Acetyl-CoA acyltransferase 1 is a potential tumor suppressor gene associated with immune cell infiltration in nasopharyngeal carcinoma

Acetyl-CoA acyltransferase 1 is a potential tumor suppressor gene associated with immune cell infiltration in nasopharyngeal carcinoma | 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 Acetyl-CoA acyltransferase 1 is a potential tumor suppressor gene associated with immune cell infiltration in nasopharyngeal carcinoma Weilin Zhao, Limei Li, Wanqi Wei, Shixing Zheng, Xiaoying Zhou, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4750465/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 Acetyl-CoA acyltransferase 1 (ACAA1), encoding the protein peroxisomal 3-ketoacyl-CoA thiolase (POT1), plays a vital role in the fatty acid beta-oxidation system. ACAA1 has been implicated in the carcinogenesis and development of various human cancers. In this study, the downregulation of ACAA1 was observed consistently throughout the progression of nasopharyngeal carcinoma (NPC) and showed a negative correlation with the expression of EBV-encoded genes. Kaplan-Meier survival analysis and time-dependent receiver operating characteristic (ROC) curve suggested the potential of ACAA1 in predicting NPC prognosis. Through in vitro and in vivo experiments, we identified that the overexpression of ACAA1 inhibited the proliferation, migration, and invasion of NPC cells, which was further confirmed by reduced Ki-67 staining and actin filaments redistribution. Gene ontology (GO) and Kyoto Encyclopedia of Gene and Genomes (KEGG) analyses indicated significant enrichment of immune-related pathways in NPC cells with higher ACAA1 expression. Furthermore, data from the xCell, ESTIMATE and Immunophenoscore analysis supported a critical role of ACAA1 in modulating immune cell infiltration and tumor immune environment of NPC. Interestingly, low expression of ACAA1 was significantly associated with NPC patients classified as tumor microenvironment (TME) subtype 1 and with poor outcome. Expression pattern analyses revealed a positive correlation between ACAA1 expression and six immune checkpoint-related genes, including CD27, PDCD1, CD86, BTLA, TIGIT, and CD28. Taken together, our study reveals that ACAA1 is a potential tumor suppressor gene, which may participate in immune evasion in NPC. ACAA1 could serve as a novel prognosis and therapeutic biomarker for NPC patients. nasopharyngeal carcinoma ACAA1 tumor suppressor immune evasion prognosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Nasopharyngeal carcinoma (NPC) is a unique head and neck squamous cell carcinoma originating from the nasopharyngeal epithelium, with a specific geographic distribution and a comprehensive etiology [ 1 ]. It is rare in most parts of the world but endemic in southern China, especially in Guangdong and Guangxi provinces, with a reported annual incidence of up to 25 per 100000 population [ 2 ]. Its distinctive etiology involves an interplay of the Epstein-Barr virus (EBV), environmental and genetic risk factors [ 3 ]. Radiotherapy remains the principal therapeutic approach for NPC. Despite the promising progress achieved by modern medicine, the persistence of local recurrences, distant metastasis, and treatment resistance are still challenges in NPC management [ 4 , 5 ]. Therefore, gaining profound insight into the intricate mechanisms underpinning NPC remains of paramount significance. Acetyl-CoA acyltransferase 1 (ACAA1), also referred to as acetyl-CoA acyltransferase (ACAA), exhibits wide expression across human liver, kidney, and various normal tissues. Its encoding yields the protein peroxisomal 3-ketoacyl-CoA thiolase (POT1). At the subcellular level, ACAA1 primarily localizes within peroxisomes. It actively participates in fatty acid metabolism, catalyzing the final step of β-oxidation of very long-chain fatty acids (VLCFAs) within peroxisomes, leading to acetyl-CoA generation[ 6 ]. ACAA1 is responsible for thiolytic cleavage of straight-chain 3-oxoacyl-CoAs. Additionally, it is engaged in the final stage of docosahexaenoic acid (DHA) synthesis and the chain shortening of dicarboxylic fatty acid, utilizing the L-bifunctional protein [ 7 ]. Evidence indicates the varying expression of ACAA1, either upregulation or downregulation, across various tumor tissues. For instance, ACAA1 is upregulated in the uterine aspirates from endometrial cancer [ 8 ], triple-negative breast cancer[ 9 ], ampullary cancer [ 10 ], and hepatocellular carcinoma [ 11 ]. In contrast, downregulation of ACAA1 is reported in hepatocellular carcinoma by Dooley et al . [ 12 ], aligning with similar findings in gastric cancer [ 13 ], lung cancer[ 14 ], colorectal cancer[ 15 ], thyroid cancer [ 16 ], and kidney renal clear cell carcinoma [ 17 ]. Nonetheless, the role of ACAA1 and its related mechanisms in cancer is complicated and remains uncovered. Of note, it is reported that ACAA1 takes part in the process of cancer immune regulation [ 18 ]. Tumor microenvironment (TME) is a key node of cancer immune modification. It is an “ecological niche” that promotes the occurrence and development of cancer [ 19 , 20 ]. The elements of TME include but not limited to tumor blood vessels and immune cells such as T cells, macrophages, tumor-associated neutrophils (TANs) with the N2 phenotype, mast cells, natural killer cells, and myeloid-derived suppressor cells (MDSC) [ 21 ]. It is well known that the immune cells in TME play as a double-edged sword in tumor progression [ 22 ]. In the early stage of tumorigenesis, immune cells in the TME target and kill the cancer cells. However, cancer cells escape the immune surveillance and even hinder the cytotoxic function of immune cells via complicated signalling crosstalk [ 23 ]. Thus, by targeting immune evasion, immunotherapy has become a hot strategy for cancer treatment. In the previous study, we’ve conducted a biological statistical analysis by employing the Gene Expression Omnibus (GEO) database, and found that ACAA1 was decreased in NPC[ 24 ]. In the present study, we sought to validate this observation by assessing the expression of ACAA1 at both mRNA and protein levels. We further investigated the potential effects of ACAA1 on NPC cells by in vivo and in vitro assays, while also exploring its diagnostic and prognostic role, as well as the underlying mechanisms driving its effects via a series of bioinformatics analyses. These findings might provide new insights into the prognosis and immune therapeutic values of ACAA1 within the context of NPC. Materials and methods Ethics statement This study was approved by the Ethics Committee of the Affiliated Tumor Hospital of Guangxi Medical University (2022-KT-243). All participants were fully informed with the aim, methodology, and possible risks of the study; written consents were obtained from each donor. Cell lines and human tissues Five NPC cell lines (CNE1, TW03, 5-8F, HONE1, and HK1) were maintained in Dulbecco’s modified medium (DMEM, Life Technologies, Gibco, USA) containing 10% fetal bovine serum (FBS; Life Technologies, Gibco, USA) at 37℃ in an atmosphere of 5% CO 2 . One non-malignant nasopharyngeal epithelial cell lines (NP460) were cultivated in a defined keratinocyte-serum free medium (KSFM, Life Technologies, Gibco, USA). A total of 19 newly diagnosed primary NPC biopsies were collected from the Department of Otolaryngology-Head and Neck Surgery, First Affiliated Hospital of Guangxi Medical University (Nanning, China). All the NPC cases were diagnosed by experienced pathologists according to the World Health Organization classification. A number of 18 normal nasopharyngeal epithelium (NNE) tissue samples diagnosed with chronic inflammation were included as controls. In total, 18 NNE and 19 NPC samples were used for RNA extraction. A tissue microarray including 125 NPC tissues samples was purchased from Shanghai Outdo Biotech Co., Ltd. (Shanghai, China; Cat No: HNasN129Su01). Plasmids, reagents, and antibodies The plasmids pCMV6-Entry-ACAA1 and pCMV6-Entry were purchased from Origene, USA. The transfection experiment was conducted using Lipofectamine 3000 Transfection (L3000015, Life Technologies, USA), adhering to the manufacturer's protocol. Antibodies and fluorescent dyes used in this study were as followed: ACAA1 (ab90647, Abcam, USA), GAPDH (HRP60004, Proteintech, USA), anti-rabbit/mouse IgG-HRP conjugate (Bio-Rad, USA), Ki67(9449, Cell Signaling, Germany), phalloidin (A12381, Invitrogen, USA), and DAPI (C0065, Solarbio, China). Raw data acquisition Microarray data, including GSE12452, GSE180272, GSE53819, GSE61218, GSE64634, and GSE102349, were downloaded from the GEO database ( http://www.ncbi.nlm.nih.gov/geo/ ) as the raw data. The dataset of GSE12452 and GSE64634 based on the GPL570 platform, GSE180272 based on the GPL16956 platform, GSE53819 based on the GPL6480, and GSE61218 based on the GPL19061, were applied to confirm the transcriptional level of ACAA1 in NPC. The dataset of GSE102349 based on the GPL11154 was used in the subsequent analyses to explore the association of ACAA1 expression with tumor stage, EBV-encoded gene expression, TME subtype, survival, immune-related indexes, etc. Transcriptional analysis of ACAA1 To validate the expression of ACAA1 in NPC, quantitative real-time polymerase chain reaction (qPCR) was performed in NPC cell lines and primary NPC tissues, with non-malignant nasopharyngeal epithelial cell line NP460 and normal nasopharyngeal epithelium (NNE) tissues as controls. In general, total RNA was extracted with TRIzol reagent (Life Technologies, Invitrogen, USA) as previously described[ 25 ]. First-strand cDNA was synthesized using RevertAid First Strand cDNA Synthesis Kit (Life Technologies, Invitrogen, USA), and qPCR was conducted using PowerUp SYBR Green PCR Master Mix (Applied Biosystems, A25777, USA). The GAPDH gene was amplified from the same cDNA sample as an internal control. The primer sequences were as follows: ACAA1 -Forward: 5’-CATCTGTGTCGGAAATGTGC-3’, ACAA1 -Reverse: 5’-TTCTGATGCCACCTGCTATG-3’, GAPDH -Forward: 5’-AAGCTCACTGGCATGGCCTT-3’, GAPDH -Reverse: 5’-CTCTCTTCCTCTTGTGCTCTTG-3’. The PCR conditions were 95℃ for 30 seconds, followed by 40 cycles at 95℃ for 5 seconds and 60℃ for 30 seconds. The relative expression level of ACAA1 was determined by the 2 −△△Ct method. The reaction was performed in triplicate. Western Blot Analysis In brief, the protein was extracted using the RIPA lysis buffer (Beyotime, China). Equal amount of protein was separated by 10% SDS-PAGE and subsequently transferred onto nitrocellulose filter membranes (Millipore, USA). The blots were then incubated with primary antibodies at 4℃ overnight, followed by appropriate secondary antibodies. Chemiluminescent signals were detected using a CCD camera in a ChemiDoc XRS instrument (Bio-Rad, USA) with Image Lab software. Immunohistochemistry (IHC) Staining Assay IHC staining was performed as previously described[ 24 ]. Briefly, paraffin embedded tissue sections were deparaffinized, rehydrated through graded alcohol. Then, the tissues were incubated with antibody against ACAA1((1:200, ab90647, Abcam, USA) at 4℃ overnight, followed by anti-rabbit-HRP at room temperature for 30 minutes. The 3,3-Diaminobenzidine (DAB, ZLI-9018, ZSGB-BIO, Beijing) was then used to visualize nuclei. Finally, counterstaining with hematoxylin was performed. Two pathologists assessed all IHC tissues in a blinded manner. The intensity of ACAA1 staining was scored and graded as described[ 26 ]. Immunofluorescence staining assay Immunofluorescence staining was performed as described previously [ 27 ]. In brief, cells were fixed with 4% formaldehyde for 15 minutes, permeabilized with 0.5% Triton X-100 for 10 minutes, and blocked with 5% BSA for 30 minutes. After incubated with primary antibodies at 4℃ overnight, cells were labeled by secondary antibodies for 1 hour at room temperature. Cells were double stained with rhodamine phalloidin for 30 mins at room temperature. Cell nuclei were finally stained with DAPI. Immunofluorescence images were obtained by a confocal microscope (FV3000, Olympus, Germany) and analyzed by ImageJ software. CCK-8 Viable Cell Counting Assay Cells were plated in 96-well plates at 2 × 10 3 cells per well and allowed to grow for five days to generate a proliferation curve. A volume of 10 µl CCK-8 solution (Dojindo Laboratories, China) was added and incubated in the detected wells at 37℃ for 2 hours in the dark before being measured by absorbance at 450 nm. All experiments were repeated in triplicate. Colony Formation Assay Cells were seeded at a density of 100 cells per well in six-well plates. During colony growth, the culture medium was replaced every three days. Fourteen days later, the colonies were stained with Giemsa, photographed, and counted using Quantity One v4.4.0 (Quantity One 1-D Analysis Software). The experiment was performed in triplicate. Wound Healing Assay Briefly, cells were seeded into six-well plates and cultured in a DMEM medium without FBS. When cells reached a monolayer and confluent state, cells were scratched using 20µl sterilized pipette tips. Images were captured with Olympus CKX-41 inverted microscope at 0 and 24 hours after scratching at 100× magnification. The width of the scratch was measured with Image J software. The experiment was performed in triplicate. Transwell Invasion Assay Cell suspensions, each containing 2.5×10 4 cells in FBS-free DMEM medium, were plated in the upper chamber of BioCoat Matrigel plates (BD, 354480), respectively. DMEM medium with 10% FBS as a chemoattractant was added to the lower chamber of the BioCoat Matrigel plate. Forty-eight hours later, cells were removed from the upper chamber, and cells that had invaded the lower surface of the membrane were fixed, stained with 1% crystal violet, and photographed with Olympus CKX-41 inverted microscope at 100× magnification. Experiments were performed in triplicate. In vivo Tumor Formation Assay in Nude Mice Six-week-old female Balb/c athymic nude mice were purchased from the Experimental Animal Center of Guangxi Medical University (Nanning, China). Stably transfected CNE1-ACAA1 cells (1×10 6 cells) were injected subcutaneously into the right flank of nude mice. An equal amount of CNE1-control cells was injected into the left flank of mice as a control. The growth of tumors was monitored for two weeks. On the 14th day, the mice were euthanized, and tumors were removed and assessed. This study was carried out following the institutional guidelines of Guangxi Medical University and approved by the Committee on the Ethics of Animal Experiments of Guangxi Medical University. GO and KEGG enrichment analysis To explore the potential biological functions and possible pathways involved in ACAA1, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were conducted via the clusterProfiler[ 28 ] packages in R. False discovery rate (FDR) < 0.05 was considered as statistically significant. Correlation of ACAA1 expression with immune cell infiltration and tumor immune environment in NPC The analyses of decode tumor microenvironment were performed using the IOBR package in R [ 29 ]. Based on the expression median of ACAA1, NPC patient from dataset GSE102349 was divided into ACAA1 high- and low- expressing groups. The xCell signature was applied to evaluate the effect of ACAA1 on the infiltration level of 64 types of immune and stroma cells, including extracellular matrix cells, epithelia cells, hematopoietic progenitors, innate and adaptive immune cells [ 30 ]. The Estimation of Stromal and Immune Cells in Malignant Tumors using the Expression Data (ESTIMATE) was applied to demonstrate the effect of ACAA1 in regulating the tumor purity, stromal and immune cells admixture of NPC[ 31 ]. Immunophenoscore was applied to evaluate the role of ACAA1 in predicting the immunotherapy response of NPC patients [ 32 ]. Further calculation was performed to reveal the correlation between the expression of ACAA1 and 16 immune checkpoint-related genes (PDCD1, CD274, CTLA4, CD80, CD86, LAG3, LMTK3, TIGIT, BTLA, CD40, CD27, CD28, CD47, SIRPA, IDO1, and IDO2). Statistical analyses All statistical evaluations were conducted using the R software (R Foundations for Statistical Computing, version 4.2.1). The ComBat function of the sva package in R was used to remove the batch effect of the merged dataset. Wilcoxon test was employed to compare the difference between two groups, while Kruskal-Wallis test was used to compare differences among three groups. Correlation analysis was adopted using Spearman’s correlation with the PerformanceAnalytics package in R. The prognostic value of ACAA1 for NPC was evaluated and plotted by the timeROC package in R[ 33 ]. The Kaplan-Meier survival analysis was carried out by the survival [ 34 ] and survminer packages in R, with the ideal cutoff point. Statistical significance was set at P < 0.05. Results ACAA1 is downregulated in NPC and negatively associated with Clinical stages Previously, a gene expression dataset (GSE12452, including 31 cases of NPC tissue and 10 normal controls) was acquired from the GEO database to investigate the alterations of gene profiles involved in reprogramming fatty acid metabolism in NPC. A total of 41 differentially expressing genes were identified, and the expression of ACAA1 was among the downregulated genes in NPC [ 24 ]. To further investigate that loss of ACAA1 mRNA is a common feature in NPC, another four gene expression datasets (GSE180272, GSE53819, GSE61218 and GSE64634) were taken into reevaluation. As shown in Fig. 1 A, a significant downregulation of ACAA1 in NPC was found (NPC = 89, controls = 56, P < 0.05). GSE102349 contains the clinical stage information of NPC patients, thus was utilized for further analysis. The transcriptional expression of ACAA1 was compared between patients at early (stage I/II) and late (stage III/IV) stage. Even though we observed the mean value of ACAA1 mRNA level was lower in patients at later stage, the difference was not statistically significant..This might indicate that the downregulation of ACAA1 is a common event in the progression of NPC (Fig. 1 B, P > \(\:\:\) 0.05). To confirm the expression pattern of ACAA1 in NPC, quantitative PCR was carried out in 19 cases of NPC and 18 NPC-free controls. As expected, the transcriptional level of ACAA1 was decreased in NPC tissues ( P < 0.05, Fig. 1 C). We next detected its protein expression level in five NPC cell lines (CNE1, TW03, 5-8F, HONE1, and HK1) and one non-malignant human nasopharyngeal epithelial cell lines (NP460). ACAA1 showed a lower expression in NPC cell lines (Fig. 1 D, E). IHC staining of ACAA1 was conducted in 125 cases of NPC and 17 cases of NNE. The average staining score of ACAA1 in NNE is 9.35, while in NPC is 4.28 ( P < 0.0001, Fig. 1 F, G). Seventeen NPC tissues among 125 NPCs contained adjacent normal epithelium. Therefore, a clearly decrease of ACAA1 in tumor tissues were shown as well. ACAA1 is a novel diagnostic and prognostic prediction biomarker of NPC Based on the mRNA of ACAA1 data, the AUC was 0.771(p < 0.01, Fig. 2 A). Meanwhile, we performed a ROC analysis based on the IHC scores, the result showed that the AUC was 0.996 ( p < 0.0001, Fig. 2 B). These data supported that both mRNA and protein expression of ACAA1 could serve as a promising diagnostic biomarker for NPC patients. To evaluate the prognostic capability of ACAA1, the same cohort of NPC patients (GSE102349) were divided into high and low ACAA1 expression groups, according to its median expression level. As indicated by Kaplan-Meier (K-M) analysis, NPC patients with higher expression of ACAA1 had a longer progression-free survival (PFS), when compared with those of lower ACAA1 expression (Fig. 2 C, P < 0.05). The Harrell’s concordance index was 0.633 (95%CI: 0.510–0.757). Time-dependent ROC curves were constructed to further confirm the prognostic value of ACAA1, with AUC values of 0.769 and 0.676, at 1 year and 2 years respectively (Fig. 2 D). Thus, it was suggested that ACAA1 was decreased in NPC and associated with poor prognosis, implying it might be involved in the tumorigenesis of NPC. The expressional correlation between ACAA1 and EBV-encoded genes EBV is a well-recognized etiological factor in NPC, and it plays an important role in the carcinogenesis and development of NPC. We tried to explore the correlation between the expression of ACAA1 and EBV-encoded genes. As shown (Fig. 2 E), the transcription of ACAA1 was negatively associated with EBNA1, RPMS1, and A73 (R=-0.64, -0.53, -0.51, respectively; all P 0.05). It was then hypothesized that NPC cells may strengthen the EBV infection via downregulating ACAA1. Exogenous Expression of ACAA1 Suppresses the Proliferation, Migration, and Invasion of NPC Cells To evaluate the effect of ACAA1 on NPC cells, stable ACAA1-overexpressing, and the corresponding pCMV6-Entry-CNE1/HK1 control cell lines were established. The expression of ACAA1 was confirmed by Western blot assay (Fig. 3 A). CCK8 assays were conducted to assess the proliferation ability, and the results demonstrated that ACAA1-CNE1/HK1 cells grew slower than pCMV6-Entry-CNE1/HK1 cells (Fig. 3 B). A significant lower expression of Ki-67 in ACAA1-CNE1/HK1 cells further supported the role of ACAA1 in suppressing cell proliferation (Fig. 4 ). Additionally, colony formation assay showed fewer and smaller colonies in ACAA1-CNE1/HK1 cells compared to pCMV6-Entry-CNE1/HK1 cells (Fig. 3 C), suggesting that ACAA1 inhibited the colony formation ability of NPC cells. To validate the tumor suppression ability exerted by ACAA1 in vivo , ACAA1-CNE1/HK1 cells and their control cells were injected into the flanks of nude mice, respectively. All nude mice generated tumor masses after transplantation, but the primary tumors derived from ACAA1-CNE1/HK1 cells were smaller than those formed by pCMV6-Entry-CNE1/HK1 cells (Fig. 3 D). Thus, exogenous expression of ACAA1 inhibited the proliferation of NPC cells in vivo . To assess the impact of ACAA1 on cell motility, wound healing assays were performed, and delayed wound closure was observed in ACAA1-CNE1/HK1 cells ( P < 0.001, Fig. 3 E). We also carried out transwell assays to evaluate the role of ACAA1 in the invasive properties of NPC cells, and a weakened invasion capability was noticed in ACAA1-CNE1/HK1 cells after observation of 48 hours (Fig. 3 F). To visualize the morphological features of microfilaments, phalloidin staining was conducted. As shown, the level of F-actin was significantly decreased in CNE1-ACAA1 cells. Interestingly, actin fluorescence was mainly accumulated at the leading edges of CNE1-ACAA1 (Fig. 3 G). We thus proposed that the enhanced migration ability of NPC cells might be the result of depolymerization and redistribution of intracellular cytoskeleton, by suppressing ACAA1. Our data suggest that ACAA1 overexpression inhibited the proliferation, migration, and invasion of NPC cells, further indicating its potential as a novel tumor suppressor. GO and KEGG enrichment analyses To uncover the potential biological functions and pathways modulated by ACAA1, GO and KEGG enrichment analysis were operated according to the dataset GSE102349. As shown, ACAA1 was highly associated with the immune-related GO terms, such as leukocyte mediated immunity, lymphocyte-mediated immunity, immune effector process, adaptive immune response, and immune response-activating/regulating cell surface receptor signaling pathway, etc. (Fig. 4 A). KEGG analysis showed that with higher expression of ACAA1, signaling pathways such as allograft rejection, graft-versus-host disease, staphylococcus aureus infection, and viral protein interaction played important roles. Reversely, in the context of lower expression of ACAA1, signaling pathways involving pluripotency of stem cells, cell cycle, hippo signaling pathway, and nucleocytoplasmic transport, Wnt signaling pathways, were significantly enriched (Fig. 4 B). Thus, it is hypothesized that NPC cells may influence the immune response by regulating ACAA1 expression. ACAA1 plays a positive regulation on the immune cell infiltration and tumor immune environment in NPC Inspired by the GO and KEGG enrichment results, we next explore the association between ACAA1 expression and immune cell infiltration. The dataset of GSE102349 was applied on the xCell platform. As shown, the levels of infiltration of activated dendritic cell (aDC), B cells, CD4 + T cells/memory T cells/naïve T cells/Tem, CD 8 + T cells/naïve T cells/Tcm/Tem, classical DC (cDC), macrophages, macrophages M1 and M2, et al ., were significantly increased in the ACAA1-high expressing NPC patients (Fig. 5 A). Through the ESTIMATE analysis, it was found that ACAA1 was positively correlated with the immune score, stromal score, and ESTIMATE score ( P < 0.001, R = 0.63; P < 0.001, R = 0.49; P < 0.001, R = 0.61, respectively) and negatively correlated with the tumor purity ( P < 0.001, R=-0.61) (Fig. 5 B). The Immunophenoscore (IPS) was further calculated to analyze the roles of immune cells. It was found that ACAA1 was positively correlated with MHC score ( P < 0.001, R = 0.53), effector cell score (e.g., activated CD8 + T cells and CD4 + cells, Tem CD8 + and Tem CD4 + cells) ( P < 0.001, R = 0.53) and immune checkpoint score ( P < 0.001, R = 0.41), and negatively correlated with immunosuppressive cell score (e.g, Tregs and MDSCs) ( P < 0.001, R=-0.55), indicating that NPC cells might escape the immune surveillance by downregulating the expression of ACAA1(Fig. 5 C). ACAA1 might be a potential immune-related prognostic and immune-therapeutic marker in NPC One interesting advantage of the dataset of GSE102349 is its classification of three NPC subtypes based on the expression pattern of immune and stromal genes, naming TME I, II, and III [ 35 ]. In the present study, it was noted that the expression of ACAA1 in TME subtype I was statistically lower than that in type II and III (TME subtype I vs. TME subtype Ⅱ, P < 0.01; TME subtype I vs. TME subtype Ⅲ, P < 0.01), suggesting that NPC patients with reduced ACAA1 expression tend to experience poorer outcomes (Fig. 5 D). It was in line with the K-M analysis, further supporting that ACAA1 might be a candidate immune-related prognostic biomarker. To assess its immunotherapeutic potential, we estimated the correlation between the expression of ACAA1 and 6 immune checkpoint-related genes. As shown, ACAA expression had a positive correlation with immune checkpoint-related genes, including CD27, PDCD1, CD86, BTLA, TIGIT, and CD28 (all P < 0.05, Fig. 5 E). Thus, it was proposed that the expression of ACAA1 might be an immune checkpoint treatment target. Discussion Evading immune destruction is another critical hallmark of cancer [ 19 ]. An immunosuppressive milieu is one of great characteristics of NPC, which facilitates its progression[ 35 ]. Recently, emerging illustrations of immune evasion and immunotherapy have been reported in NPC. However, searching for proper immune-related target and evaluating NPC patients who might benefit from immunotherapy remain a great challenge. In this study, we identified the downregulation of ACAA1 in NPC. In vitro and in vivo studies verified that overexpression of ACAA1 could inhibit the proliferation, migration, and invasion capabilities of NPC cells. Computational analysis showed that decreased ACAA1 was associated with poor survival of NPC patients. GO and KEGG analyses revealed an enrichment of immune-related pathways in the ACAA1 high-expressing NPC group. Furthermore, we found that NPC patients with higher expression of ACAA1 were tend to exhibit higher levels of infiltration of immune cells, and higher correlation with the expression of immune checkpoint-related genes, suggesting that ACAA1 might be a novel immune therapeutic target. Under-expressed ACAA1 has been found in a panel of malignancies and negatively associated with Ki-67 expression [ 15 , 17 , 18 ]. It had been noted that lower expression of ACAA1 might contribute to the occurrence of hepatocellular carcinoma [ 36 ]. A decreased transcription of ACAA1 was associated with unfavorable overall survival of breast cancer patients [ 37 ]. The single nucleotide polymorphism (SNP) rs4988453, which maps to the promoter region shared by ACAA1 and toll-like receptors (TLR) downstream effector MYD88, is associated with decreased survival of colorectal cancer [ 38 ]. In addition, ACAA1 rs2239621 is a risk factor of gastric cancer [ 39 ]. In non-small cell lung cancer, it is noted that ACAA1 is downregulated by oncogenic KRAS through MAPK pathway[ 40 ]. In line with these, our data supported that the downregulation of ACAA1 was a prevalent phenomenon in NPC, and ROC analysis showed a favorable diagnosis efficiency of ACAA1 for NPC, both in mRNA and protein levels. This is worth further evaluation in a larger size of clinical samples. Besides, overexpression of ACAA1 could inhibit the malignant behaviours of NPC cells. One of the involving mechanisms might lie in the repression and redistribution of cellular actin filaments. ACAA1 is related to several immune-related disease processes and is associated with the effect of anti-cancer treatment. For instance, a SNP (rs156265) in ACAA1 is found to modify the effect of endotoxin exposure on childhood asthma risk [ 41 ]. In lung tumor microenvironment, ACAA1 is positively associated with antigen presentation and correlated with infiltration of T cells, including CD4+, Th1, Th2, and Treg cells [ 18 , 40 ]. In addition, the expression of ACAA1 could be applied as a biomarker for personal therapeutic assessment. Patients with higher expression of ACAA1 are proposed to be more sensitive to anti-cancer drugs such as EGFR inhibitor Erlotinib and VEGFR2/3 inhibitor ZD-6474[ 18 ]. Notably, Erlotinib might be an enhancer of radiotherapy in NPC by evoking G2/M phase cell cycle arrest, as well as an enhancer of chemoradiotherapy by impeding DNA damage repairment[ 42 ]. Consistently, we found that higher ACAA1 expression was positively associated with higher infiltration of immune cells, and lower expression of ACAA1 was associated with worse overall survival of NPC patients. Indicated by these data, we hypothesis that NPC cells stimulate their malignant behaviours and survive through regulating immune cell infiltration in the tumor microenvironment. Tumor microenvironment and immune modulation are pivotal in cancer research. Numerous studies underscore the critical roles of TME, particularly infiltrating immune cells, in tumor promotion and progression [ 43 , 44 ]. T lymphocytes, particularly CD4 + and CD8 + cells, are abundant in NPC tumors [ 45 ]. CD4 + T cells, which generating chemokine ligand 13 (CXCL13), critically contribute to the formation of tertiary lymphoid structures by interacting with B cells, correlating with better survival of NPC patients[ 46 ]. CD8 + T cells, known for their ability to eliminate target cells through anti-tumor cytokines and cytotoxic molecules, exhibit exhaustion and dysfunction, and subsequently promote immune evasion of NPC cells [ 45 , 47 , 48 ]. Furthermore, recurrent NPC is associated with increased immunosuppression of T cells and exacerbated dysfunctional cytotoxicity in CD8 + T cells [ 49 ]. In addition to T cells, macrophages also contribute to the regulation of tumor infiltration. Macrophage phenotypes in NPC exhibit significant differences, with M1 macrophages mainly residing in tumor nests and M2 macrophages predominantly presenting in tumor stroma [ 50 ]. Tumor-derived fibroblast growth factor 2 (FGF-2) has been identified as a recruiter of macrophages and a promoter of M2 macrophage polarization through the upregulation of CXCL14 in NPC [ 51 ]. Notably, plasmacytoid dendritic cells (pDCs) aggregate the tumor stroma of NPC, and this phenomenon is significantly associated with improved survival outcomes [ 52 ]. Our study demonstrated a negative association between ACAA1 and immunosuppressive cells, alongside a positive association with effector cells, suggesting ACAA1’s potential role in regulating immune cells and counteracting tumor suppression in NPC. EBV maintains a persistent latent infection in the human population by establishing a balance with the host’s immune system. Interestingly, reports have documented that EBV-encoded genes, such as EBNA1, EBNA2, EBV-encoded miRNAs BART11 and BART17-3p, facilitate tumor immune evasion through a complex network of pathways [ 53 – 55 ]. For instance, EBNA1 activates the JAK2/STAT1/IRF-1 signalling pathway and suppresses the promoter activity of PD-L1[ 53 ]. EBNA2 inhibits miR-34a through the downregulation of the transcription factor EBF1, consequently enhancing the expression of PD-1[ 54 ]. EBV-miRNAs BART11 and EBV-miR-BART17-3p stimulate the expression of PD-L1 by repressing FOXP1 and PBRM1, respectively[ 55 ]. RPMS1 and A73 are also important members of EBV BRATs implicated in NPC tumorigenesis [ 56 , 57 ]. In the current study, we have described a negative association between the expression of ACAA1 and EBV-encoded genes (ie., EBNA1, RPMS1, and A73), indicating that the decrease of ACAA1 might heighten the infection of EBV. In addition, findings from the POLARIS-02 and CAPTAIN-1st trials have shown that an early decrease in plasma EBV titer or DNA is associated with a more favorable response to immunotherapy, implying that dynamic alterations in plasma EBV may serve as a promising biomarker [ 58 , 59 ]. In this regard, we propose that ACAA1 might play a role in facilitating the immune response of NPC by modulating EBV infection. However, the precise relationship between ACAA1 and EBV infection remains uncertain and warrants further investigation. The primary strategies for immunotherapy consist of immune checkpoint blockade, adoptive cell therapy, and vaccination. Among these, immune checkpoint inhibitors (ICIs) are employed to obstruct the activity of immune checkpoint proteins, boosting the immune response and alleviating immune suppression. Targeting T cell exhaustion-associated co-stimulatory signals (e.g., PD-1/PD-L1, CTLA-4, and LAG-3) and inhibiting tumor-infiltrating lymphocytes (TILs) are key objectives in immunotherapy [ 60 ]. This offers a novel and promising approach for the treatment of NPC. Clinical trials involving PD-1 inhibitors in recurrent and metastatic NPC patients have shown promising anti-tumor efficacy and favorable safety profiles [ 61 – 64 ]. Furthermore, patients receiving combined treatment with PD-1 inhibitors and chemotherapy have achieved extended overall survival (OS) and progression free survival (PFS) [ 59 , 65 ]. Besides PD-1 inhibitors, dual immune checkpoint inhibitors, such as CTLA-4/PD-L1, LAG-3/PD-L1, and TIM-3/PD-L1, have undergone developed and evaluation in clinical trials [ 66 ]. However, these novel treatment approaches have not been approved by the FDA or NMPA, and the efficacy of PD-1/PD-L1 therapies remains constrained. In our study, we have observed a positive correlation between ACAA1 expression and several immune checkpoint-related genes, including PDCD1 (encoding PD-1 protein), CTLA4, CD80, CD86, and LAG3. Therefore, modulation of ACAA1 expression represents another important target for anti-cancer therapy. In conclusion, we have identified ACAA1 as a novel tumor suppressor in NPC. ACAA1 is notably downregulated in NPC. Restoring ACAA1 effectively inhibits the proliferation, migration, and invasion of NPC cells, by suppressing Ki-67 expression and altering action filaments. Moreover, decrease of ACAA1 expression indicates poor survival of NPC patients, potentially due to immune evasion. These findings are anticipated to shed light on novel perspectives regarding NPC diagnosis markers, personalized immunotherapeutic strategies, and prognosis. Declarations ETHICS STATEMENT This study was approved by the Ethics Committee of First Affiliated Hospital of Guangxi Medical University (No. 2022-KT-243). CONFLICT OF INTEREST The authors declare no conflict of interests. FUNDING This study was supported by grants from the National Natural Science Foundation of China (No. 81560439 and 82060511), Guangxi Natural Science Foundation Program for Youths ((No. 2015GXNSFBA139143 and 2018GXNSFBA281158), Guangxi Medical University Natural Science Foundation for Youths (No. GXMUYSF2014035) and High-level Talent Introduction Plan of the First Affiliated Hospital of Guangxi Medical University (the fifth level). Author Contribution X. X., LM. L., WQ. W.: conducted experiments, data analysis, figure preparation, and manuscript writing; SX. Z, F. H., YS. L.: conducted experiments and data analysis; XY. Z., Z. Z.: experiment design and technical consultation; YL. C. and WL. Z.: experiment design, financial support, and manuscript writing. Data Availability The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.All data generated or analyzed during this study are included in this published article and its supplementary information files. The relevant data used in this study were obtained from the GEO database. References Chen YP, Chan ATC, Le QT, Blanchard P, Sun Y, Ma J. Nasopharyngeal carcinoma. Lancet. 2019;394(10192):64–80. Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, Bray F. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin. 2021;71(3):209–49. Chang ET, Adami HO. The enigmatic epidemiology of nasopharyngeal carcinoma. Cancer Epidemiol Biomarkers Prev. 2006;15(10):1765–77. Lee AW, Ma BB, Ng WT, Chan AT. Management of Nasopharyngeal Carcinoma: Current Practice and Future Perspective. J Clin Oncol. 2015;33(29):3356–64. 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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-4750465","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":335784282,"identity":"56aa1896-7087-4c84-a3df-db94060fc4f5","order_by":0,"name":"Weilin Zhao","email":"","orcid":"","institution":"Department of Otolaryngology-Head and Neck Surgery, First Affiliated Hospital of Guangxi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Weilin","middleName":"","lastName":"Zhao","suffix":""},{"id":335784283,"identity":"f8b36175-58bd-4ce3-9b05-558c36bf76f9","order_by":1,"name":"Limei 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14:29:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4750465/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4750465/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":63271793,"identity":"f784e282-8394-4666-b97f-d9ac5a6020df","added_by":"auto","created_at":"2024-08-26 11:28:44","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":9824289,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eACAA1 is downregulated in NPC primary tissues and cell lines\u003c/strong\u003e (A) Relative mRNA expression of ACAA1 was analyzed based on GEO datasets. (B) The differentially expression analysis of ACAA1 between stage III/IV and I/II of NPC. (C) qPCR analysis of the mRNA level of ACAA1 in NPC primary tumors (n=19) and NNE tissues (n=18). The scatter plots indicate 25 to 75 percentiles, and the horizontal line indicates the mean. (D-E) The protein expression of ACAA1 was detected by Western blot in five NPC cell lines (CNE1, TW03, 5-8F, HONE1, and HK1) and one non-cancerous nasopharyngeal epithelial cell lines (NP460). GAPDH was used as an internal control. Data are mean ± SD (n=3). (F-G) Immunohistochemistry staining of ACAA1 in a tissue microarray including 125 primary NPC tissues and 17 paired normal tissues. Representative images and IHC scores were shown. Magnification×100, ×400. *P\u0026lt;0.05, **P\u0026lt;0.01, and ****P\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4750465/v1/8c0d2f5b71b30efeeaa93105.jpg"},{"id":63271788,"identity":"a95313ff-f29b-4fc9-9499-d058e071611d","added_by":"auto","created_at":"2024-08-26 11:28:43","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3779299,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eACAA1 is a potential biomarker for diagnosis and prognosis of NPC\u003c/strong\u003e ROC curve was used to assess the diagnostic efficacy of ACAA1 expression level based on \u003cstrong\u003e(A) \u003c/strong\u003eqRT‐PCR, and \u003cstrong\u003e(B) \u003c/strong\u003eIHC results. (\u003cstrong\u003eC\u003c/strong\u003e) Kaplan-Mier plot illustrating the progression-free survival (PFS) difference between high and low ACAA1 expressing groups. (\u003cstrong\u003eD\u003c/strong\u003e) Time-dependent ROC curves for ACAA1 at 1- and 2-year of PFS. (\u003cstrong\u003eE\u003c/strong\u003e) The correlation analysis between the expression of ACAA1 and EBV-encoded genes.\u003c/p\u003e","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4750465/v1/09427d91b58304c466218551.jpg"},{"id":63271791,"identity":"72f75345-85fd-44b4-818c-7560021131b1","added_by":"auto","created_at":"2024-08-26 11:28:44","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":8503065,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExogenous expression of ACAA1 suppresses the proliferation, migration, and invasion of NPC cells\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003eWestern blot analysis confirming the expression of ACAA1 in CNE1 and HK1 cells stably transfected with ACAA1-pCMV6-Entry or pCMV6-Entry. \u003cstrong\u003e(B) \u003c/strong\u003eCCK8 assay detecting the proliferation of ACAA1-CNE1/HK1 and pCMV6-Entry-CNE1/HK1 cells. Data are mean ± SD (n=5). \u003cstrong\u003e(C) \u003c/strong\u003eColony formation assay of ACAA1-CNE1/HK1 and pCMV6-Entry-CNE1/HK1 cells. Bar charts show the numbers of colonies. Data are mean ± SD (n=3). Representative images are shown. \u003cstrong\u003e(D)\u003c/strong\u003eXenografts were removed from nude mice 14 days after inoculation. The bar chart shows the average volume of the removed tumors 14 days after injection. Data are mean ± SD (n=5). \u003cstrong\u003e(E) \u003c/strong\u003eWound healing assays of ACAA1-CNE1/HK1 and pCMV6-Entry-CNE1/HK1 cells. Photos were taken at the beginning and 24h later. The gap closures were measured as (width at 24h) / (width at 0h). \u003cstrong\u003e(F)\u003c/strong\u003e Transwell assays of ACAA1-CNE1/HK1 and pCMV6-Entry-CNE1/HK1 cells. Invading cells were stained with crystal violet and counted. Representative images are shown. Data are mean ± SD (n=3). \u003cstrong\u003e(G) \u003c/strong\u003eImmunofluorescence staining of Ki-67 and phalloidin. The mean grey value of Ki-67 per cells was shown in bar chart. green:Ki-67, red:phalloidin, blue:DAPI. Magnification: ×1000. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 and ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4750465/v1/9ea2fb57b887754e669f273a.jpg"},{"id":63271792,"identity":"a7f0646f-d6ed-4b90-a814-002d04c0bfa2","added_by":"auto","created_at":"2024-08-26 11:28:44","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":6283732,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGO and KEGG enrichment analysis \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) GO enrichment analysis. (\u003cstrong\u003eB\u003c/strong\u003e) KEGG enrichment analysis. GO: Gene Ontology, KEGG: Kyoto Encyclopedia of Genes and Genomes, BP: biological process, CC: cellular component, MF: molecular function.\u003c/p\u003e","description":"","filename":"Fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4750465/v1/641541f33051b5ae582b61bc.jpg"},{"id":63271790,"identity":"19cc867e-96ac-44fb-8735-ce39dba69676","added_by":"auto","created_at":"2024-08-26 11:28:43","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":7106412,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe potential role analysis of ACAA1 in NPC immune microenvironment and immunotherapy (A) \u003c/strong\u003eInfiltration levels of 64 kinds of immune cell subtypes between high and low ACAA1 expressing groups. Immune cell subtypes with statistical difference are shown (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05). (\u003cstrong\u003eB\u003c/strong\u003e) The ESTIMATE analysis of the association between ACAA1 expression and immune score, stromal score, ESTIMATE score and tumor purity. (\u003cstrong\u003eC\u003c/strong\u003e) The immunophenoscore (IPS) analysis of the association between ACAA1 expression and MHC score, effector cell score, immunosuppressive score, and immune checkpoint score. (\u003cstrong\u003eD\u003c/strong\u003e) The association between ACAA1 expression and TME-based subtypes. (\u003cstrong\u003eE\u003c/strong\u003e) The correlation analysis between the expression of ACAA1 and 6 immune checkpoint-related genes with correlation coefficient≥0.5. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 and ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001. NS, no statistically difference.\u003c/p\u003e","description":"","filename":"Fig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4750465/v1/354c88fa117619d183c75f2b.jpg"},{"id":66626093,"identity":"86b08c4b-7465-4ed1-b4cd-27f545f360fc","added_by":"auto","created_at":"2024-10-15 03:39:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":36297429,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4750465/v1/817dc7ae-f21b-4d73-b245-e389df76d2ea.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Acetyl-CoA acyltransferase 1 is a potential tumor suppressor gene associated with immune cell infiltration in nasopharyngeal carcinoma","fulltext":[{"header":"Introduction","content":"\u003cp\u003eNasopharyngeal carcinoma (NPC) is a unique head and neck squamous cell carcinoma originating from the nasopharyngeal epithelium, with a specific geographic distribution and a comprehensive etiology [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. It is rare in most parts of the world but endemic in southern China, especially in Guangdong and Guangxi provinces, with a reported annual incidence of up to 25 per 100000 population [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Its distinctive etiology involves an interplay of the Epstein-Barr virus (EBV), environmental and genetic risk factors [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Radiotherapy remains the principal therapeutic approach for NPC. Despite the promising progress achieved by modern medicine, the persistence of local recurrences, distant metastasis, and treatment resistance are still challenges in NPC management [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Therefore, gaining profound insight into the intricate mechanisms underpinning NPC remains of paramount significance.\u003c/p\u003e \u003cp\u003eAcetyl-CoA acyltransferase 1 (ACAA1), also referred to as acetyl-CoA acyltransferase (ACAA), exhibits wide expression across human liver, kidney, and various normal tissues. Its encoding yields the protein peroxisomal 3-ketoacyl-CoA thiolase (POT1). At the subcellular level, ACAA1 primarily localizes within peroxisomes. It actively participates in fatty acid metabolism, catalyzing the final step of β-oxidation of very long-chain fatty acids (VLCFAs) within peroxisomes, leading to acetyl-CoA generation[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. ACAA1 is responsible for thiolytic cleavage of straight-chain 3-oxoacyl-CoAs. Additionally, it is engaged in the final stage of docosahexaenoic acid (DHA) synthesis and the chain shortening of dicarboxylic fatty acid, utilizing the L-bifunctional protein [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eEvidence indicates the varying expression of ACAA1, either upregulation or downregulation, across various tumor tissues. For instance, ACAA1 is upregulated in the uterine aspirates from endometrial cancer [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], triple-negative breast cancer[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], ampullary cancer [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], and hepatocellular carcinoma [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In contrast, downregulation of ACAA1 is reported in hepatocellular carcinoma by Dooley \u003cem\u003eet al\u003c/em\u003e. [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], aligning with similar findings in gastric cancer [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], lung cancer[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], colorectal cancer[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], thyroid cancer [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], and kidney renal clear cell carcinoma [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Nonetheless, the role of ACAA1 and its related mechanisms in cancer is complicated and remains uncovered. Of note, it is reported that ACAA1 takes part in the process of cancer immune regulation [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTumor microenvironment (TME) is a key node of cancer immune modification. It is an \u0026ldquo;ecological niche\u0026rdquo; that promotes the occurrence and development of cancer [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The elements of TME include but not limited to tumor blood vessels and immune cells such as T cells, macrophages, tumor-associated neutrophils (TANs) with the N2 phenotype, mast cells, natural killer cells, and myeloid-derived suppressor cells (MDSC) [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. It is well known that the immune cells in TME play as a double-edged sword in tumor progression [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In the early stage of tumorigenesis, immune cells in the TME target and kill the cancer cells. However, cancer cells escape the immune surveillance and even hinder the cytotoxic function of immune cells via complicated signalling crosstalk [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Thus, by targeting immune evasion, immunotherapy has become a hot strategy for cancer treatment.\u003c/p\u003e \u003cp\u003eIn the previous study, we\u0026rsquo;ve conducted a biological statistical analysis by employing the Gene Expression Omnibus (GEO) database, and found that ACAA1 was decreased in NPC[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. In the present study, we sought to validate this observation by assessing the expression of ACAA1 at both mRNA and protein levels. We further investigated the potential effects of ACAA1 on NPC cells by \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e assays, while also exploring its diagnostic and prognostic role, as well as the underlying mechanisms driving its effects via a series of bioinformatics analyses. These findings might provide new insights into the prognosis and immune therapeutic values of ACAA1 within the context of NPC.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eEthics statement\u003c/h2\u003e \u003cp\u003eThis study was approved by the Ethics Committee of the Affiliated Tumor Hospital of Guangxi Medical University (2022-KT-243). All participants were fully informed with the aim, methodology, and possible risks of the study; written consents were obtained from each donor.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eCell lines and human tissues\u003c/h2\u003e \u003cp\u003eFive NPC cell lines (CNE1, TW03, 5-8F, HONE1, and HK1) were maintained in Dulbecco\u0026rsquo;s modified medium (DMEM, Life Technologies, Gibco, USA) containing 10% fetal bovine serum (FBS; Life Technologies, Gibco, USA) at 37℃ in an atmosphere of 5% CO\u003csub\u003e2\u003c/sub\u003e. One non-malignant nasopharyngeal epithelial cell lines (NP460) were cultivated in a defined keratinocyte-serum free medium (KSFM, Life Technologies, Gibco, USA).\u003c/p\u003e \u003cp\u003eA total of 19 newly diagnosed primary NPC biopsies were collected from the Department of Otolaryngology-Head and Neck Surgery, First Affiliated Hospital of Guangxi Medical University (Nanning, China). All the NPC cases were diagnosed by experienced pathologists according to the World Health Organization classification. A number of 18 normal nasopharyngeal epithelium (NNE) tissue samples diagnosed with chronic inflammation were included as controls. In total, 18 NNE and 19 NPC samples were used for RNA extraction.\u003c/p\u003e \u003cp\u003eA tissue microarray including 125 NPC tissues samples was purchased from Shanghai Outdo Biotech Co., Ltd. (Shanghai, China; Cat No: HNasN129Su01).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003ePlasmids, reagents, and antibodies\u003c/h2\u003e \u003cp\u003eThe plasmids pCMV6-Entry-ACAA1 and pCMV6-Entry were purchased from Origene, USA. The transfection experiment was conducted using Lipofectamine 3000 Transfection (L3000015, Life Technologies, USA), adhering to the manufacturer's protocol. Antibodies and fluorescent dyes used in this study were as followed: ACAA1 (ab90647, Abcam, USA), GAPDH (HRP60004, Proteintech, USA), anti-rabbit/mouse IgG-HRP conjugate (Bio-Rad, USA), Ki67(9449, Cell Signaling, Germany), phalloidin (A12381, Invitrogen, USA), and DAPI (C0065, Solarbio, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eRaw data acquisition\u003c/h2\u003e \u003cp\u003eMicroarray data, including GSE12452, GSE180272, GSE53819, GSE61218, GSE64634, and GSE102349, were downloaded from the GEO database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.ncbi.nlm.nih.gov/geo/\u003c/span\u003e\u003cspan address=\"http://www.ncbi.nlm.nih.gov/geo/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) as the raw data. The dataset of GSE12452 and GSE64634 based on the GPL570 platform, GSE180272 based on the GPL16956 platform, GSE53819 based on the GPL6480, and GSE61218 based on the GPL19061, were applied to confirm the transcriptional level of ACAA1 in NPC. The dataset of GSE102349 based on the GPL11154 was used in the subsequent analyses to explore the association of ACAA1 expression with tumor stage, EBV-encoded gene expression, TME subtype, survival, immune-related indexes, etc.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eTranscriptional analysis of ACAA1\u003c/h2\u003e \u003cp\u003eTo validate the expression of ACAA1 in NPC, quantitative real-time polymerase chain reaction (qPCR) was performed in NPC cell lines and primary NPC tissues, with non-malignant nasopharyngeal epithelial cell line NP460 and normal nasopharyngeal epithelium (NNE) tissues as controls. In general, total RNA was extracted with TRIzol reagent (Life Technologies, Invitrogen, USA) as previously described[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. First-strand cDNA was synthesized using RevertAid First Strand cDNA Synthesis Kit (Life Technologies, Invitrogen, USA), and qPCR was conducted using PowerUp SYBR Green PCR Master Mix (Applied Biosystems, A25777, USA). The \u003cem\u003eGAPDH\u003c/em\u003e gene was amplified from the same cDNA sample as an internal control. The primer sequences were as follows:\u003c/p\u003e \u003cp\u003e \u003cem\u003eACAA1\u003c/em\u003e-Forward: 5\u0026rsquo;-CATCTGTGTCGGAAATGTGC-3\u0026rsquo;,\u003c/p\u003e \u003cp\u003e \u003cem\u003eACAA1\u003c/em\u003e-Reverse: 5\u0026rsquo;-TTCTGATGCCACCTGCTATG-3\u0026rsquo;,\u003c/p\u003e \u003cp\u003e \u003cem\u003eGAPDH\u003c/em\u003e-Forward: 5\u0026rsquo;-AAGCTCACTGGCATGGCCTT-3\u0026rsquo;,\u003c/p\u003e \u003cp\u003e \u003cem\u003eGAPDH\u003c/em\u003e-Reverse: 5\u0026rsquo;-CTCTCTTCCTCTTGTGCTCTTG-3\u0026rsquo;.\u003c/p\u003e \u003cp\u003eThe PCR conditions were 95℃ for 30 seconds, followed by 40 cycles at 95℃ for 5 seconds and 60℃ for 30 seconds. The relative expression level of \u003cem\u003eACAA1\u003c/em\u003e was determined by the 2\u003csup\u003e\u0026minus;△△Ct\u003c/sup\u003e method. The reaction was performed in triplicate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eWestern Blot Analysis\u003c/h2\u003e \u003cp\u003eIn brief, the protein was extracted using the RIPA lysis buffer (Beyotime, China). Equal amount of protein was separated by 10% SDS-PAGE and subsequently transferred onto nitrocellulose filter membranes (Millipore, USA). The blots were then incubated with primary antibodies at 4℃ overnight, followed by appropriate secondary antibodies. Chemiluminescent signals were detected using a CCD camera in a ChemiDoc XRS instrument (Bio-Rad, USA) with Image Lab software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry (IHC) Staining Assay\u003c/h2\u003e \u003cp\u003eIHC staining was performed as previously described[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Briefly, paraffin embedded tissue sections were deparaffinized, rehydrated through graded alcohol. Then, the tissues were incubated with antibody against ACAA1((1:200, ab90647, Abcam, USA) at 4℃ overnight, followed by anti-rabbit-HRP at room temperature for 30 minutes. The 3,3-Diaminobenzidine (DAB, ZLI-9018, ZSGB-BIO, Beijing) was then used to visualize nuclei. Finally, counterstaining with hematoxylin was performed. Two pathologists assessed all IHC tissues in a blinded manner. The intensity of ACAA1 staining was scored and graded as described[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence staining assay\u003c/h2\u003e \u003cp\u003eImmunofluorescence staining was performed as described previously [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In brief, cells were fixed with 4% formaldehyde for 15 minutes, permeabilized with 0.5% Triton X-100 for 10 minutes, and blocked with 5% BSA for 30 minutes. After incubated with primary antibodies at 4℃ overnight, cells were labeled by secondary antibodies for 1 hour at room temperature. Cells were double stained with rhodamine phalloidin for 30 mins at room temperature. Cell nuclei were finally stained with DAPI. Immunofluorescence images were obtained by a confocal microscope (FV3000, Olympus, Germany) and analyzed by ImageJ software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCCK-8 Viable Cell Counting Assay\u003c/h2\u003e \u003cp\u003eCells were plated in 96-well plates at 2 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e cells per well and allowed to grow for five days to generate a proliferation curve. A volume of 10 \u0026micro;l CCK-8 solution (Dojindo Laboratories, China) was added and incubated in the detected wells at 37℃ for 2 hours in the dark before being measured by absorbance at 450 nm. All experiments were repeated in triplicate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eColony Formation Assay\u003c/h2\u003e \u003cp\u003eCells were seeded at a density of 100 cells per well in six-well plates. During colony growth, the culture medium was replaced every three days. Fourteen days later, the colonies were stained with Giemsa, photographed, and counted using Quantity One v4.4.0 (Quantity One 1-D Analysis Software). The experiment was performed in triplicate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eWound Healing Assay\u003c/h2\u003e \u003cp\u003eBriefly, cells were seeded into six-well plates and cultured in a DMEM medium without FBS. When cells reached a monolayer and confluent state, cells were scratched using 20\u0026micro;l sterilized pipette tips. Images were captured with Olympus CKX-41 inverted microscope at 0 and 24 hours after scratching at 100\u0026times; magnification. The width of the scratch was measured with Image J software. The experiment was performed in triplicate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eTranswell Invasion Assay\u003c/h2\u003e \u003cp\u003eCell suspensions, each containing 2.5\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells in FBS-free DMEM medium, were plated in the upper chamber of BioCoat Matrigel plates (BD, 354480), respectively. DMEM medium with 10% FBS as a chemoattractant was added to the lower chamber of the BioCoat Matrigel plate. Forty-eight hours later, cells were removed from the upper chamber, and cells that had invaded the lower surface of the membrane were fixed, stained with 1% crystal violet, and photographed with Olympus CKX-41 inverted microscope at 100\u0026times; magnification. Experiments were performed in triplicate.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vivo\u003c/b\u003e \u003cb\u003eTumor Formation Assay in Nude Mice\u003c/b\u003e\u003c/p\u003e \u003cp\u003eSix-week-old female Balb/c athymic nude mice were purchased from the Experimental Animal Center of Guangxi Medical University (Nanning, China). Stably transfected CNE1-ACAA1 cells (1\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells) were injected subcutaneously into the right flank of nude mice. An equal amount of CNE1-control cells was injected into the left flank of mice as a control. The growth of tumors was monitored for two weeks. On the 14th day, the mice were euthanized, and tumors were removed and assessed. This study was carried out following the institutional guidelines of Guangxi Medical University and approved by the Committee on the Ethics of Animal Experiments of Guangxi Medical University.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eGO and KEGG enrichment analysis\u003c/h2\u003e \u003cp\u003eTo explore the potential biological functions and possible pathways involved in ACAA1, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were conducted via the clusterProfiler[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] packages in R. False discovery rate (FDR)\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered as statistically significant.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eCorrelation of ACAA1 expression with immune cell infiltration and tumor immune environment in NPC\u003c/h2\u003e \u003cp\u003eThe analyses of decode tumor microenvironment were performed using the IOBR package in R [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Based on the expression median of ACAA1, NPC patient from dataset GSE102349 was divided into ACAA1 high- and low- expressing groups. The xCell signature was applied to evaluate the effect of ACAA1 on the infiltration level of 64 types of immune and stroma cells, including extracellular matrix cells, epithelia cells, hematopoietic progenitors, innate and adaptive immune cells [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The Estimation of Stromal and Immune Cells in Malignant Tumors using the Expression Data (ESTIMATE) was applied to demonstrate the effect of ACAA1 in regulating the tumor purity, stromal and immune cells admixture of NPC[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Immunophenoscore was applied to evaluate the role of ACAA1 in predicting the immunotherapy response of NPC patients [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Further calculation was performed to reveal the correlation between the expression of ACAA1 and 16 immune checkpoint-related genes (PDCD1, CD274, CTLA4, CD80, CD86, LAG3, LMTK3, TIGIT, BTLA, CD40, CD27, CD28, CD47, SIRPA, IDO1, and IDO2).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analyses\u003c/h2\u003e \u003cp\u003eAll statistical evaluations were conducted using the R software (R Foundations for Statistical Computing, version 4.2.1). The ComBat function of the sva package in R was used to remove the batch effect of the merged dataset. Wilcoxon test was employed to compare the difference between two groups, while Kruskal-Wallis test was used to compare differences among three groups. Correlation analysis was adopted using Spearman\u0026rsquo;s correlation with the PerformanceAnalytics package in R. The prognostic value of ACAA1 for NPC was evaluated and plotted by the timeROC package in R[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The Kaplan-Meier survival analysis was carried out by the survival [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] and survminer packages in R, with the ideal cutoff point. Statistical significance was set at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eACAA1 is downregulated in NPC and negatively associated with Clinical stages\u003c/h2\u003e \u003cp\u003ePreviously, a gene expression dataset (GSE12452, including 31 cases of NPC tissue and 10 normal controls) was acquired from the GEO database to investigate the alterations of gene profiles involved in reprogramming fatty acid metabolism in NPC. A total of 41 differentially expressing genes were identified, and the expression of ACAA1 was among the downregulated genes in NPC [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. To further investigate that loss of ACAA1 mRNA is a common feature in NPC, another four gene expression datasets (GSE180272, GSE53819, GSE61218 and GSE64634) were taken into reevaluation. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, a significant downregulation of ACAA1 in NPC was found (NPC\u0026thinsp;=\u0026thinsp;89, controls\u0026thinsp;=\u0026thinsp;56, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). GSE102349 contains the clinical stage information of NPC patients, thus was utilized for further analysis. The transcriptional expression of ACAA1 was compared between patients at early (stage I/II) and late (stage III/IV) stage. Even though we observed the mean value of ACAA1 mRNA level was lower in patients at later stage, the difference was not statistically significant..This might indicate that the downregulation of ACAA1 is a common event in the progression of NPC (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, P\u003cem\u003e\u0026gt;\u003c/em\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:\\)\u003c/span\u003e\u003c/span\u003e0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo confirm the expression pattern of ACAA1 in NPC, quantitative PCR was carried out in 19 cases of NPC and 18 NPC-free controls. As expected, the transcriptional level of ACAA1 was decreased in NPC tissues (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). We next detected its protein expression level in five NPC cell lines (CNE1, TW03, 5-8F, HONE1, and HK1) and one non-malignant human nasopharyngeal epithelial cell lines (NP460). ACAA1 showed a lower expression in NPC cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, E). IHC staining of ACAA1 was conducted in 125 cases of NPC and 17 cases of NNE. The average staining score of ACAA1 in NNE is 9.35, while in NPC is 4.28 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF, G). Seventeen NPC tissues among 125 NPCs contained adjacent normal epithelium. Therefore, a clearly decrease of ACAA1 in tumor tissues were shown as well.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eACAA1 is a novel diagnostic and prognostic prediction biomarker of NPC\u003c/h2\u003e \u003cp\u003eBased on the mRNA of ACAA1 data, the AUC was 0.771(p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Meanwhile, we performed a ROC analysis based on the IHC scores, the result showed that the AUC was 0.996 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). These data supported that both mRNA and protein expression of ACAA1 could serve as a promising diagnostic biomarker for NPC patients.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo evaluate the prognostic capability of ACAA1, the same cohort of NPC patients (GSE102349) were divided into high and low ACAA1 expression groups, according to its median expression level. As indicated by Kaplan-Meier (K-M) analysis, NPC patients with higher expression of ACAA1 had a longer progression-free survival (PFS), when compared with those of lower ACAA1 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The Harrell\u0026rsquo;s concordance index was 0.633 (95%CI: 0.510\u0026ndash;0.757). Time-dependent ROC curves were constructed to further confirm the prognostic value of ACAA1, with AUC values of 0.769 and 0.676, at 1 year and 2 years respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Thus, it was suggested that ACAA1 was decreased in NPC and associated with poor prognosis, implying it might be involved in the tumorigenesis of NPC.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eThe expressional correlation between ACAA1 and EBV-encoded genes\u003c/h2\u003e \u003cp\u003eEBV is a well-recognized etiological factor in NPC, and it plays an important role in the carcinogenesis and development of NPC. We tried to explore the correlation between the expression of ACAA1 and EBV-encoded genes. As shown (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE), the transcription of ACAA1 was negatively associated with EBNA1, RPMS1, and A73 (R=-0.64, -0.53, -0.51, respectively; all \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Although no statistically difference, there was a tendency toward a negative association between ACAA1 and LMP2B (\u003cem\u003eP\u0026thinsp;\u0026gt;\u003c/em\u003e\u0026thinsp;0.05). It was then hypothesized that NPC cells may strengthen the EBV infection via downregulating ACAA1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eExogenous Expression of ACAA1 Suppresses the Proliferation, Migration, and Invasion of NPC Cells\u003c/h2\u003e \u003cp\u003eTo evaluate the effect of ACAA1 on NPC cells, stable ACAA1-overexpressing, and the corresponding pCMV6-Entry-CNE1/HK1 control cell lines were established. The expression of ACAA1 was confirmed by Western blot assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). CCK8 assays were conducted to assess the proliferation ability, and the results demonstrated that ACAA1-CNE1/HK1 cells grew slower than pCMV6-Entry-CNE1/HK1 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). A significant lower expression of Ki-67 in ACAA1-CNE1/HK1 cells further supported the role of ACAA1 in suppressing cell proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Additionally, colony formation assay showed fewer and smaller colonies in ACAA1-CNE1/HK1 cells compared to pCMV6-Entry-CNE1/HK1 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), suggesting that ACAA1 inhibited the colony formation ability of NPC cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo validate the tumor suppression ability exerted by ACAA1 \u003cem\u003ein vivo\u003c/em\u003e, ACAA1-CNE1/HK1 cells and their control cells were injected into the flanks of nude mice, respectively. All nude mice generated tumor masses after transplantation, but the primary tumors derived from ACAA1-CNE1/HK1 cells were smaller than those formed by pCMV6-Entry-CNE1/HK1 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Thus, exogenous expression of ACAA1 inhibited the proliferation of NPC cells \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eTo assess the impact of ACAA1 on cell motility, wound healing assays were performed, and delayed wound closure was observed in ACAA1-CNE1/HK1 cells (\u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). We also carried out transwell assays to evaluate the role of ACAA1 in the invasive properties of NPC cells, and a weakened invasion capability was noticed in ACAA1-CNE1/HK1 cells after observation of 48 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). To visualize the morphological features of microfilaments, phalloidin staining was conducted. As shown, the level of F-actin was significantly decreased in CNE1-ACAA1 cells. Interestingly, actin fluorescence was mainly accumulated at the leading edges of CNE1-ACAA1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). We thus proposed that the enhanced migration ability of NPC cells might be the result of depolymerization and redistribution of intracellular cytoskeleton, by suppressing ACAA1. Our data suggest that ACAA1 overexpression inhibited the proliferation, migration, and invasion of NPC cells, further indicating its potential as a novel tumor suppressor.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eGO and KEGG enrichment analyses\u003c/h2\u003e \u003cp\u003eTo uncover the potential biological functions and pathways modulated by ACAA1, GO and KEGG enrichment analysis were operated according to the dataset GSE102349. As shown, ACAA1 was highly associated with the immune-related GO terms, such as leukocyte mediated immunity, lymphocyte-mediated immunity, immune effector process, adaptive immune response, and immune response-activating/regulating cell surface receptor signaling pathway, etc. (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). KEGG analysis showed that with higher expression of ACAA1, signaling pathways such as allograft rejection, graft-versus-host disease, staphylococcus aureus infection, and viral protein interaction played important roles. Reversely, in the context of lower expression of ACAA1, signaling pathways involving pluripotency of stem cells, cell cycle, hippo signaling pathway, and nucleocytoplasmic transport, Wnt signaling pathways, were significantly enriched (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Thus, it is hypothesized that NPC cells may influence the immune response by regulating ACAA1 expression.\u003c/p\u003e \u003cp\u003e \u003cb\u003eACAA1 plays a positive regulation on the immune cell infiltration and tumor immune environment in NPC\u003c/b\u003e \u003c/p\u003e \u003cp\u003eInspired by the GO and KEGG enrichment results, we next explore the association between ACAA1 expression and immune cell infiltration. The dataset of GSE102349 was applied on the xCell platform. As shown, the levels of infiltration of activated dendritic cell (aDC), B cells, CD4\u003csup\u003e+\u003c/sup\u003e T cells/memory T cells/na\u0026iuml;ve T cells/Tem, CD 8\u003csup\u003e+\u003c/sup\u003e T cells/na\u0026iuml;ve T cells/Tcm/Tem, classical DC (cDC), macrophages, macrophages M1 and M2, \u003cem\u003eet al\u003c/em\u003e., were significantly increased in the ACAA1-high expressing NPC patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThrough the ESTIMATE analysis, it was found that ACAA1 was positively correlated with the immune score, stromal score, and ESTIMATE score (\u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001, R\u0026thinsp;=\u0026thinsp;0.63; \u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001, R\u0026thinsp;=\u0026thinsp;0.49; \u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001, R\u0026thinsp;=\u0026thinsp;0.61, respectively) and negatively correlated with the tumor purity (\u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001, R=-0.61) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eThe Immunophenoscore (IPS) was further calculated to analyze the roles of immune cells. It was found that ACAA1 was positively correlated with MHC score (\u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001, R\u0026thinsp;=\u0026thinsp;0.53), effector cell score (e.g., activated CD8\u003csup\u003e+\u003c/sup\u003e T cells and CD4\u003csup\u003e+\u003c/sup\u003e cells, Tem CD8\u003csup\u003e+\u003c/sup\u003e and Tem CD4\u003csup\u003e+\u003c/sup\u003e cells) (\u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001, R\u0026thinsp;=\u0026thinsp;0.53) and immune checkpoint score (\u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001, R\u0026thinsp;=\u0026thinsp;0.41), and negatively correlated with immunosuppressive cell score (e.g, Tregs and MDSCs) (\u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001, R=-0.55), indicating that NPC cells might escape the immune surveillance by downregulating the expression of ACAA1(Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eACAA1 might be a potential immune-related prognostic and immune-therapeutic marker in NPC\u003c/h2\u003e \u003cp\u003eOne interesting advantage of the dataset of GSE102349 is its classification of three NPC subtypes based on the expression pattern of immune and stromal genes, naming TME I, II, and III [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. In the present study, it was noted that the expression of ACAA1 in TME subtype I was statistically lower than that in type II and III (TME subtype I vs. TME subtype Ⅱ, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; TME subtype I vs. TME subtype Ⅲ, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), suggesting that NPC patients with reduced ACAA1 expression tend to experience poorer outcomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). It was in line with the K-M analysis, further supporting that ACAA1 might be a candidate immune-related prognostic biomarker.\u003c/p\u003e \u003cp\u003eTo assess its immunotherapeutic potential, we estimated the correlation between the expression of ACAA1 and 6 immune checkpoint-related genes. As shown, ACAA expression had a positive correlation with immune checkpoint-related genes, including CD27, PDCD1, CD86, BTLA, TIGIT, and CD28 (all \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Thus, it was proposed that the expression of ACAA1 might be an immune checkpoint treatment target.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eEvading immune destruction is another critical hallmark of cancer [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. An immunosuppressive milieu is one of great characteristics of NPC, which facilitates its progression[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Recently, emerging illustrations of immune evasion and immunotherapy have been reported in NPC. However, searching for proper immune-related target and evaluating NPC patients who might benefit from immunotherapy remain a great challenge.\u003c/p\u003e \u003cp\u003eIn this study, we identified the downregulation of ACAA1 in NPC. \u003cem\u003eIn vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e studies verified that overexpression of ACAA1 could inhibit the proliferation, migration, and invasion capabilities of NPC cells. Computational analysis showed that decreased ACAA1 was associated with poor survival of NPC patients. GO and KEGG analyses revealed an enrichment of immune-related pathways in the ACAA1 high-expressing NPC group. Furthermore, we found that NPC patients with higher expression of ACAA1 were tend to exhibit higher levels of infiltration of immune cells, and higher correlation with the expression of immune checkpoint-related genes, suggesting that ACAA1 might be a novel immune therapeutic target.\u003c/p\u003e \u003cp\u003eUnder-expressed ACAA1 has been found in a panel of malignancies and negatively associated with Ki-67 expression [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. It had been noted that lower expression of ACAA1 might contribute to the occurrence of hepatocellular carcinoma [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. A decreased transcription of ACAA1 was associated with unfavorable overall survival of breast cancer patients [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. The single nucleotide polymorphism (SNP) rs4988453, which maps to the promoter region shared by ACAA1 and toll-like receptors (TLR) downstream effector MYD88, is associated with decreased survival of colorectal cancer [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. In addition, ACAA1 rs2239621 is a risk factor of gastric cancer [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. In non-small cell lung cancer, it is noted that ACAA1 is downregulated by oncogenic KRAS through MAPK pathway[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. In line with these, our data supported that the downregulation of ACAA1 was a prevalent phenomenon in NPC, and ROC analysis showed a favorable diagnosis efficiency of ACAA1 for NPC, both in mRNA and protein levels. This is worth further evaluation in a larger size of clinical samples. Besides, overexpression of ACAA1 could inhibit the malignant behaviours of NPC cells. One of the involving mechanisms might lie in the repression and redistribution of cellular actin filaments.\u003c/p\u003e \u003cp\u003eACAA1 is related to several immune-related disease processes and is associated with the effect of anti-cancer treatment. For instance, a SNP (rs156265) in ACAA1 is found to modify the effect of endotoxin exposure on childhood asthma risk [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. In lung tumor microenvironment, ACAA1 is positively associated with antigen presentation and correlated with infiltration of T cells, including CD4+, Th1, Th2, and Treg cells [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. In addition, the expression of ACAA1 could be applied as a biomarker for personal therapeutic assessment. Patients with higher expression of ACAA1 are proposed to be more sensitive to anti-cancer drugs such as EGFR inhibitor Erlotinib and VEGFR2/3 inhibitor ZD-6474[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Notably, Erlotinib might be an enhancer of radiotherapy in NPC by evoking G2/M phase cell cycle arrest, as well as an enhancer of chemoradiotherapy by impeding DNA damage repairment[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Consistently, we found that higher ACAA1 expression was positively associated with higher infiltration of immune cells, and lower expression of ACAA1 was associated with worse overall survival of NPC patients. Indicated by these data, we hypothesis that NPC cells stimulate their malignant behaviours and survive through regulating immune cell infiltration in the tumor microenvironment.\u003c/p\u003e \u003cp\u003eTumor microenvironment and immune modulation are pivotal in cancer research. Numerous studies underscore the critical roles of TME, particularly infiltrating immune cells, in tumor promotion and progression [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. T lymphocytes, particularly CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e cells, are abundant in NPC tumors [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. CD4\u003csup\u003e+\u003c/sup\u003e T cells, which generating chemokine ligand 13 (CXCL13), critically contribute to the formation of tertiary lymphoid structures by interacting with B cells, correlating with better survival of NPC patients[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. CD8\u003csup\u003e+\u003c/sup\u003e T cells, known for their ability to eliminate target cells through anti-tumor cytokines and cytotoxic molecules, exhibit exhaustion and dysfunction, and subsequently promote immune evasion of NPC cells [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Furthermore, recurrent NPC is associated with increased immunosuppression of T cells and exacerbated dysfunctional cytotoxicity in CD8\u003csup\u003e+\u003c/sup\u003e T cells [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. In addition to T cells, macrophages also contribute to the regulation of tumor infiltration. Macrophage phenotypes in NPC exhibit significant differences, with M1 macrophages mainly residing in tumor nests and M2 macrophages predominantly presenting in tumor stroma [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Tumor-derived fibroblast growth factor 2 (FGF-2) has been identified as a recruiter of macrophages and a promoter of M2 macrophage polarization through the upregulation of CXCL14 in NPC [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Notably, plasmacytoid dendritic cells (pDCs) aggregate the tumor stroma of NPC, and this phenomenon is significantly associated with improved survival outcomes [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Our study demonstrated a negative association between ACAA1 and immunosuppressive cells, alongside a positive association with effector cells, suggesting ACAA1\u0026rsquo;s potential role in regulating immune cells and counteracting tumor suppression in NPC.\u003c/p\u003e \u003cp\u003eEBV maintains a persistent latent infection in the human population by establishing a balance with the host\u0026rsquo;s immune system. Interestingly, reports have documented that EBV-encoded genes, such as EBNA1, EBNA2, EBV-encoded miRNAs BART11 and BART17-3p, facilitate tumor immune evasion through a complex network of pathways [\u003cspan additionalcitationids=\"CR54\" citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. For instance, EBNA1 activates the JAK2/STAT1/IRF-1 signalling pathway and suppresses the promoter activity of PD-L1[\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. EBNA2 inhibits miR-34a through the downregulation of the transcription factor EBF1, consequently enhancing the expression of PD-1[\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. EBV-miRNAs BART11 and EBV-miR-BART17-3p stimulate the expression of PD-L1 by repressing FOXP1 and PBRM1, respectively[\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. RPMS1 and A73 are also important members of EBV BRATs implicated in NPC tumorigenesis [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. In the current study, we have described a negative association between the expression of ACAA1 and EBV-encoded genes (ie., EBNA1, RPMS1, and A73), indicating that the decrease of ACAA1 might heighten the infection of EBV. In addition, findings from the POLARIS-02 and CAPTAIN-1st trials have shown that an early decrease in plasma EBV titer or DNA is associated with a more favorable response to immunotherapy, implying that dynamic alterations in plasma EBV may serve as a promising biomarker [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. In this regard, we propose that ACAA1 might play a role in facilitating the immune response of NPC by modulating EBV infection. However, the precise relationship between ACAA1 and EBV infection remains uncertain and warrants further investigation.\u003c/p\u003e \u003cp\u003eThe primary strategies for immunotherapy consist of immune checkpoint blockade, adoptive cell therapy, and vaccination. Among these, immune checkpoint inhibitors (ICIs) are employed to obstruct the activity of immune checkpoint proteins, boosting the immune response and alleviating immune suppression. Targeting T cell exhaustion-associated co-stimulatory signals (e.g., PD-1/PD-L1, CTLA-4, and LAG-3) and inhibiting tumor-infiltrating lymphocytes (TILs) are key objectives in immunotherapy [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. This offers a novel and promising approach for the treatment of NPC. Clinical trials involving PD-1 inhibitors in recurrent and metastatic NPC patients have shown promising anti-tumor efficacy and favorable safety profiles [\u003cspan additionalcitationids=\"CR62 CR63\" citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. Furthermore, patients receiving combined treatment with PD-1 inhibitors and chemotherapy have achieved extended overall survival (OS) and progression free survival (PFS) [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. Besides PD-1 inhibitors, dual immune checkpoint inhibitors, such as CTLA-4/PD-L1, LAG-3/PD-L1, and TIM-3/PD-L1, have undergone developed and evaluation in clinical trials [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. However, these novel treatment approaches have not been approved by the FDA or NMPA, and the efficacy of PD-1/PD-L1 therapies remains constrained. In our study, we have observed a positive correlation between ACAA1 expression and several immune checkpoint-related genes, including PDCD1 (encoding PD-1 protein), CTLA4, CD80, CD86, and LAG3. Therefore, modulation of ACAA1 expression represents another important target for anti-cancer therapy.\u003c/p\u003e \u003cp\u003eIn conclusion, we have identified ACAA1 as a novel tumor suppressor in NPC. ACAA1 is notably downregulated in NPC. Restoring ACAA1 effectively inhibits the proliferation, migration, and invasion of NPC cells, by suppressing Ki-67 expression and altering action filaments. Moreover, decrease of ACAA1 expression indicates poor survival of NPC patients, potentially due to immune evasion. These findings are anticipated to shed light on novel perspectives regarding NPC diagnosis markers, personalized immunotherapeutic strategies, and prognosis.\u003c/p\u003e"},{"header":"Declarations","content":" \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003eETHICS STATEMENT\u003c/h2\u003e \u003cp\u003eThis study was approved by the Ethics Committee of First Affiliated Hospital of Guangxi Medical University (No. 2022-KT-243).\u003c/p\u003e \u003c/div\u003e\u003ch2\u003eCONFLICT OF INTEREST\u003c/h2\u003e \u003cp\u003eThe authors declare no conflict of interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFUNDING\u003c/h2\u003e \u003cp\u003eThis study was supported by grants from the National Natural Science Foundation of China (No. 81560439 and 82060511), Guangxi Natural Science Foundation Program for Youths ((No. 2015GXNSFBA139143 and 2018GXNSFBA281158), Guangxi Medical University Natural Science Foundation for Youths (No. GXMUYSF2014035) and High-level Talent Introduction Plan of the First Affiliated Hospital of Guangxi Medical University (the fifth level).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eX. X., LM. L., WQ. W.: conducted experiments, data analysis, figure preparation, and manuscript writing; SX. Z, F. H., YS. L.: conducted experiments and data analysis; XY. Z., Z. Z.: experiment design and technical consultation; YL. C. and WL. Z.: experiment design, financial support, and manuscript writing.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.All data generated or analyzed during this study are included in this published article and its supplementary information files. The relevant data used in this study were obtained from the GEO database.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eChen YP, Chan ATC, Le QT, Blanchard P, Sun Y, Ma J. Nasopharyngeal carcinoma. 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Ther Adv Med Oncol. 2022;14:17588359221096214.\u003c/span\u003e\u003c/li\u003e\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, ACAA1, tumor suppressor, immune evasion, prognosis","lastPublishedDoi":"10.21203/rs.3.rs-4750465/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4750465/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAcetyl-CoA acyltransferase 1 (ACAA1), encoding the protein peroxisomal 3-ketoacyl-CoA thiolase (POT1), plays a vital role in the fatty acid beta-oxidation system. ACAA1 has been implicated in the carcinogenesis and development of various human cancers. In this study, the downregulation of ACAA1 was observed consistently throughout the progression of nasopharyngeal carcinoma (NPC) and showed a negative correlation with the expression of EBV-encoded genes. Kaplan-Meier survival analysis and time-dependent receiver operating characteristic (ROC) curve suggested the potential of ACAA1 in predicting NPC prognosis. Through \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e experiments, we identified that the overexpression of ACAA1 inhibited the proliferation, migration, and invasion of NPC cells, which was further confirmed by reduced Ki-67 staining and actin filaments redistribution. Gene ontology (GO) and Kyoto Encyclopedia of Gene and Genomes (KEGG) analyses indicated significant enrichment of immune-related pathways in NPC cells with higher ACAA1 expression. Furthermore, data from the xCell, ESTIMATE and Immunophenoscore analysis supported a critical role of ACAA1 in modulating immune cell infiltration and tumor immune environment of NPC. Interestingly, low expression of ACAA1 was significantly associated with NPC patients classified as tumor microenvironment (TME) subtype 1 and with poor outcome. Expression pattern analyses revealed a positive correlation between ACAA1 expression and six immune checkpoint-related genes, including CD27, PDCD1, CD86, BTLA, TIGIT, and CD28. Taken together, our study reveals that ACAA1 is a potential tumor suppressor gene, which may participate in immune evasion in NPC. ACAA1 could serve as a novel prognosis and therapeutic biomarker for NPC patients.\u003c/p\u003e","manuscriptTitle":"Acetyl-CoA acyltransferase 1 is a potential tumor suppressor gene associated with immune cell infiltration in nasopharyngeal carcinoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-26 11:28:38","doi":"10.21203/rs.3.rs-4750465/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":"c213df34-2214-49eb-a623-15cb029911e7","owner":[],"postedDate":"August 26th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-10-15T03:38:15+00:00","versionOfRecord":[],"versionCreatedAt":"2024-08-26 11:28:38","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4750465","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4750465","identity":"rs-4750465","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}