DTL promotes glycolysis and tumor progression in nasopharyngeal carcinoma cells by degrading KAT2B and activating the PI3K/AKT/mTOR pathway

DTL promotes glycolysis and tumor progression in nasopharyngeal carcinoma cells by degrading KAT2B and activating the PI3K/AKT/mTOR pathway | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article DTL promotes glycolysis and tumor progression in nasopharyngeal carcinoma cells by degrading KAT2B and activating the PI3K/AKT/mTOR pathway Jingwen Sun, Chaoping Huang, Wentao Zou, Shuang Zhou, Haibo Ye, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7061714/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Nasopharyngeal carcinoma (NPC) is a prevalent malignancy in East and Southeast Asia, with limited effective treatment options due to late-stage diagnosis. E3 ubiquitin ligase DTL has been implicated in various cancers, but its role in NPC remains obscure. This study aimed to investigate the regulatory mechanisms of DTL in NPC and its potential as a therapeutic target. Methods We conducted a comprehensive analysis combining bioinformatics, immunohistochemistry on clinical specimens, and a series of in vitro and in vivo experiments. Gene expression was analyzed through the GEO database, and the impact of DTL on NPC cell lines was assessed using qRT-PCR, western blotting, and various cellular assays. The interaction between DTL and KAT2B was explored, and the role of the PI3K/AKT/mTOR pathway in DTL-mediated NPC progression was investigated. Results DTL expression was significantly higher in NPC tissues and associated with poor prognosis. DTL knockdown inhibited NPC cell proliferation, migration, and glycolysis, while its overexpression promoted these phenotypes. Mechanistically, DTL interacted with and ubiquitinated KAT2B, leading to its degradation and subsequent activation of the PI3K/AKT/mTOR pathway, which in turn enhanced glycolysis and NPC progression. Conclusions Our findings identify DTL as a critical promoter of NPC, highlighting its potential as a therapeutic target. By targeting the KAT2B-PI3K/AKT/mTOR axis, interventions of DTL could offer a promising strategy for NPC treatment. nasopharyngeal carcinoma DTL KAT2B molecular mechanism glycolysis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Nasopharyngeal carcinoma (NPC) is an epithelial malignancy that occurs in the nasopharyngeal mucosa and is characterized by significant regional, racial, and gender differences in its incidence ( 1 ). Globally, 129,000 new cases of NPC were reported in 2018, accounting for only 0.7% of all new malignancies. However, the geographic distribution of NPC incidence is extremely unbalanced, with over 70% of new cases concentrated in East and Southeast Asia. The age-standardized rates in China and in populations that are mainly white are 3.0 and 0.4 per 100,000 ( 2 , 3 ). In China, the prevalence of NPC is particularly elevated in the southern and southwestern regions. Moreover, the occurrence of NPC is more prevalent in men compared to women, with a ratio (men/women) of approximately 2.5 in China in 2015 ( 4 ). The etiology of NPC is unknown and is currently considered to be a genetic disorder involving multiple genes, with a high racial and familial predisposition. Pathogenic factors include EB virus infection, chemical carcinogens, environmental influences, and genetic factors ( 5 ). Radiotherapy remains the primary treatment for NPC. However, 70% of patients are diagnosed in advanced stages due to hidden symptoms, and 10–30% still experience recurrence or distant metastasis after standard treatment. These are important factors affecting overall survival of NPC patients ( 3 , 4 ). Despite advances in treatment, clinical trials on targeted therapies for NPC are not abundant, which are mainly targeting epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor (VEGFR). Different targeted drugs show varying degrees of efficacy in delaying disease progression and extending patient survival ( 6 ). Therefore, exploring novel gene regulatory mechanisms and therapeutic targets could contribute to the diagnosis and treatment of NPC. DTL, also called DNA replication factor 2 (Cdt2) or retinoic acid-regulated nuclear matrix-associated protein (RAMP), is a member of the DCAF family of genes that encode the Cullin-Ring E3 ubiquitin ligase substrate receptor protein. DTL forms the E3 complex CRL4A with CUL4A, DDB1, and RBX1 proteins. As a denticleless E3 ubiquitin protein ligase homolog gene, DTL is essential to regulate the cell cycle and ensure proper DNA replication by mediating the ubiquitination and subsequent degradation of Cdt1 ( 7 ). Additionally, it is also involved in biological processes through ubiquitination of target proteins and is important in the occurrence, progression and metastasis of tumors. Numerous studies in recent years have demonstrated the potential role of DTL in various malignancies such as ovarian cancer, melanoma, colon cancer, breast cancer and hepatocellular carcinoma ( 8 – 13 ). Nevertheless, the precise mechanisms by which DTL influences NPC development remain largely unexplored. Malignant tumor cells rely on a large supply of macromolecules and energy to sustain their rapid proliferation and growth. Enhanced aerobic glycolysis not only allows tumor cells to produce ATP, but also maintains redox balance, and generates the metabolites needed for macromolecular synthesis ( 14 ). Several studies showed that the overexpression or increased activity of several critical regulatory enzymes promotes the glycolytic pathway in NPC cells, resulting in elevated glucose uptake and lactate production ( 15 – 17 ). Since the relationship between DTL and glycolysis is still unclear, exploring the potential mechanisms regulating glycolysis is of great significance for developing new therapies of NPC. In this study, differential genes and signaling pathways associated with NPC were identified through analysis of GSE12452 and GSE102349 from the GEO database. Immunohistochemistry of clinical specimens showed a significant increase in DTL levels in NPC patients, which was correlated with disease progression and poor prognosis. DTL promoted NPC proliferation in vitro and in vivo. Additionally, DTL activated PI3K/AKT/mTOR pathway through ubiquitination-mediated degradation of KAT2B, thereby promoting the glycolysis and tumor progression of NPC. These findings provided new insights into the role of DTL in NPC pathogenesis and highlighted its potential as a therapeutic target. 2. Materials and Methods 2.1 Bioinformatics analysis To screen for genes associated with NPC and analyze gene expression correlation, publicly available datasets from Gene Expression Omnibus (GEO, https://www.ncbi.nlm.nih.gov/geo ) database were utilized, including GSE12452 and GSE102349. GSE12452 dataset comprises samples from both NPC and normal tissues, which were used to analyze the differential expression of genes between these groups. Data normalization was conducted using the RMA (Robust Multi-array Average) method, and differential expressed analysis was performed with the R package limma. Genes were considered significantly differentially expressed if they exhibited a log2 fold change and an adjusted p-value of less than 0.05. The GSE102349 dataset was utilized for survival analysis. The optimal cutoff value of DTL expression in all NPC samples was used as the threshold to divide samples into high and low DTL expression groups. The progression-free survival (PFS) outcomes between these two groups were then compared using the log-rank test. Additionally, the clinical variables was categorized based on relevant clinical criteria, and intergroup differences in DTL expression among groups were analyzed using ANOVA. This allowed for the evaluation of DTL expression patterns across distinct clinical stages. Gene set enrichment analysis (GSEA) was performed using the GSEA software (GSEA_Linux_4.2.3), with the HALLMARK gene set defined in the MSigDB database ( https://www.gsea-msigdb.org/gsea/msigdb/download_file.jsp?filePath=/msigdb/release/2023.2.Hs/h.all.v2023.2.Hs.symbols.gmt ) as the reference. NPC samples from the GSE102349 dataset, retrieved from the GEO database ( https://www.ncbi.nlm.nih.gov/geo/download/?type=rnaseq_counts&acc=GSE102349 ), were stratified into high and low expression groups based on the median DTL gene expression level. The normalized expression matrix obtained using the R package DESeq2 was used for GSEA analysis. Gene sets with a NP < 0.05 and FDR < 0.25 were considered statistically significant. To further investigate DTL-related interactions at the protein level, the STRING database (version 11.5) was used to identify putative DTL-interacting proteins. The expression-levels correlations between DTL and its interacting molecules was assessed using the Spearman correlation analysis. 2.2 Antibodies and reagents The following antibodies were used for western blotting (WB), immunoprecipitation (IP) and immunohistochemistry in this study. Primary antibodies included DTL (#orb1529346, Biorbyt, UK), GAPDH (#60004-1-Ig, Proteintech, USA), PCAF (also called KAT2B, #sc-13124, Santa Cruz, USA), Ubiquitin (#sc-8017, Santa Cruz, USA), PKM2 (#15822-1-AP, Proteintech, USA), HK2 (#22029-1-AP, Proteintech, USA), GLUT1 (#21829-1-AP, Proteintech, USA), ADH4 (#ab137077, Abcam, UK), ADH6 (#67709-1-Ig, Proteintech, USA), LDHA (#ab52488, Abcam, UK), AKT (#10176-2-AP, Proteintech, USA), p-AKT (#66444-1-Ig, Proteintech, USA), mTOR (#66888-1-Ig, Proteintech, USA), p-mTOR (#67778-1-Ig, Proteintech, USA), Cyclin B1 (#ab32053, Abcam, UK), P21 (#ab109520, Abcam, UK), CDK4 ( #11026-1-AP, Proteintech, USA), KI67 (#ab16667, Abcam, UK). Secondary antibodies included Goat Anti-Rabbit (#A0208, Beyotime, China), Goat Anti-Mouse (#A0216, Beyotime, China), Goat Anti-Rabbit IgG H&L (HRP) preabsorbed (#ab97080, Abcam, UK). SC79 (#SF2730, Beyotime, China) is an AKT activator. In our experiments, cells were treated with SC79 at a concentration of 8 µg/mL for a duration of 24 to 48 h. MK-2206 2HCI (#S1078, Selleck, USA) is an AKT inhibitor and AZ-33 (#S0108, Selleck, USA) is a glycolysis inhibitor. Their dose was 10 µM and the time was 24 h. 2.3 Tissue microarray and immunohistochemical analysis Approval for the use of human tissues was obtained from the ethics committee of Shanghai Tenth People’s Hospital. Tissue microarray comprising 116 cases of NPC tissue and 14 cases of paraneoplastic tissue was purchased from the Shanghai Peide Biotechnology Center (#NPC1401, Shanghai, China). Clinicopathologic data included patient sex, age, clinical stage, TMN stage, metastasis status, and recurrence information. Immunohistochemistry of target proteins was performed as standard protocol. After deparaffinization and antigen retrieval, endogenous peroxides were quenched by incubating the sections in 3% H2O2. The sections were incubated with a primary anti-DTL antibody at 4℃ overnight. Subsequently, sections were incubated with a secondary antibody, cleaned, and stained with DAB in dark, followed by counterstaining with hematoxylin. DTL expression in cytoplasm, membrane and nucleus was estimated. The immunohistochemistry results were quantified by multiplying the staining intensity (dark brown = 3, brown and yellow = 2, pale yellow = 1, and negative = 0) with the percentage of positive cells (75–100% = 4, 50–74% = 3, 25–49% = 2, 1–24% = 1, and 0% = 0). The final scores were defined as strongly positive (+++, 9–12), moderately positive (++, 5–8), weakly positive (+, 1–4), and negative (-, 0). 2.4 Cell lines and culture The cell lines were from iCell Bioscience Inc (China). The human immortalized nasopharyngeal epithelial cell line NP69 was cultured in keratinocyte/serum-free medium (Invitrogen, USA) supplemented with bovine pituitary extract (BD Biosciences, USA). Human NPC cell lines (NPC/HK-1, 5-8F, HONE-1, C666-1) were cultured in DMEM or RPMI 1640 medium (Invitrogen, USA) containing 10% fetal bovine serum (Gibco, USA). The temperature required for cell culture was 37℃ and the air condition was air containing 5% CO2. 2.5 Quantitative real-time polymerase chain reaction (qRT-PCR) The Trizol purchased from Sigma was used to extract total RNA from NPC cells following the manufacturer’s instructions. Then, the reverse transcription reaction was performed using the Hiscript QRT supermix for qPCR (+ gDNA WIPE) (Vazyme, China). Finally, cDNA, forward and reverse primers and AceQ qPCR SYBR Green master mix (Vazyme, China) were mixed, and qRT-PCR was performed on the Real-Time PCR system (ABI, USA). The primer sequences used for GAPDH were 5’-TGACTTCAACAGCGACACCCA-3’ and 3’-CACCCTGTTGCTGTAGCCAAA-5’. The primer sequences for DTL were 5’-TAAAAGCTGGTGAGCTGATTGG-3’ and 5’-TCTTCCACCCGTACAGAATACA-3’. 2.6 Construction of plasmid vector, package of lentivirus and, transfection Three RNA interference sequences targeting the DTL gene were designed, and the following sequences were used as RNA interference targets: 5’-CTGCACATACTTCCATAGAAA-3’, 5’-TGGCGCTTGAATAGAGGCTTA-3’, and 5’-AGGGTCTGAAATGGTAGGCAA-3’. Lentivirus vectors carrying short hairpin RNAs (shRNAs) specific for DTL (shDTL-1, shDTL-2 and shDTL-3 lentivirus) and lentiviral constructs overexpressing DTL and KAT2B overexpression were constructed and packaged by Yibeirui (Shanghai, China). Cell transfections were performed using Puromycin (Gibco, USA) according to the manufacturer’s instructions, and the transfection efficiency was determined 72 h post-infection using RT-qPCR, fluorescence observation and western blotting. 2.7 Western blotting Western blotting was performed to detect protein expression levels. Cells were lysed with the RIPA lysis buffer (Beyotime, China) supplemented with protease inhibitors. Lysates were centrifuged at 12,000 rpm for 10 min (4℃) to collect the supernatant. Protein concentrations were quantified using a BCA protein assay kit (Beyotime, China). After gel electrophoresis, the protein was transferred to PVDF membrane. 5% skim milk was added for 1 h at room temperature. Membranes were incubated with primary antibodies overnight at 4℃, followed by incubation with secondary antibodies for 1 h at room temperature. Protein bands were visualized using Chemiluminescent HRP Substrote (Beyotime, China). 2.8 CCK-8 assay, flow cytometry, colony formation, wound-healing assay, transwell assay Annexin V-APC/PI Apoptosis Detection Kit (eBioscience, USA) was used to evaluate apoptosis of NPC cells transfected with lentivirus according to the manufacturer’s instructions. Cells were collected with trypsinization, centrifuged, washed with PBS and made into a suspension of 1×10 6 cell/mL. 100 µL of cell suspension was stained by 5 µL of Annexin V-APC for 15 min at room temperature in the dark, and then added 5 µL of PI. Cell apoptosis was detected by a flow cytometer (Millipore, USA). CCK-8 assay and colony formation assay were performed to measure cell proliferation. For the CCK-8 assay, NPC cells in the logarithmic growth phase were inoculated in 96-well plates (2000 cells/well). After specified incubation periods, 10 µL of CCK-8 (Sigma, USA) was added to each well and the absorbance at 450 nm per well was read on a spectrophotometer. For the colony formation assay, NPC cells were plated in 6-well plates (400 cells/well) and maintained for 14 days. Then, colonies were fixed with 4% paraformaldehyde, stained with Giemsa stain, and counted. Wound-healing assay was used to assess the migration ability of cells. Cells were cultured in 96-well plates. Linear wounds were created using a 96 Wounding Replicator (VP scientific, USA) and cells were incubated for another 24 h in starvation medium. Images of the wound area were captured using Cellomics (Thermo, USA) at 0 h and 24 h. The migration rate was calculated by comparing the wound area at the two time points. Transwell assay was conducted using Transwell chambers (3422 Corning, NY, USA) with an 8-µm pore size to assess the migration capability of NPC cells. 2×10 5 cells suspended in 100 µL of serum-free medium were seeded into transwell chambers (3422 Corning, NY, USA). The transwell chambers were placed into the lower chambers with 600 µL of complete media containing 30% FBS. After 24 h of incubation at 37°C, non-migratory cells were removed, while cells that had migrated to the underside of the membrane were fixed in 4% paraformaldehyde and stained with 0.05% crystal violet. Migratory cells were counted under a microscope (Olympus, Japan). 2.9 Glucose and lactate assay and measurement of extracellular acidification rate (ECAR) To assess glucose metabolism and lactate production, cells were washed with cold PBS and lysed in 0.1% Triton X-100. The cell lysates were filtered using an Amicon Ultra-3 kDa cutoff (Merck). Glucose and lactate levels were quantitated using a Glucose Assay kit-WST and Lactate Assay kit-WST (Dojindo) following the manufacturer's instructions. The glycolysis stress test was conducted in a XF24 Extracellular Flux Analyzer (Seahorse Bioscience, North Billerica, MA). Glucose (10 mM; Sigma), oligomycin (1 µM; Sigma), and 2-deoxyglucose (2-DG, 100 mM; Sigma) were sequentially added to the assay medium at the indicated time points during ECAR measurement. ECAR values were normalized to protein contents quantified using Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific). 2.10 In vivo xenograft models All animal experiments were approved by Shanghai Tenth People's Hospital Laboratory Animal Ethics Committee (Approval number: SHDSYY-2024-3832). 4-week-old female NXG nude mice (Cavens, Changzhou, China) were used for animal studies. The animal experiment was conducted using a double-blind design and randomly divided into two groups. To establish mouse xenograft models, NPC/HK-1 cells (1×10 7 /200 µL) with DTL knockdown or negative control were injected subcutaneously into the right flank of each mouse. The tumor volume was monitored every three or four days by measuring the length (L) and width (W) of tumors using calipers. Tumor volume (mm 3 ) was calculated as length × width 2 /2. Mice were sacrificed after 18 days, and the tumors were excised, weighed, and processed for further analyses. Samples were embedded in paraffin and subjected to immunohistochemistry for histological evaluation of KI67 and DTL expression. Western blotting detected PI3K/AKT/mTOR pathway proteins. 2.11 Statistical analysis All statistical analyses were performed using SPSS (version 22.0, IBM Corp., USA) and GraphPad Prism (version 10, CA, USA). Data are expressed as mean ± standard deviation (SD). Comparison among groups was performed by t-test and One-way ANOVA. Mann-Whitney U test and Spearman’ s rank correlation test were used to evaluate the correlation between DTL expression and pathological features of NPC patients. Survival curves were analyzed using Kaplan-Meier analysis. Statistical significance was defined as P < 0.05. 3. Results 3.1 DTL is highly expressed in NPC tissues and predicts poor prognosis. To identify genes associated with the occurrence and development of NPC, the expression of genes in NPC tissues and the relationship between gene expression and prognosis were analyzed. Data from GSE12452 dataset revealed that DTL expression was significantly higher in NPC tissues than in normal tissues (Fig. 1 A). Further investigation of the GSE102349 dataset showed that high DTL expression was associated with a significantly shorter progression-free survival (PFS) in NPC patients, as indicated by Kaplan-Meier survival curves (Fig. 1 B). Additionally, DTL expression was found to be significantly higher in stage IV NPC tissues compared to stage I tissues (Fig. 1 C). These results indicated that DTL was not only highly expressed in NPC, but also associated with advanced stages and poor prognosis, making it a potential target for therapeutic intervention. To learn about the clinical significance of DTL expression in NPC, immunohistochemistry was carried out on a tissue microarray consisting of 116 cases of NPC tissue and 14 cases of paraneoplastic tissue. Compared with paraneoplasitc tissue, DTL expression was significantly higher in NPC tissue (Fig. 1 D, Table 1 ). Mann-Whitney U test further confirmed that DTL expression was markedly associated with pathological stage of NPC (Table 2 ). Furthermore, the analysis of Spearman’s rank correlation revealed a positive correlation between DTL expression and pathological stage (P = 0.022) (Table 3 ). Kaplan-Meier survival analysis showed that high DTL expression was significantly associated with poor overall survival and disease-free survival in NPC patients (Fig. 1 E). These findings suggested that elevated DTL expression was significantly associated with poorer overall survival in NPC and may serve as a new therapeutic target in managing the disease. Table 1 Expression patterns in nasopharyngeal carcinoma tissues and para-carcinoma tissues revealed in immunohistochemistry analysis DTL expression Tumor tissue Para-carcinoma tissue p value Cases Percentage Cases Percentage P < 0.001 Low 62 53.4% 13 92.9% High 54 46.6% 1 7.1% Table 2 Relationship between DTL expression and tumor characteristics in patients with nasopharyngeal carcinoma Features No. of patients DTL expression p value low high All patients 116 62 54 Gender 0.577 Male 74 41 33 Female 42 21 21 Tumor size 0.489 ≤ 1.2 cm 62 35 27 >1.2 cm 54 27 27 T Infiltrate 0.414 T1 42 24 18 T2 43 23 20 T3 29 15 14 T4 2 0 2 lymphatic metastasis (N) 0.308 N0 79 45 34 N1 34 15 19 N2 3 2 1 Metastasis 0.091 M0 92 53 39 M1 23 8 15 M4 1 1 0 Stage 0.023 I 32 20 12 II 31 19 12 III 25 14 11 IV 28 9 19 Age 0.980 > 52 years 60 32 28 ≤ 52 years 56 30 26 Table 3 Relationship between DTL expression and tumor characteristics in patients with nasopharyngeal carcinoma DTL Stage Spearman’s rank correlation 0.213 Significance 0.022 N 116 3.2 DTL knockdown suppresses malignant phenotypes of NPC in vitro. To investigate biological functions of DTL in NPC, shRNA-mediated knockdown of DTL was performed in NPC cell lines. Compared with nasal mucosal epithelial cells (NP69 cells), DTL is highly expressed in NPC/HK-1, HONE-1 and C666-1 cells. Among the four NPC cell lines tested, DTL was most highly expressed in NPC/HK-1 and C666-1 cells (Fig. 1 F), which were selected for subsequent experiments. Transfection efficiency was verified by qRT-PCR, which showed that shDTL-1 and shDTL-2 lentiviruses achieved efficient knockdown of DTL (Supplementary Fig. 1A). qRT-PCR and western blotting results confirmed the successful construction of DTL knockdown cell models (Supplementary Fig. 1B). The results of CCK-8 assay (Fig. 2 A), cell apoptosis (Fig. 2 B) and colony formation assay (Fig. 2 C) showed that proliferation and colony numbers of NPC/HK-1 and C666-1 cells were suppressed by knockdown of DTL. Wound-healing assay (Fig. 2 D) and transwell assay (Fig. 2 E) indicated that DTL knockdown resulted in a decrease in migration rate of NPC/HK-1 and C666-1 cells. These results implied that downregulation of DTL might inhibit the malignant phenotype of NPC cells in vitro. It is necessary to further explore underlying mechanisms of DTL regulation in the progression of NPC. 3.3 DTL promotes the ubiquitination and degradation of KAT2B. To explore the regulatory mechanism underlying DTL in NPC, bioinformatic analysis using STRING and GEO database (GSE12452 dataset) were performed to screen genes that may interact with DTL and are significantly downregulated in NPC samples. The potential downstream molecules of DTL were screened by some criteria. The downstream gene was a tumor suppressor gene, which interacted with DTL and showed negative correlation in expression. KAT2B (also known as PCAF) was found to be a potential downstream molecule of DTL, exhibiting the highest negative correlation coefficient with DTL expression (Supplementary Table S1). Since DTL is an E3 ubiquitin ligase ( 7 ), it is hypothesized that DTL may affect the protein stability of KAT2B through ubiquitination. The regulatory effect of DTL on KAT2B was verified by western blot. KAT2B protein was increased in DTL-knockdown NPC cells (Fig. 3 A). As an E3 ubiquitin ligase, DTL is known to connect to its substrate and promote the degradation of the substrate. The interaction between DTL and KAT2B was further confirmed by co-immunoprecipitation (Co-IP) assays (Fig. 3 B). Subsequently, NPC cells were treated with cycloheximide (CHX) to inhibit the synthesis of proteins, and KAT2B in the DTL-knockdown group declined more slowly compared to control cells, indicating that DTL had a negative regulatory effect on KAT2B by affecting its degradation rate (Fig. 3 C). Furthermore, ubiquitination assay showed that DTL overexpression increased polyubiquitinated KAT2B (Fig. 3 D). These results suggested that DTL promoted KAT2B degradation by increasing KAT2B ubiquitination. 3.4 DTL inhibits KAT2B and activates PI3K/AKT/mTOR pathway to promote aerobic glycolysis and NPC progression. Previous studies reported that DTL might promote bladder cancer progression through the AKT/mTOR pathway ( 18 ). Besides, PI3K/AKT signaling pathway was modulated by miR-17-5p/KAT2B axis in liver fibrosis ( 19 ). To further investigate whether DTL and KAT2B affect the progression of NPC through the PI3K/AKT/mTOR pathway, gene set enrichment analysis (GSEA) was performed using samples from the GEO database (GSE102349 dataset), stratified by DTL expression. This analysis revealed that DTL expression was positively related to the activation of PI3K/AKT/mTOR pathway (Fig. 4 A). Downregulation of DTL suppressed the expression and phosphorylation of key proteins in PI3K/AKT/mTOR pathway, including p-AKT and p-mTOR, in NPC/HK-1 and C666-1 cells (Fig. 4 B). Then, to further demonstrate whether DTL activated PI3K/AKT/mTOR pathway by inhibiting KAT2B, KAT2B overexpression was simultaneously conducted in NPC cells with DTL overexpression. Overexpression of KAT2B restored the stimulative effect of DTL on the activation of the above pathway to a certain extent (Fig. 4 C). Subsequently, SC79 (the AKT activator) was used to explore if DTL promoted NPC progression through PI3K/AKT/mTOR pathway. SC79 partially reversed the inhibitory effect of DTL knockdown on the PI3K/AKT/mTOR pathway (Fig. 4 D). Using apoptosis assays, we also observed that the inhibitory effect of DTL knockdown on NPC cell proliferation was partially restored by SC79 (Fig. 4 E). Furthermore, the GSEA analysis revealed a relationship between DTL and glycolysis (Fig. 4 A, Fig. 5 A). To evaluate the impact of DTL on aerobic glycolysis, we examined the expression of glycolytic proteins, glucose uptake, lactate production, and ECAR. DTL knockdown contributed to a reduced expression of glycolytic-related proteins such as ADH4, ADH6, GLUT1, HK2, LDHA, PKM2 (Fig. 5 B) and a decrease in glucose uptake, lactate production, ECAR (Fig. 5 C). In contrast, overexpression of DTL increased these glycolytic parameters. Importantly, KAT2B, MK-2206 2HCI (the AKT inhibitor) and AZ-33 (the glycolysis inhibitor) restored the effects of DTL on glycolysis, indicating that DTL regulated glycolysis through KAT2B and PI3K/AKT/mTOR pathway (Fig. 5 D, E, F). Moreover, CCK-8 and cell apoptosis assays demonstrated that AZ-33 partially restored the effect of DTL overexpression on NPC cell proliferation (Fig. 5 G). Therefore, we concluded that DTL facilitated aerobic glycolysis and NPC progression by inhibiting KAT2B and activating the PI3K/AKT/mTOR pathway. 3.5 DTL promotes NPC tumor growth in vivo. To assess the role of DTL in tumor progression of NPC in vivo, we carried out xenograft experiments in mice. As expected, DTL depletion significantly reduced the size and weight of NPC xenografts (Fig. 6 A, B). In addition, the levels of p-AKT and p-mTOR of PI3K/AKT/mTOR pathway was markedly decreased in DTL-knockdown xenografts (Fig. 6 C). Immunohistochemistry demonstrated that the levels of Ki67, a marker of cell proliferation, and DTL were greatly reduced in tumors from NPC/HK-1 xenografts with depleted DTL (Fig. 6 D). Collectively, these data suggested that DTL promoted NPC tumor growth in vivo by modulating the PI3K/AKT/mTOR pathway and enhancing proliferation. 4. Discussion As previously mentioned, the expression of DTL was found to stimulate several kinds of malignancies ( 18 ). For instance, Cui H et al. reported that DTL degraded PDCD4 through ubiquitination, which in turn enhanced proliferation and migration abilities of breast and lung cancers ( 12 ). Moreover, by identifying 46 differentially expressed genes (DEGs), enrichment analysis results showed that DTL was closely associated with the development and progression of NPC, and it has been recognized as a potential biomarker of NPC. However, these conclusions were not supported by experiments such as immunohistochemistry ( 20 ). The study of Chang X et al. indicated that under the regulation of ZFPM2-AS1/miR-3612, the expression of DTL was upregulated in NPC and drove the progression of NPC ( 21 ). These findings were consistent with our study, which highlighted the role of DTL in promoting NPC tumor growth both in vitro and in vivo. In addition to proving that DTL drives progression of NPC, we screened downstream genes and signaling pathways involved in this process. As a lysine acetyltransferase, KAT2B catalyzes lysine acetylation on both histones and non-histone proteins and regulates gene expression ( 22 , 23 ). Zhang J et al. substantiated KAT2B as a key effector of breast tumorigenesis, which contributed to cancer progression by synergizing with NELF-E-SLUG to promote EMT ( 24 ). In contrast, KAT2B expression was downregulated in non-small cell lung cancer (NSCLC), and low KAT2B expression predicted poor prognosis of patients. KAT2B was mainly linked to the regulation of immune cells and IFN-γ mediated signaling pathways ( 23 ), suggesting that KAT2B was involved in the immune response and tumor microenvironment. In human aortic endothelial cell line HAEC cells, KAT2B was found to be degraded by UBE2D2 through ubiquitination ( 25 ). Similar to the mechanism of previous studies, our study showed that KAT2B was degraded by DTL through ubiquitination. Notably, the effects of KAT2B and DTL were opposite, with DTL promoting tumor progression through its regulation of KAT2B. PI3K/AKT/mTOR pathway plays a crucial role in fundamental cellular functions, such as cell proliferation, metabolism, and motility. Aberrant activation of this pathway is frequently observed in various cancers ( 26 ). Notably, PI3K/AKT/mTOR signaling pathway is active in over 90% of head and neck squamous cell carcinomas. Luo Y et al. reported that DTL might promote bladder cancer progression through the AKT/mTOR pathway ( 18 ). However, there is a lack of experiments to prove that DTL induces the activation of the PI3K/AKT/mTOR pathway. Our results demonstrated that DTL knockdown inhibited the phosphorylation of AKT and mTOR proteins, while overexpression of DTL increased the phosphorylation levels of these two proteins. A study of liver fibrosis showed that human bone marrow mesenchymal stem cells-derived exosome circCDK13 inhibited PI3K/AKT and NF-κB pathways through regulating the miR-17-5p/KAT2B axis, thereby mitigating liver fibrosis ( 19 ). In this research, we found that DTL activated the PI3K/AKT/mTOR pathway by disrupting the stability of KAT2B protein, thereby promoting the proliferation of NPC cells. Aerobic glycolysis or the “Warburg effect” refers to the tendency of tumor cells to convert pyruvate derived from glucose into lactate through lactate dehydrogenase (LDH) ( 27 ). In NPC cells, glucose uptake is due to the activation of mTORC1/NF-κB signaling pathway and the transcription of glucose transporter 1 (GLUT1) ( 28 ). Hexokinase and pyruvate kinase are critical regulatory enzymes in the glycolytic pathway ( 29 ). FOXC2-YAP pathway increases hexokinase expression in NPC cells ( 16 ), and significantly upregulate the expression of PKM2 (a subtype of pyruvate kinase) by activating the PI3K/AKT signaling pathway ( 17 ). The overexpression of these enzymes induces the Warburg effect, resulting in elevated the lactate production in NPC cells even in the presence of oxygen. Our research indicated that DTL, by mediating the ubiquitination and degradation of KAT2B, not only activated the PI3K/AKT/mTOR pathway but also promoted the expression of HK2, LDHA, and PKM2, thereby enhancing glycolysis. Moreover, DTL also promoted the proliferation of NPC cells through glycolysis. In conclusion, our study revealed that DTL was remarkably upregulated in NPC tumor tissues, which was closely associated with disease progression and poor prognosis. Knockdown of DTL suppressed several malignant phenotypes of NPC cells, including proliferation and migration. Mechanistically, DTL interacted with KAT2B and downregulated its expression, thereby activating the PI3K/AKT/mTOR pathway, which promoted the glycolysis and led to the development of NPC. These results indicated that DTL could be a potential therapeutic target for NPC. However, this study has certain limitations. First, the tissue microarray was used for clinicopathological analysis and a larger sample size of NPC is required to verify the prognostic value of DTL. Second, while the role of DTL in glycolysis has been suggested, further investigation is required to fully elucidate the mechanism of DTL in this process. Declarations Author Contribution Jingwen Sun and Chaoping Huang contributed equally to this study. Jingwen Sun and Chaoping Huang wrote the main manuscript text. Wentao Zou and Shuang Zhou prepared figures 1-3, supplementary figure 1 and all the tables. Haibo Ye and Jingshuo Wang prepared figure 4-6. All authors reviewed the manuscript. References Bossi P, Chan AT, Licitra L, Trama A, Orlandi E, Hui EP, et al. Nasopharyngeal carcinoma: ESMO-EURACAN Clinical Practice Guidelines for diagnosis, treatment and follow-up(†). Ann Oncol. 2021;32(4):452-65. Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2018;68(6):394-424. Ferlay J, Colombet M, Soerjomataram I, Mathers C, Parkin DM, Piñeros M, et al. Estimating the global cancer incidence and mortality in 2018: GLOBOCAN sources and methods. Int J Cancer. 2019;144(8):1941-53. Chen YP, Chan ATC, Le QT, Blanchard P, Sun Y, Ma J. Nasopharyngeal carcinoma. Lancet. 2019;394(10192):64-80. Huang H, Yao Y, Deng X, Huang Z, Chen Y, Wang Z, et al. Immunotherapy for nasopharyngeal carcinoma: Current status and prospects (Review). Int J Oncol. 2023;63(2). Kang Y, He W, Ren C, Qiao J, Guo Q, Hu J, et al. Advances in targeted therapy mainly based on signal pathways for nasopharyngeal carcinoma. Signal Transduct Target Ther. 2020;5(1):245. Jin J, Arias EE, Chen J, Harper JW, Walter JC. A family of diverse Cul4-Ddb1-interacting proteins includes Cdt2, which is required for S phase destruction of the replication factor Cdt1. Mol Cell. 2006;23(5):709-21. Pan WW, Zhou JJ, Yu C, Xu Y, Guo LJ, Zhang HY, et al. Ubiquitin E3 ligase CRL4(CDT2/DCAF2) as a potential chemotherapeutic target for ovarian surface epithelial cancer. J Biol Chem. 2013;288(41):29680-91. Ding Y, Li M, Tayier T, Zhang M, Chen L, Feng S. Bioinformatics analysis of lncRNA‑associated ceRNA network in melanoma. J Cancer. 2021;12(10):2921-32. Ma J, Cai X, Kang L, Chen S, Liu H. Identification of novel biomarkers and candidate small-molecule drugs in cutaneous melanoma by comprehensive gene microarrays analysis. J Cancer. 2021;12(5):1307-17. Chen W, Gao C, Liu Y, Wen Y, Hong X, Huang Z. Bioinformatics Analysis of Prognostic miRNA Signature and Potential Critical Genes in Colon Cancer. Front Genet. 2020;11:478. Cui H, Wang Q, Lei Z, Feng M, Zhao Z, Wang Y, et al. DTL promotes cancer progression by PDCD4 ubiquitin-dependent degradation. J Exp Clin Cancer Res. 2019;38(1):350. Zhou Z, Li Y, Hao H, Wang Y, Zhou Z, Wang Z, et al. Screening Hub Genes as Prognostic Biomarkers of Hepatocellular Carcinoma by Bioinformatics Analysis. Cell Transplant. 2019;28(1_suppl):76s-86s. Szwed A, Kim E, Jacinto E. Regulation and metabolic functions of mTORC1 and mTORC2. Physiol Rev. 2021;101(3):1371-426. Zschaeck S, Li Y, Lin Q, Beck M, Amthauer H, Bauersachs L, et al. Prognostic value of baseline [18F]-fluorodeoxyglucose positron emission tomography parameters MTV, TLG and asphericity in an international multicenter cohort of nasopharyngeal carcinoma patients. PLoS One. 2020;15(7):e0236841. Song L, Tang H, Liao W, Luo X, Li Y, Chen T, et al. FOXC2 positively regulates YAP signaling and promotes the glycolysis of nasopharyngeal carcinoma. Exp Cell Res. 2017;357(1):17-24. Chen S, Youhong T, Tan Y, He Y, Ban Y, Cai J, et al. EGFR-PKM2 signaling promotes the metastatic potential of nasopharyngeal carcinoma through induction of FOSL1 and ANTXR2. Carcinogenesis. 2020;41(6):723-33. Luo Y, He Z, Liu W, Zhou F, Liu T, Wang G. DTL Is a Prognostic Biomarker and Promotes Bladder Cancer Progression through Regulating the AKT/mTOR axis. Oxid Med Cell Longev. 2022;2022:3369858. Ma J, Li Y, Chen M, Wang W, Zhao Q, He B, et al. hMSCs-derived exosome circCDK13 inhibits liver fibrosis by regulating the expression of MFGE8 through miR-17-5p/KAT2B. Cell Biol Toxicol. 2023;39(2):1-22. Wang H, Zhang J. Identification of DTL as Related Biomarker and Immune Infiltration Characteristics of Nasopharyngeal Carcinoma via Comprehensive Strategies. Int J Gen Med. 2022;15:2329-45. Chang X, Jian L. LncRNA ZFPM2-AS1 drives the progression of nasopharyngeal carcinoma via modulating the downstream miR-3612/DTL signaling. Anticancer Drugs. 2022;33(6):523-33. Nagy Z, Tora L. Distinct GCN5/PCAF-containing complexes function as co-activators and are involved in transcription factor and global histone acetylation. Oncogene. 2007;26(37):5341-57. Zhou X, Wang N, Zhang Y, Yu H, Wu Q. KAT2B is an immune infiltration-associated biomarker predicting prognosis and response to immunotherapy in non-small cell lung cancer. Invest New Drugs. 2022;40(1):43-57. Zhang J, Hu Z, Chung HH, Tian Y, Lau KW, Ser Z, et al. Dependency of NELF-E-SLUG-KAT2B epigenetic axis in breast cancer carcinogenesis. Nat Commun. 2023;14(1):2439. Qi X, Wang H, Xia L, Lin R, Li T, Guan C, et al. miR-30b-5p releases HMGB1 via UBE2D2/KAT2B/HMGB1 pathway to promote pro-inflammatory polarization and recruitment of macrophages. Atherosclerosis. 2021;324:38-45. Alzahrani AS. PI3K/Akt/mTOR inhibitors in cancer: At the bench and bedside. Semin Cancer Biol. 2019;59:125-32. Vander Heiden MG, Cantley LC, Thompson CB. Understanding the Warburg effect: the metabolic requirements of cell proliferation. Science. 2009;324(5930):1029-33. Anderson NM, Mucka P, Kern JG, Feng H. The emerging role and targetability of the TCA cycle in cancer metabolism. Protein Cell. 2018;9(2):216-37. Liu Q, Bode AM, Chen X, Luo X. Metabolic reprogramming in nasopharyngeal carcinoma: Mechanisms and therapeutic opportunities. Biochim Biophys Acta Rev Cancer. 2023;1878(6):189023. Additional Declarations No competing interests reported. Supplementary Files Supplementarymaterial.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7061714","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":483980860,"identity":"1e3f4df6-b1b9-4a8a-8a95-799b5803a3d4","order_by":0,"name":"Jingwen Sun","email":"","orcid":"","institution":"Shanghai Tenth People's Hospital, Tongji Unerversity School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Jingwen","middleName":"","lastName":"Sun","suffix":""},{"id":483980861,"identity":"89b357b5-e2d3-4cc2-9851-993ea2a7d2e8","order_by":1,"name":"Chaoping Huang","email":"","orcid":"","institution":"Chengdu Sixth People’s Hospital","correspondingAuthor":false,"prefix":"","firstName":"Chaoping","middleName":"","lastName":"Huang","suffix":""},{"id":483980862,"identity":"671755dd-c33c-4e44-bdde-3626561f6276","order_by":2,"name":"Wentao Zou","email":"","orcid":"","institution":"Shanghai Tenth People's Hospital, Tongji Unerversity School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Wentao","middleName":"","lastName":"Zou","suffix":""},{"id":483980863,"identity":"1069374f-ed22-4e95-ad37-74605a897eca","order_by":3,"name":"Shuang Zhou","email":"","orcid":"","institution":"Shanghai Tenth People's Hospital, Tongji Unerversity School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Shuang","middleName":"","lastName":"Zhou","suffix":""},{"id":483980864,"identity":"8c98201e-a121-4930-8fb1-edcd8d2d36ab","order_by":4,"name":"Haibo Ye","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5UlEQVRIie3PsWrDMBCA4QsGZ7k2qwzBfQWVTAVDXkWC4C4JZNQQaIyNNCSlr+ItGSUEnq57xuQRSpcMGZK9pXK2Dvrm+7k7gCj6h9Jhbf1FFfnUVNVRqFU4ecROWqRywtHX/EhdOMnZfGIfGi9b9qqzU5P0OAyJ22xdDnbotJLrFEZmIwK/6KV93hfJi6n0Qe7HwOizDWzxrZVUpkDullAKnC0CCRPcOu0RDlIvpU76JDPuKu0ZvyXQL8FOeKCSZ1tXM0EdBn95MrX/BlW8fQzN6eusVvnIvP+d/ID3jUdRFEW/ugKzl1Rpgf9XzQAAAABJRU5ErkJggg==","orcid":"","institution":"Shanghai Tenth People's Hospital, Tongji Unerversity School of Medicine","correspondingAuthor":true,"prefix":"","firstName":"Haibo","middleName":"","lastName":"Ye","suffix":""},{"id":483980865,"identity":"0852650b-5d67-4ffa-aa24-01b46a5e5521","order_by":5,"name":"Jingshuo Wang","email":"","orcid":"","institution":"Renji Hospital, School of Medicine, Shanghai Jiao Tong University","correspondingAuthor":false,"prefix":"","firstName":"Jingshuo","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2025-07-07 06:08:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7061714/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7061714/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":86748937,"identity":"beab2daa-529d-49f4-95b6-a93ce95a08a6","added_by":"auto","created_at":"2025-07-15 08:14:35","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1335419,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of DTL in NPC. A. Comparison of the gene expression levels of DTL in NPC and normal samples using public dataset (GSE12452). B. Survival analysis for NPC in the GEO database (GSE102349). C. The expression levels of DTL across different pathologic stages of NPC were assessed. D. Immunohistochemistry of DTL was performed on NPC tissue microarrays, which included both NPC and paraneoplastic tissues (scale bar, 50 μm in 200×, 20 μm in 400×). E. Kaplan-Meier survival curves illustrating overall survival and disease-free survival of NPC patients, based on tissue microarray data. F. RT-qPCR was used to detect the expression of DTL in different NPC cell lines. *P \u0026lt; 0.05; **P \u0026lt; 0.01; ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7061714/v1/09cee496cc4a8dce37e5328c.png"},{"id":86748934,"identity":"05ff7ad3-4bf5-4f57-8db0-4ada1b9942e6","added_by":"auto","created_at":"2025-07-15 08:14:35","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1005698,"visible":true,"origin":"","legend":"\u003cp\u003eDTL knockdown suppresses malignant phenotypes of NPC in vitro. A. CCK-8 assay was used to assess the cell viability. B. Apoptosis of NPC/HK-1 and C666-1 cells after knockdown of DTL was analyzed by flow cytometry. C. The colony formation assay was conducted to detect the cell reproductive capacity. D and E. Wound-healing and transwell assays were performed to investigate migratory ability of NPC/HK-1 and C666-1 cells post- lentiviral infection.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7061714/v1/78401881a4accc0c4360bb24.png"},{"id":86748938,"identity":"73d05b8c-975b-4762-9bdf-9322ecc47ec6","added_by":"auto","created_at":"2025-07-15 08:14:35","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":557977,"visible":true,"origin":"","legend":"\u003cp\u003eDTL promotes the ubiquitination and degradation of KAT2B. A. Western blotting analysis verified the regulatory effect of DTL on KAT2B expression. B. Co-immunoprecipitation (Co-IP) assays confirmed that DTL was immunoprecipitated with KAT2B antibody. C. The half-life of KAT2B in C666-1 and NPC/HK-1 cells, with or without DTL knockdown, was assessed. Cells were treated with cycloheximide (CHX) for the indicated times, and western blotting showed that KAT2B in the DTL-knockdown group declined more slowly. D. Co-IP assays were performed to detect polyubiquitinated KAT2B.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7061714/v1/f8780ae944fa5baf32fc2609.png"},{"id":86748943,"identity":"f3c9e913-de91-4bba-9eb8-96fbe7c06856","added_by":"auto","created_at":"2025-07-15 08:14:35","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1353420,"visible":true,"origin":"","legend":"\u003cp\u003eDTL inhibits KAT2B and activates the PI3K/AKT/mTOR pathway in NPC cells. A. GSEA of the RNA-seq data from the GEO database (GSE102349 dataset) revealed a positive correlation between DTL expression and the PI3K/AKT/mTOR pathway, as well as glycolysis. B. Key proteins involved in PI3K/AKT/mTOR pathway were determined by western blotting in NPC cell lines. C. Overexpression of both DTL and KAT2B resulted in reduced phosphorylation of AKT and mTOR compared to DTL expression alone. D. Treatment with SC79 (an AKT activator) increased levels of p-AKT and p-mTOR when DTL was knocked down. E. Apoptosis assays were performed to evaluate the apoptotic response of NPC cells. *P \u0026lt; 0.05; **P \u0026lt; 0.01; ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7061714/v1/4fb3c9efe22684c23b84d3f7.png"},{"id":86748939,"identity":"c257132a-7754-455b-95cf-61d852979035","added_by":"auto","created_at":"2025-07-15 08:14:35","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":932498,"visible":true,"origin":"","legend":"\u003cp\u003eDTL facilitates aerobic glycolysis and NPC progression. A. The Enrichment Score of the glycolysis pathway. B. Western blotting was used to assess the efficiency of DTL knockdown on the expression of glycolysis-related proteins. C. Glucose uptake, lactate production and ECAR were measured following DTL knockdown in NPC cells. D. Overexpression of both DTL and KAT2B inhibited the phosphorylation of AKT and mTOR, as well as the expression of glycolysis-related proteins, compared with DTL overexpression alone. The similar results were observed with the overexpression of DTL and treatment with MK-2206 2HCI (an AKT inhibitor). E. Following the overexpression of DTL, either KAT2B was overexpressed or MK-2206 2HCI was administered, after which glucose uptake and lactic acid production were detected. F. Glucose uptake and lactate production were measured with DTL overexpression and the addition of glycolysis inhibitor AZ-33. G. The CCK-8 assay and apoptosis analysis were conducted to evaluate NPC cell proliferation with DTL overexpression and glycolysis inhibitor treatment. *P \u0026lt; 0.05; **P \u0026lt; 0.01; ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7061714/v1/32b6298bd8d44edfcaac0be5.png"},{"id":86749306,"identity":"6fc4b3a5-5245-40a6-842b-05ca59094813","added_by":"auto","created_at":"2025-07-15 08:22:35","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":640999,"visible":true,"origin":"","legend":"\u003cp\u003eDTL promotes NPC tumor progression in nude mice. A. NPC/HK-1 cells with DTL knockdown or negative control were injected subcutaneously into the right flank of each mouse. Photographs of xenograft tumors isolated from mice were taken 21 days post-inoculation. B. Tumor weight and volume were compared between the DTL knockdown and shCtrl groups. C. Molecules involved in the PI3K/AKT/mTOR pathway were analyzed by western blotting from three tumors in each group. D. The expression of DTL and Ki67 were determined by immunohistochemistry (scale bar, 20 μm). *P \u0026lt; 0.05; **P \u0026lt; 0.01; ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7061714/v1/637c6f3e583bbe6ef1176b3a.png"},{"id":109599104,"identity":"93152358-e2f4-4c31-b898-329251b88631","added_by":"auto","created_at":"2026-05-20 05:11:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6090810,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7061714/v1/7d922756-80bd-477c-91e5-32314212a0ba.pdf"},{"id":86748936,"identity":"2e98af29-be39-4385-a3b6-354a71ed5ed2","added_by":"auto","created_at":"2025-07-15 08:14:35","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":368479,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-7061714/v1/67b242d31b764a8015e000fb.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"DTL promotes glycolysis and tumor progression in nasopharyngeal carcinoma cells by degrading KAT2B and activating the PI3K/AKT/mTOR pathway","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eNasopharyngeal carcinoma (NPC) is an epithelial malignancy that occurs in the nasopharyngeal mucosa and is characterized by significant regional, racial, and gender differences in its incidence (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Globally, 129,000 new cases of NPC were reported in 2018, accounting for only 0.7% of all new malignancies. However, the geographic distribution of NPC incidence is extremely unbalanced, with over 70% of new cases concentrated in East and Southeast Asia. The age-standardized rates in China and in populations that are mainly white are 3.0 and 0.4 per 100,000 (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). In China, the prevalence of NPC is particularly elevated in the southern and southwestern regions. Moreover, the occurrence of NPC is more prevalent in men compared to women, with a ratio (men/women) of approximately 2.5 in China in 2015 (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). The etiology of NPC is unknown and is currently considered to be a genetic disorder involving multiple genes, with a high racial and familial predisposition. Pathogenic factors include EB virus infection, chemical carcinogens, environmental influences, and genetic factors (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Radiotherapy remains the primary treatment for NPC. However, 70% of patients are diagnosed in advanced stages due to hidden symptoms, and 10\u0026ndash;30% still experience recurrence or distant metastasis after standard treatment. These are important factors affecting overall survival of NPC patients (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Despite advances in treatment, clinical trials on targeted therapies for NPC are not abundant, which are mainly targeting epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor (VEGFR). Different targeted drugs show varying degrees of efficacy in delaying disease progression and extending patient survival (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Therefore, exploring novel gene regulatory mechanisms and therapeutic targets could contribute to the diagnosis and treatment of NPC.\u003c/p\u003e\u003cp\u003eDTL, also called DNA replication factor 2 (Cdt2) or retinoic acid-regulated nuclear matrix-associated protein (RAMP), is a member of the DCAF family of genes that encode the Cullin-Ring E3 ubiquitin ligase substrate receptor protein. DTL forms the E3 complex CRL4A with CUL4A, DDB1, and RBX1 proteins. As a denticleless E3 ubiquitin protein ligase homolog gene, DTL is essential to regulate the cell cycle and ensure proper DNA replication by mediating the ubiquitination and subsequent degradation of Cdt1 (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Additionally, it is also involved in biological processes through ubiquitination of target proteins and is important in the occurrence, progression and metastasis of tumors. Numerous studies in recent years have demonstrated the potential role of DTL in various malignancies such as ovarian cancer, melanoma, colon cancer, breast cancer and hepatocellular carcinoma (\u003cspan additionalcitationids=\"CR9 CR10 CR11 CR12\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Nevertheless, the precise mechanisms by which DTL influences NPC development remain largely unexplored.\u003c/p\u003e\u003cp\u003eMalignant tumor cells rely on a large supply of macromolecules and energy to sustain their rapid proliferation and growth. Enhanced aerobic glycolysis not only allows tumor cells to produce ATP, but also maintains redox balance, and generates the metabolites needed for macromolecular synthesis (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Several studies showed that the overexpression or increased activity of several critical regulatory enzymes promotes the glycolytic pathway in NPC cells, resulting in elevated glucose uptake and lactate production (\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Since the relationship between DTL and glycolysis is still unclear, exploring the potential mechanisms regulating glycolysis is of great significance for developing new therapies of NPC.\u003c/p\u003e\u003cp\u003eIn this study, differential genes and signaling pathways associated with NPC were identified through analysis of GSE12452 and GSE102349 from the GEO database. Immunohistochemistry of clinical specimens showed a significant increase in DTL levels in NPC patients, which was correlated with disease progression and poor prognosis. DTL promoted NPC proliferation in vitro and in vivo. Additionally, DTL activated PI3K/AKT/mTOR pathway through ubiquitination-mediated degradation of KAT2B, thereby promoting the glycolysis and tumor progression of NPC. These findings provided new insights into the role of DTL in NPC pathogenesis and highlighted its potential as a therapeutic target.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Bioinformatics analysis\u003c/h2\u003e\u003cp\u003eTo screen for genes associated with NPC and analyze gene expression correlation, publicly available datasets from Gene Expression Omnibus (GEO, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/geo\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/geo\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) database were utilized, including GSE12452 and GSE102349. GSE12452 dataset comprises samples from both NPC and normal tissues, which were used to analyze the differential expression of genes between these groups. Data normalization was conducted using the RMA (Robust Multi-array Average) method, and differential expressed analysis was performed with the R package limma. Genes were considered significantly differentially expressed if they exhibited a log2 fold change and an adjusted p-value of less than 0.05. The GSE102349 dataset was utilized for survival analysis. The optimal cutoff value of DTL expression in all NPC samples was used as the threshold to divide samples into high and low DTL expression groups. The progression-free survival (PFS) outcomes between these two groups were then compared using the log-rank test. Additionally, the clinical variables was categorized based on relevant clinical criteria, and intergroup differences in DTL expression among groups were analyzed using ANOVA. This allowed for the evaluation of DTL expression patterns across distinct clinical stages. Gene set enrichment analysis (GSEA) was performed using the GSEA software (GSEA_Linux_4.2.3), with the HALLMARK gene set defined in the MSigDB database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.gsea-msigdb.org/gsea/msigdb/download_file.jsp?filePath=/msigdb/release/2023.2.Hs/h.all.v2023.2.Hs.symbols.gmt\u003c/span\u003e\u003cspan address=\"https://www.gsea-msigdb.org/gsea/msigdb/download_file.jsp?filePath=/msigdb/release/2023.2.Hs/h.all.v2023.2.Hs.symbols.gmt\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) as the reference. NPC samples from the GSE102349 dataset, retrieved from the GEO database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/geo/download/?type=rnaseq_counts\u0026amp;acc=GSE102349\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/geo/download/?type=rnaseq_counts\u0026amp;acc=GSE102349\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), were stratified into high and low expression groups based on the median DTL gene expression level. The normalized expression matrix obtained using the R package DESeq2 was used for GSEA analysis. Gene sets with a NP\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.25 were considered statistically significant. To further investigate DTL-related interactions at the protein level, the STRING database (version 11.5) was used to identify putative DTL-interacting proteins. The expression-levels correlations between DTL and its interacting molecules was assessed using the Spearman correlation analysis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Antibodies and reagents\u003c/h2\u003e\u003cp\u003eThe following antibodies were used for western blotting (WB), immunoprecipitation (IP) and immunohistochemistry in this study. Primary antibodies included DTL (#orb1529346, Biorbyt, UK), GAPDH (#60004-1-Ig, Proteintech, USA), PCAF (also called KAT2B, #sc-13124, Santa Cruz, USA), Ubiquitin (#sc-8017, Santa Cruz, USA), PKM2 (#15822-1-AP, Proteintech, USA), HK2 (#22029-1-AP, Proteintech, USA), GLUT1 (#21829-1-AP, Proteintech, USA), ADH4 (#ab137077, Abcam, UK), ADH6 (#67709-1-Ig, Proteintech, USA), LDHA (#ab52488, Abcam, UK), AKT (#10176-2-AP, Proteintech, USA), p-AKT (#66444-1-Ig, Proteintech, USA), mTOR (#66888-1-Ig, Proteintech, USA), p-mTOR (#67778-1-Ig, Proteintech, USA), Cyclin B1 (#ab32053, Abcam, UK), P21 (#ab109520, Abcam, UK), CDK4 ( #11026-1-AP, Proteintech, USA), KI67 (#ab16667, Abcam, UK). Secondary antibodies included Goat Anti-Rabbit (#A0208, Beyotime, China), Goat Anti-Mouse (#A0216, Beyotime, China), Goat Anti-Rabbit IgG H\u0026amp;L (HRP) preabsorbed (#ab97080, Abcam, UK).\u003c/p\u003e\u003cp\u003eSC79 (#SF2730, Beyotime, China) is an AKT activator. In our experiments, cells were treated with SC79 at a concentration of 8 \u0026micro;g/mL for a duration of 24 to 48 h. MK-2206 2HCI (#S1078, Selleck, USA) is an AKT inhibitor and AZ-33 (#S0108, Selleck, USA) is a glycolysis inhibitor. Their dose was 10 \u0026micro;M and the time was 24 h.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Tissue microarray and immunohistochemical analysis\u003c/h2\u003e\u003cp\u003e Approval for the use of human tissues was obtained from the ethics committee of Shanghai Tenth People\u0026rsquo;s Hospital. Tissue microarray comprising 116 cases of NPC tissue and 14 cases of paraneoplastic tissue was purchased from the Shanghai Peide Biotechnology Center (#NPC1401, Shanghai, China). Clinicopathologic data included patient sex, age, clinical stage, TMN stage, metastasis status, and recurrence information. Immunohistochemistry of target proteins was performed as standard protocol. After deparaffinization and antigen retrieval, endogenous peroxides were quenched by incubating the sections in 3% H2O2. The sections were incubated with a primary anti-DTL antibody at 4℃ overnight. Subsequently, sections were incubated with a secondary antibody, cleaned, and stained with DAB in dark, followed by counterstaining with hematoxylin. DTL expression in cytoplasm, membrane and nucleus was estimated. The immunohistochemistry results were quantified by multiplying the staining intensity (dark brown\u0026thinsp;=\u0026thinsp;3, brown and yellow\u0026thinsp;=\u0026thinsp;2, pale yellow\u0026thinsp;=\u0026thinsp;1, and negative\u0026thinsp;=\u0026thinsp;0) with the percentage of positive cells (75\u0026ndash;100% = 4, 50\u0026ndash;74% = 3, 25\u0026ndash;49% = 2, 1\u0026ndash;24% = 1, and 0% = 0). The final scores were defined as strongly positive (+++, 9\u0026ndash;12), moderately positive (++, 5\u0026ndash;8), weakly positive (+, 1\u0026ndash;4), and negative (-, 0).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Cell lines and culture\u003c/h2\u003e\u003cp\u003eThe cell lines were from iCell Bioscience Inc (China). The human immortalized nasopharyngeal epithelial cell line NP69 was cultured in keratinocyte/serum-free medium (Invitrogen, USA) supplemented with bovine pituitary extract (BD Biosciences, USA). Human NPC cell lines (NPC/HK-1, 5-8F, HONE-1, C666-1) were cultured in DMEM or RPMI 1640 medium (Invitrogen, USA) containing 10% fetal bovine serum (Gibco, USA). The temperature required for cell culture was 37℃ and the air condition was air containing 5% CO2.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Quantitative real-time polymerase chain reaction (qRT-PCR)\u003c/h2\u003e\u003cp\u003eThe Trizol purchased from Sigma was used to extract total RNA from NPC cells following the manufacturer\u0026rsquo;s instructions. Then, the reverse transcription reaction was performed using the Hiscript QRT supermix for qPCR (+\u0026thinsp;gDNA WIPE) (Vazyme, China). Finally, cDNA, forward and reverse primers and AceQ qPCR SYBR Green master mix (Vazyme, China) were mixed, and qRT-PCR was performed on the Real-Time PCR system (ABI, USA). The primer sequences used for GAPDH were 5\u0026rsquo;-TGACTTCAACAGCGACACCCA-3\u0026rsquo; and 3\u0026rsquo;-CACCCTGTTGCTGTAGCCAAA-5\u0026rsquo;. The primer sequences for DTL were 5\u0026rsquo;-TAAAAGCTGGTGAGCTGATTGG-3\u0026rsquo; and 5\u0026rsquo;-TCTTCCACCCGTACAGAATACA-3\u0026rsquo;.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Construction of plasmid vector, package of lentivirus and, transfection\u003c/h2\u003e\u003cp\u003eThree RNA interference sequences targeting the DTL gene were designed, and the following sequences were used as RNA interference targets: 5\u0026rsquo;-CTGCACATACTTCCATAGAAA-3\u0026rsquo;, 5\u0026rsquo;-TGGCGCTTGAATAGAGGCTTA-3\u0026rsquo;, and 5\u0026rsquo;-AGGGTCTGAAATGGTAGGCAA-3\u0026rsquo;. Lentivirus vectors carrying short hairpin RNAs (shRNAs) specific for DTL (shDTL-1, shDTL-2 and shDTL-3 lentivirus) and lentiviral constructs overexpressing DTL and KAT2B overexpression were constructed and packaged by Yibeirui (Shanghai, China). Cell transfections were performed using Puromycin (Gibco, USA) according to the manufacturer\u0026rsquo;s instructions, and the transfection efficiency was determined 72 h post-infection using RT-qPCR, fluorescence observation and western blotting.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Western blotting\u003c/h2\u003e\u003cp\u003eWestern blotting was performed to detect protein expression levels. Cells were lysed with the RIPA lysis buffer (Beyotime, China) supplemented with protease inhibitors. Lysates were centrifuged at 12,000 rpm for 10 min (4℃) to collect the supernatant. Protein concentrations were quantified using a BCA protein assay kit (Beyotime, China). After gel electrophoresis, the protein was transferred to PVDF membrane. 5% skim milk was added for 1 h at room temperature. Membranes were incubated with primary antibodies overnight at 4℃, followed by incubation with secondary antibodies for 1 h at room temperature. Protein bands were visualized using Chemiluminescent HRP Substrote (Beyotime, China).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8 CCK-8 assay, flow cytometry, colony formation, wound-healing assay, transwell assay\u003c/h2\u003e\u003cp\u003eAnnexin V-APC/PI Apoptosis Detection Kit (eBioscience, USA) was used to evaluate apoptosis of NPC cells transfected with lentivirus according to the manufacturer\u0026rsquo;s instructions. Cells were collected with trypsinization, centrifuged, washed with PBS and made into a suspension of 1\u0026times;10\u003csup\u003e6\u003c/sup\u003e cell/mL. 100 \u0026micro;L of cell suspension was stained by 5 \u0026micro;L of Annexin V-APC for 15 min at room temperature in the dark, and then added 5 \u0026micro;L of PI. Cell apoptosis was detected by a flow cytometer (Millipore, USA).\u003c/p\u003e\u003cp\u003eCCK-8 assay and colony formation assay were performed to measure cell proliferation. For the CCK-8 assay, NPC cells in the logarithmic growth phase were inoculated in 96-well plates (2000 cells/well). After specified incubation periods, 10 \u0026micro;L of CCK-8 (Sigma, USA) was added to each well and the absorbance at 450 nm per well was read on a spectrophotometer.\u003c/p\u003e\u003cp\u003eFor the colony formation assay, NPC cells were plated in 6-well plates (400 cells/well) and maintained for 14 days. Then, colonies were fixed with 4% paraformaldehyde, stained with Giemsa stain, and counted.\u003c/p\u003e\u003cp\u003eWound-healing assay was used to assess the migration ability of cells. Cells were cultured in 96-well plates. Linear wounds were created using a 96 Wounding Replicator (VP scientific, USA) and cells were incubated for another 24 h in starvation medium. Images of the wound area were captured using Cellomics (Thermo, USA) at 0 h and 24 h. The migration rate was calculated by comparing the wound area at the two time points.\u003c/p\u003e\u003cp\u003eTranswell assay was conducted using Transwell chambers (3422 Corning, NY, USA) with an 8-\u0026micro;m pore size to assess the migration capability of NPC cells. 2\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells suspended in 100 \u0026micro;L of serum-free medium were seeded into transwell chambers (3422 Corning, NY, USA). The transwell chambers were placed into the lower chambers with 600 \u0026micro;L of complete media containing 30% FBS. After 24 h of incubation at 37\u0026deg;C, non-migratory cells were removed, while cells that had migrated to the underside of the membrane were fixed in 4% paraformaldehyde and stained with 0.05% crystal violet. Migratory cells were counted under a microscope (Olympus, Japan).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9 Glucose and lactate assay and measurement of extracellular acidification rate (ECAR)\u003c/h2\u003e\u003cp\u003eTo assess glucose metabolism and lactate production, cells were washed with cold PBS and lysed in 0.1% Triton X-100. The cell lysates were filtered using an Amicon Ultra-3 kDa cutoff (Merck). Glucose and lactate levels were quantitated using a Glucose Assay kit-WST and Lactate Assay kit-WST (Dojindo) following the manufacturer's instructions.\u003c/p\u003e\u003cp\u003eThe glycolysis stress test was conducted in a XF24 Extracellular Flux Analyzer (Seahorse Bioscience, North Billerica, MA). Glucose (10 mM; Sigma), oligomycin (1 \u0026micro;M; Sigma), and 2-deoxyglucose (2-DG, 100 mM; Sigma) were sequentially added to the assay medium at the indicated time points during ECAR measurement. ECAR values were normalized to protein contents quantified using Pierce\u0026trade; BCA Protein Assay Kit (Thermo Fisher Scientific).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.10 In vivo xenograft models\u003c/h2\u003e\u003cp\u003e All animal experiments were approved by Shanghai Tenth People's Hospital Laboratory Animal Ethics Committee (Approval number: SHDSYY-2024-3832). 4-week-old female NXG nude mice (Cavens, Changzhou, China) were used for animal studies. The animal experiment was conducted using a double-blind design and randomly divided into two groups. To establish mouse xenograft models, NPC/HK-1 cells (1\u0026times;10\u003csup\u003e7\u003c/sup\u003e/200 \u0026micro;L) with DTL knockdown or negative control were injected subcutaneously into the right flank of each mouse. The tumor volume was monitored every three or four days by measuring the length (L) and width (W) of tumors using calipers. Tumor volume (mm\u003csup\u003e3\u003c/sup\u003e) was calculated as length \u0026times; width\u003csup\u003e2\u003c/sup\u003e /2. Mice were sacrificed after 18 days, and the tumors were excised, weighed, and processed for further analyses. Samples were embedded in paraffin and subjected to immunohistochemistry for histological evaluation of KI67 and DTL expression. Western blotting detected PI3K/AKT/mTOR pathway proteins.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e2.11 Statistical analysis\u003c/h2\u003e\u003cp\u003eAll statistical analyses were performed using SPSS (version 22.0, IBM Corp., USA) and GraphPad Prism (version 10, CA, USA). Data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Comparison among groups was performed by t-test and One-way ANOVA. Mann-Whitney U test and Spearman\u0026rsquo; s rank correlation test were used to evaluate the correlation between DTL expression and pathological features of NPC patients. Survival curves were analyzed using Kaplan-Meier analysis. Statistical significance was defined as P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.1 DTL is highly expressed in NPC tissues and predicts poor prognosis.\u003c/h2\u003e\u003cp\u003eTo identify genes associated with the occurrence and development of NPC, the expression of genes in NPC tissues and the relationship between gene expression and prognosis were analyzed. Data from GSE12452 dataset revealed that DTL expression was significantly higher in NPC tissues than in normal tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Further investigation of the GSE102349 dataset showed that high DTL expression was associated with a significantly shorter progression-free survival (PFS) in NPC patients, as indicated by Kaplan-Meier survival curves (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Additionally, DTL expression was found to be significantly higher in stage IV NPC tissues compared to stage I tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). These results indicated that DTL was not only highly expressed in NPC, but also associated with advanced stages and poor prognosis, making it a potential target for therapeutic intervention.\u003c/p\u003e\u003cp\u003eTo learn about the clinical significance of DTL expression in NPC, immunohistochemistry was carried out on a tissue microarray consisting of 116 cases of NPC tissue and 14 cases of paraneoplastic tissue. Compared with paraneoplasitc tissue, DTL expression was significantly higher in NPC tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Mann-Whitney U test further confirmed that DTL expression was markedly associated with pathological stage of NPC (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Furthermore, the analysis of Spearman\u0026rsquo;s rank correlation revealed a positive correlation between DTL expression and pathological stage (P\u0026thinsp;=\u0026thinsp;0.022) (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Kaplan-Meier survival analysis showed that high DTL expression was significantly associated with poor overall survival and disease-free survival in NPC patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). These findings suggested that elevated DTL expression was significantly associated with poorer overall survival in NPC and may serve as a new therapeutic target in managing the disease.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eExpression patterns in nasopharyngeal carcinoma tissues and para-carcinoma tissues revealed in immunohistochemistry analysis\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eDTL expression\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eTumor tissue\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003ePara-carcinoma tissue\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ep value\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCases\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePercentage\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCases\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ePercentage\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLow\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e62\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e53.4%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e92.9%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHigh\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e54\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e46.6%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e7.1%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eRelationship between DTL expression and tumor characteristics in patients with nasopharyngeal carcinoma\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eFeatures\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eNo. of patients\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003eDTL expression\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003ep value\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003elow\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003ehigh\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAll patients\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e116\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e62\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e54\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGender\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.577\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e74\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e41\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFemale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTumor size\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.489\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u0026le;\u0026thinsp;1.2 cm\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e62\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u0026gt;1.2 cm\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e54\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT Infiltrate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.414\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e43\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e29\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003elymphatic\u0026nbsp;metastasis (N)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.308\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eN0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e79\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e45\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e34\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eN1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e34\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eN2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMetastasis\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.091\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eM0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e92\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e53\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e39\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eM1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eM4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eStage\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.023\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eI\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eII\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIII\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIV\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAge\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.980\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;52 years\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u0026le;\u0026thinsp;52 years\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eRelationship between DTL expression and tumor characteristics in patients with nasopharyngeal carcinoma\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eDTL\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eStage\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSpearman\u0026rsquo;s rank correlation\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.213\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSignificance\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.022\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eN\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e116\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.2 DTL knockdown suppresses malignant phenotypes of NPC in vitro.\u003c/h2\u003e\u003cp\u003eTo investigate biological functions of DTL in NPC, shRNA-mediated knockdown of DTL was performed in NPC cell lines. Compared with nasal mucosal epithelial cells (NP69 cells), DTL is highly expressed in NPC/HK-1, HONE-1 and C666-1 cells. Among the four NPC cell lines tested, DTL was most highly expressed in NPC/HK-1 and C666-1 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF), which were selected for subsequent experiments. Transfection efficiency was verified by qRT-PCR, which showed that shDTL-1 and shDTL-2 lentiviruses achieved efficient knockdown of DTL (Supplementary Fig.\u0026nbsp;1A). qRT-PCR and western blotting results confirmed the successful construction of DTL knockdown cell models (Supplementary Fig.\u0026nbsp;1B). The results of CCK-8 assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), cell apoptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) and colony formation assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC) showed that proliferation and colony numbers of NPC/HK-1 and C666-1 cells were suppressed by knockdown of DTL. Wound-healing assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD) and transwell assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE) indicated that DTL knockdown resulted in a decrease in migration rate of NPC/HK-1 and C666-1 cells. These results implied that downregulation of DTL might inhibit the malignant phenotype of NPC cells in vitro. It is necessary to further explore underlying mechanisms of DTL regulation in the progression of NPC.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.3 DTL promotes the ubiquitination and degradation of KAT2B.\u003c/h2\u003e\u003cp\u003eTo explore the regulatory mechanism underlying DTL in NPC, bioinformatic analysis using STRING and GEO database (GSE12452 dataset) were performed to screen genes that may interact with DTL and are significantly downregulated in NPC samples. The potential downstream molecules of DTL were screened by some criteria. The downstream gene was a tumor suppressor gene, which interacted with DTL and showed negative correlation in expression. KAT2B (also known as PCAF) was found to be a potential downstream molecule of DTL, exhibiting the highest negative correlation coefficient with DTL expression (Supplementary Table S1). Since DTL is an E3 ubiquitin ligase (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e), it is hypothesized that DTL may affect the protein stability of KAT2B through ubiquitination. The regulatory effect of DTL on KAT2B was verified by western blot. KAT2B protein was increased in DTL-knockdown NPC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). As an E3 ubiquitin ligase, DTL is known to connect to its substrate and promote the degradation of the substrate. The interaction between DTL and KAT2B was further confirmed by co-immunoprecipitation (Co-IP) assays (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Subsequently, NPC cells were treated with cycloheximide (CHX) to inhibit the synthesis of proteins, and KAT2B in the DTL-knockdown group declined more slowly compared to control cells, indicating that DTL had a negative regulatory effect on KAT2B by affecting its degradation rate (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Furthermore, ubiquitination assay showed that DTL overexpression increased polyubiquitinated KAT2B (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). These results suggested that DTL promoted KAT2B degradation by increasing KAT2B ubiquitination.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e3.4 DTL inhibits KAT2B and activates PI3K/AKT/mTOR pathway to promote aerobic glycolysis and NPC progression.\u003c/h2\u003e\u003cp\u003ePrevious studies reported that DTL might promote bladder cancer progression through the AKT/mTOR pathway (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Besides, PI3K/AKT signaling pathway was modulated by miR-17-5p/KAT2B axis in liver fibrosis (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). To further investigate whether DTL and KAT2B affect the progression of NPC through the PI3K/AKT/mTOR pathway, gene set enrichment analysis (GSEA) was performed using samples from the GEO database (GSE102349 dataset), stratified by DTL expression. This analysis revealed that DTL expression was positively related to the activation of PI3K/AKT/mTOR pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Downregulation of DTL suppressed the expression and phosphorylation of key proteins in PI3K/AKT/mTOR pathway, including p-AKT and p-mTOR, in NPC/HK-1 and C666-1 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Then, to further demonstrate whether DTL activated PI3K/AKT/mTOR pathway by inhibiting KAT2B, KAT2B overexpression was simultaneously conducted in NPC cells with DTL overexpression. Overexpression of KAT2B restored the stimulative effect of DTL on the activation of the above pathway to a certain extent (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Subsequently, SC79 (the AKT activator) was used to explore if DTL promoted NPC progression through PI3K/AKT/mTOR pathway. SC79 partially reversed the inhibitory effect of DTL knockdown on the PI3K/AKT/mTOR pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Using apoptosis assays, we also observed that the inhibitory effect of DTL knockdown on NPC cell proliferation was partially restored by SC79 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE).\u003c/p\u003e\u003cp\u003eFurthermore, the GSEA analysis revealed a relationship between DTL and glycolysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). To evaluate the impact of DTL on aerobic glycolysis, we examined the expression of glycolytic proteins, glucose uptake, lactate production, and ECAR. DTL knockdown contributed to a reduced expression of glycolytic-related proteins such as ADH4, ADH6, GLUT1, HK2, LDHA, PKM2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB) and a decrease in glucose uptake, lactate production, ECAR (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). In contrast, overexpression of DTL increased these glycolytic parameters. Importantly, KAT2B, MK-2206 2HCI (the AKT inhibitor) and AZ-33 (the glycolysis inhibitor) restored the effects of DTL on glycolysis, indicating that DTL regulated glycolysis through KAT2B and PI3K/AKT/mTOR pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD, E, F). Moreover, CCK-8 and cell apoptosis assays demonstrated that AZ-33 partially restored the effect of DTL overexpression on NPC cell proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG). Therefore, we concluded that DTL facilitated aerobic glycolysis and NPC progression by inhibiting KAT2B and activating the PI3K/AKT/mTOR pathway.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e3.5 DTL promotes NPC tumor growth in vivo.\u003c/h2\u003e\u003cp\u003eTo assess the role of DTL in tumor progression of NPC in vivo, we carried out xenograft experiments in mice. As expected, DTL depletion significantly reduced the size and weight of NPC xenografts (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, B). In addition, the levels of p-AKT and p-mTOR of PI3K/AKT/mTOR pathway was markedly decreased in DTL-knockdown xenografts (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Immunohistochemistry demonstrated that the levels of Ki67, a marker of cell proliferation, and DTL were greatly reduced in tumors from NPC/HK-1 xenografts with depleted DTL (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Collectively, these data suggested that DTL promoted NPC tumor growth in vivo by modulating the PI3K/AKT/mTOR pathway and enhancing proliferation.\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eAs previously mentioned, the expression of DTL was found to stimulate several kinds of malignancies (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). For instance, Cui H et al. reported that DTL degraded PDCD4 through ubiquitination, which in turn enhanced proliferation and migration abilities of breast and lung cancers (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Moreover, by identifying 46 differentially expressed genes (DEGs), enrichment analysis results showed that DTL was closely associated with the development and progression of NPC, and it has been recognized as a potential biomarker of NPC. However, these conclusions were not supported by experiments such as immunohistochemistry (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). The study of Chang X et al. indicated that under the regulation of ZFPM2-AS1/miR-3612, the expression of DTL was upregulated in NPC and drove the progression of NPC (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). These findings were consistent with our study, which highlighted the role of DTL in promoting NPC tumor growth both in vitro and in vivo. In addition to proving that DTL drives progression of NPC, we screened downstream genes and signaling pathways involved in this process.\u003c/p\u003e\u003cp\u003eAs a lysine acetyltransferase, KAT2B catalyzes lysine acetylation on both histones and non-histone proteins and regulates gene expression (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). Zhang J et al. substantiated KAT2B as a key effector of breast tumorigenesis, which contributed to cancer progression by synergizing with NELF-E-SLUG to promote EMT (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). In contrast, KAT2B expression was downregulated in non-small cell lung cancer (NSCLC), and low KAT2B expression predicted poor prognosis of patients. KAT2B was mainly linked to the regulation of immune cells and IFN-γ mediated signaling pathways (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e), suggesting that KAT2B was involved in the immune response and tumor microenvironment. In human aortic endothelial cell line HAEC cells, KAT2B was found to be degraded by UBE2D2 through ubiquitination (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). Similar to the mechanism of previous studies, our study showed that KAT2B was degraded by DTL through ubiquitination. Notably, the effects of KAT2B and DTL were opposite, with DTL promoting tumor progression through its regulation of KAT2B.\u003c/p\u003e\u003cp\u003ePI3K/AKT/mTOR pathway plays a crucial role in fundamental cellular functions, such as cell proliferation, metabolism, and motility. Aberrant activation of this pathway is frequently observed in various cancers (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Notably, PI3K/AKT/mTOR signaling pathway is active in over 90% of head and neck squamous cell carcinomas. Luo Y et al. reported that DTL might promote bladder cancer progression through the AKT/mTOR pathway (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). However, there is a lack of experiments to prove that DTL induces the activation of the PI3K/AKT/mTOR pathway. Our results demonstrated that DTL knockdown inhibited the phosphorylation of AKT and mTOR proteins, while overexpression of DTL increased the phosphorylation levels of these two proteins. A study of liver fibrosis showed that human bone marrow mesenchymal stem cells-derived exosome circCDK13 inhibited PI3K/AKT and NF-κB pathways through regulating the miR-17-5p/KAT2B axis, thereby mitigating liver fibrosis (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). In this research, we found that DTL activated the PI3K/AKT/mTOR pathway by disrupting the stability of KAT2B protein, thereby promoting the proliferation of NPC cells.\u003c/p\u003e\u003cp\u003eAerobic glycolysis or the \u0026ldquo;Warburg effect\u0026rdquo; refers to the tendency of tumor cells to convert pyruvate derived from glucose into lactate through lactate dehydrogenase (LDH) (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). In NPC cells, glucose uptake is due to the activation of mTORC1/NF-κB signaling pathway and the transcription of glucose transporter 1 (GLUT1) (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Hexokinase and pyruvate kinase are critical regulatory enzymes in the glycolytic pathway (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). FOXC2-YAP pathway increases hexokinase expression in NPC cells (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e), and significantly upregulate the expression of PKM2 (a subtype of pyruvate kinase) by activating the PI3K/AKT signaling pathway (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). The overexpression of these enzymes induces the Warburg effect, resulting in elevated the lactate production in NPC cells even in the presence of oxygen. Our research indicated that DTL, by mediating the ubiquitination and degradation of KAT2B, not only activated the PI3K/AKT/mTOR pathway but also promoted the expression of HK2, LDHA, and PKM2, thereby enhancing glycolysis. Moreover, DTL also promoted the proliferation of NPC cells through glycolysis.\u003c/p\u003e\u003cp\u003eIn conclusion, our study revealed that DTL was remarkably upregulated in NPC tumor tissues, which was closely associated with disease progression and poor prognosis. Knockdown of DTL suppressed several malignant phenotypes of NPC cells, including proliferation and migration. Mechanistically, DTL interacted with KAT2B and downregulated its expression, thereby activating the PI3K/AKT/mTOR pathway, which promoted the glycolysis and led to the development of NPC. These results indicated that DTL could be a potential therapeutic target for NPC. However, this study has certain limitations. First, the tissue microarray was used for clinicopathological analysis and a larger sample size of NPC is required to verify the prognostic value of DTL. Second, while the role of DTL in glycolysis has been suggested, further investigation is required to fully elucidate the mechanism of DTL in this process.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eJingwen Sun and Chaoping Huang contributed equally to this study. Jingwen Sun and Chaoping Huang wrote the main manuscript text. Wentao Zou and Shuang Zhou prepared figures 1-3, supplementary figure 1 and all the tables. Haibo Ye and Jingshuo Wang prepared figure 4-6. All authors reviewed the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBossi P, Chan AT, Licitra L, Trama A, Orlandi E, Hui EP, et al. Nasopharyngeal carcinoma: ESMO-EURACAN Clinical Practice Guidelines for diagnosis, treatment and follow-up(\u0026dagger;). Ann Oncol. 2021;32(4):452-65.\u003c/li\u003e\n\u003cli\u003eBray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2018;68(6):394-424.\u003c/li\u003e\n\u003cli\u003eFerlay J, Colombet M, Soerjomataram I, Mathers C, Parkin DM, Pi\u0026ntilde;eros M, et al. Estimating the global cancer incidence and mortality in 2018: GLOBOCAN sources and methods. Int J Cancer. 2019;144(8):1941-53.\u003c/li\u003e\n\u003cli\u003eChen YP, Chan ATC, Le QT, Blanchard P, Sun Y, Ma J. Nasopharyngeal carcinoma. Lancet. 2019;394(10192):64-80.\u003c/li\u003e\n\u003cli\u003eHuang H, Yao Y, Deng X, Huang Z, Chen Y, Wang Z, et al. Immunotherapy for nasopharyngeal carcinoma: Current status and prospects (Review). Int J Oncol. 2023;63(2).\u003c/li\u003e\n\u003cli\u003eKang Y, He W, Ren C, Qiao J, Guo Q, Hu J, et al. Advances in targeted therapy mainly based on signal pathways for nasopharyngeal carcinoma. Signal Transduct Target Ther. 2020;5(1):245.\u003c/li\u003e\n\u003cli\u003eJin J, Arias EE, Chen J, Harper JW, Walter JC. A family of diverse Cul4-Ddb1-interacting proteins includes Cdt2, which is required for S phase destruction of the replication factor Cdt1. Mol Cell. 2006;23(5):709-21.\u003c/li\u003e\n\u003cli\u003ePan WW, Zhou JJ, Yu C, Xu Y, Guo LJ, Zhang HY, et al. Ubiquitin E3 ligase CRL4(CDT2/DCAF2) as a potential chemotherapeutic target for ovarian surface epithelial cancer. J Biol Chem. 2013;288(41):29680-91.\u003c/li\u003e\n\u003cli\u003eDing Y, Li M, Tayier T, Zhang M, Chen L, Feng S. Bioinformatics analysis of lncRNA‑associated ceRNA network in melanoma. J Cancer. 2021;12(10):2921-32.\u003c/li\u003e\n\u003cli\u003eMa J, Cai X, Kang L, Chen S, Liu H. Identification of novel biomarkers and candidate small-molecule drugs in cutaneous melanoma by comprehensive gene microarrays analysis. J Cancer. 2021;12(5):1307-17.\u003c/li\u003e\n\u003cli\u003eChen W, Gao C, Liu Y, Wen Y, Hong X, Huang Z. Bioinformatics Analysis of Prognostic miRNA Signature and Potential Critical Genes in Colon Cancer. Front Genet. 2020;11:478.\u003c/li\u003e\n\u003cli\u003eCui H, Wang Q, Lei Z, Feng M, Zhao Z, Wang Y, et al. DTL promotes cancer progression by PDCD4 ubiquitin-dependent degradation. J Exp Clin Cancer Res. 2019;38(1):350.\u003c/li\u003e\n\u003cli\u003eZhou Z, Li Y, Hao H, Wang Y, Zhou Z, Wang Z, et al. Screening Hub Genes as Prognostic Biomarkers of Hepatocellular Carcinoma by Bioinformatics Analysis. Cell Transplant. 2019;28(1_suppl):76s-86s.\u003c/li\u003e\n\u003cli\u003eSzwed A, Kim E, Jacinto E. Regulation and metabolic functions of mTORC1 and mTORC2. Physiol Rev. 2021;101(3):1371-426.\u003c/li\u003e\n\u003cli\u003eZschaeck S, Li Y, Lin Q, Beck M, Amthauer H, Bauersachs L, et al. Prognostic value of baseline [18F]-fluorodeoxyglucose positron emission tomography parameters MTV, TLG and asphericity in an international multicenter cohort of nasopharyngeal carcinoma patients. PLoS One. 2020;15(7):e0236841.\u003c/li\u003e\n\u003cli\u003eSong L, Tang H, Liao W, Luo X, Li Y, Chen T, et al. FOXC2 positively regulates YAP signaling and promotes the glycolysis of nasopharyngeal carcinoma. Exp Cell Res. 2017;357(1):17-24.\u003c/li\u003e\n\u003cli\u003eChen S, Youhong T, Tan Y, He Y, Ban Y, Cai J, et al. EGFR-PKM2 signaling promotes the metastatic potential of nasopharyngeal carcinoma through induction of FOSL1 and ANTXR2. Carcinogenesis. 2020;41(6):723-33.\u003c/li\u003e\n\u003cli\u003eLuo Y, He Z, Liu W, Zhou F, Liu T, Wang G. DTL Is a Prognostic Biomarker and Promotes Bladder Cancer Progression through Regulating the AKT/mTOR axis. Oxid Med Cell Longev. 2022;2022:3369858.\u003c/li\u003e\n\u003cli\u003eMa J, Li Y, Chen M, Wang W, Zhao Q, He B, et al. hMSCs-derived exosome circCDK13 inhibits liver fibrosis by regulating the expression of MFGE8 through miR-17-5p/KAT2B. Cell Biol Toxicol. 2023;39(2):1-22.\u003c/li\u003e\n\u003cli\u003eWang H, Zhang J. Identification of DTL as Related Biomarker and Immune Infiltration Characteristics of Nasopharyngeal Carcinoma via Comprehensive Strategies. Int J Gen Med. 2022;15:2329-45.\u003c/li\u003e\n\u003cli\u003eChang X, Jian L. LncRNA ZFPM2-AS1 drives the progression of nasopharyngeal carcinoma via modulating the downstream miR-3612/DTL signaling. Anticancer Drugs. 2022;33(6):523-33.\u003c/li\u003e\n\u003cli\u003eNagy Z, Tora L. Distinct GCN5/PCAF-containing complexes function as co-activators and are involved in transcription factor and global histone acetylation. Oncogene. 2007;26(37):5341-57.\u003c/li\u003e\n\u003cli\u003eZhou X, Wang N, Zhang Y, Yu H, Wu Q. KAT2B is an immune infiltration-associated biomarker predicting prognosis and response to immunotherapy in non-small cell lung cancer. Invest New Drugs. 2022;40(1):43-57.\u003c/li\u003e\n\u003cli\u003eZhang J, Hu Z, Chung HH, Tian Y, Lau KW, Ser Z, et al. Dependency of NELF-E-SLUG-KAT2B epigenetic axis in breast cancer carcinogenesis. Nat Commun. 2023;14(1):2439.\u003c/li\u003e\n\u003cli\u003eQi X, Wang H, Xia L, Lin R, Li T, Guan C, et al. miR-30b-5p releases HMGB1 via UBE2D2/KAT2B/HMGB1 pathway to promote pro-inflammatory polarization and recruitment of macrophages. Atherosclerosis. 2021;324:38-45.\u003c/li\u003e\n\u003cli\u003eAlzahrani AS. PI3K/Akt/mTOR inhibitors in cancer: At the bench and bedside. Semin Cancer Biol. 2019;59:125-32.\u003c/li\u003e\n\u003cli\u003eVander Heiden MG, Cantley LC, Thompson CB. Understanding the Warburg effect: the metabolic requirements of cell proliferation. Science. 2009;324(5930):1029-33.\u003c/li\u003e\n\u003cli\u003eAnderson NM, Mucka P, Kern JG, Feng H. The emerging role and targetability of the TCA cycle in cancer metabolism. Protein Cell. 2018;9(2):216-37.\u003c/li\u003e\n\u003cli\u003eLiu Q, Bode AM, Chen X, Luo X. Metabolic reprogramming in nasopharyngeal carcinoma: Mechanisms and therapeutic opportunities. Biochim Biophys Acta Rev Cancer. 2023;1878(6):189023.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"nasopharyngeal carcinoma, DTL, KAT2B, molecular mechanism, glycolysis","lastPublishedDoi":"10.21203/rs.3.rs-7061714/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7061714/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eNasopharyngeal carcinoma (NPC) is a prevalent malignancy in East and Southeast Asia, with limited effective treatment options due to late-stage diagnosis. E3 ubiquitin ligase DTL has been implicated in various cancers, but its role in NPC remains obscure. This study aimed to investigate the regulatory mechanisms of DTL in NPC and its potential as a therapeutic target.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eWe conducted a comprehensive analysis combining bioinformatics, immunohistochemistry on clinical specimens, and a series of in vitro and in vivo experiments. Gene expression was analyzed through the GEO database, and the impact of DTL on NPC cell lines was assessed using qRT-PCR, western blotting, and various cellular assays. The interaction between DTL and KAT2B was explored, and the role of the PI3K/AKT/mTOR pathway in DTL-mediated NPC progression was investigated.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eDTL expression was significantly higher in NPC tissues and associated with poor prognosis. DTL knockdown inhibited NPC cell proliferation, migration, and glycolysis, while its overexpression promoted these phenotypes. Mechanistically, DTL interacted with and ubiquitinated KAT2B, leading to its degradation and subsequent activation of the PI3K/AKT/mTOR pathway, which in turn enhanced glycolysis and NPC progression.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eOur findings identify DTL as a critical promoter of NPC, highlighting its potential as a therapeutic target. By targeting the KAT2B-PI3K/AKT/mTOR axis, interventions of DTL could offer a promising strategy for NPC treatment.\u003c/p\u003e","manuscriptTitle":"DTL promotes glycolysis and tumor progression in nasopharyngeal carcinoma cells by degrading KAT2B and activating the PI3K/AKT/mTOR pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-15 08:14:30","doi":"10.21203/rs.3.rs-7061714/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9af0e781-b9de-4b1f-a767-11ab9b88fc34","owner":[],"postedDate":"July 15th, 2025","published":true,"recentEditorialEvents":[{"type":"decision","content":"Withdrawn","date":"2026-05-20T05:06:29+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-05-20T05:10:16+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-15 08:14:30","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7061714","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7061714","identity":"rs-7061714","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}