CHRNA5 drives pancreatic cancer progression by promoting tumorigenesis and remodeling immune invasion microenvironment via CAMKII/AKT/NF-κB-CCL20 axis

CHRNA5 drives pancreatic cancer progression by promoting tumorigenesis and remodeling immune invasion microenvironment via CAMKII/AKT/NF-κB-CCL20 axis | 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 CHRNA5 drives pancreatic cancer progression by promoting tumorigenesis and remodeling immune invasion microenvironment via CAMKII/AKT/NF-κB-CCL20 axis Yue Zhang, Liang Li, Jiao Zhu, Kai Wei, Min Ma, Kunming Tao, Xuerong Miao, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7418128/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy with poor prognosis, characterized by a distinct tumor microenvironment featuring perineural invasion (PNI), high tumor malignancy, and immune evasion. This study aims to identify the crucial cholinergic receptor involved in PDAC progression and elucidate its underlying mechanisms. Methods Transcriptomic analyses of public PDAC datasets, immunohistochemical staining, and cellular assays were conducted to investigate the enrichment of cholinergic signaling and the tumor-promoting effects of acetylcholine (ACh) in PDAC. We integrated three machine learning models, single-cell RNA-seq reanalysis, in vitro and in vivo experiments to identify alpha5-nicotinic acetylcholine receptor (CHRNA5) as a central mediator of PDAC progression. RNA sequencing and rescue experiments were carried out to elucidate the mechanisms of CHRNA5. Furthermore, KN93, a CaMKII inhibitor, was used to assess the therapeutic potential of targeting CHRNA5-mediated pathways in the PDAC tumor microenvironment. Results Integrated multi-omics analysis, cellular and experimental assays demonstrated the tumor-promoting roles of ACh and CHRNA5, while rescue experiments confirmed that CHRNA5 knockdown abrogated ACh-induced effects. Notably, CHRNA5 inhibition produced more pronounced anti-tumor responses in C57BL/6 mice compared to BALB/c mice. Mechanistically, CHRNA5 shaped an immunosuppressive tumor microenvironment by transcriptionally upregulating CCL20 via CAMKII/AKT/NF-κB signaling, thereby promoting the recruitment of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs). Furthermore, the CAMKII inhibitor KN93 effectively suppressed both tumorigenesis and immune evasion. Conclusion Collectively, our findings identify CHRNA5 as a critical driver of PDAC progression by promoting malignant behaviors and reshaping the immune microenvironment, highlighting its potential as a promising therapeutic target in pancreatic cancer. CHRNA5 tumor immune microenvironment tumor malignancy PDAC ACh Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Pancreatic ductal adenocarcinoma (PDAC) is one of the most devastating and lethal malignancies [ 1 ]. Despite advances in therapeutic approaches, the overall survival (OS) rate of PDAC remains dismally low, with only about 10% of patients surviving beyond five years post-diagnosis [ 1 , 2 ]. The incidence of PDAC continues to rise annually by approximately 0.5–1.0%, largely driven by risk factors such as smoking, diabetes, and chronic pancreatitis, making it as a leading cause of cancer-related mortality worldwide [ 3 ]. The clinical aggressiveness of PDAC is strongly influenced by its highly dynamic tumor microenvironment, which is characterized by hypoxia, immune evasion, and enhanced tumor cell stemness signatures [ 4 , 5 ]. Therefore, a deeper understanding of the tumor microenvironment and the mechanisms driving tumor cell malignancy is essential for developing more effective therapeutic strategies against PDAC. Perineural invasion (PNI) is a prominent clinical feature of PDAC, observed in nearly all cases, and contributes to tumor aggressiveness, distant metastasis, poor prognosis, and diminished patient quality of life [ 6 ]. Emerging evidence suggests that PDAC patients with extensive PNI exhibit elevated levels of acetylcholine (ACh) in tumor tissues compared to norepinephrine (NE) [ 7 ], underscoring the potential involvement of cholinergic signaling in PDAC progression. However, the roles of ACh in PDAC remain controversial. While some study has reported that acetylcholine may exert an inhibitory effect on tumor growth [ 8 ], others have shown that parasympathetic neurogenesis facilitates tumor progression and is associated with worse clinical outcomes [ 7 , 9 ]. These conflicting findings highlight the urgent need to clarify the precise functions and mechanisms of ACh signaling in PDAC. Unraveling the role of this pathway may facilitate the development of targeted therapeutic strategies to improve clinical management and patient prognosis. Alpha5-nicotinic acetylcholine receptor (CHRNA5), a ​key ​member of the nicotinic acetylcholine receptor (nAChR) family, functions as a ​ligand-gated ion channel that ​mediates calcium influx upon activation. As a critical effector of cholinergic signaling, ​CHRNA5 has been implicated in regulating tumorigenesis, invasion, and metastasis across multiple cancers. ​In non-small cell lung cancer (NSCLC), ​CHRNA5 activation enhances tumor migration through TGF-β1/Smad pathway [ 10 ]. ​In hepatocellular carcinoma (HCC), ​CHRNA5 overexpression is ​significantly associated with enhanced metastasis and increased tumor stemness [ 11 ]. Additionally, CHRNA5 regulates CES1 expression through the MEK/ERK signaling, ​thereby contributing to tumor recurrence and metastasis in head and neck squamous cell carcinoma [ 12 ]. Despite these findings, ​the specific roles and mechanistic contributions of CHRNA5 in PDAC progression, particularly in remodeling the tumor microenvironment, remain largely unclear. In this study, we integrated multi-omics and cellular assays to confirm the enrichment of cholinergic signaling and the tumor-promoting role of ACh in PDAC. Machine learning and single-cell RNA-seq reanalysis identified CHRNA5 as a key driver, whose knockdown mitigated ACh-induced malignancy. Mechanistically, CHRNA5 promoted PMN-MDSC recruitment via the CAMKII/AKT/NF-κB–CCL20 axis. Furthermore, KN93, a CaMKII inhibitor, effectively suppressed both tumorigenesis and immune invasion, highlighting the translational potential of targeting CHRNA5-mediated pathways in PDAC. These findings offer valuable insights into nerve–tumor–immune crosstalk and support CHRNA5 as a promising therapeutic target in pancreatic cancer. Materials and Methods Clinical Patients and samples Formalin-fixed PDAC tumor tissue sections were collected from patients with PDAC at the Third Affiliated Hospital of Naval Military Medical University. Sections were stained with PGP9.5, ChAT, and TH antibodies to assess the distribution of parasympathetic and sympathetic nerves in three patients. Detailed clinical information is provided in Table S1. Written informed consent was obtained from all participants. The study protocol was approved by the Medical Ethics Committee of the Third Affiliated Hospital of Naval Military Medical University (Approval No. EHBHKY2025-K026-P001). Bulk and single cell RNA sequencing data acquisition and enrichment analysis We sourced the integrated and batch-effect-corrected The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) datasets from the UCSC Xena database (https://xena.ucsc.edu/) [13]. This combined dataset comprises transcriptomic and clinical data for 147 PDAC samples and 167 normal tissue samples. Differentially expressed genes (DEGs) between PDAC and normal tissues were identified using the DESeq2 package. Genes with an adjusted P -value of < 0.05 and a |log₂FC| ≥ 1 were deemed significantly differentially expressed. scRNA-seq data, including three adjacent normal tissues and seventeen PDAC tumor samples, were downloaded from the GEO database (GSE155698) [14] to investigate alterations in ACh receptor expression across different cell types. Based on canonical marker genes, fourteen distinct cell types were annotated. Enrichment analyses were conducted using the clusterProfiler package, including Gene Set Enrichment Analysis (GSEA), Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses [15]. Machine learning modeling, SHAP interpretation, and stemness analysis To identify key contributors to tumorigenesis, three machine learning models, Random Forest (RF), LightGBM (LGBM), and Least Absolute Shrinkage and Selection Operator (LASSO) regression, were constructed using the randomForest [16], lightgbm [17], and glmnet [18] R packages. All TCGA-PDAC samples were randomly divided into training (70%) and validation (30%) sets. LASSO was first applied for feature selection, and features with non-zero coefficients were retained for model building. Optimal parameters were tuned by grid search, and model performance was evaluated using 5-fold cross-validation. Accuracy, recall, precision, F1 score, Cohen's kappa, Matthews correlation coefficient (MCC), and the area under the ROC curve (AUC) were used as evaluation metrics. To interpret model outputs, SHapley Additive exPlanations (SHAP) values were calculated using the fastshap and shapviz packages [19]. Summary plots, heatmaps, and other visualizations were generated to depict feature importance and their influence on individual predictions, thereby clarifying the contribution of each variable to overall performance. ROC curves were generated using the pROC package to assess sensitivity and specificity. To calculate the mRNA-based stemness score (mRNAsi), we obtained the human stem cell/progenitor cell dataset from the Progenitor Cell Biology Consortium (PCBC, https://www.synapse.org). Subsequently, the "gelnet" package was utilized to apply one-class logistic regression (OCLR) for calculating the mRNAsi of each TCGA-PDAC sample.[20] Spearman correlation analysis was performed to assess the relationship between gene expression and the stemness score. Cell culture and treatment Human PANC-01 cells (Jiangsu Nobio Biotechnology Co., Ltd.) and murine PANC-02 cells (Servicebio Biotechnology Co., Ltd.) were cultured in DMEM and RPMI-1640 medium, respectively, supplemented with 10% FBS and 1% penicillin–streptomycin at 37 °C in a 5% CO₂ humidified incubator. To assess the effects of ACh (A2661, Sigma-Aldrich) on proliferation, invasion, migration, colony formation, and sphere formation, cells were treated with 10 or 100 μM ACh or DMSO as control. Three siRNAs targeting CHRNA5 were transfected into PANC-01 and PANC-02 cells using Lipofectamine 2000 (Thermo Fisher Scientific). Knockdown efficiency was confirmed by qRT-PCR and western blotting, with the most effective siRNA used to generate shRNA plasmids. siRNA and shRNA sequences are listed in Table S2. Lentiviruses encoding CHRNA5 shRNA (Lv-Sh-CHRNA5) or control (Lv-Sh-NC) were provided by Wuhan Shumì Neuroscience Technology Co., Ltd. PANC-02 cells were infected and selected with puromycin (2 mg/L) to establish stable knockdown lines. These cells, along with controls, were used to establish orthotopic tumor models to evaluate the contribution of CHRNA5 to PDAC progression. Mice experiments Male C57BL/6 and BALB/c nude mice (six weeks old, Gempharmatech Co., Ltd.) were used to establish orthotopic PDAC models. Mice were anesthetized with 1.25% tribromoethanol, and a 1-cm incision was made below the left rib cage. Approximately 2 × 10⁶ PANC-02 cells (Lv-Sh-CHRNA5 or Lv-Sh-NC) were injected into the pancreatic tail. After two weeks, mice were sacrificed, and pancreatic tumors were collected for histological staining, qRT-PCR, western blotting, and flow cytometry. Tumor volume was calculated using the formula: 0.52 × length × width². One week after tumor implantation, mice were randomly assigned to receive intraperitoneal KN93 (20 mg/kg, HY-15465, MCE) once daily. After one week of KN93 treatment, mice were euthanized, and tumors were collected for further analysis. All mice were housed in a specific pathogen-free (SPF) facility with controlled temperature and light, maintaining a 12-hour light/dark cycle. Ethical approval for the animal experiments was granted by the Third Affiliated Hospital of Naval Military Medical University Ethics Committee (EDWLL-2025-002). Enzyme-Linked Immunosorbent Assay (ELISA) ACh and NE levels in pancreatic and tumor tissues from PDAC mice were quantified using ELISA kits (Cat. YJ063805, YJ401805, Shanghai Enzyme-linked Biotechnology Co., Ltd.) following the manufacturer’s instructions. Cell proliferation Cell proliferation of PDAC cells was measured using the CCK-8 kit (A311-01, Vazyme, Nanjing, China). PANC-01 or PANC-02 cells (2,000/well) were seeded in 96-well plates and treated every three days with 10 or 100 μM ACh or DMSO. To assess the role of CHRNA5, cells were transfected with Sh-NC or Sh-CHRNA5 plasmids. Each day, 10 μL of CCK-8 solution was added, followed by 1 h incubation at 37°C. Absorbance at 450 nm was recorded daily using a Thermo Scientific microplate reader. Cell invasion and migration For migration and invasion assays, 2×10⁴ PDAC cells were seeded into 24-well transwell chambers (8.0-μm pore, BD, USA). For invasion, filters were pre-coated with Matrigel (Invitrogen, USA). Cells were treated with 10 or 100 μM ACh or DMSO. To assess CHRNA5 function, cells were transfected with Sh-NC or Sh-CHRNA5 plasmids in 6-well plates for 24 h, then transferred to transwell chambers. After 72 h, migrated/invaded cells were stained with 0.1% crystal violet and quantified using ImageJ. Colony formation For colony formation, 100 PDAC cells were plated in 60-mm dishes and treated with 100 μM ACh or DMSO, with fresh medium replaced every three days. To assess CHRNA5 function, PANC-01 and PANC-02 cells were transfected with Sh-NC or Sh-CHRNA5 plasmids for 24 h, digested, and reseeded. After 14 days, colonies were stained with 0.1% crystal violet. Sphere-formation assay Approximately 1,000 PANC-01 or PANC-02 cells were seeded into 12-well ultra-low attachment plates and cultured in FBS-free DMEM/F12 supplemented with 2% B-27(17504044, Gibco, USA), 20 ng/ml epidermal growth factor (EGF, C029/CH28, Novoprotein, Suzhou, China), and 20 ng/ml basic fibroblast growth factor (bFGF, C046/C044, Novoprotein, Suzhou, China). For ACh treatment, 100 μM ACh or the control DMSO was added to the cells twice a week. After 2 weeks, spheres were observed under a light microscope, and the total number of spheres was counted. To assess the effect of CHRNA5 on sphere formation, Sh-NC and Sh-CHRNA5 plasmids were transfected into PDAC cells. qRT-PCR Total RNA was extracted from cell or tissue samples using Trizol reagent (R401-01, Vazyme, Nanjing, China). Total RNA was reverse transcribed into complementary DNA (cDNA) using the reverse transcription kit (R333, Vazyme, Nanjing, China). The cDNA was diluted and subjected to qRT-PCR using an amplification kit (Q711, Vazyme, Nanjing, China) to analyze gene expression under appropriate reaction conditions. The primers used in this study were shown in Table S3. Western blotting Proteins were extracted from mice pancreatic cancer tissues or cell samples using RIPA lysis buffer (G2002, Servicebio, Wuhan, China). For detection signals, the membrane was processed with ECL chemiluminescence reagent. The antibodies used in this study were shown in Table S4. Immunohistochemical staining 4 μm paraffin sections were deparaffinized and rehydrated to remove paraffin and allow proper reagent penetration. After treatment with hydrogen peroxide, sections were boiled in citrate buffer (pH 6.0) for 20 minutes for antigen retrieval, followed by blocking with 5% bovine serum albumin (BSA). Sections were then incubated with primary antibodies at 4°C overnight. The next day, sections were incubated with the secondary antibody for 1 hour. Images were captured using an Olympus microscope. The antibodies used are listed in Table S4. The positive staining area was quantified using Image J. RNA seq in the study To explore the downstream targets and pathway of ACh/CHRNA5, PANC-02 transfected with Sh-CHRNA5 and Sh-NC plasmids (n=3) were applied to perform RNA sequencing by LC-Bio Technology Co., Ltd. (Hangzhou, China). Based on the manufacturer’s protocol, RNA was extracted and transcriptome library was constructed using the VAHTS Universal V8 RNA-seq Library Prep Kit for Illumina. The constructed library was sequenced on the Illumina Novaseq platform using PE150 sequencing mode. Quantification was performed using the StringTie software, and edgeR was employed for differential expression analysis. The default criteria for screening significantly differentially expressed genes were FDR < 0.05 and |log₂FC| ≥ 1. Calcium flow detection Calcium concentration ([Ca²⁺]) in PANC-02 cells was measured using Fluo-4 AM dye (S1061S, Beyotime, Suzhou, China). After treatment with ACh or Sh-CHRNA5, cells were incubated with Fluo-4 AM solution at 37°C for 30 minutes. Fluorescence was observed using fluorescence microscopy to detect spontaneous green fluorescence. Additionally, flow cytometry was used to quantify fluorescence intensity, enabling assessment of the effects of ACh and CHRNA5 on intracellular calcium levels. Cleavage under targets and tagmentation (Cut&Tag) Cut&Tag was performed to explore the direction binding of p65 on the promotor of CCL20 with Cut&Tag assay kit (TD904, Vazyme, Nanjing, China). PANC-02 cells were harvested, counted, and centrifuged at 600 × g for 10 min at room temperature. A total of 100,000 cells were subjected to Cut&Tag. According to manufacturer's protocol, cells were first incubated with NE buffer, and then mixed with concanavalin A-coated magnetic beads and p65 antibody overnight. The second day, the mixture was incubated with second antibody for one hour followed by incubation with pA/G-Tnp for one hour at room temperature. The bead/nucleus pellet was resuspended in tagmentation buffer and incubated with DNA extract Beads to capture DNA for final qRT-PCR experiment. Flow cytometry Tumor tissues were digested with Type IV collagenase (C5138, Sigma-aldrich, Darmstadt, Germany) for 20 minutes. The isolated cells were then treated with red blood cell lysis, followed by staining with a viability marker (564406, BD Bioscience, USA). Cells were stained with CD45, CD11B, Ly6C, Ly6G, CD3, CD4, and CD8. The number of immune cells subtypes was quantified to assess the impact of CHRNA5 on tumor microenvironment. The antibodies used were shown in Table S4. Statistical analysis Statistical analyses were conducted using R software (version 4.4.1), SPSS version 23.0, or GraphPad Prism version 9.0 (GraphPad Software, La Jolla, CA). Results are presented as mean ± standard deviation (SD). Comparisons between two datasets were performed using the Wilcoxon rank-sum test or Student's t-test. For comparisons involving three or more groups, one-way or two-way analysis of variance (ANOVA) was employed. Spearman's rank correlation test was used to assess correlations between variables. A P -value of less than 0.05 was considered statistically significant (* P < 0.05, ** P < 0.01, *** P < 0.001). Results ACh promotes malignant phenotype of PDAC in vitro To investigate the role of cholinergic signaling in PDAC, we reanalyzed RNA sequencing data from PDAC and normal tissue samples using the integrated TCGA-GTEx dataset. KEGG, GO, and GSEA analyses revealed significant enrichment of neural-related pathways (Fig. 1A, S1A-B). Expression of CHAT (ACh synthesis) [21] and SLC18A3 (encodes the VAChT) [22] was markedly elevated in PDAC tissues, with strong predictive power for distinguishing PDAC from normal samples (Fig. S1C-D). Moreover, in PDAC patients with PNI, PGP9.5 co-localized with the cholinergic marker CHAT but not with the sympathetic marker TH (Fig. 1B). In our murine orthotopic model, both ACh/NE levels and CHAT/VAChT protein expression were significantly upregulated (Fig. 1C-D), suggesting a tumor-promoting role of ACh. To further investigate the role of ACh in vitro , we explored its effects at different concentrations in PANC-01 and PANC-02 cell lines. In PANC-01 cells, CCK8 and transwell assays revealed that 100 μM ACh significantly promoted cell proliferation, invasion, and migration (Fig. S2A- B). In addition, ACh enhanced these malignant characteristics in PANC-02 cells (Fig. 1E-F). Accordingly, 100 μM was used in subsequent experiments. ACh also increased colony formation (Fig. 1G, S2C), sphere formation, and cancer stem cell (CSC) marker genes expression (Fig. 1H-I, S2D-E), confirming its role in driving aggressive PDAC phenotypes. CHRNA5 is the crucial contributor in PDAC As muscarinic and nicotinic receptors are established downstream targets of ACh, we aim to determine which receptor plays a pivotal role in PDAC. Venn diagram analysis was conducted to identify differentially expressed genes of cholinergic receptors in human PDAC tissues (Fig. 2A). To further determine key contributors during PDAC progression, we employed three machine learning models—LASSO, RF and LGBM—evaluating their performance based on accuracy, recall, F1 score, kappa, MCC, AUC and precision (Fig. 2B). SHAP analysis provided a detailed and transparent interpretation of the decision-making process of machine learning models, elucidating the crucial contributions of CHRNA5 in PDAC, each demonstrating the highest mean SHAP values (Fig. 2C-E, S3A-C). A heatmap revealed that CHRNA5 was the most significantly upregulated receptor in PDAC relative to normal tissues (Fig. 2F). ROC analysis (AUC = 0.964, Fig. S3D) supported its diagnostic value, while Kaplan–Meier survival analysis revealed high CHRNA5 expression predicted poorer overall survival ( P = 0.02, Fig. 2G). Moreover, CHRNA5 expression correlated positively with tumor stemness (Fig. S3E). To define CHRNA5-expressing cell populations, we reanalyzed scRNA-seq data (GSE155698) [14], identifying 14 cell clusters (Fig. 2H). Violin plots showed that CHRNA5 was predominantly enriched in epithelial/tumor cells (Fig. S4), with significantly higher expression in tumor epithelium than adjacent normal tissue (Fig. 2I-J). Immunofluorescence confirmed CHRNA5 co-localized with Ki-67 positive proliferating tumor cells (Fig. 2K), and high expression was also validated in PDAC cell lines (Fig. 2L). Together, these results identify CHRNA5 as a critical driver of PDAC progression and a potential diagnostic and prognostic biomarker. CHRNA5 exerts tumorigenic effects in vitro and in vivo Since the role of CHRNA5 in PDAC remains largely unexplored, we designed siRNAs to knock down CHRNA5 in PANC-01 and PANC-02 cells. qRT-PCR and western blotting confirmed knockdown efficiency (Fig. 3A-B), with Si-1 showing the strongest suppression, and its sequence was used to construct Sh-CHRNA5 plasmids. CHRNA5 silencing significantly reduced PANC-02 cell proliferation (Fig. 3C), invasion and migration (Fig. 3D), and colony formation (Fig. 3E). Importantly, CHRNA5 inhibition also diminished sphere formation and downregulated CSC marker genes, indicating reduced tumor stemness (Fig. 3F-G). Similar results were observed in PANC-01 cells (Fig. S5A–G). To investigate the role of CHRNA5 in vivo , PANC-02 cells were transduced with lentivirus carrying either Sh-NC or Sh-CHRNA5 to establish stable knockdown cell lines. These cells were then orthotopically implanted into C57BL/6 mice to establish PDAC orthotopic tumor models. Tumors derived from CHRNA5-silenced cells exhibited significantly reduced tumorigenic capacity compared to controls (Fig. 3H). The knockdown efficiency was validated by qRT-PCR and western blotting (Fig. 3I-J). Consistently, mice in the Sh-CHRNA5 group showed markedly decreased tumor volume and weight (Fig. 3K-L). Immunohistochemical staining further confirmed a lower Ki-67 expression, indicating decreased tumor proliferation in the Sh-CHRNA5 group (Fig. 3M). CHRNA5 mediates the oncogenic effects of ACh To determine whether CHRNA5 mediates the effects of ACh, we conducted a rescue experiment involving ACh treatment following CHRNA5 knockdown. qRT-PCR and western blotting experiments confirmed that Sh-CHRNA5 transfection suppressed the ACh-induced upregulation of CHRNA5 at both RNA and protein levels (Fig. 4A, B, D, E). Notably, CHRNA5 knockdown significantly blocked ACh-induced cell proliferation in both PANC-01 and PANC-02 cells (Fig. 4C, F). Similarly, inhibition of CHRNA5 reduced ACh-mediated colony formation (Fig. 4G-H). More importantly, ACh-enhanced tumorsphere formation was markedly inhibited following CHRNA5 knockdown (Fig. 4I-J), indicating that CHRNA5 mediates the tumor-promoting effects of ACh. CHRNA5 targets CaMKII/AKT/NF-κB pathway RNA sequencing of PANC-02 cells transduced with either Sh-NC or Sh-CHRNA5 was performed to explore the downstream targets of CHRNA5. Differential expression analysis revealed 351 upregulated and 299 downregulated genes in the Sh-CHRNA5 group compared to the Sh-NC group, as illustrated by a volcano plot (Fig. 5A). KEGG pathway enrichment analysis indicated that CHRNA5 knockdown affected several key pathways, including neuroactive ligand–receptor interaction, calcium signaling, PI3K–AKT, and NF-κB signaling (Fig. 5B), suggesting that CHRNA5 may influence calcium homeostasis. Given that CHRNA5 functions as a ligand-gated calcium channel [23], we hypothesized that it modulates intracellular Ca²⁺ levels. To verify this, we measured Ca²⁺ flux using Fluo-4 AM via immunofluorescence staining and flow cytometry. The results showed that ACh stimulation and CHRNA5 activation increased intracellular Ca²⁺ flux, whereas CHRNA5 knockdown abrogated ACh-induced calcium signaling (Fig. 5C-D). As Ca²⁺ binds to calmodulin (CaM) to activate the CaMK pathway, particularly CaMKII [24], we further investigated this signaling cascade. Western blotting confirmed that ACh and CHRNA5 mediated the phosphorylation of CaMKII (Fig. 5E-F). Since CaMKII is known to activate AKT [25], which subsequently regulates the NF-κB pathway [26, 27], we examined the expression of downstream effectors. Notably, CHRNA5 suppression mitigated the ACh-induced activation of p-AKT, p-p65 and p-IκBα (Fig. 5E-F). These results suggest that the CAMKII/AKT/NF-κB pathway is a major downstream effector of the ACh/CHRNA5 axis, contributing to PDAC progression. CHRNA5 remodels the tumor immune microenvironment Given the critical role of immune cell infiltration in the tumor microenvironment, we then investigate whether CHRNA5 could regulate tumor-associated immune response. To explore this, orthotopic tumor model was constructed in both immunodeficient nude mice and immunocompetent C57BL/6 mice. CHRNA5 knockdown markedly suppressed tumor formation, reduced tumor volume, and decreased Ki-67 expression in both models, with a more pronounced inhibitory effect observed in C57BL/6 mice (Fig. 6A-E), suggesting that CHRNA5 might exert immunomodulatory effects in PDAC tumorigenesis. GO analysis revealed the enrichment of immune-related pathways, such as leukocyte migration, further supporting the involvement of CHRNA5 in immune regulation (Fig. 6F). A heatmap analysis of genes related to leukocyte migration showed that CHRNA5 could affect chemokines like CCL20, CXCL9, CTSG, etc., which might mediate the chemotaxis and infiltration of myeloid cells (Fig. 6G). Additionally, qRT-PCR in PANC-02 cells and tumor tissues validated the decreased expression of CCL20 upon CHRNA5 inhibition (Fig. 6H-I). As reported [28], the promotor region of CCL20 enriched a NF-κB binding site (Fig. 6J). Cut&Tag results further vindicated the direct binding of p65 on the promotor region of CCL20 (Fig. 6K). CCL20 is a critical mediator in immune cell recruitment, particularly for myeloid-derived suppressor cells (MDSCs) and CD4 + T cells [29, 30]. Flow cytometry confirmed that CHRNA5 knockdown resulted in a significant decrease in MDSCs, especially polymorphonuclear MDSCs (PMN-MDSCs), and an increase ratio of CD8 + T cell (Fig. 6L-M, S6A-B). These results demonstrate that CHRNA5 modulates the tumor immune microenvironment by promoting CCL20-mediated recruitment of MDSCs, thereby contributing to immune evasion in PDAC. CAMKII inhibitor KN93 suppresses tumorigenesis and immune invasion in PDAC Considering the lack of selective CHRNA5 inhibitors, we employed KN93, a small-molecule CaMKII inhibitor, to assess the therapeutic impact of downregulating the CHRNA5-mediated signaling pathway on tumor malignancy and the immune microenvironment. As expected, KN93-treated mice exhibited significantly smaller tumor size and volume (Fig. 7A–C). Immunohistochemical staining of Ki-67 revealed a marked reduction in tumor proliferative capacity (Fig. 7D). qRT-PCR analysis demonstrated decreased CCL20 expression following KN93 treatment (Fig. 7E), and western blotting confirmed inhibition of the AKT/NF-κB signaling pathway in the KN93-treated group (Fig. 7F). Importantly, flow cytometry revealed that KN93 significantly reduced the proportion of MDSCs, particularly PMN-MDSCs, while enhancing CD8⁺ T cell infiltration (Fig. 7G-H), indicating its immunomodulatory effect in PDAC. Taken together, these above findings highlight the pivotal role of CHRNA5 in driving tumorigenesis and immune evasion through the CAMKII/AKT/NF-κB–CCL20 axis, underscoring its potential as a promising therapeutic target in PDAC (Fig. 7I). Discussion PDAC is an aggressive malignancy with enhanced ACh levels, a pathological feature related to tumor progression, resistance to therapy, and poor prognosis [ 31 ]. Although extensive investigations have been conducted to elucidate the role of cholinergic signaling in cancer [ 32 ], the specific nAChR subtypes involved in PDAC remain insufficiently defined, and effective therapeutic strategies are still limited. Moreover, immunotherapy has shown limited efficacy in PDAC patients, underscoring the urgent need to clarify the mechanisms underlying tumor–immune interactions [ 33 ]. In this study, we identify CHRNA5 as a critical oncogenic driver in PDAC, and demonstrate that CHRNA5 not only facilitates malignant phenotypes but also plays a pivotal role in remodeling the immune microenvironment. These observations offer significant insights into the nerve-tumor-immune environment network and support CHRNA5 as a promising therapeutic target for pancreatic cancer. The role of ACh in PDAC remains controversial. Some study suggested that ACh contributed to an immunosuppressive microenvironment by inhibiting immune cell recruitment and reducing interferon-gamma (IFN-γ) production, thereby leading to a decrease in the Th1/Th2 ratio [ 7 ]. Conversely, other research indicated that subdiaphragmatic vagotomy accelerated PDAC progression, while systemic administration of the muscarinic agonist bethanechol restored the malignant phenotype via cholinergic receptor muscarinic 1 (CHRM1) activation [ 8 ]. These conflicting findings imply that ACh may exert distinct effects depending on the specific receptor subtype involved. Given that ACh has multiple receptors, it is plausible that different subtypes mediate different roles in tumor progression. In the current study, our in vitro experiments demonstrated that 100 µM ACh significantly enhanced PDAC cell proliferation, invasion, migration, colony formation, and tumor stemness, consistent with its tumor-promoting effects previously observed in lung cancer [ 34 ] and intrahepatic cholangiocarcinoma (ICC) [ 23 ]. As a subtype of nAChR, CHRNA5 has been implicated in tumor progression across multiple cancers. In HCC, CHRNA5 has been shown to promote cell proliferation, metastasis, stemness, and even enhance sensitivity to sorafenib [ 11 ]. Similarly, in ICC, the ACh/CHRNA5 axis activates CAMKII/GSK3β signaling, leading to β-catenin upregulation, which promotes metastasis and resistance to gemcitabine [ 23 ]. However, little is known about the function of CHRNA5 in PDAC. In this study, we identified CHRNA5 as a key mediator of ACh’s tumor-promoting effects in PDAC through bulk RNA-seq analysis using three machine learning models. Notably, our re-analysis of scRNA-seq data revealed that CHRNA5 was highly expressed in epithelial cells, with significantly elevated level in tumor epithelial cells compared to adjacent normal epithelial cells, suggesting its potential role in tumorigenesis. We further demonstrated that CHRNA5 promoted PDAC cell proliferation, invasion, migration, colony formation, and tumor stemness in vitro , and enhanced tumor formation in vivo . To our knowledge, this is the first comprehensive investigation of CHRNA5 in PDAC, providing substantial evidence for its tumor-promoting function in tumorigenesis and highlighting CHRNA5 as a potential therapeutic target for PDAC treatment. The immune microenvironment plays a crucial role in tumor progression and treatment resistance [ 35 ]. PDAC is characterized by an immunosuppressive microenvironment, with low infiltration of CD8 + T cells, high levels of immunosuppressive cells (e.g., regulatory T cells, MDSCs, tumor-associated macrophages), and upregulation of immune checkpoint molecules [ 36 ]. This immunosuppressive state contributes to poor responses to immunotherapy, posing a great threat to the treatment of PDAC. Intriguingly, we observed that CHRNA5 exerted more pronounced effects in C57BL/6 mice compared to nude mice, suggesting its involvement in shaping the immune microenvironment. Flow cytometry analysis further revealed that CHRNA5 inhibition significantly reduced the proportions of total MDSCs and PMN-MDSCs, while enhancing CD8⁺ T cell infiltration. PMN-MDSCs, a major subset of MDSCs, are known immunosuppressive regulators that modulate both the number and function of CD8⁺ T cells [ 37 ]. These current findings suggest that CHRNA5 acts not only as an intrinsic driver of tumor progression but also as a key modulator of the immunosuppressive tumor microenvironment. CaMKII, a serine/threonine kinase, plays a crucial role in regulating various cellular processes, including tumor proliferation, metastasis, and drug resistance, by maintaining Ca²⁺ homeostasis [ 38 – 40 ]. Its activation is primarily dependent on Ca²⁺/CaM binding. However, once phosphorylated at Thr286, CaMKII becomes constitutively active, allowing it to function independently of Ca²⁺/CaM signaling [ 41 ]. Given that CHRNA5 functions as a calcium ion channel, it may influence Ca²⁺/CaM binding and subsequently regulate p-CaMKII and CaMKII levels. Our RNA sequencing analysis of sh-CHRNA5-treated cells revealed significant enrichment of the calcium signaling pathway. Further in vitro experiments, including western blot analysis and calcium flow detection, confirmed that ACh/CHRNA5 modulates intracellular Ca²⁺ levels and p-CaMKII expression. Additionally, we observed the activation of PI3K-AKT pathway in sh-CHRNA5-treated cells based on RNA-seq. As previous studies have demonstrated that CaMKII can activate PI3K, leading to AKT phosphorylation at Ser473[ 42 , 43 ], we hypothesize that the CHRNA5 promotes PDAC progression through the CaMKII/PI3K-AKT signaling pathway. The PI3K-AKT signaling pathway plays a critical role in PDAC progression by regulating tumor cell proliferation, stemness, and immune microenvironment remodeling [ 44 , 45 ]. As a central node in multiple oncogenic processes, AKT activation promotes tumor growth by enhancing cell survival, inhibiting apoptosis, and sustaining metabolic adaptation [ 44 ]. In addition to its direct effects on tumor cells, PI3K-AKT signaling also plays a pivotal role in shaping the immune microenvironment [ 46 ]. Activation of AKT can modulate immune cell recruitment, differentiation, and function, creating an immunosuppressive niche that facilitates tumor evasion from immune surveillance. One important downstream target of AKT is CCL20 [ 47 ], a chemokine that recruits regulatory immune cells, including Tregs [ 48 ] and MDSCs [ 30 ], to the tumor microenvironment. P65, the crucial transcription factor in the NF-κB pathway, is reported to be translocated into the nucleus via the AKT/IκBα signaling pathway [ 26 , 27 , 49 , 50 ]. As previously reported, NF-κB binding sites are enriched on the promotor region of CCL20 [ 28 , 51 ]. Therefore, we speculate that CAMKII/AKT/NF-κB-CCL20 might be the downstream pathway of CHRNA5. Supporting this in our Cut&Tag results, p65 directly binds to the promoter region of CCL20, thereby upregulating its transcription level. Collectively, these observations suggest that CHRNA5 regulates the CAMKII/AKT/NF-κB pathway to mediate CCL20 expression, thereby contributing to the formation of an immunosuppressive microenvironment. Based on these findings, CHRNA5 plays dual roles in promoting tumorigenesis and modulating immune microenvironment in PDAC, suggesting that targeting CHRNA5 may offer therapeutic potential in clinical management. However, the lack of a specific CHRNA5 inhibitor remains a major barrier to translational application and represents a limitation of this study. Emerging evidence from studies in ICC [ 23 ], head and neck squamous cell carcinoma (HNSC) [ 12 ], and lung cancer [ 52 ] has highlighted the therapeutic relevance of CHRNA5 inhibition, though large-scale, multi-ethnic clinical studies are also warranted to validate these observations. Beyond direct CHRNA5 inhibitors, targeting downstream signaling pathways also holds promise. For instance, KN93, a CaMKII inhibitor, has shown protective effects in diabetic retinal vascular injury [ 53 ]. In our in vivo experiments, KN93 significantly suppressed tumor progression, reduced PMN-MDSC infiltration, and enhanced CD8⁺ T cell presence, supporting the translational potential of targeting CHRNA5-mediated pathways in PDAC. In summary, we confirmed the upregulation of ACh in public PDAC datasets and demonstrated its tumor-promoting effects in vitro . Importantly, our study provides the first evidence that CHRNA5 plays dual roles in PDAC by promoting tumor malignancy and modulating the immune microenvironment, thereby mediating the oncogenic effects of ACh. Mechanistically, CHRNA5 exerts its oncogenic functions through CAMKII/AKT/NF-κB-CCL20 axis, contributing to immune regulation within the tumor microenvironment. Furthermore, KN93, an inhibitor targeting CHRNA5-mediated pathways, showed significant therapeutic efficacy in suppressing tumorigenesis and immune evasion. Collectively, these findings deepen our understanding of nerve–tumor–immune interactions and highlight CHRNA5 as a promising and translational target for therapeutic intervention in pancreatic cancer. Abbreviations PDAC, pancreatic ductal adenocarcinoma; OS, overall survival; PNI, perineural invasion; ACh, acetylcholine; NE, norepinephrine; EMT, epithelial-mesenchymal transition; CHRNA5, cholinergic receptor nicotinic alpha 5; DEGs, differentially expressed genes; KEGG, Kyoto Encyclopedia of Genes and Genomes; GO, Gene Ontology; GSEA, Gene Set Enrichment Analysis; FBS, fetal bovine serum; DMEM, Dulbecco’s modified Eagle’s medium; SPF, specific pathogen-free; LGBM, LightGBM; RF, Random Forest; MCC, Matthews correlation coefficient; AUC, area under the receiver operating characteristic curve; SHAP, SHapley Additive exPlanations; EGF, epidermal growth factor; bFGF, basic fibroblast growth factor; BSA, bovine serum albumin; DAB, 3,3'-diaminobenzidine; CSC, cancer stem cell; CAM, calmodulin; CaMKII, calcium-calmodulin CaM-dependent protein kinase II; HCC, hepatocellular carcinoma; ICC, intrahepatic cholangiocarcinoma; GEO, Gene Expression Omnibus; IFN-γ, interferon-gamma; CHRM1, cholinergic receptor muscarinic 1. Declarations Ethics approval and consent to participate Clinical tissue samples were obtained from the Third Affiliated Hospital of Naval Military Medical University with approval from the Ethics Committee (EHBHKY2025-K026-P001). The animal studies were conducted under approval from the Ethics Committee of the Third Affiliated Hospital of Naval Military Medical University (Approval ID: EDWLL-2025-002) and adhered to the official guidelines set forth by the Institutional Animal Care and Use Committee (IACUC). All procedures were implemented with strict adherence to protocols designed to ensure animal welfare and minimize discomfort, in compliance with the principles of ethical experimentation. Consent for publication Not applicable. Competing interests The authors have no conflict of interest regarding this study. Authors contributions Zhijie Lu, Xuerong Miao and Kunming Tao: participated in the study design, coordination, supervision, and manuscript review/editing; Yue Zhang: prepared the original draft, established animal models, and drafted the manuscript; Liang Li: analyzed data and wrote code for the study; Jiao Zhu, Kai Wei, and Min Ma: performed data analysis and interpretation of results. All authors critically revised the manuscript and approved the final version. Availability of data and materials All sequencing data generated in this study have been deposited in the Gene Expression Omnibus (GEO) database and are publicly accessible as of the publication date. The code and research materials utilized in this article can be obtained from the corresponding author upon reasonable request. Funding This work was supported by the National Natural Science Foundation of China (82171232, 82371241, 31900716), the Program of Shanghai Academic/Technology Research Leader (22XD1404900), the Clinical Research Special Project of the Shanghai Health Commission (20204Y0047, 2020YJZX0133), and Science and Technology Innovation Plan of Shanghai Science and Technology Commission (21Y11903200). Acknowledgements Not applicable. References Blackford AL, Canto MI, Dbouk M, Hruban RH, Katona BW, Chak A, Brand RE, Syngal S, Farrell J, Kastrinos F, et al. Pancreatic Cancer Surveillance and Survival of High-Risk Individuals. JAMA Oncol. 2024;10:1087–96. Siegel RL, Miller KD, Wagle NS, Jemal A, Cancer statistics. 2023. CA: a cancer journal for clinicians. 2023;73:17–48. Park W, Chawla A, O'Reilly EM. Pancreat Cancer: Rev Jama. 2021;326:851–62. Yamasaki A, Yanai K, Onishi H. Hypoxia and pancreatic ductal adenocarcinoma. Cancer Lett. 2020;484:9–15. Ho WJ, Jaffee EM, Zheng L. 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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-7418128","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":512192007,"identity":"ad9b52c9-1618-4e1d-9477-e930ca4a7ed5","order_by":0,"name":"Yue Zhang","email":"","orcid":"","institution":"Fudan University Minhang Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yue","middleName":"","lastName":"Zhang","suffix":""},{"id":512192008,"identity":"6b6879a1-8d15-42a7-912e-b4c376b40dc9","order_by":1,"name":"Liang Li","email":"","orcid":"","institution":"Tongji University Affilliated Yangpu Hospital: Shanghai Yangpu District Central Hospital","correspondingAuthor":false,"prefix":"","firstName":"Liang","middleName":"","lastName":"Li","suffix":""},{"id":512192009,"identity":"35de6f16-4719-477e-8a59-f4d38f7e8806","order_by":2,"name":"Jiao Zhu","email":"","orcid":"","institution":"Shanghai Eastern Hepatobiliary Surgery Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jiao","middleName":"","lastName":"Zhu","suffix":""},{"id":512192010,"identity":"dd981246-406c-4d63-9244-9c89b3e87696","order_by":3,"name":"Kai Wei","email":"","orcid":"","institution":"Shanghai Eastern Hepatobiliary Surgery Hospital","correspondingAuthor":false,"prefix":"","firstName":"Kai","middleName":"","lastName":"Wei","suffix":""},{"id":512192011,"identity":"d5fdea62-e6b6-43d2-8464-8b3805b862c7","order_by":4,"name":"Min Ma","email":"","orcid":"","institution":"Shanghai Eastern Hepatobiliary Surgery Hospital","correspondingAuthor":false,"prefix":"","firstName":"Min","middleName":"","lastName":"Ma","suffix":""},{"id":512192012,"identity":"03389f76-c6f9-4525-940d-665973ba1651","order_by":5,"name":"Kunming Tao","email":"","orcid":"","institution":"Shanghai Eastern Hepatobiliary Surgery Hospital","correspondingAuthor":false,"prefix":"","firstName":"Kunming","middleName":"","lastName":"Tao","suffix":""},{"id":512192013,"identity":"5e28a68a-97fe-40c1-bef0-f73be918d91a","order_by":6,"name":"Xuerong Miao","email":"","orcid":"","institution":"Shanghai Eastern Hepatobiliary Surgery Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xuerong","middleName":"","lastName":"Miao","suffix":""},{"id":512192014,"identity":"245edaa4-aa63-4e4e-ad17-9b9489d213e8","order_by":7,"name":"Zhijie Lu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5ElEQVRIie3RsQqCQBjA8U+Ea7l0PbHsFRRBinqYEwfXoL2C4Cbb6y2KIBpruZYewKZqcWtyySFIhRpP24LuPx3H/fg+OACZ7AfTABBQTEBX90DKq30FQW9iMPoNKbJ5bULC5HptdS2XYyfOdmBpMVXSoZDQ0M4Xcz2O3d78BK4RU9VciAknOfG3l8gjTQb+KqZIxULis4JMNgx7xpPBpAYJUEGojbBn5lOoXUlwUhJnwdGo32bEWZ5uM1NE9EaYGFk07uhMXZ/vbNDRjsEhFZEyJfoci69RplUg71HjjUwmk/1vL0JTQBKAHCt4AAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-0561-0591","institution":"Fudan University Minhang Hospital","correspondingAuthor":true,"prefix":"","firstName":"Zhijie","middleName":"","lastName":"Lu","suffix":""}],"badges":[],"createdAt":"2025-08-20 13:52:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7418128/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7418128/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":91448540,"identity":"12444456-02f6-4d7b-a8c4-6a89a522bfd2","added_by":"auto","created_at":"2025-09-16 15:03:05","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2371571,"visible":true,"origin":"","legend":"\u003cp\u003eACh promotes malignant phenotype of PDAC\u003cem\u003e in vitro\u003c/em\u003e.\u003cem\u003e \u003c/em\u003e\u003cstrong\u003eA\u003c/strong\u003e KEGG pathway analysis revealed the enrichment of Neuroactive ligand-receptor interaction signaling in PDAC patients. \u003cstrong\u003eB\u003c/strong\u003e HE and immunohistochemical staining analysis of PDAC patient tumor tissues (n = 3) exhibiting PNI. Black arrows indicate areas of co-localization between the pan-neuronal marker PGP9.5 and the cholinergic marker CHAT. Scar bar=100μm. \u003cstrong\u003eC\u003c/strong\u003e ELISA assay verified an obvious increased level of ACh in mice PDAC tissues, rather than NE, compared with normal pancreatic tissues. \u003cstrong\u003eD\u003c/strong\u003e The increased levels of VACHT and CHAT were observed in PDAC mice model (n=5). \u003cstrong\u003eE-F\u003c/strong\u003e ACh, especially with a concentration of 100μM, could promote cell proliferation, invasion and migration in PANC-02. \u003cstrong\u003eG\u003c/strong\u003e100μM ACh enhanced tumor cells colony formation. \u003cstrong\u003eH\u003c/strong\u003e Sphere formation assay revealedan increase of tumor cell stemness after ACh treatment.\u003cstrong\u003e I\u003c/strong\u003e qRT-PCR results demonstrated the upregulation of cancer stem cell (CSC) genes upon treatment with 100 μM ACh. Data with error bars represented as mean ± SD and analyzed using one-way ANOVA (\u003cstrong\u003eE\u003c/strong\u003e, \u003cstrong\u003eF\u003c/strong\u003e) and two-tailed Student’s t-test (\u003cstrong\u003eC\u003c/strong\u003e, \u003cstrong\u003eG\u003c/strong\u003e, \u003cstrong\u003eH\u003c/strong\u003e, \u003cstrong\u003eI\u003c/strong\u003e). * represented \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, ** represented \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, *** represented \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7418128/v1/36a61dd50032586e38ca1465.png"},{"id":91446619,"identity":"7c0c6cec-9917-4faa-9b6f-9c6f444f589b","added_by":"auto","created_at":"2025-09-16 14:47:05","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1871749,"visible":true,"origin":"","legend":"\u003cp\u003eCHRNA5 is the crucial contributor in PDAC. \u003cstrong\u003eA\u003c/strong\u003e Venn diagram showing the overlap between differentially expressed genes and cholinergic-related genes. \u003cstrong\u003eB\u003c/strong\u003e Radar chart revealed the evaluation on accuracy, recall, F1 score, kappa, MCC, AUC and precision of three machine learning models. \u003cstrong\u003eC-E\u003c/strong\u003e SHAP value-based analysis identifying key contributors to PDAC progression. \u003cstrong\u003eF\u003c/strong\u003e Heatmap illustrated the expression levels of muscarinic and nicotinic receptors in PDAC patients compared to the normal group. \u003cstrong\u003eG\u003c/strong\u003e Kaplan-Meier survival analysis suggested that high CHRNA5 expression is positively related to poor prognosis. \u003cstrong\u003eH-J\u003c/strong\u003e Re-analysis of the scRNA-seq dataset (GSE154778). Fourteen cell types were annotated based on canonical cell markers (\u003cstrong\u003eH\u003c/strong\u003e). Violin plots showed the expression levels of CHRNA5 across different cell populations (\u003cstrong\u003eI\u003c/strong\u003e). CHRNA5 expression was significantly higher in tumor epithelial cells compared to adjacent normal epithelial cells (\u003cstrong\u003eJ\u003c/strong\u003e). \u003cstrong\u003eK\u003c/strong\u003e Immunofluorescence staining of PDAC tumor tissues (from Patient1#) displayed the co-localization of CHRNA5 and Ki-67-positive tumor cells. CHRNA5, green. Ki-67, magenta. DAPI, blue. Scar bar=20μm. \u003cstrong\u003eL\u003c/strong\u003e qRT-PCR results demonstrated the high expression of CHRNA5 in both PANC-01 and PANC-02 cells. Data with error bars represented as mean ± SD, and analyzed using Kaplan-Meier survival analysis (\u003cstrong\u003eH\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7418128/v1/8d3c1f837ce7f440ff281c75.png"},{"id":91446620,"identity":"17946d7c-67fb-4b70-91bf-baf31a20a11d","added_by":"auto","created_at":"2025-09-16 14:47:05","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1809831,"visible":true,"origin":"","legend":"\u003cp\u003eCHRNA5 exerts tumorigenic effects \u003cem\u003ein vitro\u003c/em\u003eand\u003cem\u003e in vivo.\u003c/em\u003e \u003cstrong\u003eA-B\u003c/strong\u003e qRT-PCR and western blotting results verified the knockdown efficiency of three pairs siRNA in PANC-02. Si-1 was chosen to construct sh-CHRNA5 plasmids and lentivirus. \u003cstrong\u003eC\u003c/strong\u003e CCK8 assay vindicated the inhibition of cell proliferation after knock-downing CHRNA5 in PANC-02 cells. \u003cstrong\u003eD\u003c/strong\u003eTranswell assay demonstrated the suppression of invasion and migration in PANC-02 cells treated with Sh-CHRNA5. \u003cstrong\u003eE\u003c/strong\u003e Silencing CHRNA5 reduced the colony formation. \u003cstrong\u003eF\u003c/strong\u003e Sh-CHRNA5 treated PANC-02 cells exhibited the lower sphere formation. \u003cstrong\u003eG\u003c/strong\u003e Repression of CHRNA5 downregulated the CSC genes. \u003cstrong\u003eH\u003c/strong\u003eLv-Sh-CHRNA5 infected PANC-02 cells showed the lower tumor formation ability in an orthotopic tumor model in C57BL/6 mice (n=6). \u003cstrong\u003eI-J\u003c/strong\u003e qRT-PCR and western blotting results confirmed the downregulation of CHRNA5 in Lv-Sh-CHRNA5 group. \u003cstrong\u003eK-L\u003c/strong\u003eInhibiting CHRNA5 had a lower tumor volume and weight \u003cem\u003ein vivo\u003c/em\u003e. \u003cstrong\u003eM\u003c/strong\u003e Ki-67 staining of tumor tissues sections of the two groups. Scar bar=400μm. The quantification of Ki-67 positive area was analyzed by Image J. Data with error bars represented as mean ± SD, and analyzed using one-way ANOVA (\u003cstrong\u003eA\u003c/strong\u003e) and two-tailed Student’s t-test (\u003cstrong\u003eC\u003c/strong\u003e,\u003cstrong\u003e D\u003c/strong\u003e,\u003cstrong\u003e E\u003c/strong\u003e,\u003cstrong\u003e F\u003c/strong\u003e,\u003cstrong\u003e G\u003c/strong\u003e,\u003cstrong\u003eI\u003c/strong\u003e,\u003cstrong\u003e K\u003c/strong\u003e,\u003cstrong\u003e L\u003c/strong\u003e,\u003cstrong\u003e M\u003c/strong\u003e). * represented \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, ** represented \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, *** represented \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7418128/v1/0e49f57e121b05c9d4284dbf.png"},{"id":91446625,"identity":"b11392b9-7a83-4e6e-91a4-616b388423cb","added_by":"auto","created_at":"2025-09-16 14:47:05","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1178017,"visible":true,"origin":"","legend":"\u003cp\u003eCHRNA5 mediates the oncogenic effects of ACh. \u003cstrong\u003eA-B\u003c/strong\u003e qRT-PCR and western blotting results demonstrated that Sh-CHRNA5 could reverse the upregulation of CHRNA5 induced by ACh in PANC-01 cells. \u003cstrong\u003eC\u003c/strong\u003e CCK8 assay verified that inhibiting CHRNA5 mitigated the promotion of cell proliferation by ACh in PANC-01 cells. \u003cstrong\u003eD-E\u003c/strong\u003eqRT-PCR and western blotting results showed that blocking CHRNA5 expression restrained the increase levels of CHRNA5 upon ACh treatment in PANC-02 cells. \u003cstrong\u003eF\u003c/strong\u003eDownregulating CHRNA5 blunted the effects of ACh on cell proliferation. \u003cstrong\u003eG-H\u003c/strong\u003eSuppression of CHRNA5 decreased the colony formation ability of ACh in two PDAC cells. \u003cstrong\u003eI-J\u003c/strong\u003e Inhibition of CHRNA5 block the tumor stemness after ACh delivery, as determined by sphere formation experiments. Data with error bars represented as mean ± SD, and analyzed using two-way ANOVA. * represented \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, ** represented \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, *** represented \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7418128/v1/993dc548e16f60f946202ec2.png"},{"id":91448535,"identity":"3853f863-4d20-48ba-a462-e52ba2bd752d","added_by":"auto","created_at":"2025-09-16 15:03:05","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":878329,"visible":true,"origin":"","legend":"\u003cp\u003eCHRNA5 targets CaMKII/AKT/NF-κB pathway. \u003cstrong\u003eA\u003c/strong\u003e Heatmap illuminated the differential expressed genes in Sh-CHRNA5 and Sh-NC treated PANC-02 cells in our RNA sequencing (n=3). \u003cstrong\u003eB\u003c/strong\u003e KEGG analysis indicated the enriched pathways. \u003cstrong\u003eC-D\u003c/strong\u003e Ca²⁺ flux detection using Flu4-AM in PANC-02 based on immunofluorescence staining and flow cytometry. \u003cstrong\u003eE\u003c/strong\u003e Western blotting demonstrated the regulation of ACh/CHRNA5 axis on p-CaMKII, p-AKT, p-p65 and p-IκBα. \u003cstrong\u003eF\u003c/strong\u003e Western blotting vindicated the effect of CHRNA5 on CaMKII, AKT and NF-κB pathway in mice (n=5). Data with error bars represented as mean ± SD, and analyzed using two-way ANOVA.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7418128/v1/7b673806b94b7ad867a7c80f.png"},{"id":91446627,"identity":"7c64c33a-98cb-4975-9baa-d61aa1f8a559","added_by":"auto","created_at":"2025-09-16 14:47:05","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1372010,"visible":true,"origin":"","legend":"\u003cp\u003eCHRNA5 remodels the tumor immune microenvironment. \u003cstrong\u003eA-D\u003c/strong\u003e Knocking down CHRNA5 significantly reduced tumor formation in nude mice, and this inhibitory effect was more obvious in C57BL/6 mice. \u003cstrong\u003eE\u003c/strong\u003e Ki-67 staining of mice tumor tissues. Scar bar=100μm. \u003cstrong\u003eF\u003c/strong\u003e GO analysis showed the enrichment of immune-related pathways. \u003cstrong\u003eG\u003c/strong\u003e Heatmap of genes involved in the leukocyte migration pathway. \u003cstrong\u003eH\u003c/strong\u003e Inhibition of CHRNA5 decreased CCL20 RNA levels in PANC-02 cells. \u003cstrong\u003eI\u003c/strong\u003e Reduction of CCL20 in mice following CHRNA5 suppression. \u003cstrong\u003eJ\u003c/strong\u003e The binding site of p65 on the promotor region of CCL20. \u003cstrong\u003eK\u003c/strong\u003e Cut\u0026amp;Tag analysis further confirmed the direct binding of p65 to this site. \u003cstrong\u003eL-M\u003c/strong\u003e Flow cytometry demonstrated a significant decrease in the ratio of PMN-MDSCs, and an increase of CD8\u003csup\u003e+\u003c/sup\u003e T cell in the CHRNA5 knockdown group (n=5). Data were analyzed and presented using FCS Express 7. Data with error bars represented as mean ± SD, and analyzed using two-tailed Student’s t-test. * represented \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, ** represented \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, *** represented \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7418128/v1/37b42d7d55990078c58b5ab8.png"},{"id":91449620,"identity":"db2ef9d6-1407-403c-ba68-a23b83fd51f2","added_by":"auto","created_at":"2025-09-16 15:11:05","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1470492,"visible":true,"origin":"","legend":"\u003cp\u003eCAMKII inhibitor KN93 suppresses tumorigenesis and immune invasion in PDAC. \u003cstrong\u003eA–C\u003c/strong\u003e KN93 treatment significantly reduced tumor size and volume in an orthotopic PDAC mouse model (n = 6). \u003cstrong\u003eD\u003c/strong\u003e Immunohistochemical staining of Ki-67 revealed decreased tumor cell proliferation following KN93 treatment. Scar bar =100μm. \u003cstrong\u003eE\u003c/strong\u003eqRT-PCR analysis showed reduced CCL20 mRNA expression in tumor tissues from KN93-treated mice. \u003cstrong\u003eF\u003c/strong\u003e Western blotting demonstrated downregulation of p-AKT and p-p65 levels in the KN93 group. \u003cstrong\u003eG–H\u003c/strong\u003e Flow cytometry analysis confirmed that KN93 decreased the proportion of MDSCs and PMN-MDSCs, while increasing CD8⁺ T cell infiltration (n = 6). Data were analyzed using FCS Express 7. \u003cstrong\u003eI\u003c/strong\u003e Schematic illustration of the proposed mechanism by which the ACh/CHRNA5 axis promotes PDAC tumorigenesis and immune modulation via the CAMKII/AKT/NF-κB–CCL20 pathway. Data with error bars represented as mean ± SD, and analyzed using two-tailed Student’s t-test. * represented \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, ** represented \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, *** represented \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7418128/v1/62e40e714360ed5c01b85a5b.png"},{"id":93905230,"identity":"4aaaeef4-7813-4f67-999f-ff26d708e773","added_by":"auto","created_at":"2025-10-20 06:54:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":11609675,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7418128/v1/01689cac-1f9c-4364-8c6a-0fa0fbbf0ea8.pdf"},{"id":91448536,"identity":"4a4a677a-945c-444b-9d9c-252e31e7ff7f","added_by":"auto","created_at":"2025-09-16 15:03:05","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":3278591,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryuncroppedGels.docx","url":"https://assets-eu.researchsquare.com/files/rs-7418128/v1/c572f013c349a9861479122e.docx"}],"financialInterests":"","formattedTitle":"CHRNA5 drives pancreatic cancer progression by promoting tumorigenesis and remodeling immune invasion microenvironment via CAMKII/AKT/NF-κB-CCL20 axis","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePancreatic ductal adenocarcinoma (PDAC) is one of the most devastating and lethal malignancies [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Despite advances in therapeutic approaches, the overall survival (OS) rate of PDAC remains dismally low, with only about 10% of patients surviving beyond five years post-diagnosis [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The incidence of PDAC continues to rise annually by approximately 0.5\u0026ndash;1.0%, largely driven by risk factors such as smoking, diabetes, and chronic pancreatitis, making it as a leading cause of cancer-related mortality worldwide [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The clinical aggressiveness of PDAC is strongly influenced by its highly dynamic tumor microenvironment, which is characterized by hypoxia, immune evasion, and enhanced tumor cell stemness signatures [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Therefore, a deeper understanding of the tumor microenvironment and the mechanisms driving tumor cell malignancy is essential for developing more effective therapeutic strategies against PDAC.\u003c/p\u003e\u003cp\u003ePerineural invasion (PNI) is a prominent clinical feature of PDAC, observed in nearly all cases, and contributes to tumor aggressiveness, distant metastasis, poor prognosis, and diminished patient quality of life [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Emerging evidence suggests that PDAC patients with extensive PNI exhibit elevated levels of acetylcholine (ACh) in tumor tissues compared to norepinephrine (NE) [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], underscoring the potential involvement of cholinergic signaling in PDAC progression. However, the roles of ACh in PDAC remain controversial. While some study has reported that acetylcholine may exert an inhibitory effect on tumor growth [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], others have shown that parasympathetic neurogenesis facilitates tumor progression and is associated with worse clinical outcomes [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. These conflicting findings highlight the urgent need to clarify the precise functions and mechanisms of ACh signaling in PDAC. Unraveling the role of this pathway may facilitate the development of targeted therapeutic strategies to improve clinical management and patient prognosis.\u003c/p\u003e\u003cp\u003eAlpha5-nicotinic acetylcholine receptor (CHRNA5), a ​key ​member of the nicotinic acetylcholine receptor (nAChR) family, functions as a ​ligand-gated ion channel that ​mediates calcium influx upon activation. As a critical effector of cholinergic signaling, ​CHRNA5 has been implicated in regulating tumorigenesis, invasion, and metastasis across multiple cancers. ​In non-small cell lung cancer (NSCLC), ​CHRNA5 activation enhances tumor migration through TGF-β1/Smad pathway [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. ​In hepatocellular carcinoma (HCC), ​CHRNA5 overexpression is ​significantly associated with enhanced metastasis and increased tumor stemness [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Additionally, CHRNA5 regulates CES1 expression through the MEK/ERK signaling, ​thereby contributing to tumor recurrence and metastasis in head and neck squamous cell carcinoma [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Despite these findings, ​the specific roles and mechanistic contributions of CHRNA5 in PDAC progression, particularly in remodeling the tumor microenvironment, remain largely unclear.\u003c/p\u003e\u003cp\u003eIn this study, we integrated multi-omics and cellular assays to confirm the enrichment of cholinergic signaling and the tumor-promoting role of ACh in PDAC. Machine learning and single-cell RNA-seq reanalysis identified CHRNA5 as a key driver, whose knockdown mitigated ACh-induced malignancy. Mechanistically, CHRNA5 promoted PMN-MDSC recruitment via the CAMKII/AKT/NF-κB\u0026ndash;CCL20 axis. Furthermore, KN93, a CaMKII inhibitor, effectively suppressed both tumorigenesis and immune invasion, highlighting the translational potential of targeting CHRNA5-mediated pathways in PDAC. These findings offer valuable insights into nerve\u0026ndash;tumor\u0026ndash;immune crosstalk and support CHRNA5 as a promising therapeutic target in pancreatic cancer.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eClinical Patients and samples\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFormalin-fixed PDAC tumor tissue sections were collected from patients with PDAC at the Third Affiliated Hospital of Naval Military Medical University. Sections were stained with PGP9.5, ChAT, and TH antibodies to assess the distribution of parasympathetic and sympathetic nerves in three patients. Detailed clinical information is provided in Table S1. Written informed consent was obtained from all participants. The study protocol was approved by the Medical Ethics Committee of the Third Affiliated Hospital of Naval Military Medical University (Approval No. EHBHKY2025-K026-P001).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBulk and single cell RNA sequencing data acquisition and enrichment analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe sourced the integrated and batch-effect-corrected The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) datasets from the UCSC Xena database (https://xena.ucsc.edu/) [13]. This combined dataset comprises transcriptomic and clinical data for 147 PDAC samples and 167 normal tissue samples. Differentially expressed genes (DEGs) between PDAC and normal tissues were identified using the DESeq2 package. Genes with an adjusted \u003cem\u003eP\u003c/em\u003e-value of \u0026lt; 0.05 and a |log₂FC| \u0026ge; 1 were deemed significantly differentially expressed.\u003c/p\u003e\n\u003cp\u003escRNA-seq data, including three adjacent normal tissues and seventeen PDAC tumor samples, were downloaded from the GEO database (GSE155698) [14] to investigate alterations in ACh receptor expression across different cell types. Based on canonical marker genes, fourteen distinct cell types were annotated.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEnrichment analyses were conducted using the clusterProfiler package, including Gene Set Enrichment Analysis (GSEA), Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses [15].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMachine learning modeling, SHAP interpretation, and stemness analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo identify key contributors to tumorigenesis, three machine learning models, Random Forest (RF), LightGBM (LGBM), and Least Absolute Shrinkage and Selection Operator (LASSO) regression, were constructed using the randomForest [16], lightgbm [17], and glmnet [18] R packages. All TCGA-PDAC samples were randomly divided into training (70%) and validation (30%) sets. LASSO was first applied for feature selection, and features with non-zero coefficients were retained for model building. Optimal parameters were tuned by grid search, and model performance was evaluated using 5-fold cross-validation. Accuracy, recall, precision, F1 score, Cohen\u0026apos;s kappa, Matthews correlation coefficient (MCC), and the area under the ROC curve (AUC) were used as evaluation metrics.\u003c/p\u003e\n\u003cp\u003eTo interpret model outputs, SHapley Additive exPlanations (SHAP) values were calculated using the fastshap and shapviz packages [19]. Summary plots, heatmaps, and other visualizations were generated to depict feature importance and their influence on individual predictions, thereby clarifying the contribution of each variable to overall performance. ROC curves were generated using the pROC package to assess sensitivity and specificity.\u003c/p\u003e\n\u003cp\u003eTo calculate the mRNA-based stemness score (mRNAsi), we obtained the human stem cell/progenitor cell dataset from the Progenitor Cell Biology Consortium (PCBC, https://www.synapse.org). Subsequently, the \u0026quot;gelnet\u0026quot; package was utilized to apply one-class logistic regression (OCLR) for calculating the mRNAsi of each TCGA-PDAC sample.[20] Spearman correlation analysis was performed to assess the relationship between gene expression and the stemness score.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell culture and treatment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman PANC-01 cells (Jiangsu Nobio Biotechnology Co., Ltd.) and murine PANC-02 cells (Servicebio Biotechnology Co., Ltd.) were cultured in DMEM and RPMI-1640 medium, respectively, supplemented with 10% FBS and 1% penicillin\u0026ndash;streptomycin at 37 \u0026deg;C in a 5% CO₂ humidified incubator. To assess the effects of ACh (A2661, Sigma-Aldrich) on proliferation, invasion, migration, colony formation, and sphere formation, cells were treated with 10 or 100 \u0026mu;M ACh or DMSO as control.\u003c/p\u003e\n\u003cp\u003eThree siRNAs targeting CHRNA5 were transfected into PANC-01 and PANC-02 cells using Lipofectamine 2000 (Thermo Fisher Scientific). Knockdown efficiency was confirmed by qRT-PCR and western blotting, with the most effective siRNA used to generate shRNA plasmids. siRNA and shRNA sequences are listed in Table S2.\u003c/p\u003e\n\u003cp\u003eLentiviruses encoding CHRNA5 shRNA (Lv-Sh-CHRNA5) or control (Lv-Sh-NC) were provided by Wuhan Shum\u0026igrave; Neuroscience Technology Co., Ltd. PANC-02 cells were infected and selected with puromycin (2 mg/L) to establish stable knockdown lines. These cells, along with controls, were used to establish orthotopic tumor models to evaluate the contribution of CHRNA5 to PDAC progression.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMice experiments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMale C57BL/6 and BALB/c nude mice (six weeks old, Gempharmatech Co., Ltd.) were used to establish orthotopic PDAC models. Mice were anesthetized with 1.25% tribromoethanol, and a 1-cm incision was made below the left rib cage. Approximately 2 \u0026times; 10⁶ PANC-02 cells (Lv-Sh-CHRNA5 or Lv-Sh-NC) were injected into the pancreatic tail. After two weeks, mice were sacrificed, and pancreatic tumors were collected for histological staining, qRT-PCR, western blotting, and flow cytometry. Tumor volume was calculated using the formula: 0.52 \u0026times; length \u0026times; width\u0026sup2;.\u003c/p\u003e\n\u003cp\u003eOne week after tumor implantation, mice were randomly assigned to receive intraperitoneal KN93 (20 mg/kg, HY-15465, MCE) once daily. After one week of KN93 treatment, mice were euthanized, and tumors were collected for further analysis.\u003c/p\u003e\n\u003cp\u003eAll mice were housed in a specific pathogen-free (SPF) facility with controlled temperature and light, maintaining a 12-hour light/dark cycle. Ethical approval for the animal experiments was granted by the Third Affiliated Hospital of Naval Military Medical University Ethics Committee (EDWLL-2025-002).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEnzyme-Linked Immunosorbent Assay (ELISA)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eACh and NE levels in pancreatic and tumor tissues from PDAC mice were quantified using ELISA kits (Cat. YJ063805, YJ401805, Shanghai Enzyme-linked Biotechnology Co., Ltd.) following the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell proliferation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCell proliferation of PDAC cells was measured using the CCK-8 kit (A311-01, Vazyme, Nanjing, China). PANC-01 or PANC-02 cells (2,000/well) were seeded in 96-well plates and treated every three days with 10 or 100 \u0026mu;M ACh or DMSO. To assess the role of CHRNA5, cells were transfected with Sh-NC or Sh-CHRNA5 plasmids. Each day, 10 \u0026mu;L of CCK-8 solution was added, followed by 1 h incubation at 37\u0026deg;C. Absorbance at 450 nm was recorded daily using a Thermo Scientific microplate reader.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell invasion and migration\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor migration and invasion assays, 2\u0026times;10⁴ PDAC cells were seeded into 24-well transwell chambers (8.0-\u0026mu;m pore, BD, USA). For invasion, filters were pre-coated with Matrigel (Invitrogen, USA). Cells were treated with 10 or 100 \u0026mu;M ACh or DMSO. To assess CHRNA5 function, cells were transfected with Sh-NC or Sh-CHRNA5 plasmids in 6-well plates for 24 h, then transferred to transwell chambers. After 72 h, migrated/invaded cells were stained with 0.1% crystal violet and quantified using ImageJ.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eColony formation\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor colony formation, 100 PDAC cells were plated in 60-mm dishes and treated with 100 \u0026mu;M ACh or DMSO, with fresh medium replaced every three days. To assess CHRNA5 function, PANC-01 and PANC-02 cells were transfected with Sh-NC or Sh-CHRNA5 plasmids for 24 h, digested, and reseeded. After 14 days, colonies were stained with 0.1% crystal violet.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSphere-formation assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eApproximately 1,000 PANC-01 or PANC-02 cells were seeded into 12-well ultra-low attachment plates and cultured in FBS-free DMEM/F12 supplemented with 2% B-27(17504044, Gibco, USA), 20 ng/ml epidermal growth factor (EGF, C029/CH28, Novoprotein, Suzhou, China), and 20 ng/ml basic fibroblast growth factor (bFGF, C046/C044, Novoprotein, Suzhou, China). For ACh treatment, 100 \u0026mu;M ACh or the control DMSO was added to the cells twice a week. After 2 weeks, spheres were observed under a light microscope, and the total number of spheres was counted. To assess the effect of CHRNA5 on sphere formation, Sh-NC and Sh-CHRNA5 plasmids were transfected into PDAC cells.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eqRT-PCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted from cell or tissue samples using Trizol reagent (R401-01, Vazyme, Nanjing, China). Total RNA was reverse transcribed into complementary DNA (cDNA) using the reverse transcription kit (R333, Vazyme, Nanjing, China). The cDNA was diluted and subjected to qRT-PCR using an amplification kit (Q711, Vazyme, Nanjing, China) to analyze gene expression under appropriate reaction conditions. The primers used in this study were shown in Table S3.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blotting\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProteins were extracted from mice pancreatic cancer tissues or cell samples using RIPA lysis buffer (G2002, Servicebio, Wuhan, China). For detection signals, the membrane was processed with ECL chemiluminescence reagent. The antibodies used in this study were shown in Table S4.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunohistochemical staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e4 \u0026mu;m paraffin sections were deparaffinized and rehydrated to remove paraffin and allow proper reagent penetration. After treatment with hydrogen peroxide, sections were boiled in citrate buffer (pH 6.0) for 20 minutes for antigen retrieval, followed by blocking with 5% bovine serum albumin (BSA). Sections were then incubated with primary antibodies at 4\u0026deg;C overnight. The next day, sections were incubated with the secondary antibody for 1 hour. Images were captured using an Olympus microscope. The antibodies used are listed in Table S4. The positive staining area was quantified using Image J.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA seq in the study\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo explore the downstream targets and pathway of ACh/CHRNA5, PANC-02 transfected with Sh-CHRNA5 and Sh-NC plasmids (n=3) were applied to perform RNA sequencing by LC-Bio Technology Co., Ltd. (Hangzhou, China). Based on the manufacturer\u0026rsquo;s protocol, RNA was extracted and transcriptome library was constructed using the VAHTS Universal V8 RNA-seq Library Prep Kit for Illumina. The constructed library was sequenced on the Illumina Novaseq platform using PE150 sequencing mode. Quantification was performed using the StringTie software, and edgeR was employed for differential expression analysis. The default criteria for screening significantly differentially expressed genes were FDR \u0026lt; 0.05 and |log₂FC| \u0026ge; 1.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCalcium flow detection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCalcium concentration ([Ca\u0026sup2;⁺]) in PANC-02 cells was measured using Fluo-4 AM dye (S1061S, Beyotime, Suzhou, China). After treatment with ACh or Sh-CHRNA5, cells were incubated with Fluo-4 AM solution at 37\u0026deg;C for 30 minutes. Fluorescence was observed using fluorescence microscopy to detect spontaneous green fluorescence. Additionally, flow cytometry was used to quantify fluorescence intensity, enabling assessment of the effects of ACh and CHRNA5 on intracellular calcium levels.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCleavage under targets and tagmentation (Cut\u0026amp;Tag)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCut\u0026amp;Tag was performed to explore the direction binding of p65 on the promotor of CCL20 with Cut\u0026amp;Tag assay kit (TD904, Vazyme, Nanjing, China). PANC-02 cells were harvested, counted, and centrifuged at 600 \u0026times; g for 10 min at room temperature.\u0026nbsp;A total of 100,000 cells were subjected to Cut\u0026amp;Tag. According to manufacturer\u0026apos;s protocol, cells were first incubated with NE buffer, and then mixed with concanavalin A-coated magnetic beads and p65 antibody overnight. The second day, the mixture was incubated with second antibody for one hour\u0026nbsp;followed by incubation with pA/G-Tnp for one hour at room temperature. The bead/nucleus pellet was resuspended in tagmentation buffer and incubated with DNA extract Beads to capture DNA for final qRT-PCR experiment.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFlow cytometry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTumor tissues were digested with Type IV collagenase (C5138, Sigma-aldrich, Darmstadt, Germany) for 20 minutes. The isolated cells were then treated with red blood cell lysis, followed by staining with a viability marker (564406, BD Bioscience, USA). Cells were stained with CD45, CD11B, Ly6C, Ly6G, CD3, CD4, and CD8. The number of immune cells subtypes was quantified to assess the impact of CHRNA5 on tumor microenvironment. The antibodies used were shown in Table S4.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analyses were conducted using R software (version 4.4.1), SPSS version 23.0, or GraphPad Prism version 9.0 (GraphPad Software, La Jolla, CA). Results are presented as mean \u0026plusmn; standard deviation (SD). Comparisons between two datasets were performed using the Wilcoxon rank-sum test or Student\u0026apos;s t-test. For comparisons involving three or more groups, one-way or two-way analysis of variance (ANOVA) was employed. Spearman\u0026apos;s rank correlation test was used to assess correlations between variables. A \u003cem\u003eP\u003c/em\u003e-value of less than 0.05 was considered statistically significant (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026lt; 0.001).\u0026nbsp;\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eACh promotes malignant phenotype of PDAC\u003cem\u003e\u0026nbsp;in vitro\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the role of cholinergic signaling in PDAC, we reanalyzed RNA sequencing data from PDAC and normal tissue samples using the integrated TCGA-GTEx dataset. KEGG, GO, and GSEA analyses revealed significant enrichment of neural-related pathways (Fig. 1A, S1A-B). Expression of CHAT (ACh synthesis) [21] and SLC18A3 (encodes the VAChT) [22] was markedly elevated in PDAC tissues, with strong predictive power for distinguishing PDAC from normal samples\u0026nbsp;(Fig. S1C-D). Moreover, in PDAC patients with PNI, PGP9.5 co-localized with the cholinergic marker CHAT but not with the sympathetic marker TH (Fig. 1B). In our murine orthotopic model, both ACh/NE levels and CHAT/VAChT protein expression were significantly upregulated (Fig. 1C-D), suggesting a tumor-promoting role of ACh.\u003c/p\u003e\n\u003cp\u003eTo further investigate the role of ACh \u003cem\u003ein vitro\u003c/em\u003e, we explored its effects at different concentrations in PANC-01 and PANC-02 cell lines. In PANC-01 cells, CCK8 and transwell assays revealed that 100 \u0026mu;M ACh significantly promoted cell proliferation, invasion, and migration (Fig. S2A- B). In addition, ACh enhanced these malignant characteristics in PANC-02 cells (Fig. 1E-F). Accordingly, 100 \u0026mu;M was used in subsequent experiments. ACh also increased colony formation (Fig. 1G, S2C), sphere formation, and cancer stem cell (CSC) marker genes expression (Fig. 1H-I, S2D-E), confirming its role in driving aggressive PDAC phenotypes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCHRNA5 is the crucial contributor in PDAC\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs muscarinic and nicotinic receptors are established downstream targets of ACh, we aim to determine which receptor plays a pivotal role in PDAC. Venn diagram analysis was conducted to identify differentially expressed genes of cholinergic receptors in human PDAC tissues (Fig. 2A). To further determine key contributors during PDAC progression, we employed three machine learning models\u0026mdash;LASSO, RF and LGBM\u0026mdash;evaluating their performance based on accuracy, recall, F1 score, kappa, MCC, AUC and precision (Fig. 2B). SHAP analysis provided a detailed and transparent interpretation of the decision-making process of machine learning models, elucidating the crucial contributions of CHRNA5 in PDAC, each demonstrating the highest mean SHAP values (Fig. 2C-E, S3A-C). A heatmap revealed that CHRNA5 was the most significantly upregulated receptor in PDAC relative to normal tissues (Fig. 2F).\u0026nbsp;ROC analysis (AUC = 0.964, Fig. S3D) supported its diagnostic value, while Kaplan\u0026ndash;Meier survival analysis revealed high CHRNA5 expression predicted poorer overall survival (\u003cem\u003eP\u003c/em\u003e = 0.02, Fig. 2G). Moreover, CHRNA5 expression correlated positively with tumor stemness (Fig. S3E).\u003c/p\u003e\n\u003cp\u003eTo define CHRNA5-expressing cell populations, we reanalyzed scRNA-seq data (GSE155698) [14], identifying 14 cell clusters (Fig. 2H). Violin plots showed that CHRNA5 was predominantly enriched in epithelial/tumor cells (Fig. S4), with significantly higher expression in tumor epithelium than adjacent normal tissue (Fig. 2I-J). Immunofluorescence confirmed CHRNA5 co-localized with Ki-67 positive proliferating tumor cells (Fig. 2K), and high expression was also validated in PDAC cell lines (Fig. 2L). Together, these results identify CHRNA5 as a critical driver of PDAC progression and a potential diagnostic and prognostic biomarker.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCHRNA5 exerts tumorigenic effects\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003ein vitro\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;and\u003cem\u003e\u0026nbsp;in vivo\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSince the role of CHRNA5 in PDAC remains largely unexplored, we designed siRNAs to knock down CHRNA5 in PANC-01 and PANC-02 cells. qRT-PCR and western blotting confirmed knockdown efficiency (Fig. 3A-B), with Si-1 showing the strongest suppression, and its sequence was used to construct Sh-CHRNA5 plasmids. CHRNA5 silencing significantly reduced PANC-02 cell proliferation (Fig. 3C), invasion and migration (Fig. 3D), and colony formation (Fig. 3E). Importantly, CHRNA5 inhibition also diminished sphere formation and downregulated CSC marker genes, indicating reduced tumor stemness (Fig. 3F-G). Similar results were observed in PANC-01 cells (Fig. S5A\u0026ndash;G).\u003c/p\u003e\n\u003cp\u003eTo investigate the role of CHRNA5 \u003cem\u003ein vivo\u003c/em\u003e, PANC-02 cells were transduced with lentivirus carrying either Sh-NC or Sh-CHRNA5 to establish stable knockdown cell lines. These cells were then orthotopically implanted into C57BL/6 mice to establish PDAC orthotopic tumor models. Tumors derived from CHRNA5-silenced cells exhibited significantly reduced tumorigenic capacity compared to controls (Fig. 3H). The knockdown efficiency was validated by qRT-PCR and western blotting (Fig. 3I-J). Consistently, mice in the Sh-CHRNA5 group showed markedly decreased tumor volume and weight (Fig. 3K-L). Immunohistochemical staining further confirmed a lower Ki-67 expression, indicating decreased tumor\u0026nbsp;proliferation in the Sh-CHRNA5 group (Fig. 3M).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCHRNA5 mediates the oncogenic effects of ACh\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine whether CHRNA5 mediates the effects of ACh, we conducted a rescue experiment involving ACh treatment following CHRNA5 knockdown. qRT-PCR and western blotting experiments confirmed that Sh-CHRNA5 transfection suppressed the ACh-induced upregulation of CHRNA5 at both RNA and protein levels (Fig. 4A, B, D, E). Notably, CHRNA5 knockdown significantly blocked ACh-induced cell proliferation in both PANC-01 and PANC-02 cells (Fig. 4C, F). Similarly, inhibition of CHRNA5 reduced ACh-mediated colony formation (Fig. 4G-H). More importantly, ACh-enhanced tumorsphere formation was markedly inhibited following CHRNA5 knockdown (Fig. 4I-J), indicating that CHRNA5 mediates the tumor-promoting effects of ACh.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCHRNA5 targets CaMKII/AKT/NF-\u0026kappa;B pathway\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRNA sequencing of PANC-02 cells transduced with either Sh-NC or Sh-CHRNA5 was performed to explore the downstream targets of CHRNA5. Differential expression analysis revealed 351 upregulated and 299 downregulated genes in the Sh-CHRNA5 group compared to the Sh-NC group, as illustrated by a volcano plot (Fig. 5A). KEGG pathway enrichment analysis indicated that CHRNA5 knockdown affected several key pathways, including neuroactive ligand\u0026ndash;receptor interaction, calcium signaling, PI3K\u0026ndash;AKT, and NF-\u0026kappa;B signaling (Fig. 5B), suggesting that CHRNA5 may influence calcium homeostasis. Given that CHRNA5 functions as a ligand-gated calcium channel [23], we hypothesized that it modulates intracellular Ca\u0026sup2;⁺ levels. To verify this, we measured Ca\u0026sup2;⁺ flux using Fluo-4 AM via immunofluorescence staining and flow cytometry. The results showed that ACh stimulation and CHRNA5 activation increased intracellular Ca\u0026sup2;⁺ flux, whereas CHRNA5 knockdown abrogated ACh-induced calcium signaling (Fig. 5C-D). As Ca\u0026sup2;⁺ binds to calmodulin (CaM) to activate the CaMK pathway, particularly CaMKII [24], we further investigated this signaling cascade. Western blotting confirmed that ACh and CHRNA5 mediated the phosphorylation of CaMKII (Fig. 5E-F). Since CaMKII is known to activate AKT [25], which subsequently regulates the NF-\u0026kappa;B pathway\u0026nbsp;[26, 27], we examined the expression of downstream effectors. Notably, CHRNA5 suppression mitigated the ACh-induced activation of p-AKT, p-p65 and p-I\u0026kappa;B\u0026alpha; (Fig. 5E-F). These results suggest that the CAMKII/AKT/NF-\u0026kappa;B pathway is a major downstream effector of the ACh/CHRNA5 axis, contributing to PDAC progression.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCHRNA5 remodels the tumor immune microenvironment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGiven the critical role of immune cell infiltration in the tumor microenvironment, we then investigate whether CHRNA5 could regulate tumor-associated immune response. To explore this, orthotopic tumor model was constructed in both immunodeficient nude mice and immunocompetent C57BL/6 mice. CHRNA5 knockdown markedly suppressed tumor formation, reduced tumor volume, and decreased Ki-67 expression in both models, with a more pronounced inhibitory effect observed in C57BL/6 mice (Fig. 6A-E), suggesting that CHRNA5 might exert immunomodulatory effects in PDAC tumorigenesis. GO analysis revealed the enrichment of immune-related pathways, such as leukocyte migration, further supporting the involvement of CHRNA5 in immune regulation (Fig. 6F). A heatmap analysis of genes related to leukocyte migration showed that CHRNA5 could affect chemokines like CCL20, CXCL9, CTSG, etc., which might mediate the chemotaxis and infiltration of myeloid cells (Fig. 6G). Additionally, qRT-PCR in PANC-02 cells and tumor tissues validated the decreased expression of CCL20 upon CHRNA5 inhibition (Fig. 6H-I). As reported [28], the promotor region of CCL20 enriched a NF-\u0026kappa;B binding site (Fig. 6J). Cut\u0026amp;Tag results further vindicated the direct binding of p65 on the promotor region of CCL20 (Fig. 6K). CCL20 is a critical mediator in immune cell recruitment, particularly for myeloid-derived suppressor cells (MDSCs) and CD4\u003csup\u003e+\u003c/sup\u003e T cells [29, 30]. Flow cytometry confirmed that CHRNA5 knockdown resulted in a significant decrease in MDSCs, especially polymorphonuclear MDSCs (PMN-MDSCs), and an increase ratio of CD8\u003csup\u003e+\u003c/sup\u003e T cell (Fig. 6L-M, S6A-B). These results demonstrate that CHRNA5 modulates the tumor immune microenvironment by promoting CCL20-mediated recruitment of MDSCs, thereby contributing to immune evasion in PDAC.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCAMKII inhibitor KN93 suppresses tumorigenesis and immune invasion in PDAC\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConsidering the lack of selective CHRNA5 inhibitors, we employed KN93, a small-molecule CaMKII inhibitor, to assess the therapeutic impact of downregulating the CHRNA5-mediated signaling pathway on tumor malignancy and the immune microenvironment. As expected, KN93-treated mice exhibited significantly smaller tumor size and volume (Fig. 7A\u0026ndash;C). Immunohistochemical staining of Ki-67 revealed a marked reduction in tumor proliferative capacity (Fig. 7D). qRT-PCR analysis demonstrated decreased CCL20 expression following KN93 treatment (Fig. 7E), and western blotting confirmed inhibition of the AKT/NF-\u0026kappa;B signaling pathway in the KN93-treated group (Fig. 7F). Importantly, flow cytometry revealed that KN93 significantly reduced the proportion of MDSCs, particularly PMN-MDSCs, while enhancing CD8⁺ T cell infiltration (Fig. 7G-H), indicating its immunomodulatory effect in PDAC. Taken together, these above findings highlight the pivotal role of CHRNA5 in driving tumorigenesis and immune evasion through the CAMKII/AKT/NF-\u0026kappa;B\u0026ndash;CCL20 axis, underscoring its potential as a promising therapeutic target in PDAC (Fig. 7I).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003ePDAC is an aggressive malignancy with enhanced ACh levels, a pathological feature related to tumor progression, resistance to therapy, and poor prognosis [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Although extensive investigations have been conducted to elucidate the role of cholinergic signaling in cancer [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], the specific nAChR subtypes involved in PDAC remain insufficiently defined, and effective therapeutic strategies are still limited. Moreover, immunotherapy has shown limited efficacy in PDAC patients, underscoring the urgent need to clarify the mechanisms underlying tumor\u0026ndash;immune interactions [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. In this study, we identify CHRNA5 as a critical oncogenic driver in PDAC, and demonstrate that CHRNA5 not only facilitates malignant phenotypes but also plays a pivotal role in remodeling the immune microenvironment. These observations offer significant insights into the nerve-tumor-immune environment network and support CHRNA5 as a promising therapeutic target for pancreatic cancer.\u003c/p\u003e\u003cp\u003eThe role of ACh in PDAC remains controversial. Some study suggested that ACh contributed to an immunosuppressive microenvironment by inhibiting immune cell recruitment and reducing interferon-gamma (IFN-γ) production, thereby leading to a decrease in the Th1/Th2 ratio [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Conversely, other research indicated that subdiaphragmatic vagotomy accelerated PDAC progression, while systemic administration of the muscarinic agonist bethanechol restored the malignant phenotype via cholinergic receptor muscarinic 1 (CHRM1) activation [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. These conflicting findings imply that ACh may exert distinct effects depending on the specific receptor subtype involved. Given that ACh has multiple receptors, it is plausible that different subtypes mediate different roles in tumor progression. In the current study, our \u003cem\u003ein vitro\u003c/em\u003e experiments demonstrated that 100 \u0026micro;M ACh significantly enhanced PDAC cell proliferation, invasion, migration, colony formation, and tumor stemness, consistent with its tumor-promoting effects previously observed in lung cancer [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] and intrahepatic cholangiocarcinoma (ICC) [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAs a subtype of nAChR, CHRNA5 has been implicated in tumor progression across multiple cancers. In HCC, CHRNA5 has been shown to promote cell proliferation, metastasis, stemness, and even enhance sensitivity to sorafenib [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Similarly, in ICC, the ACh/CHRNA5 axis activates CAMKII/GSK3β signaling, leading to β-catenin upregulation, which promotes metastasis and resistance to gemcitabine [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. However, little is known about the function of CHRNA5 in PDAC. In this study, we identified CHRNA5 as a key mediator of ACh\u0026rsquo;s tumor-promoting effects in PDAC through bulk RNA-seq analysis using three machine learning models. Notably, our re-analysis of scRNA-seq data revealed that CHRNA5 was highly expressed in epithelial cells, with significantly elevated level in tumor epithelial cells compared to adjacent normal epithelial cells, suggesting its potential role in tumorigenesis. We further demonstrated that CHRNA5 promoted PDAC cell proliferation, invasion, migration, colony formation, and tumor stemness \u003cem\u003ein vitro\u003c/em\u003e, and enhanced tumor formation \u003cem\u003ein vivo\u003c/em\u003e. To our knowledge, this is the first comprehensive investigation of CHRNA5 in PDAC, providing substantial evidence for its tumor-promoting function in tumorigenesis and highlighting CHRNA5 as a potential therapeutic target for PDAC treatment.\u003c/p\u003e\u003cp\u003eThe immune microenvironment plays a crucial role in tumor progression and treatment resistance [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. PDAC is characterized by an immunosuppressive microenvironment, with low infiltration of CD8\u003csup\u003e+\u003c/sup\u003e T cells, high levels of immunosuppressive cells (e.g., regulatory T cells, MDSCs, tumor-associated macrophages), and upregulation of immune checkpoint molecules [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. This immunosuppressive state contributes to poor responses to immunotherapy, posing a great threat to the treatment of PDAC. Intriguingly, we observed that CHRNA5 exerted more pronounced effects in C57BL/6 mice compared to nude mice, suggesting its involvement in shaping the immune microenvironment. Flow cytometry analysis further revealed that CHRNA5 inhibition significantly reduced the proportions of total MDSCs and PMN-MDSCs, while enhancing CD8⁺ T cell infiltration. PMN-MDSCs, a major subset of MDSCs, are known immunosuppressive regulators that modulate both the number and function of CD8⁺ T cells [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. These current findings suggest that CHRNA5 acts not only as an intrinsic driver of tumor progression but also as a key modulator of the immunosuppressive tumor microenvironment.\u003c/p\u003e\u003cp\u003eCaMKII, a serine/threonine kinase, plays a crucial role in regulating various cellular processes, including tumor proliferation, metastasis, and drug resistance, by maintaining Ca\u0026sup2;⁺ homeostasis [\u003cspan additionalcitationids=\"CR39\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Its activation is primarily dependent on Ca\u0026sup2;⁺/CaM binding. However, once phosphorylated at Thr286, CaMKII becomes constitutively active, allowing it to function independently of Ca\u0026sup2;⁺/CaM signaling [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Given that CHRNA5 functions as a calcium ion channel, it may influence Ca\u0026sup2;⁺/CaM binding and subsequently regulate p-CaMKII and CaMKII levels. Our RNA sequencing analysis of sh-CHRNA5-treated cells revealed significant enrichment of the calcium signaling pathway. Further \u003cem\u003ein vitro\u003c/em\u003e experiments, including western blot analysis and calcium flow detection, confirmed that ACh/CHRNA5 modulates intracellular Ca\u0026sup2;⁺ levels and p-CaMKII expression. Additionally, we observed the activation of PI3K-AKT pathway in sh-CHRNA5-treated cells based on RNA-seq.\u0026nbsp;As previous studies have demonstrated that CaMKII can activate PI3K, leading to AKT phosphorylation at Ser473[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], we hypothesize that the CHRNA5 promotes PDAC progression through the CaMKII/PI3K-AKT signaling pathway.\u003c/p\u003e\u003cp\u003eThe PI3K-AKT signaling pathway plays a critical role in PDAC progression by regulating tumor cell proliferation, stemness, and immune microenvironment remodeling [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. As a central node in multiple oncogenic processes, AKT activation promotes tumor growth by enhancing cell survival, inhibiting apoptosis, and sustaining metabolic adaptation [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. In addition to its direct effects on tumor cells, PI3K-AKT signaling also plays a pivotal role in shaping the immune microenvironment [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Activation of AKT can modulate immune cell recruitment, differentiation, and function, creating an immunosuppressive niche that facilitates tumor evasion from immune surveillance. One important downstream target of AKT is CCL20 [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], a chemokine that recruits regulatory immune cells, including Tregs [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e] and MDSCs [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], to the tumor microenvironment. P65, the crucial transcription factor in the NF-κB pathway, is reported to be translocated into the nucleus via the AKT/IκBα signaling pathway [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. As previously reported, NF-κB binding sites are enriched on the promotor region of CCL20 [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Therefore, we speculate that CAMKII/AKT/NF-κB-CCL20 might be the downstream pathway of CHRNA5. Supporting this in our Cut\u0026amp;Tag results, p65 directly binds to the promoter region of CCL20, thereby upregulating its transcription level. Collectively, these observations suggest that CHRNA5 regulates the CAMKII/AKT/NF-κB pathway to mediate CCL20 expression, thereby contributing to the formation of an immunosuppressive microenvironment.\u003c/p\u003e\u003cp\u003eBased on these findings, CHRNA5 plays dual roles in promoting tumorigenesis and modulating immune microenvironment in PDAC, suggesting that targeting CHRNA5 may offer therapeutic potential in clinical management. However, the lack of a specific CHRNA5 inhibitor remains a major barrier to translational application and represents a limitation of this study. Emerging evidence from studies in ICC [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], head and neck squamous cell carcinoma (HNSC) [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], and lung cancer [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e] has highlighted the therapeutic relevance of CHRNA5 inhibition, though large-scale, multi-ethnic clinical studies are also warranted to validate these observations. Beyond direct CHRNA5 inhibitors, targeting downstream signaling pathways also holds promise. For instance, KN93, a CaMKII inhibitor, has shown protective effects in diabetic retinal vascular injury [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. In our \u003cem\u003ein vivo\u003c/em\u003e experiments, KN93 significantly suppressed tumor progression, reduced PMN-MDSC infiltration, and enhanced CD8⁺ T cell presence, supporting the translational potential of targeting CHRNA5-mediated pathways in PDAC.\u003c/p\u003e\u003cp\u003eIn summary, we confirmed the upregulation of ACh in public PDAC datasets and demonstrated its tumor-promoting effects \u003cem\u003ein vitro\u003c/em\u003e. Importantly, our study provides the first evidence that CHRNA5 plays dual roles in PDAC by promoting tumor malignancy and modulating the immune microenvironment, thereby mediating the oncogenic effects of ACh. Mechanistically, CHRNA5 exerts its oncogenic functions through CAMKII/AKT/NF-κB-CCL20 axis, contributing to immune regulation within the tumor microenvironment. Furthermore, KN93, an inhibitor targeting CHRNA5-mediated pathways, showed significant therapeutic efficacy in suppressing tumorigenesis and immune evasion. Collectively, these findings deepen our understanding of nerve\u0026ndash;tumor\u0026ndash;immune interactions and highlight CHRNA5 as a promising and translational target for therapeutic intervention in pancreatic cancer.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003ePDAC, pancreatic ductal adenocarcinoma; OS, overall survival; PNI, perineural invasion; ACh, acetylcholine; NE, norepinephrine; EMT, epithelial-mesenchymal transition; CHRNA5, cholinergic receptor nicotinic alpha 5; DEGs, differentially expressed genes; KEGG, Kyoto Encyclopedia of Genes and Genomes; GO, Gene Ontology; GSEA, Gene Set Enrichment Analysis; FBS, fetal bovine serum; DMEM, Dulbecco’s modified Eagle’s medium; SPF, specific pathogen-free; LGBM, LightGBM; RF, Random Forest; MCC, Matthews correlation coefficient; AUC, area under the receiver operating characteristic curve; SHAP, SHapley Additive exPlanations; EGF, epidermal growth factor; bFGF, basic fibroblast growth factor; BSA, bovine serum albumin; DAB, 3,3'-diaminobenzidine; CSC, cancer stem cell; CAM, calmodulin; CaMKII, calcium-calmodulin CaM-dependent protein kinase II; HCC, hepatocellular carcinoma; ICC, intrahepatic cholangiocarcinoma; GEO, Gene Expression Omnibus;\u0026nbsp;IFN-γ, interferon-gamma; CHRM1, cholinergic receptor muscarinic 1.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eClinical tissue samples were obtained from the Third Affiliated Hospital of Naval Military Medical University\u0026nbsp;with approval from the Ethics Committee (EHBHKY2025-K026-P001).\u0026nbsp;The animal studies were conducted under approval from the Ethics Committee of the Third Affiliated Hospital of Naval Military Medical University (Approval ID: EDWLL-2025-002) and adhered to the official guidelines set forth by the Institutional Animal Care and Use Committee (IACUC). All procedures were implemented with strict adherence to protocols designed to ensure animal welfare and minimize discomfort, in compliance with the principles of ethical experimentation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors have no conflict of interest regarding this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eZhijie Lu, Xuerong Miao and Kunming Tao: participated in the study design, coordination, supervision, and manuscript review/editing; Yue Zhang: prepared the original draft, established animal models, and drafted the manuscript; Liang Li: analyzed data and wrote code for the study;\u0026nbsp;Jiao Zhu,\u0026nbsp;Kai Wei, and Min Ma: performed data analysis and interpretation of results. All authors critically revised the manuscript and approved the final version.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll sequencing data generated in this study have been deposited in the Gene Expression Omnibus (GEO) database and are publicly accessible as of the publication date. The code and research materials utilized in this article can be obtained from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (82171232, 82371241, 31900716), the Program of Shanghai Academic/Technology Research Leader (22XD1404900), the Clinical Research Special Project of the Shanghai Health Commission (20204Y0047, 2020YJZX0133), and Science and Technology Innovation Plan of Shanghai Science and Technology Commission (21Y11903200).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBlackford AL, Canto MI, Dbouk M, Hruban RH, Katona BW, Chak A, Brand RE, Syngal S, Farrell J, Kastrinos F, et al. 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Cellular physiology and biochemistry: international journal of experimental cellular physiology, biochemistry, and pharmacology. 2016;39:1196\u0026ndash;208.\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":"CHRNA5, tumor immune microenvironment, tumor malignancy, PDAC, ACh","lastPublishedDoi":"10.21203/rs.3.rs-7418128/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7418128/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cb\u003eBackground\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy with poor prognosis, characterized by a distinct tumor microenvironment featuring perineural invasion (PNI), high tumor malignancy, and immune evasion. This study aims to identify the crucial cholinergic receptor involved in PDAC progression and elucidate its underlying mechanisms.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMethods\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTranscriptomic analyses of public PDAC datasets, immunohistochemical staining, and cellular assays were conducted to investigate the enrichment of cholinergic signaling and the tumor-promoting effects of acetylcholine (ACh) in PDAC. We integrated three machine learning models, single-cell RNA-seq reanalysis, \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e experiments to identify alpha5-nicotinic acetylcholine receptor (CHRNA5) as a central mediator of PDAC progression. RNA sequencing and rescue experiments were carried out to elucidate the mechanisms of CHRNA5. Furthermore, KN93, a CaMKII inhibitor, was used to assess the therapeutic potential of targeting CHRNA5-mediated pathways in the PDAC tumor microenvironment.\u003c/p\u003e\u003cp\u003e\u003cb\u003eResults\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIntegrated multi-omics analysis, cellular and experimental assays demonstrated the tumor-promoting roles of ACh and CHRNA5, while rescue experiments confirmed that CHRNA5 knockdown abrogated ACh-induced effects. Notably, CHRNA5 inhibition produced more pronounced anti-tumor responses in C57BL/6 mice compared to BALB/c mice. Mechanistically, CHRNA5 shaped an immunosuppressive tumor microenvironment by transcriptionally upregulating CCL20 via CAMKII/AKT/NF-κB signaling, thereby promoting the recruitment of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs). Furthermore, the CAMKII inhibitor KN93 effectively suppressed both tumorigenesis and immune evasion.\u003c/p\u003e\u003cp\u003e\u003cb\u003eConclusion\u003c/b\u003e\u003c/p\u003e\u003cp\u003eCollectively, our findings identify CHRNA5 as a critical driver of PDAC progression by promoting malignant behaviors and reshaping the immune microenvironment, highlighting its potential as a promising therapeutic target in pancreatic cancer.\u003c/p\u003e","manuscriptTitle":"CHRNA5 drives pancreatic cancer progression by promoting tumorigenesis and remodeling immune invasion microenvironment via CAMKII/AKT/NF-κB-CCL20 axis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-16 14:47:00","doi":"10.21203/rs.3.rs-7418128/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":"fb3aa533-04c3-4b59-a70f-e73452320536","owner":[],"postedDate":"September 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-20T06:46:45+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-16 14:47:00","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7418128","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7418128","identity":"rs-7418128","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}