Correlation between the decreased expression of NUDT18 and tumor progression in endometrial cancer

Introduction

Endometrial cancer (EC) is the most prevalent gynecologic cancer. It is the fourth most prevalent cancer among women, following breast cancer, lung cancer, and colorectal cancer (1,2). Since 2008, the incidence of EC has grown by 21% (3). The mortality rate per 100,000 population has grown by more than 100% over the previous two decades and by 8% since 2008 (1,3). It is doubtful that the incidence and mortality of EC will decline significantly over the next several years (2), given that the methods available for early diagnosis and treatment do not have a substantial impact on mortality (4). Therefore, investigating the pathogenesis of EC and identifying new therapeutic targets is essential to facilitating personalized treatment and improving patient outcomes.

In the tumor microenvironment, tumor cells undergo significant metabolic and redox alterations, leading to increased release of reactive oxygen species (ROS). For cancer cells with dysregulated ROS levels, nucleotidase is critical because damaged deoxynucleotide triphosphate (dNTPs) can be incorporated into DNA, inducing mutations and DNA damage. Nudix hydrolases derive from their conserved catalytic motif, nudix box [GX(5)EX(7)REUXEEXGU, where X is any amino acid, and U is a hydrophobic residue] (5). This motif serves as the enzyme’s catalytic center. Thus far, 22 types of human nudix hydrolases have been identified. Among them, NUDT1 (MTH1) has been extensively studied. Under physiological conditions, MTH1 hydrolyzes oxidized nucleotides (e.g., 8-oxo-dGTP) to maintain genomic stability. However, tumors exhibit elevated oxidative stress, generating excessive oxidized nucleotides that would otherwise suppress cancer cell growth. Paradoxically, upregulated MTH1 expression in cancers (e.g., melanoma, colon cancer, and breast cancer) removes these damaged nucleotides, promoting tumor cell survival and malignancy (6,7). Notably, MTH1 inhibitors (e.g., TH588 and TH287) selectively kill cancer cells with minimal effects on normal cells and can enhance chemo- and radiotherapy efficacy (8,9). In contrast, nudix hydrolase 18 (NUDT18) remains poorly characterized. As NUDT18 has a similar sequence to that of MTH1 (NUDT1) and MTH2 (NUDT15), the name “MTH3” has been proposed for this enzyme. Despite these structural similarities, NUDT18 exhibits distinct substrate specificity: while MTH1 hydrolyzes 8-oxo-dGTP but not 8-oxo-dGDP, NUDT18 preferentially cleaves 8-oxo-dGDP and its ribonucleotide counterpart 8-oxo-GDP, with minimal activity toward 8-oxo-dGTP (10). It also degrades other oxidized diphosphates, such as 2-hydroxy-dADP and 8-hydroxy-dADP (11).

As one of the most well-characterized oxidative DNA lesions, 8-oxo-7,8-dihydroguanine (8-oxo-Gua) is a key product of oxidative DNA damage (12) that may result in C:G→A:T transversion mutations (13). Hydrolysis of nucleotides containing 8-oxoGua is essential for maintaining genome stability during oxidative stress (13). The NUDT18 protein can degrade 8-oxo-Gua-containing nucleoside diphosphates into monophosphates, thereby reducing the mutational potential of 8-oxo-Gua (10). As a member of the nucleotide pool sanitization enzymes that prevent oxidized nucleotide integration into DNA, NUDT18 has been associated with oxidative DNA protection (13). Apart from its involvement in the cleavage of 8-oxoG-containing deoxyribonucleotide, the much slower pace of growth of the NUDT18-KO Hela S3 cell line implies that NUDT18 plays an essential cell proliferation function (10). NUDT18 is expressed in respiratory epithelium and hydrolyzes remdesivir-TP, the bioactive triphosphate form of remdesivir, potentially compromising the efficacy of ribavirin and related antivirals (14).

Since expression and function in EC have not been clarified, we examined the expression of profile and of NUDT18 and its clinical implications in EC. Additionally, the association between NUDT18 expression level and DNA copy gain, methylation, and TP53 mutation was determined, and microRNAs (miRNAs) were screened to characterize the mechanism underlying NUDT18’s upregulation in EC and its biological function (15). Furthermore, gene set enrichment analysis (GSEA) and immune cell abundance analysis were conducted to characterize the immune cell alterations and biological pathways associated with NUDT18-related EC pathogenesis. We present this article in accordance with the REMARK reporting checklist (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2024-2538/rc).

Methods

RNA sequencing and bioinformatics analysis of The Cancer Genome Atlas (TCGA) patient data

A total of 572 cases belonging to either the HTSeq-FPKM-UQ or the HTSeq-Counts workflow type, along with their respective medical data, were downloaded from the Uterine Corpus Endometrial Carcinoma (UCEC) project on the recognized website (https://portal.gdc.cancer.gov) of TCGA via the “TCGAbiolinks” package (16) as data for gene expression analysis. This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments.

Relative NUDT18 expression level between UCEC and ordinary tissues

Using Gene Expression Profiling Interactive Analysis (GEPIA) platform (http://gepia.cancer-pku.cn), we evaluated NUDT18 messenger RNA (mRNA) expression among several human normal and malignant tissues (17). In addition, TCGA datasets were used to visualize expression differences between normal and cancerous tissues.

Investigation of NUDT18 protein expressions via the Human Protein Atlas (HPA)

The HPA database (https://www.proteinatlas.org/) (18) was used to validate the protein level of NUDT18 in normal endometrium and EC tissues. As specified on the website, protein expression scores included not detected, low, medium, and high.

Immunohistochemical analysis of NUDT18 protein

Paraffin-embedded tissues were baked at 65 °C for 30 min, dewaxed in xylene (3×5 min), and sequentially rehydrated through graded ethanol solutions (100%, 95%, and 75%, each 5 min). Antigen retrieval was performed in a pressure cooker in sodium citrate antigen retrieval solution (1:50; MVS-0099; Fuzhou Maixin Biotech, Fuzhou, China). Endogenous peroxidase activity was blocked via incubation in endogenous peroxidase blocking agent (210707S391ad; Fuzhou Maixin Biotech) for 10 min at room temperature. Following washes with phosphate-buffered saline (3×5 min), samples were treated with 0.3% Triton X-100 (T9284; Sigma-Aldrich, St. Louis, America) for 20 minutes at room temperature to achieve membrane permeabilization. Phosphate Buffered Saline (PBS) + 0.3% Triton X-100 was used for blocking with 1% bovine serum albumin (BSA) + 5% fetal bovine serum. Sections were then treated with anti-NUDT18 antibody (A8514; ABclonal, Wuhan, China) diluted to a working concentration of 1:150 via antibody diluent (TPB003218; Nanjing Typing Biotech, Nanjing, China) and stored at 4 °C overnight. On the following day, after washes with PBS (3×5 min), tissue sections were incubated with a universal horseradish peroxidase (HRP)-conjugated polymer secondary antibody (goat anti-mouse/rabbit IgG) (TPB-0015; Nanjing Typing Biotech) for 8 min at room temperature. Immunoreactivity was visualized with 3,3'-diaminobenzidine (DAB; TPB-13; Nanjing Typing Biotech) for 3 min. Counterstaining was performed with hematoxylin for 1 min, followed by a 10-min rinse under running water. Finally, slides were dehydrated through a graded ethanol series, cleared in xylene, and mounted with permount mounting medium (S3006; Shanghai Specimen and Model Factory, Shanghai, China). The expression of NUDT18 was analyzed under the microscope and scored according to the staining intensity and the proportion of positive tumor cells. The staining intensity was scored as follows: 0 for no staining, 1 for weak staining, 2 for moderate staining, and 3 for strong staining. Meanwhile, the proportion of positive tumor cells was scored as follows: percentage of stained tumor cells ≤25%, 1 point; >25–50%, 2 points; >50–75%, 3 points; and >75%, 4 points. The histological score was calculated as follows: staining intensity (grade 0–3) × percentage of positive cells (grade 0–4). We considered a score of ≤6 as a low expression of NUDT18 and >6 as a high expression.

Cell line establishment and real-time quantitative polymerase chain reaction

To establish stable NUDT18-overexpression cell lines, two kinds of EC cells (Ishikawa and MFE-296) were transfected with an overexpression plasmid targeting human NUDT18 or an empty vector control with Lipofectamine 3000; the sequence of the NUDT18-overexpression plasmid is provided in Appendix 1. Transfected cells were selected with puromycin dihydrochloride (2 µg/mL; ST551; Beyotime Biotechnology, Shanghai, China) for 2 weeks. Stable clones were validated by real-time quantitative polymerase chain reaction (RT-qPCR) and Western blotting.

Total RNA was extracted from cells with TRIzol reagent (R401-01; Vazyme, Nanjing, China) and reverse-transcribed into complement DNA (cDNA) with HiScript III RT SuperMix for qPCR(+g DNA wiper) (R323-01; Vazyme) under the following conditions: 42 °C for 2 min, 37 °C for 15 min, and 85 °C for 5 s. RT-qPCR was performed with ChamQ Universal SYBR qPCR Master Mix (Q711; Vazyme) on a QuantStudio 6 Flex Real-Time PCR System under the following cycling conditions: 95 °C for 30 s, 95 °C for 10 s, and 60 °C for 30 s, followed by 40 cycles of 95 °C for 15 s, 60 °C for 60 s, and 95 °C for 15 s.

The primer sequences are provided in Table S1.

GSEA

Based on the TCGA-UCEC dataset, GSEA version 4.1.0) (19,20) was used to determine the signaling pathways associated with NUDT18 in EC. In addition, as reference gene sets, those annotated from c2.cp.kegg.v7.4.symbols.gmt were selected, and the normalized enrichment score (NES) was computed. To rank the enriched pathways in each group, nominal P value, false-discovery rate (FDR) q value, and NES were applied.

Bioinformatics prediction of NUDT18-regulating microRNAs

Using the miRwalk (21) database, we predicted the potential regulatory microRNAs (miRNAs) of NUDT18, which we then intersected with differentially expressed miRNAs between EC tissue and normal controls based on TCGA datasets. The common miRNAs represented the possible regulatory miRNAs of NUDT18 in EC (22). Through the use of the cBio Cancer Genomics Portal (cBioPortal), copy number alteration (CNA), DNA methylation, and the association between NUDT18 expression and mutations of TP53 in TCGA-UCEC were analyzed.

Analysis of immune cell abundance

ImmuCellAI, which estimates the profusion of 24 immune cells based on the datasets of gene expression (23), was used to examine immune cell infiltration variations under different NUDT18 expression levels.

Statistical analysis

All statistical analyses were conducted with R software version 4.0.2 (The R Foundation of Statistical Computing). The significance level was set at a P value <0.05. To compare the overall survival (OS), disease-free interval (DFI), and progression-free interval (PFI) between the low- and high-NUDT18 expression groups, the log-rank test and Kaplan-Meier analysis were applied. The χ² test and Fisher exact test were used to evaluate the associations between NUDT18 expression levels and clinicopathological factors. To determine the independent clinical indicators with a statistically significant association with PFI in TCGA patients, multivariate Cox regression analysis was used. Hazard ratios (HRs) and 95% confidence intervals (CIs) were further calculated. Finally, linear regression analysis was used to identify the correlation between NUDT18 mRNA expression and NUDT18 DNA methylation levels (or the expression of miRNAs).

All methods were carried out in accordance with the relevant guidelines and regulations, and consent for participation in this study was provided by all patients and/or their legal guardian(s).

Results

Difference in the expression of NUDT18 between UCEC and physiological endometrium

GEPIA was used to evaluate the mRNA expression of NUDT18 among UCEC and physiological endometrial tissues, which indicated that NUDT18 expression was significantly higher in UCEC (Figure 1A). Using both clinical and gene expression data downloaded from TCGA in December 2021, we found that the expression levels of NUDT18 in 35 normal endometrium tissues were much lower than those in 537 EC tumors (Figure 1B). The NUDT18 protein expression level in normal and EC tissues was compared via the HPA database (Figure 1C). The diagnostic accuracy of NUDT18 for UCEC was also confirmed via the area under the receiver operating characteristic (ROC) curve, which was 0.796, with P<0.0001 indicating diagnostic accuracy (Figure 1D).

Figure 1 NUDT18 expression (mRNA and protein) was significantly upregulated in EC tissues compared to normal endometrium. (A) NUDT18 mRNA expression in various pairs of cancer and normal tissues. (B) NUDT18 mRNA expression in UCEC tissues (n=537) was significantly higher than that in physiological endometrium (n=35). (C) NUDT18 protein expression was higher in EC tissues (image available from https://www.proteinatlas.org/ENSG00000275074-NUDT18/cancer/endometrial+cancer#img) compared to normal endometrial tissues (image available from https://www.proteinatlas.org/ENSG00000275074-NUDT18/tissue/Endometrium#img) according to the HPA dataset (https://www.proteinatlas.org/). Scale bar =100 µm. (D) ROC curve analysis indicated that NUDT18 upregulation had good diagnostic accuracy for EC. (E) Analysis of overall survival of dichotomized NUDT18 expression in patients with UCEC according to the GEPIA platform. (F-H) High NUDT18 expression was associated with superior OS, DFI, and PFI in patients with EC according to data downloaded from TCGA database. DFI, disease-free interval; HPA, Human Protein Atlas; HR, hazard ratio; NUDT18, nudix hydrolase 18; OS, overall survival; PFI, progression-free interval; ROCs, receiver operating characteristics.

High expression of NUDT18 in UCEC correlated with prolonged OS

Based on the TCGA database and the GEPIA platform, the Kaplan-Meier curve was used to analyze the correlation between NUDT18 expression and OS of patients with UCEC. We discovered that the group with high expression had a considerably longer OS than the group with low expression (Figure 1E). Gene expression and clinical data acquired from TCGA produced comparable findings. Patients with UCEC with elevated NUDT18 expression also demonstrated a superior OS and DFI (all P values <0.05) (Figure 1F-1H).

Expression of NUDT18 protein in UCEC and clinicopathological correlations

To compare the expression of NUDT18 between normal endometrium and EC tissues, we detected the expression of NUDT18 protein in paraffin tissue specimens via immunohistochemistry (normal endometrium: n=5; EC stage I: n=32; EC stage II: n=10; EC stage III: n=10; EC stage IV: n=4). As is in Figure 2, the level of NUDT18 protein expression was elevated in the early stage of EC and then gradually decreased, and its expression in highly differentiated tumors was also higher than that in intermediate and poorly differentiated tumors. We further analyzed the relationship between NUDT18 expression and clinicopathological factors in EC. We found that the expression level of NUDT18 was significantly correlated with EC stage, grade, and myometrial invasion (P<0.05; Table 1), and NUDT18 protein expression exhibited distinct patterns across different clinicopathological parameters. In Federation of Gynecology and Obstetrics (FIGO) stage I EC, NUDT18 expression was significantly elevated but progressively declined with advancing tumor stage. Similarly, higher NUDT18 levels were observed in low-grade EC as compared to high-grade EC, demonstrating an inverse correlation with histological grade. Notably, tumors with elevated NUDT18 expression were associated with reduced myometrial invasion depth, suggesting a potential link between NUDT18 and favorable prognostic outcomes.

Figure 2 IHC staining of NUDT18 protein expression in tissues from EC patients (scale bar =100 µm). NUDT18 was positively stained mainly in the cytoplasm of the tumor cells. The expression of NUDT18 decreased with the increase in stage and decrease in differentiation. *, P<0.05; **, P<0.01; ***, P<0.001. EC, endometrial cancer; IHC, immunohistochemistry; NUDT18, nudix hydrolase 18.

NUDT18 overexpression independently predicted prolonged PFI in UCEC

Initially, the predictive value of NUDT18 in UCEC was determined via the optimum threshold-based Kaplan-Meier curve. It was found that patients with UCEC and a high NUDT18 expression had a longer PFI (P<0.05) (Figure 1H). In addition, Cox regression analysis was applied to investigate independent indicators of PFI in UCEC. Histological type, FIGO stage, histologic grade, status, peritoneal wash, para-aortic lymph nodes, pelvic lymph nodes, and NUDT18 expression were all significantly associated with PFI in UCEC based on the univariate model (all P values <0.05). After correction for other prognostic factors, the subsequent multivariate analyses indicated that NUDT18 overexpression was an independent predictor of PFI in UCEC (HR =0.49, 95% CI: 0.25–0.98; P=0.04) (Table 2).

Identification of signaling pathways for NUDT18 through GSEA and RT-qPCR

GSEA was used to identify the biological pathways significantly downregulated in UCEC samples with high NUDT18 expression. GSEA revealed that high NUDT18 expression was significantly associated with enrichment of gene sets involved in the cell cycle, TGF-β signaling pathway, RNA degradation, adherens junction, basal transcription factors, and DNA replication (all P values <0.05) (Figure 3A-3F). The genes affected in these pathways are also listed in https://cdn.amegroups.cn/static/public/tcr-2024-2538-1.xlsx. In order to verify the results from GSEA, we constructed two NUDT18-overexpression cell lines with stable transformation in Ishikawa and MFE-296, two EC cell lines. Western blotting (Figure S1) and RT-qPCR (Figure 3G,3H) experiments were used to verify that the cell lines with overexpression of NUDT18 were successfully constructed. Subsequently, we used RT-qPCR to verify the expression of several key factors in the TGF-β pathway and found that TGF-β, TGF-β receptor, SMAD2, SMAD3, and SMAD4 were all upregulated when NUDT18 was overexpressed (Figure 3G,3H).

Figure 3 GSEA of the TCGA-UCEC dataset. The GSEA results indicated that the overexpression of NUDT18 was significantly associated with (A) cytologic cycle, (B) TGF-β signaling pathway, (C) RNA degradation, (D) adherens junction, (E) basal transcription factors, and (F) DNA replication pathways. The NUDT18-overexpression and vector plasmid were transfected into two endometrial cancer cell lines, and the expression of several key factors of TGF-β pathway were detected. (G) RT-qPCR analysis of NUDT18 expression in MFE-296 oeNUDT18 and vector cell lines and test the expression level of TGF-β, TGF-β R, SMAD2, SMAD3, and SMAD4. (H) RT-qPCR analysis of NUDT18, TGF-β, TGF-β R, SMAD2, SMAD3, and SMAD4 expression in Ishikawa oeNUDT18 and vector cell lines. *, P<0.05; **, P<0.01; ***, P<0.001. FDR, false-discovery rate; GSEA, gene set enrichment analysis; KEGG, Kyoto Encyclopedia of Genes and Genomes; NES, normalized enrichment; NOM p-val, normalized P value; NUDT18, nudix hydrolase 18; RT-qPCR, real-time quantitative polymerase chain reaction; TCGA, The Cancer Genome Atlas; UCEC, Uterine Corpus Endometrial Carcinoma.

DNA copy gain and miR‑758‑3p downregulation toward the upregulation of NUDT18 in UCEC

The genetic and epigenetic changes potentially responsible for NUDT18 upregulation in UCEC were investigated. Among patients with UCEC and full CNA, mRNA, and methylation data within the TCGA-UCEC dataset, 16 patients exhibited low-level copy loss (deep deletion) in NUDT18, 38 shallow deletions, and 28 low-level copy-number increases (gains). In contrast, only one patient exhibited NUDT18 amplification, which revealed that NUDT18 is more frequently subject to copy-number loss (54 cases of deep and shallow deletions) than amplification among patients with UCEC. NUDT18 mRNA overexpression substantially correlated with NUDT18 copy number increase (P<0.05) (Figure 4A). We also analyzed the mRNA expression of NUDT18 in the same patient’s tissue when evaluated across CNA gene database (Figure S2). Meanwhile, Pearson correlation revealed a significant correlation between the NUDT18 DNA methylation and mRNA expression, which was further corroborated by linear regression analysis (Pearson r=−0.32; P=1.8e−05) (Figure 4B).

Figure 4 DNA copy gain, DNA methylation, miR-758-3p downregulation, and TP53 mutation were associated with NUDT18 overexpression in EC. (A) NUDT18 mRNA expression when evaluated across CNA. (B) NUDT18 mRNA expression decreased with the increase in the corresponding methylation of DNA. (C) miR-758-3p expression was downregulated in EC cases as opposed to normal endometrial tissues. (D) miR758-3p expression was negatively associated with NUDT18 mRNA levels. (E) High miR-758-3p expression was associated with poor OS in EC. (F) Heatmap of the association between NUDT18 mRNA expression and TP53 genetic change for TCGA-UCEC. (G,H) NUDT18 mRNA expression was significantly reduced in tumors with the TP53 mutation type. (I) The TP53 mutation was associated with unfavorable OS in EC. *, P<0.05; **, P<0.01; ***, P<0.001. CNA, copy number alteration; EC, endometrial cancer; HR, hazard ratio; NT, normal tissues; NUDT18, nudix hydrolase 18; OS, overall survival; TCGA, The Cancer Genome Atlas; UCEC, Uterine Corpus Endometrial Carcinoma.

We further sought to ascertain the impact of microRNAs on gene expression by repressing mRNA translation or inducing mRNA degradation. The miRwalk databases were used to predict potential regulatory NUDT18 microRNAs, while TCGA datasets were applied to identify microRNAs differentially expressed between EC tissues and normal controls. miR-758-3p was the candidate microRNA target for validation and was found to be substantially downregulated in EC tissues (n=531) as compared to control tissues (n=33) (P=1.32e−07) (Figure 4C). Meanwhile, there was a negative association between miR-758-3p expression and that of NUDT18 mRNA (Pearson r=−0.22; P=2.1e−07) (Figure 4D). Moreover, Kaplan-Meier analysis indicated that the increased miR-758-3p expression correlated with less satisfactory OS among patients with EC (P=0.02) (Figure 4E).

Association of the TP53 mutation of with lower NUDT18 expression in UCEC

Further evaluation of the relationship between NUDT18 expression and TP53 mutation in UCEC revealed that NUDT18 expression was significantly reduced in tumors with the TP53 mutation (P<0.001) (Figure 4F,4G). It was further found that TP53 in-frame mutations, missense mutations, and truncating mutations, as compared to the TP53 wild type, were associated with decreased NUDT18 expression, while no significant difference was observed for TP53 multiple mutations or splice mutations (Figure 4H). cBioPortal datasets from the category “Uterine Corpus Endometrial Carcinoma (TCGA, Nature 2013)” were analyzed for TP53 mutations. In the TCGA-UCEC dataset, patients with TP53 mutations had significantly poorer survival (P=0.03) (Figure 4I).

Association of NUDT18 with the tumor-immune microenvironment

The correlation between NUDT18 and the tumor-immune microenvironment was determined to clarify the underlying mechanism of NUDT18’s function in UCEC. Using the Tumor-Immune System Interactions and Drug Bank Database (TISIDB), we discovered that NUDT18 was associated with longer survival, particularly in UCEC and that a number of immune cells were upregulated in the high-NUDT18 expression group as compared to the low-expression group (Figure 5A,5B). In UCEC, NUDT18 expression was significantly associated with immune subtypes (Figure 5C,5D) and molecular subtypes (Figure 5E,5F). ImmunoCellAI was also used to determine which types of immune cells are regulated by NUDT18. Immune cell abundance examination indicated the higher abundance of several immune cell types [e.g., CD4⁺ T cells, dendritic cells (DCs), type 1 regulatory T cells (Tr1 cells), CD8⁺ T cells, T helper 1 cells (Th1 cells), T helper 2 cells (Th2 cells), induced T-regulatory cells (iTregs), T helper 17 cells (Th17 cells), mucosal-associated invariant T cells (MAIT cells), and T cell exhaustion (Tex) cells] in cases with high NUDT18 expression. In addition, this group exhibited a higher percentage of antitumor immune cells, including CD4⁺ T cells, CD8⁺ T cells, and DCs, (all P values <0.05) and a lower percentage protumor-type immune cells, including monocytes (P<0.001) (Figure 5E). The results indicated the association of NUDT18 with antitumor immunity in EC, which somewhat explains the connection between NUDT18 and a favorable prognosis.

Figure 5 Association between NUDT18 expression and the tumor-immune microenvironment in UCEC. (A) High NUDT18 expression correlated with improved survival across multiple cancers, most significantly in UCEC. (B) High NUDT18 expression correlated with elevated immune cell infiltration. (C) NUDT18 expression and its relationship with cancers in terms of immune subtypes. (D) Comparison of NUDT18 mRNA expression between various EC immune subtypes. (E) The high NUDT18-expression group demonstrated enhanced immune cell abundance. (F) NUDT18 expression and its association with cancers in terms of molecular subtypes. (G) Comparison of NUDT18 mRNA expression among various EC molecular subtypes. (Y axis means Infiltration_EnrichScore; X axis: 1 represent NUDT18-high group, 2 represent NUDT18-low group). CPM, counts per million mapped reads; EC, endometrial cancer; NUDT18, nudix hydrolase 18; NS, no significance; UCEC, Uterine Corpus Endometrial Carcinoma.

Discussion

The MTH family plays an important role in maintaining genomic stability (24-26). To our knowledge, this is the first study to examine the role of NUDT18 in EC, although other NUDT family members have been implicated in other tumor types. For example, it was found that NUDT1 is closely associated with increased invasiveness in glioblastoma (27) and to significantly upregulated in gastric cancer and melanoma (28,29). NUDT5 serves as an independent prognostic factor for colorectal cancer and is linked to poor prognosis in renal clear-cell carcinoma and esophageal carcinoma (30,31). Analysis of TCGA breast cancer database revealed that NUDT1, 2, 5, and 16 are significantly overexpressed in breast cancer tissues and correlate with poor prognosis in hormone receptor (HR)-positive breast cancer (32). Furthermore, inhibitors targeting these enzymes have shown promising results in preclinical trials. For instance, the MTH1 inhibitor (TH588) combined with the PI3K inhibitor (BKM120) synergistically enhances therapeutic efficacy against glioblastoma multiforme (33).

Through the analysis of NUDT18 expression levels and clinicopathological characteristics in EC tissues and healthy endometrial tissues via public databases, we found that NUDT18 expression is significantly upregulated in EC compared to a normal endometrium. However, in EC, NUDT18 expression did not show increased expression with tumor progression: stage I EC demonstrated a relatively high NUDT18 expression, but as the tumor stage advanced, NUDT18 levels incrementally decreased. Moreover, high NUDT18 expression correlated with better OS and DFI in patients with EC. This atypical pattern may be linked to epigenetic changes, transcriptional regulation, immune cell infiltration, genomic instability, or other factors specific to EC at different stages. Additionally, considering the role of NUDT18 as a nudix hydrolase in hydrolyzing damaged dNTPs in cancer cells with dysregulated ROS levels, its expression may be compensatively elevated during the early stages of tumorigenesis. Our findings suggest that NUDT18 may play a unique role in EC progression.

The observed decline in NUDT18 expression during advanced disease stages contrasts with other oncogenes that typically exhibit increasing expression with tumor progression. This distinct expression pattern suggests that NUDT18 may act as an environmentally dependent tumor modulator, potentially exerted a tumor-suppressive effect in early-stage disease through multiple pathways. However, its function may be compromised in later stages due to accumulated genomic instability or epigenetic modifications. TGF-β is a pleiotropic secreted cytokine whose signaling pathway activation plays a critical role in tumorigenesis and cancer progression. Studies suggest a dual, context-dependent role for TGF-β in tumor biology. In normal and premalignant cells, it can inhibit cell proliferation and induce differentiation to exert a tumor-suppressive effect. However, during the advanced tumor stages, TGF-β signaling undergoes a functional switch, promoting epithelial-mesenchymal transition (EMT), tumor cell invasion, and metastatic dissemination, thereby driving tumor progression (34). DNA methylation is one of the epigenetic regulatory mechanisms, which figures prominently in gene regulation and in cell growth and proliferation in the normal menstrual cycle (35). Abnormal activation of methylation may promote endometrial atypical hyperplasia (36) and even the occurrence and development of EC (37). TP53 is a critical tumor-suppressor gene, and the TP53 mutation serves as a potential biomarker for poor prognosis in EC (38,39). The TP53 mutation drives tumor progression through multiple oncogenic mechanisms, including the promotion of EMT, secretion of enhanced matrix metalloproteinase (MMP), and angiogenesis, leading to chromosome damage and instability (40,41). These coordinated effects contribute to tumor aggressiveness and therapeutic resistance in EC. The tumor microenvironment comprises diverse cellular and noncellular components, including blood vessels, immune cells, fibroblasts, signaling molecules, and the extracellular matrix (ECM), which engage in dynamic bidirectional crosstalk with tumor cells. Through the secretion of soluble factors, induction of angiogenesis, and establishment of immune tolerance, tumors actively remodel their microenvironment. Conversely, immune populations within the tumor microenvironment critically influence neoplastic progression through complex immunoediting processes (42). During early tumorigenesis, robust immune infiltration initially mounts an antitumor inflammatory response. However, progressive tumor microenvironment reprogramming drives the functional polarization of these immune effectors toward protumorigenic phenotypes. Specifically, tumor-associated macrophages (TAMs) predominantly tend to polarize toward M2 macrophages instead of the M1 type (43), regulatory T cells (Tregs) undergo significant expansion (44), tumor-associated neutrophils (TANs) shift from antitumor N1 to protumor N2 states (45,46), and DCs exhibit functional impairment, with immature DC populations promoting Treg expansion rather than the activation of effector T cells (47). Our findings suggest that NUDT18 may contribute to early tumor promotion through the modulation of these pathways. NUDT18 could serve as a novel prognostic biomarker for EC, particularly in early-stage patients, but further validation is necessary. Its high expression might identify a subgroup with favorable outcomes, potentially guiding adjuvant therapy decisions. Furthermore, restoring NUDT18 function (e.g., via demethylating agents or gene therapy) may serve as a therapeutic strategy for advanced EC.

However, the specific mechanism by which NUDT18 exerts its effects in EC was not extensively investigated in our study, which rather focused on identifying potential downstream regulatory pathways of NUDT18 through public database analysis. Further in vivo and in vitro experiments are still needed for verification.

Conclusions

NUDT18 expression is closely associated with the prognosis of EC and can serve as a prognostic factor for patients with EC, which may provide a clinical strategy for early prevention.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the REMARK reporting checklist. Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2024-2538/rc

Data Sharing Statement: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2024-2538/dss

Peer Review File: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2024-2538/prf

Funding: This work was supported by the National Natural Science Foundation of China (No. NSFC 82172819 to H.Z.).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2024-2538/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. This study has been granted an exemption from requiring ethics approval and an exemption from requiring written informed consent by Nanjing Drum Tower Hospital.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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