and resistance to therapy. Because of this, integrating NCOA4 and TME research may provide novel insights into tumor treatment.
Finally, we conducted GO and KEGG enrichment analyses of NCOA4-interacting proteins identified using the STRING tools and GEPIA2 in pan-cancer samples to determine their functional significance. Using the GO|KEGG pathway analysis, we discovered that "DNA damage checkpoint," "negative regulation of translation," and "mRNA catabolic process" were the top hits, indicating that these pathways are engaged in NCOA4's role in tumor pathogenesis regulation. Our results may provide insight into the mechanism through which NCOA4 exerts its downstream effects.

Conclusions
In conclusion, our thorough pan-cancer analysis of NCOA4 demonstrated that it was related to a poor prognosis in individuals who had it downregulated. NCOA4 is an oncogene and a prognostic marker in COAD. Targeting NCOA4 may be a potential method for COAD therapy. Additional factors such as DNA methylation, immune cell infiltration, and NCOA4-interacting proteins may be of assistance in explaining the role of NCOA4 in the development of cancer from a variety of perspectives.
Data Availability e data supporting the findings of this study are available within the article. Further inquiries can be directed to the corresponding authors.

Conflicts of Interest
e authors declare that they have no conflicts of interest.

Authors' Contributions
C.G., W.C., and J.W. designed and performed experiments and drafted the manuscript. A.S. and D.L. conceived and designed the study. G.L. and Y.Y. performed the data analysis. Y.Y., W.Q., and Z.S. provided technical support. D.L. designed and performed experiments and wrote the manuscript. All authors read and approved the manuscript. C.G., W.C., and J.W. contributed equally to this work as cofirst authors. Figure S1. NCOA4-related gene hallmark enrichment pathway of COAD in the TCGA database. Figure S2. NCOA4-related gene hallmark enrichment pathway of KIRC in the TCGA database.