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α5-nAChR/NETO2 contributed to chronic stress-promoted lung adenocarcinoma progression

Cancer Cell International volume 25, Article number: 67 (2025) Cite this article

Abstract

Background

α5-nicotinic acetylcholine receptor (α5‐nAChR) participates in chronic stress-promoted lung adenocarcinoma (LUAD) progression. Neuropilin and tolloid-like 2 (NETO2) contributes to fear expression and extinction, which is related to tumorigenesis. CHRNA5 (encoding α5‐nAChR) gene profiling revealed a reduction in NETO2 expression following CHRNA5 knockdown. Nevertheless, the connection between α5-nAChR and NETO2 in LUAD progression induced by chronic stress remains unclear.

Methods

RNA-Seq and bioinformatics database were used for analyzing the expression as well as correlation of α5-nAChR, together with NETO2 in LUAD. α5-nAChR and NETO2 expression were detected using immunohistochemistry in LUAD tissue microarrays, chronic restraint stress (CRS) and chronic unpredictable stress (CUMS) mice tissues. In lung adenocarcinoma A549 and H1299 cells, the expression of α5-nAChR, NETO2, p-CAMKII, p-STAT3 and vimentin induced by acetylcholine/nicotine was examined by western blot. The interaction of α5-nAChR with NETO2 in lung adenocarcinoma cells was detected by Co-immunoprecipitation assay and modeled using molecular docking. EdU assay and colony formation assay were conducted to evaluate cell proliferation, while wound healing assay as well as transwell assay assessed the migration and invasion of lung adenocarcinoma cells.

Results

α5-nAChR expression was related to NETO2 expression, low survival rate, staging as well as smoking status in LUAD dataset as well as tissue microarrays. The correlation between α5-nAChR and NETO2 was validated in nude mice xenograft tissues. α5-nAChR as well as NETO2 expression correlated in CRS and CUMS mice tissues. In vitro, acetylcholine/nicotine mediated NETO2, p-CAMKII, p-STAT3 and vimentin expression via α5-nAChR. α5-nAChR interacted with NETO2 as well as CAMKII in LUAD cells. α5-nAChR/NETO2 signaling contributed to LUAD cell proliferation, migration and invasion.

Conclusions

The above results uncover a new chronic stress-promoted LUAD signaling pathway: α5-nAChR/NETO2 axis contributes to chronic stress-promoted LUAD cell proliferation, migration and invasion.

Background

Lung cancer is an extremely invasive and highly prevalent disease. It is estimated that 2.20 million persons are diagnosed with lung cancer, while 1.79 million patients succumb to it each year [1]. In the whole world, lung cancer tops the list of tumor-linked fatalities among men and ranks second in women, trailing only breast cancer [2, 3]. Non-small cell lung cancer (NSCLC) constitutes the principal lung cancer subtype (comprising 80 ~ 85%). Within this category, lung adenocarcinoma (LUAD) accounting for 40% is the most leading type [4, 5].

Emotional changes (fear, anxiety, depression, despair, etc.) that occur in oncology patients triggered by the diagnosis, progression and treatment of the disease put the organism in the state of chronic stress [6, 7]. Chronic stress contributes to tumorigenesis, progression as well as metastasis by triggering the production of stress hormones, initiating inflammation as well as promoting body into a state of immunosuppression [8,9,10,11]. Chronic stress mediates tumor progression through stimulating the sympathetic nervous system to release norepinephrine, but there is limited study on the function and mechanism of the parasympathetic nervous system in tumor progression resulting from chronic stress [12, 13].

Nicotinic acetylcholine receptors (nAChRs) combine with the endogenous neurotransmitter acetylcholine (ACh) from the parasympathetic nervous system to mediate a range of physiological processes, such as neurotransmission, muscle contraction and sensory transduction [14, 15]. In addition, both ACh and nAChRs are important regulators of tumorigenesis and progression [16, 17]. Pharmacologically, nAChRs drive tobacco addiction [15, 18]. Genome-wide association studies (GWASs) showed that variants in nAChRs gene cluster CHRNA5/A3/B4 were strongly associated with tobacco-associated cancers, and among them, variants in the CHRNA5 gene (encoding α5-nAChR) were particularly related to lung carcinogenesis, which may be a candidate gene for lung cancer development [19]. α5-nAChR can activate multiple signaling pathways: STAT3/Jab1-PD-L1, Jab1/Csn5, TGF-β1/Smad, and so on, which plays important roles in lung adenocarcinoma progression and immune escape [20,21,22,23]. The nAChRs play a crucial role in various neurobiological mechanisms linked to the underlying causes of depression. Consequently, medications designed to interact with nAChRs could open up a promising new frontier in the treatment of depression [24]. Studies have shown that chronic stress results in elevated ACh in lung culture supernatant and the increase in ACh leads to the development of anxiety and depressive symptoms in human subjects and mouse models [25, 26]. Our previous study showed that chronic stress increases murine serum ACh concentrations, which in turn combine with α5-nAChR on LUAD cells to enhance tumor progression and metastasis [27].

Neuropilin and tolloid-like 2 (NETO2) serves as a single-channel transmembrane protein, which consists a transmembrane helix, a N-terminal extracellular domain and an intracellular C-terminal region [28, 29]. Several studies have shown that the LDLa structural domain, along with N-terminal and C-terminal regions of NETO2 are important for the regulation of the kainate receptors (KARs) [29]. KAR constitutes a subset of ionotropic glutamate receptor families, regulating neurotransmitter release, mediating excitatory synaptic transmission and influencing neuronal excitability and network dynamics [30]. Stress contributes to the elimination of fear [31], while NETO2 is necessary for fear expression and abatement [32]. NETO2 is a significant oncogene, which is highly linked to the progression of various tumors (gastric, esophageal, pancreatic, nasopharyngeal cancer & osteosarcoma, etc.) [33,34,35,36,37,38]. It has been shown that α5-nAChR and NETO2 activated the Ca2+/CaMKII signaling pathway mediated by Ca2+ inward flow in cancer [38, 39]. CaMKII is activated by phosphorylation, contributing to increased phosphorylation of STAT3 and inducing nuclear translocation [40]. Significantly, CHRNA5 gene expression profiling study revealed that silencing CHRNA5 markedly reduced the expression of NETO2 (dataset submitted to GEO, No. GSE101979) [41]. What is the relevance between α5-nAChR and NETO2 in chronic stress-promoted LUAD progression? No relevant reports have been retrieved.

In this research, we evaluated the expression of α5-nAChR and NETO2 in LUAD dataset and found that α5-nAChR expression was related to NETO2 expression, low survival, pathologic staging and smoking status. α5-nAChR and NETO2 expression were related in CRS as well as CUMS mice. In vitro, acetylcholine/nicotine mediated NETO2, p-CAMKII, p-STAT3 and vimentin expression via α5-nAChR. The α5-nAChR/NETO2 signaling pathway mediated the proliferation, migration as well as invasion of LUAD cells. This research sheds light on the roles and mechanisms of α5-nAChR and NETO2 in chronic stress-promoted LUAD progression, providing fresh strategies and rationale for LUAD prevention and therapy.

Materials and methods

Online database analysis

The GEPIA 2 online database was utilized to examine the correlation between CHRNA5 and NETO2 expression in lung adenocarcinoma and its association with the prognosis of LUAD patients. The UALCAN online database was used for assessing the expression of CHRNA5 and NETO2 in a range of human tumors, in different pathological stages of lung adenocarcinoma and in normal people, LUAD smoking besides non-smoking patients.

Cell culture

A549 and H1299 human lung adenocarcinoma cell lines were sourced from the Cell Resource Center of the Chinese Academy of Sciences (Shanghai, China), which were verified by STR profiling and confirmed mycoplasma-free. Cells were cultured in RIPM 1640 medium (HyClone) with 1% penicillin and streptomycin (Macgene) and 10% fetal bovine serum (Gibco), which were exposed to nicotine (1 µM, 16 h; Sigma) or ACh (10 µM, 48 h; Solarbio).

Lentiviral infection

To carry out experiments, we established stable cell lines with CHRNA5 knockdown and overexpression via CHRNA5-targeted lentivirus (Shanghai Jikai Genome Technology Co., Ltd). The infected cells underwent puromycin selection for a minimum of one week. Subsequently, q-PCR or WB was conducted to detect target mRNA or protein expression.

Small interfering RNA transfection

Upon reaching a cell fusion rate of 30 ~ 50%, siRNAs of NETO2 or the control (50 nM, Keyybio, Shandong, China) with Opti-MEM medium and Lipofectamine™ 2000 (Thermo Fisher Scientific) were added in 6-well plates. Cells were incubated with the mixture for six hours. After that, substitute with culture medium and proceed with incubation for 1 ~ 3 days to detect the mRNA expression using q-PCR, or 2 ~ 4 days to detect the protein expression by western blot.

Tissue microarray

The tissue microarray included 55 adenocarcinoma specimens and 53 adjacent non-tumor tissues (No. OD-CT‐RsLug04‐003). Each set of carcinomas specimen and adjacent non-tumor tissue was categorized based on the clinical data. Among the 55 samples, 25 were female and 30 were male, with ages spanning 45 to 82 years.

Immunohistochemistry (IHC)

Tissue sections, 4 micrometers thick, were baked in a baking machine for 2 h, cleared of wax in xylene, progressively hydrated using ethanol concentrations and subsequently soaked in citrate solution. At 4 °C, treat the sections with diluted primary antibodies overnight. Next day, remove the primary antibody diluent and incubate the secondary antibodies according to the instructions provided (Origene, PV-9001 and PV-9002). When staining, begin with the diaminobenzidine working solution, followed by hematoxylin for the cell nucleus. The stained tissue slices were dehydrated in gradient alcohol, cleaned with xylene and finally packaged with neutral gum. After drying, the sections were scanned and photographed using a digital pathology slide scanner (Pannoramic SCAN II).

Western blot (WB)

After washing the cells twice with PBS buffer, lyse and extract the protein. Perform 2.5 h SDS-PAGE electrophoresis and 1.5 h membrane transfer. At room temperature, 5% BSA was used to block for 2 h. At 4 °C, incubate with diluted primary antibodies overnight. The following day, wash away the primary antibodies and incubate the secondary antibodies at 25 °C for 45 min. Drip the chromogenic solution and use the FluorChenEgel imaging system to detect proteins.

Co-immunoprecipitation assay (CO-IP)

After cell lysis, mix it with α5-nAChR antibody (5 µg; Bio-Techne, NBP3-22231) or NETO2 antibody (5 µg; Thermo Fisher Scientific, PA5-48040) and incubate them overnight at 4 °C to detect endogenous protein interactions. Protein A/G-MagBeads were added and incubated for 1 ~ 2 h using a vertical mixer with shaking at 4 °C to bind antigen-antibody complex. Wash the magnetic beads 5 times with washing buffer, collect the supernatant after elution, followed by Western blotting to detect.

Molecular docking study

The protein models utilized for docking were CHRNA5 (Uniprot ID: P30532), NETO2 (Uniprot ID: Q8NC67) and CAMKIIα (Uniprot ID: Q9UQM7). HDOCK SERVER was utilized for protein-protein molecular docking. Protein pre-processing (addition of hydrogen atoms, deletion of water molecules and redundant ligands) was done by PyMol 2.4. Docking Score, Confidence Score and Ligand RMSD were used as the evaluation criteria for docking. The output was configured to display the top 10 optimal docking locations. The top-scoring model was chosen as the optimal docking model. At last‌, we utilized Pymol 2.4 to render the docking outcomes visually. Information about molecular docking interaction sites is listed in Table S1.

EdU assay

In this study, a 96-well plate as well as a kit (RiboBio) were used for EdU assay. According to the instructions, stain the cells with DAPI and EdU. The inverted-contrast microscope with High Content was applied to observe as well as take pictures.

Colony formation assay

Count approximately 800 cells in logarithmic growth phase per well and culture that in a 6-well plate for 1 ~ 2 weeks. During the cultivation process, change the culture medium approximately every 3 days and pay attention to observing the cell status. Observe whether these cells can proliferate and eventually form a colony containing at least 50 cells. Wash the cells with PBS buffer 2 ~ 3 times. At last, fix cells with methanol and stain them by 0.1% crystalline violet dye.

Wound healing assay

Count 200 thousand cells in logarithmic growth phase per well and inoculate them in a 6-well plate. In this experiment, low concentration serum medium (< 2%) was used to alleviate the negative effects of cell proliferation. When cell fusion was around 90%, use a sterile pipette tip of 20 or 200 µL to vertically scratch the cells. Observe the cells under a microscope and take photos at 0, 24, and 48 h after scratching.

Transwell assay

A 24-well plate as well as several 24-well transwell chambers (Corning) were applied to transwell assay. Approximately 50,000 cells in logarithmic growth phase per well were counted and inoculated. For transwell invasion assay, cover the chamber with Matrigel to simulate the extracellular matrix environment. After incubation in the incubator for 1 ~ 2 days, fix and stain the cells. Non-migratory or non-invasive cells were wiped away. The microscope with inverted contrast was used to observe and take photos for recording.

Chronic restraint stress (CRS) and Chronic unpredictable stress (CUMS) nude mice model

BALB/c nude mouses (28 ~ 42 days old) were subjected to environmental adaptation for one week before being separately modeled. Behavioral tests, including the elevated plus maze test, tail suspension test and the Y‑maze test were used to detect that the mice exhibited stress-like behaviors. Paraffin sections of nude mice CRS and CUMS models were obtained from our laboratory [27].

Xenograft tumor model

Nude mouses were kept in the animal laboratory, where humidity ranging between 30 ~ 40%, at 25 °C, with unrestricted access to nourishment and hydration. We obtained lung cancer xenografts paraffin sections of nude mouses from our laboratory, including NC group, NC with nicotine group (NC + NIC), CHRNA5 knockout group (KD) as well as KD + NIC group.

Statistical analysis

Data were processed and visualized with GraphPad Prism 9.0, Image J or SPSS v27.0, which were presented as the means ± SDs. Two-group contrasts were assessed using Student’s t test, whereas comparisons among multiple groups were conducted with one-way ANOVA followed by either two-tailed Dunnett’s or Tukey’s test. Pearson chi-square test was used for analyzing the correlation of α5-nAChR and NETO2 expression with gender, age and smoking history in LUAD patients. P < 0.05 was considered as statistically significant.

Results

CHRNA5 and NETO2 expression correlates with poor prognosis in LUAD datasets

RNA-Seq results showed that 1, 972 differentially expressed genes were found in A549 cells with CHRNA5 knockdown by comparison to control cells (GEO: GSE101979) [41], including NETO2 with reduced expression (Fig. 1A). Survival analysis using the GEPIA 2 online database showed that high expression of CHRNΑ5 or NETO2 in LUAD was linked to poor prognosis (Fig. 1B-C). In addition, KM curve of CHRNA5 and NETO2 high expression in LUAD stage I, II, III and IV, showing a trend that CHRNA5 or NETO2 high expression was associated with lower survival in later-stage patients compared to early-stage patients (Supplementary Fig. 1). CHRNA5 and NETO2 showed elevated expression in numerous tumors including 515 LUAD samples in comparison to corresponding normal tissues (Fig. 1D-G). CHRNA5 and NETO2 overexpression in LUAD patients were related to clinical stage based on the UALCAN online database (Fig. 1H-I). What’s more, the expression of CHRNA5 and NETO2 in LUAD was positively correlated in the GEPIA 2 online database (P < 0.05) (Fig. 1J).

Fig. 1
figure 1

Expression of CHRNA5 and NETO2 in the LUAD dataset. (A) Volcano plot displaying differentially expressed genes in A549 cells with CHRNA5-knockdown versus control cells (P < 0.01). (B, C) CHRNA5 and NETO2 expression correlated to poor prognosis in LUAD. (D, F) CHRNA5 and NETO2 expression in diverse cancers. (E, G) CHRNA5 and NETO2 expression in LUAD. (H, I) CHRNA5 and NETO2 expression associated with pathologic stage in LUAD. (J) Positive correlation between CHRNA5 and NETO2 expression in LUAD

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Expression of α5‑nAChR and NETO2 in LUAD tissue microarrays

α5-nAChR and NETO2 expression in tissue microarrays with 55 LUAD and 53 adjacent non-tumor tissue samples were examined using immunohistochemical methods. The foundings revealed that α5-nAChR and NETO2 expression were significantly more in LUAD tissues (63.6%, 35/55; 67.3%, 37/55) than in adjacent non-cancerous tissues, which was correlated to whether smoking or not (Fig. 2A-B). According to Pearson chi square test or scatter plot, it was found that a positive correlation between α5-nAChR expression and NETO2 expression in LUAD patients (Fig. 2C-E). In addition, the expression of α5-nAChR and NETO2 was not linked to gender or age but related to smoking status (Table 1). At the same time, that’s consistent with the UALCAN online database indicating LUAD smokers have higher expression of CHRNΑ5 and NETO2 than non-smokers (Fig. 2F-G).

Fig. 2
figure 2

Expression of α5-nAChR and NETO2 in LUAD tissue microarray. (A, B) α5-nAChR and NETO2 expression in LUAD carcinoma and adjacent non-tumor tissues of nonsmokers and smokers. Scare bar, 100 μm. (C, D) Correlation between α5-nAChR and NETO2 expression in 55 LUAD specimens. Pearson chi square test (P < 0.05). (E) Scatter plot of the correlation between α5-nAChR expression and NETO2 expression in LUAD tissues (r = 0.298, P < 0.05). (F, G) CHRNA5 and NETO2 expression in normal people, LUAD nonsmokers as well as smokers

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Table 1 Correlations of α5-nAChR and NETO2 expression with clinical parameters in lung adenocarcinoma specimens
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Expression levels of α5-nAChR and NETO2 are correlated in LUAD xenograft tissues of BALB/c nude mice

Since α5-nAChR expression has been shown to correlate to NETO2 expression in the LUAD datasets as well as in tissue microarrays, we further examined their expression levels in paraffin sections of LUAD xenograft tissues using immunohistochemical experiments. ‌In comparison to the NC group, α5-nAChR as well as NETO2 expression were notably lower in the KD group. Additionally, the expression of α5-nAChR and NETO2 was higher in the nicotine-treated xenograft tissue NC + NIC group than the NC group. The KD and KD + NIC groups confirmed this result (Fig. 3). These foundings indicate a positive correlation between the expression of α5-nAChR and NETO2 in LUAD xenograft tissues.

Fig. 3
figure 3

Expression of α5-nAChR and NETO2 in LUAD tumor xenograft tissues. (A, B) α5-nAChR, NETO2 expression in NC, NC + NIC, KD and KD + NIC groups of LUAD tumour xenograft tissues. Scale bar, 100 μm. (C, D) Quantitative outcomes of α5-nAChR as well as NETO2 expression in LUAD xenograft tissues. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

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Expression levels of α5-nAChR and NETO2 are correlated in chronic stress-promoted LUAD tissues

To explore the ‌relation between α5-nAChR and NETO2 in chronic stress-promoted LUAD, we detected the expression levels of both in the CRS and CUMS models. We found that in the CRS model, the expression of α5-nAChR as well as NETO2 were markedly higher in the stress group than in controls (Fig. 4A-B). Similarly, in CUMS model, the expression levels of α5-nAChR and NETO2 in stress group tissues were more than those in controls. Additionally, the overexpression of CHRNA5 (OE-CHRNA5) group had higher α5-nAChR and NETO2 expression than the control group. ‌By comparison with the untreated group, the expression of α5-nAChR and NETO2 were prominently reduced in the mecamylamine-treated group (Fig. 4C-F). These foundings indicate that both α5-nAChR and NETO2 expression levels are elevated and positively correlated in chronic stress-promoted LUAD tissues, and mecamylamine can reduce the expression of both simultaneously.

Fig. 4
figure 4

Expression of α5-nAChR and NETO2 in CRS and CUMS mice tumor tissues. (A, B) α5-nAChR, NETO2 expression in Control and CRS groups of CRS model mouse tumor tissues. Scale bar, 100 μm. The right panel displays the quantitative results. (C) α5-nAChR, NETO2 expression in Control, OE-CHRNA5 and OE-CHRNA5 + Mec groups of CUMS model mouse tumor tissues. Scale bar, 100 μm. (D) α5-nAChR, NETO2 expression in CUMS, CUMS + OE-CHRNA5 and CUMS + OE-CHRNA5 + Mec groups of CUMS model mouse tumor tissues. Scale bar, 100 μm. (E, F) Quantitative outcomes of α5-nAChR as well as NETO2 expression in CUMS mouses. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. There is no statistical significance for groups with the same letter markings, otherwise the differences are significant. The significance level is set at 0.05

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Nicotine/ACh mediates the expression of NETO2, CAMKII, STAT3 and Vimentin via α5-nAChR in LUAD cells

In order to further understand the relationship between smoking, chronic stress, α5-nAChR and NETO2 in LUAD cells, WB was applied to detect the protein levels of α5-nAChR, NETO2, T-STAT3, P-STAT3, P-CAMKII and Vimentin in A549 and H1299 cells with CHRNA5 knockout, ‌ coupled with‌ nicotine/ACh treatment. Based on prior studies, experimental conditions were set at 16 h with 1 µM nicotine and 48 h with 10 µM ACh [27, 42]. The findings indicated that the expression levels of α5-nAChR, NETO2, P-STAT3, P-CAMKII and Vimentin were up-regulated in nicotine/ACh-stimulated group compared to the controls; on the contrary, the expression of α5-nAChR, NETO2, P-STAT3, P-CAMKII and Vimentin were notably downregulated in cells expressing CHRNA5-siRNA. More importantly, compared to the CHRNA5 knockdown group, nicotine/ACh restored the expression of α5-nAChR, NETO2, P-STAT3, P-CAMKII and Vimentin after CHRNA5 knockout (Fig. 5). These findings indicate that nicotine/ACh regulates the expression of NETO2, P-STAT3, P-CAMKII and Vimentin in LUAD cells by activating α5-nAChR.

Fig. 5
figure 5

Nicotine/ACh mediates NETO2, CAMKII, STAT3 and Vimentin expression via α5-nAChR in vitro. (A-D) Nicotine mediates NETO2, T-STAT3, P-STAT3, P-CAMKII and Vimentin expression via α5-nAChR in LUAD cells. (E-H) ACh mediates NETO2, T-STAT3, P-STAT3, P-CAMKII and Vimentin expression via α5-nAChR in LUAD cells. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

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α5-nAChR interacts with NETO2 in LUAD cells

Given the link between α5-nAChR and NETO2, we detected the potential binding between these two proteins. Molecular docking showed that both of α5-nAChR and NETO2 proteins had high affinity with CAMKIIα, with docking scores of -304.11 (Fig. 6A) and  -267.99 (Fig. 6B). In addition, α5-nAChR strongly bound to NETO2 protein with a docking fraction of -302.11 (Fig. 6C). These results suggest the interactions between α5-nAChR, NETO2 and P-CAMKII in LUAD cells (Fig. 6D). We further examined the protein-protein interactions by Co-immunoprecipitation assays. The foundings showed that α5-nAChR, NETO2 co-immunoprecipitated in A549 cells and also respectively bound to P-CAMKII (Fig. 6E, G). Similar results were detected in H1299 cells (Fig. 6F, H). In addition, multicolor immunofluorescence assay confirmed the co-localization of α5-nAChR and NETO2 (Supplementary Fig. 2).

Fig. 6
figure 6

α5-nAChR interacts with NETO2 in vitro. (A) Molecular docking of α5-nAChR and CAMKIIα. Docking Score: -304.11; Confidence Score: 0.9562; Ligand RMSD: 70.33. (B) Molecular docking of NETO2 and CAMKIIα. Docking Score: -267.99; Confidence Score: 0.9137; Ligand RMSD: 72.15. (C) Molecular docking of α5-nAChR and NETO2. Docking Score: -302.11; Confidence Score: 0.9544; Ligand RMSD: 59.38. (D) α5-nAChR, NETO2 and P-CAMKII interactions in vitro. (E, G) Based on WB to detect the interactions of α5-nAChR, NETO2 and P-CAMKII in A549 cells. (F, H) Based on WB to detect the interactions of α5-nAChR, NETO2 and P-CAMKII in H1299 cells

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α5-nAChR/NETO2 signaling promotes LUAD cell proliferation, migration and invasion

We evaluated the effect of α5-nAChR/NETO2 signaling on cell proliferation using colony formation assay and EdU assay. The results showed that cell proliferation was significantly increased in the OE-CHRNA5 group and decreased in the Si-NETO2 group in comparison with the controls. Moreover, the OE-CHRNA5 + Si-NETO2 group could upregulate cell proliferation in the Si-NETO2 group (Figs. 7A-D and 8A-D). In addition, transwell assay as well as wound healing assay using to detect cell migration and invasion functions showed that the OE-CHRNA5 group had the fastest wound healing rate and the most cells undergoing migration and invasion, while the Si-NETO2 group had the slowest healing rate and the least cells, whereas the OE-CHRN5 + Si-NETO2 group could rescue the alteration in the Si-NETO2 group. (Figures 7E-J and 8E-H). These findings indicate that α5-nAChR/NETO2 signaling involves in and promotes the proliferation, migration as well as invasion of LUAD cells.

Fig. 7
figure 7

α5-nAChR/NETO2 signaling promotes LUAD cell proliferation, migration and invasion. (A, C) Colony formation assay demonstrating that NETO2 knockout inhibits proliferation of A549/H1299 cells. (B, D) Quantitative outcomes of colony formation assay. (E, H) Transwell assay demonstrating that NETO2 suppression restricts A549/H1299 cell migration and invasion. Scale bar, 100 μm. (F, I) Quantitative outcomes of Transwell migration assay. (G, J) Quantitative outcomes of Transwell invasion assay. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

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Fig. 8
figure 8

α5-nAChR/NETO2 signaling promotes LUAD cell proliferation and migration. (A, C) EdU assay on A549 and H1299 cells, positive spots labeling nascent DNA in red and DAPI-stained nuclei in blue. Scale bar, 50 μm. (B, D) Quantitative results of EdU assay. (E, G) Wound healing assay on LUAD cells, and migrating cells were imaged at 0, 24 and 48 h post-scratching. Scale bar, 200 μm. (F, H) Quantitative results of wound healing assay. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

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Discussion

Chronic stress arises from environmental or psychosocial triggers ([10]. Patients with cancer have numerous serious sources of stress and around one third of cancer patients also suffer from depression without sufficient attention ([43]. Thus, there is an urgent need to unravel the molecular mechanisms between depression and cancer progression and develop effective treatments for cancer patients suffering from depression. In this research, we revealed an underlying mechanism between α5-nAChR and NETO2 by which depression may facilitate lung cancer progression. The results showed that chronic stress promotes LUAD proliferation, migration as well as invasion through α5-nAChR/NETO2 activating the CAMKII/STAT3 pathway.

Most researches on the role of chronic stress in promoting tumors have focused on the sympathetic nervous system ([44, 45], but we still know little about the effect of the parasympathetic nervous system. Acetylcholine serves as a key neurotransmitter linking postganglionic neurons to effector cells in parasympathetic nervous system ([46]. Previously, we have demonstrated that chronic stress facilitates tumor growth by activating ACh released from the parasympathetic nervous system to promote the progression of chronic stress-induced LUAD through the α5-nAChR/FHIT signaling ([27]. In addition, the α5-nAChR subunit gene harbors the rs16969968 polymorphism, which is recognized as an important indicator of smoking risk ([47, 48]. In this study, we found through UALCAN online analysis that CHRNA5 was significantly expressed in different types of cancers, including LUAD, which was strongly associated with smoking in LUAD. This result aligns with previous reports that nicotine combines with nAChR, triggering various downstream signaling cascades that bring about cancer initiation and progression ([49]. Next, we identified the key role of α5-nAChR in nicotine-induced lung carcinogenesis through in vivo as well as vitro experiments.

Given that CHRNA5 is both a key gene in chronic stress-promoted LUAD development and closely relates to nicotine-induced lung carcinogenesis. In this study, RNA-Seq was conducted on A549 cells with CHRNA5 knockout as well as the controls. The findings indicated that CHRNA5 suppression notably reduced the expression of NETO2 gene, which was identified as an interaction partner of KAR through a proteomic method combining immunoprecipitation and mass spectrometry in 2009 ([28]. KAR has been shown to be linked to epilepsy as well as other neuropsychiatric disorders ([30]. Research has shown that NETO2 exhibits significant expression within the neonatal dorsal root ganglia (DRG) of mice, influencing KAR gating within these neurons through a developmentally controlled mechanism ([50]. As the significant impact of NETO2 on KAR function, NETO2 as an auxiliary protein of the KAR can provide an important therapeutic target. What’s more, NETO2 expression is indispensable in the elimination of fear by the organism, while stress is equally beneficial for fear extinction ([31, 32]. α5-nAChR can enhance the penetration of calcium ions, thereby increasing the intracellular calcium concentration in the presence of acetylcholine ([51]. Acetylcholine/α5-nAChR axis-mediated Ca²⁺ inward flow activates CAMKII in intrahepatic cholangiocarcinoma ([39]. The crucial role of α5-nAChR in mediating Ca²⁺ fluxes was also previously reported in nerve cells ([52]. NETO2 is involved in mediating calcium inward flow in normal cells ([53]. NETO2 promoted melanoma progression via activation of the Ca²⁺/CaMKII signaling pathway ([38]. In addition, in this study, our clinical tissue microarray revealed that both CHRNA5 and NETO2 were abundantly expressed in lung cancer tissues by comparison to paraneoplastic tissues, demonstrating a positive correlation. And their expression was not related to gender or age, but to smoking. Moreover, we found that NETO2 was also demonstrated to be highly expressed in all kinds of‌ cancers and involved in tumor progression. For example, NETO2 was dysregulated and participated in cellular signaling in colorectal cancer ([54].

Based on the currently available data (55 LUAD patients), the Pearson chi-square test showed no correlation between α5-nAChR and NETO2 expression with gender and age. This finding does not exclude sample size limitations. We plan to address this point in our next studies: on the one hand, to increase the clinical sample size and validate it in a larger cohort; on the other hand, to continue to follow the same participants to obtain more data to validate the existing conclusions.

Our study showed that in vitro experiments, nicotine or acetylcholine mediated NETO2, CAMKII expression in LUAD cells through α5-nAChR. Moreover, α5-nAChR/NETO2 signaling was found to facilitate LUAD cell proliferation, migration as well as invasion through EdU assay, colony formation assay, wound healing assay, together with transwell assay. The next step is to construct an in vitro cellular chronic stress model to further study α5-nAChR interacts with NETO2 as well as CAMKII in chronic stress-promoted lung adenocarcinoma progression. Here, we utilized two mouse models widely used in stress-related disease research to study the expression as well as relationship of α5-nAChR and NETO2 under the context of chronic stress. The findings revealed that we observed the accumulation of α5-nAChR and NETO2 in cancer tissues of stressed animals, which were positively correlated. The above results suggest that chronic stress activates the CAMKII/STAT3 pathway through α5-nAChR/NETO2 signaling, which promotes the proliferation, migration as well as invasion of LUAD.

Conclusions

‌To sum up‌, this research offers fresh insights on the potential molecular mechanisms of α5-nAChR/NETO2 in the progression of chronic stress-promoted LUAD. Chronic stress-induced ACh or nicotine and its analogs combine with cell surface α5-nAChR that interacts with NETO2, activating CAMKII/STAT3 signaling mediated by Ca2+ influx, which subsequently promotes LUAD cell proliferation, migration as well as invasion (Fig. 9). ‌As far as we know, our research for the first time supports the correlation between the involvement of α5-nAChR and NETO2 in the progression of chronic stress-promoted LUAD that could serve as an innovative target for treatment of depressed LUAD co-patients.

Fig. 9
figure 9

Proposed signalling cascades of α5-nAChR and NETO2 participate in chronic stress-promoted LUAD progression

Full size image

Chronic stress-induced ACh or nicotine and its analogs (NNK, etc.) bind to cell surface α5-nAChR that interacts with NETO2, activating CAMKII/STAT3 signaling mediated by Ca2+influx, which subsequently promotes LUAD cell proliferation, migration as well as invasion.

Data availability

All relevant data are available in the manuscript and supplementary information.

Abbreviations

α5-nAChR:

α5-nicotinic acetylcholine receptor

NETO2:

Neuropilin and tolloid-like 2

nAChRs:

Nicotinic acetylcholine receptors

ACh:

Acetylcholine

CRS:

Chronic restraint stress

CUMS:

Chronic unpredictable stress

LUAD:

Lung adenocarcinoma

KARs:

Kainate receptors

NSCLC:

Non-small cell lung cancer

GWASs:

Genome-wide association studies

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Funding

This study was funded by the Natural Science Foundation of Shandong Province (ZR2021MH322 and ZR2022QC156) and the National Natural Science Foundation of China (32170496).

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Authors and Affiliations

  1. Research Center of Basic Medicine, Central Hospital Affiliated to Shandong First Medical University, Jinan, Shandong, 250013, China

    Jingting Wang, Jiaying Cai, Shuran Yang, Jing Wang, Yanfei Jia & Xiaoli Ma

  2. Department of Medical Laboratory, Shandong Second Medical University, Weifang, Shandong, 261053, China

    Zengping Wang & Xiaoli Ma

  3. College of Life Science, Shandong Normal University, Shandong, 250014, China

    Haiji Sun

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Contributions

JW and XM: Design the research and writing. JW, JC, ZW, SY and JW: Conduct experiments and process data. YJ, HS and XM: Draft the manuscript. XM: Provide funding, revise and supervise. All authors have read and approved the final manuscript.

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Correspondence to Xiaoli Ma.

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This research was approved by the Ethics committee of the Institutional Animal Care and Use Committee of Central Hospital Affiliated to Shandong First Medical University and conformed to the Declaration of Helsinki (approval NO. JNCH2021-77). The research utilizing human tissue samples was conducted in accordance with the approval of the ethics committee of Shanghai Outdo Biotech (approval NO. SHXC2021YF01). Written informed consent was obtained from all patients prior to participation.

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Wang, J., Cai, J., Wang, Z. et al. α5-nAChR/NETO2 contributed to chronic stress-promoted lung adenocarcinoma progression. Cancer Cell Int 25, 67 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12935-025-03701-5

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Cancer Cell International

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