Cholinergic signals promote esophageal squamous cell carcinoma metastasis by activating EGFR/AKT signaling pathway

doi:10.21203/rs.3.rs-1534161/v3

Purpose Increasing evidence has shown that the nervous system plays a vital role in the regulation of tumorigenesis. However, the regulatory mechanism of the parasympathetic nervous system in the progression of esophageal squamous cell carcinoma (ESCC) has not been elucidated.

Methods Vesicular acetylcholine transporter (VAChT) protein expression was detected by immunohistochemistry (IHC) and parasympathetic nerve density was analyzed in human ESCC tissues. The co-culture model of ESCC cells and dorsal root ganglia (DRG) was established in vitro. The effects of cholinergic stimulation on cell growth, migration and invasion were investigated through CCK-8, Transwell and wound healing assays using acetylcholine (ACh). PCR and western blotting were utilized respectively for the purpose of detecting Epithelial-to-mesenchymal transition (EMT) related transcripts proteins. M3 muscarinic receptor (CHRM3) was knocked down using small interfering RNA (siRNA). RNA sequencing explored the possible altered gene transcriptome and signal pathways, which were further verified by Western blotting and CO-IP analysis.

Results The intensity of parasympathetic nerve in ESCC was significantly higher than that in adjacent tissues, and was positively correlated with higher clinical staging and poorer prognosis. Co-incubation with DRG enhanced the migration and invasion ability of ESCC cells in vitro. Moreover, exogenous choline stimulation enhanced the migration, invasion ability together with EMT of ESCC cells, while CHRM3 knockdown were opposite. Mechanistically, cholinergic activation enhanced the phosphorylation levels of EGFR and AKT through CHRM3, which bound directly to EGFR.

Conclusion We revealed a critical role of cholinergic signals ACh/CHRM3 in mediating the effects of parasympathetic nerve activity on ESCC through the EGFR/AKT signaling pathway, which might provide a novel therapeutic target for ESCC.

Parasympathetic system

Acetylcholine

CHRM3

Esophageal squamous cell carcinoma

Metastasis

Esophageal cancer is the seventh-most commonly diagnosed cancer and sixth leading cause of cancer-related mortality worldwide [1]. The two most common histologic subtypes are esophageal adenocarcinoma and esophageal squamous cell carcinoma (ESCC), the latter of which is dominant in China. Conventional treatment based on surgery, radiotherapy, chemotherapy and targeted therapy represent the main strategy for esophageal cancer treatment, but it nevertheless has a five-year survival rate of < 30% in most countries [2]. Despite combination treatment, most patients still experience late recurrence and metastasis. The discovery of new therapeutic targets for ESCC is a key issue to be urgently addressed.

The nervous system plays a leading role in modulating development, homeostasis, tissue repair, regeneration, and plasticity [3]. Recently, increasing evidence has shown that neurogenesis and axogenesis play positive roles in tumor development. Tumor tissue contains a large number of nerve fibers in various malignant tissues, such as prostate cancer [4], breast cancer [5], gastric cancer [6], glioma [7] and pancreatic cancer [8], and higher nerve infiltration is closely related to higher clinical staging and poorer prognosis. Direct or indirect cross-talk between neurons and tumors mediates malignant tumor phenotypes, including promoting tumor proliferation and growth [7], inducing lymph node metastasis [9], secreting soluble factors to promote angiogenesis [10] and regulating immune cells to induce immune escape [11].

The autonomic nervous system consists of sympathetic and parasympathetic nerves. Local and systemic effects of parasympathetic nerve fibers are closely related to the occurrence and development of tumors, relying on the cancer type and receptors expressed within the tumor microenvironment [12]. Several studies have confirmed that the parasympathetic nerve plays an important role in the malignant progression of gastrointestinal tumors [13, 14]. The esophagus is innervated by both sympathetic and parasympathetic nerves, while the latter are dominant. A pioneering study employing immunohistochemical methods to detect nerves in 260 cases of esophageal cancer (both esophageal adenocarcinoma and ESCC) found that nerves were detected in 38% of esophageal cancers, which is correlated to a greater extent in ESCC patients. Further studies have confirmed that cancer cells secrete nerve growth factor to drive nerve infiltration [15]. Nevertheless, the authors did not clarify how neural activity affects ESCC development.

In summary, the effects and underlying mechanisms involving the parasympathetic nervous system in the development of ESCC have not been uncovered. In this study, exogenous acetylcholine (ACh) stimulation promoted ESCC metastasis and epithelial-to-mesenchymal transition (EMT) in vitro. Inhibition of M3 muscarinic receptor (CHRM3) via small interfering RNA (siRNA) effectively inhibited ESCC cell migration and invasion. Furthermore, RNA-seq showed that ACh/CHRM3 promoted ESCC development by activating the EGFR/AKT signaling pathway, by which CHRM3 combined with EGFR and promoted its phosphorylation. Overall, we provide clues that parasympathetic infiltration is essential for ESCC progression and suggest that targeting cholinergic signaling may represent a therapeutic strategy for ESCC patient treatment.

Reagents and antibodies

Small interference RNAs (siRNAs) were from Ribobio (Guangzhou, China). Primers used for qPCR sequence were from Ribobio (Guangzhou, China), which are as follows: GAPDH (Forward: 5′-GGTCCTCAGTGTAGCCCAAG-3′, Reverse: 5′-AATGTGTC CGTCGTGGATCT-3′), E-cadherin (Forward: 5′-GTCTCTCTCACCACCTCCACA-3′, Reverse: 5′-CTCGGACACTTCCACTCTCTTT-3′), N-cadherin (Forward: 5′-TGCTA CTTTCCTTGCTTCTGAC-3′, Reverse: 5′-TAACACTTGAGGGGCATTGTC-3′), Vimentin (Forward: 5′-GAAGAGAACTTTGCCGTTGAAG-3′, Reverse: 5′-GAAGG TGACGAGCCATTTC-3′), Snail (Forward: 5′-TCGGAAGCCTAACTACACGA-3′, Reverse: 5′-AGATGAGCATTGGCAGCGAG-3′), Twist (Forward: 5′-GCAAGAAGT CGAGCGAAGAT-3′, Reverse: 5′-GCTCTGCAGCTCCTCGAA -3′).

Antibodies for GAPDH (60004-1-Ig), EGFR (18986-1-AP), AKT (10176-2-Ig), p-AKT (66444-1-Ig), N-cadherin (22018-1-AP), E-cadherin (20874-1-AP), Vimentin (10366-1-AP) were purchased from proteintech. Antibodies for p-EGFR (DF2969) were purchased from Affinity. Antibodies for CHRM3 (GTX111637) were purchased from Gene Tex. Reagents of 4-DAMP (1952-15-4), ACh (60-31-1) were purchased from Sigma Aldrich.

Cell lines and cell culture

Human ESCC cell lines, including TE1 and KYSE30, were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Both of them were cultured in RPMI-1640 medium supplemented containing 10% fetal bovine serum (Clark, AUS). All cell lines were maintained in a humidified cell incubator at 37°C with an atmosphere of 5% CO₂.

Clinical tissue microarray and Immunohistochemical staining

76 ESCC tissues and pair-matched para-carcinoma tissues were obtained from ESCC patients who underwent radical surgery at the Affiliated Huai’an No.1 People’s Hospital of Nanjing Medical University (Huai’an, China). Each of them was evaluated histologically by two experienced pathologists. This study was approved by the Committee for Ethical Review of Research involving Human Subjects of Nanjing Medical University and informed consent was obtained from all patients.Tissues were paraffin-embedded and cut into 4 μm think sections, which were then deparaffinized and rehydrated. All sections were incubated with anti-VAChT antibodies at 4°C in the refrigerator overnight. After incubating with HRP-conjugated secondary antibody (Dako, Glostrup, Denmark), the sections were counterstained with Mayer’s hematoxylin. The neural density calculation was independently evaluated by two pathologists.

RNA interference

Sequences of siRNA used for knockdown of CHRM3 were previously described [16] and synthesized by OBiO Technology (Shanghai, China). Cells were transfected with siRNAs using Lipofectamine 3000 (Invitrogen, USA), according to the manufacturer’s protocol. The interference efficiency was determined by western blotting after transfection for 48 h.

Wound-healing assay

Cells were seeded into 6-well plate and scratches across the cell layers were made by the 200 μL pipet. Floating cells were washed 2 times with PBS. Photos were taken under microscope at 0, 24, 48, 72 and 96 h. The wound healing of each area was measured by ImageJ.

Migration and invasion assay

Cell migration and invasion ability were analyzed using transwell chambers (8-μm pore size, Corning Costar, Cambridge, MA, USA). 100 μL resuspended cells (0.8×10⁵per mL) were inoculated in each transwell, then cultured in 600 μL complete medium for 72 h. After 72 h incubation, cells were washed with PBS. Then penetrated cells were fixed by 4% paraformaldehyde for 25 minutes and stained with 600 μL 0.1% crystal violet solution for another 25 minutes. Pictures were collected under microscope by randomly selecting five fields of vision and the migration ability of cells was analyzed. Unlike migration, in invasion assay, the upper-chamber needed to add 100 μL matrigel gel (diluted with medium without FBS) in advance.

Real-time quantitative PCR (qRT-PCR)

Total RNA was extracted using Trizol reagent (Thermo, USA) as the manufacturer’s instructions. The cDNA was reversed with FastQuant RT Kit (Tiangen, Beijing, China). Then, PCR reaction was performed in triplicate with SuperReal PreMix Plus (Tiangen, Beijing, China) on the Real-Time PCR Detection System (Roche, California, USA). The formula 2^-^△△^CT was used for relative expression, GAPDH as negative control. Western blot and Immunoprecipitation assay

Cells were dissolved in lysis buffer (RIPA, KeyGEN, China) and the concentrations were examined by BCA Protein Assay kit (KeyGEN, China). For each sample,30 μg total protein was applied for 10% or 7.5% SDS-PAGE. The proteins were transferred to PVDF membranes (Roche, Mannheim, Germany). The membranes were sealed with 5% BSA and incubated with primary antibodies in 4°C overnight and corresponding secondary antibodies at normal atmospheric temperature for 1 h the next day. Protein bands were visualized with ECL substrate (Affinity). For CO-IP, Protein A/G PLUS-Agarose (SANTA CRUZ BIOTECHNOLOGY, INC) was used. Protein bands were visualized with Automatic chemiluminescence image analysis system (Bio-Rad, USA).

RNA-seq and data analysis

Cell gene expression profile (choline stimulation groups and controls) was detected by RNA screening analysis in Novogenen Company (Tianjin, China). Raw data statistical significance assessment was accomplished using a Welch t-test with Benjamini-Hochberg FDR (|fold change | > 1 and padj < 0.05 as significant). Significant difference analysis and function analysis based on Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was executed, and |Z - score | > 2 is considered meaningful.

Dorsal root ganglia (DRG) co-cultured with ESCC cells

The DRGs were extracted according to the method previously described [17]. The mice were carefully necked and expose the position of the spine. Cut off the diaphragm, viscera and ribs from the anterior side of the spine, then cut the femur with a bone shear, and finally make a transverse cut at the femur level to remove the entire spine. From the head to the tail, peel off the spine to find the lower dorsal root ganglion and expose it. Carefully lift the dorsal root ganglion upward, using fine spring shears to trim the small axon bundles and soft tissues around DGRs, then transfer them to precooled HBSS solution. ESCC cells in logarithmic growth phase were digested with trypsin, terminated by complete medium, centrifuged and resuscitated, then transferred to 6-well plate or 24-well plate. DRGs were planted in the upper or lower layer of transwell chamber as needed to establish a co-incubation model.

Statistical analysis

All the cell experiments above were performed in triplicate. All data were analyzed by SPSS26.0 (SPSS, Chicago, IL, USA) or GraphPad PrismV8 (GraphPad Prism, Inc., La Jolla, CA, USA) and presented in the form of mean ± standard deviation (SD). Student’s t-test or one-way ANOVA was used for statistical analysis and p < 0.05 was considered to be significant. Nerve density and the patient clinicopathological parameters was performed by χ2-test and Kaplan-Meier survival analysis with log-rank test.

1. Higher parasympathetic nerve infiltration is associated with malignant phenotype and poor prognosis of ESCC patients

To better understand the role of parasympathetic nerve innervation in ESCC, IHC analysis was performed to visualize parasympathetic nerve density by detecting the expression of VAChT, a specific marker of parasympathetic nerves [18, 19], in ESCC tissues in comparison with adjacent paracancerous tissues, uncovering the upregulated expression of VAChT in ESCC (Fig. 1A). As shown by the data analyzed from paired samples of 76 patients, there was significantly higher parasympathetic nerve density in tumor tissues than in adjacent paracancerous tissues in ESCC (Fig. 1B). Correlation analysis involving parasympathetic nerve density and clinical characteristics revealed that parasympathetic nerve infiltration was positively correlated with ESCC malignancy, including TNM stage and lymph node metastasis (Table 1). Moreover, patients with higher parasympathetic nerve infiltration had a poorer survival rate, as determined by Kaplan–Meier analysis (Fig. 1C). Together, these data demonstrated that parasympathetic nerve infiltration may be a critical promoter of ESCC progression.

2. Co-culture with DRG enhances cell migration and invasion in vitro

We first probed the effects of neural activity in the phenotype of ESCC cells based on the research above. An in vitro culture system consisting of DRG and ESCC cells was established because it has been proven that DRG neurons can synthesize and excrete ACh and are cholinoceptive [20, 21]. Compared to the control, the proliferation ability of KYSE30 and TE1 cells co-cultured with DRGs did not change (Supplementary file 1). However, we confirmed that cell migration and invasion were significantly enhanced by co-culturing with DRGs (Fig. 2A-D).

3. Cholinergic signals facilitate metastatic attributes of ESCC cells

The parasympathetic nerve mainly secretes acetylcholine to activate cholinergic signals that are linked with a range of physiological and pathological processes [22]. We used acetylcholine, a stable cholinergic agonist, to interfere with KYSE30 and TE1 cells. To further explore the influence of cholinergic signaling on ESCC progression, we found that exogenous choline stimulation significantly increased cell migration and invasion abilities in a dose-dependent manner, as detected by wound healing and Transwell assays (Fig. 3A-H). We performed CCK-8 and colony formation assays, which indicated that exogenous choline stimulation had no effect on proliferation of ESCC cells (Supplementary file 1).

EMT plays a central role in the metastatic ability of tumor cells, resulting in tissue infiltration and metastatic foci formation [23]. We performed PCR and western blotting to detect the expression of mesenchymal markers in KYSE30 cells. The results showed that exogenous choline stimulation decreased the levels of E-cadherin but increased those of N-cadherin, vimentin, Snail, and Twist (Fig. 4A and B). Consistently, immunofluorescence assays showed decreased membrane localization of E-cadherin, but increased expression of N-cadherin and vimentin, while the cells displayed a spindle-like morphology (Fig. 4C). Together, these findings indicated that cholinergic signaling facilitates metastasis and EMT in ESCC.

4. Knockdown of CHRM3 inhibits ACh-induced cell migration and invasion

Acetylcholine mainly acts through M-type receptors [24]. CHRM3, one of the most important M-type receptors, dominates cancer progression [25-27]. Thus, we investigated whether CHRM3 mediates ACh-induced migration and invasion of ESCC cells. Our study revealed that the increased migration and invasion abilities observed in KYSE30 cells via exogenous choline stimulation in wound healing and Transwell assays were partially restrained when CHRM3 was silenced (Fig. 5A–F). CHRM3 knockdown also inhibited N-cadherin and vimentin protein levels, but upregulated the expression of E-cadherin (Fig. 5G). These results indicated that CHRM3 mediates ACh-induced cell metastasis and EMT in ESCC.

5. ACh activates intracellular EGFR/AKT signaling pathway through the binding of CHRM3 to EGFR

To investigate which signaling pathway ACh produced a marked effect on ESCC cells through CHRM3, we performed RNA-seq to identify differentially expressed genes (DEGs). A total of 1,207 genes had higher expression in the choline stimulation group than in the control group, while 1,471 genes showed lower expression (Fig. 6A). KEGG pathway analysis revealed several impaired pathways, including protein processing in endoplasmic reticulum miRNAs, tight junction, non-small cell lung cancer and so on (Fig. 6B). The signaling pathways such as microRNAs in cancer, ErbB signaling pathway and focal adhesion were significantly up-regulated (Fig. 6C). Then we calculated the significantly activated molecular pathway with -log10 (padj) and found that ErbB pathway ranked first (Fig. 6D).

Epidermal growth factor receptor (EGFR), one of the most important members of the ErbB family, is a transmembrane glycoprotein belonging to the receptor tyrosine kinase group [28, 29]. Based on the important role EGFR plays in tumors [30-32], we verified how the EGFR/AKT pathway was activated. Further work showed increased protein levels of p-EGFR and p-AKT in choline-stimulated groups from 1 h to 4 h (Fig. 7A). Consistently, CHRM3 knockdown partially reversed their expression (Fig. 7B). Previous studies have shown that EGFR is a transmembrane glycoprotein that binds to various ligands, including EGF. Linage of the protein with a ligand induces EGFR dimerization and tyrosine autophosphorylation, contributing to activation of key downstream molecules [29]. To further explore whether CHRM3 could interact with EGFR to activate the signaling pathway, we performed a CO-IP assay. The results showed that CHRM3 could bind to EGFR (Fig. 7C) and increase autophosphorylation. Together, CHRM3 mediated cholinergic signaling in ESCC progression by combining with EGFR, promoting EGFR autophosphorylation, and activating p-AKT.

The autonomic nervous system is composed of sympathetic and parasympathetic nerves, the influence of parasympathetic nerves on gastrointestinal tumors has been preliminarily recognized. Previous studies have shown that increased parasympathetic activity has pro-tumorigenic effects, and the use of muscarinic receptor inhibitors or vagotomy can reduce tumorigenesis and metastasis. However, some researchers have argued that cholinergic signals exhibit tissue-dependent effects. CHRM3 is upregulated in gastric cancers, and surgical or pharmacological denervation suppresses gastric tumorigenesis [13]. However, parasympathetic deletion accelerates cancer progression in pancreatic cancer, and the use of the non-selective muscarinic agonist bethanechol, which stimulates cholinergic signaling, suppresses pancreatic cancer progression in transgenic and orthotopic xenograft models [33, 34]. It can be seen that parasympathetic nerve can promote and inhibit tumor growth in the digestive system, and its mechanism needs to be further clarified. The present study is the first to investigate parasympathetic cholinergic signaling in ESCC progression and the possible mechanisms involved.

In this study, we noticed that parasympathetic nerve innervation in ESCC tissues was higher than that in adjacent non-cancerous tissues and had a significant clinical correlation with ESCC TNM stage and lymph node metastasis, indicating that parasympathetic activity might be a novel diagnostic marker of ESCC. Our results further demonstrated that cholinergic activation contributed to the migration and invasion of ESCC cells in a dose-dependent manner, but not proliferation in vitro. EMT is a primary process that involves increased cancer progression and metastasis with the loss of epithelial markers and gain of mesenchymal markers [35], such as in cervical cancer [36]. In turn, we detected several EMT-related genes in KYSE30 cells with or without cholinergic stimulation, including downregulated expression of E-cadherin and upregulated expression of N-cadherin, vimentin, and Twist. Our data demonstrated that cholinergic signaling could promote ESCC metastasis and EMT.

The parasympathetic cholinergic system is essentially based on ACh as a mediator. ACh is synthesized from choline and acetylCoA by the choline acetyltransferase (ChAT) enzyme and stored in vesicles through vesicular ACh transporter (VAChT) activity. It binds to two distinctive types of receptors, the nicotinic acetylcholine receptor (nAChR) and muscarinic acetylcholine receptor (mAChR) [37]. CHRM3, a mAChR, is a leading transmitting element involved in signal transmission in the parasympathetic vagus nerve. Previous studies have shown that the vagus nerve can increase cell proliferation and tumor progression in the brain and stomach through CHRM3 [38, 39]. Consistent with our hypothesis, we found that CHRM3 knockdown by siRNA decreased the metastatic attributes of ESCC cells. At the same time, we observed a gain in epithelial markers and a loss of mesenchymal markers.

To determine the mechanisms underlying these phenomena, we performed RNA-seq and found that the ErbB signaling pathway was the most upregulated pathway by KEGG pathway analysis. The ERBB family of transmembrane receptor tyrosine kinases (RTKs) consists of EGFR (ERBB1), HER2 (ERBB2), HER3 (ERBB3), and HER4 (ERBB4) [28]. EGFR is also involved in cell proliferation, mitosis, and cancer progression [29]. We explored whether ACh could work through CHRM3 to activate EGFR signaling in ESCC cells. This is supported by our findings that the expression of p-EGFR was significantly increased after ACh stimulation, and the activation levels of p-EGFR and p-AKT were reversed by siRNA-mediated CHRM3 knockdown. In addition, CO-IP analysis confirmed that CHRM3 could combine with EGFR and increase p-EGFR levels by activating dimerization and tyrosine autophosphorylation. Further investigations are required to assess why CHRM3 binds to EGFR and which binding sites or domains are involved in binding. In addition to the phosphorylation of downstream AKT, it is also necessary to investigate what other effects their reciprocal combinations might have.

Previous studies have observed that neural activity can enhance the proliferation of tumor cells in vitro [4, 6, 7, 13]. Notably, our results were in contrast to the expectation that parasympathetic cholinergic stimulation promotes ESCCs proliferation via co-culture and exogenous ACh stimulation, even if the ACh concentration was 1000 μM.

Similar to our results, capsaicin ablation of sensory neurons resulted in significantly more lung and heart metastasis in mice, but the growth of primary tumors was not affected in a breast cancer study[40]. In the same group of breast cancer studies, the number of lung, liver and kidney metastasis increased after unilateral vagotomy in mice. However, the tumor volume of vagotomy mice did not change compared with the sham operation group [41]. There are several possible explanations for this finding. First, to a large extent, given the heterogeneity among different types of cancers, histological typing of ESCC tissues indicates that they are less affected by cholinergic stimulation. Second, either the different cells in TME or TME in different cancers might have an effect on cancer cells directly or indirectly. Therefore, in vitro experiments can only explain part of the problem because we have not explored this phenomenon in murine models. It also corroborates with the observation that parasympathetic activity might contribute to ESCC metastasis through other mechanisms in the TME, in addition to cell growth. Consequently, we will further study whether the parasympathetic nerve has any effect on ESCC cell proliferation by constructing orthotopic transplanted tumor mouse models.

The deficiency of this study lies in direct data support of experiments in vivo. We did not establishe the model of mouse esophageal carcinoma in situ successfully, and we could not observe its effect on the growth, invasion and metastasis of ESCC by disconnecting the parasympathetic nerve. In addition, for different types of cholinergic receptors on ESCC cell membrane, we did not analyze their expression after ACh stimulation, therefore, we cannot rule out the possibility that other receptors might play a more important role. From the perspective of TME integrity, the expression of different cholinergic receptors on cancer cells and other cell membranes, the activation of different intracellular pathways, different immune modes of immune cells, the different rate of ACh secretion and host susceptibility all lead to challenges in the study of the effect of parasympathetic nerve on tumor. Moreover, the complex interactions of the parasympathetic nervous system at the systemic and local levels also need to be considered.

In conclusion, parasympathetic activity plays a key role for ESCC progression, and highlights a component of the TME that contributes to metastasis. These data suggest that a critical role of cholinergic signals ACh/CHRM3 in mediating the effects of parasympathetic nerve activity on ESCC through the EGFR/AKT signaling pathway, which might provide a novel therapeutic target for ESCC.

Acknowledgements We would like to thank Editage (www.editage.cn) for English language editing.

Funding This research was sponsored by the Jiangsu Natural Science Foundation of China (grant no. BK20211112).

Authors’ contributions Y.G., B.W., and T.W. conceived and designed this study. T.W., B.G., and C.L. performed experiments. T. W. and B. G. wrote the manuscript. Y. G. and B.W. provided insightful discussions and comments regarding the manuscript. All authors have approved the final submitted manuscript.

Availability of data and materials The data that support the fndings of this study are openly available in GEO databases and The Cancer Genome Atlas database (https://www.ncbi.nlm.nih. gov/gds).

Ethics approval and consent to participate All methods were carried out in accordance with the Declaration of Helsinki. No ethics approval was required for this work. All utilized public data sets were generated by others who obtained ethical approval. This study was approved by the Committee for Ethical Review of Research involving Human Subjects of Nanjing Medical University and informed consent was obtained from all patients.

Competing interests The authors state that they have no conflict of interest.

Consent for publication No applicable.

Author’ information Department of Medical Oncology, Cancer Center, The Affiliated Huaian No.1 People’s Hospital of Nanjing Medical University, Huai’an 223300, China.

Sung, H., et al., Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin, 2021. 71(3): p. 209–249. https://doi.org/10.3322/caac.21660
Allemani, C., et al., Global surveillance of trends in cancer survival 2000-14 (CONCORD-3): analysis of individual records for 37†513†025 patients diagnosed with one of 18 cancers from 322 population-based registries in 71 countries. Lancet, 2018. 391(10125): p. 1023–1075. https://doi.org/10.1016/s0140-6736(17)33326-3
Venkatesh, H. and M. Monje, Neuronal Activity in Ontogeny and Oncology. Trends Cancer, 2017. 3(2): p. 89–112. https://doi.org/10.1016/j.trecan.2016.12.008. https://www.ncbi.nlm.nih.gov/pubmed/28718448.
Magnon, C., et al., Autonomic nerve development contributes to prostate cancer progression. Science, 2013. 341(6142): p. 1236361. https://doi.org/10.1126/science.1236361. https://www.ncbi.nlm.nih.gov/pubmed/23846904.
Kappos, E.A., et al., Denervation leads to volume regression in breast cancer. J Plast Reconstr Aesthet Surg, 2018. 71(6): p. 833–839. https://doi.org/10.1016/j.bjps.2018.03.012. https://www.ncbi.nlm.nih.gov/pubmed/29653898.
Hayakawa, Y., et al., Nerve Growth Factor Promotes Gastric Tumorigenesis through Aberrant Cholinergic Signaling. Cancer Cell, 2017. 31(1): p. 21–34. https://doi.org/10.1016/j.ccell.2016.11.005. https://www.ncbi.nlm.nih.gov/pubmed/27989802.
Venkatesh, H.S., et al., Neuronal Activity Promotes Glioma Growth through Neuroligin-3 Secretion. Cell, 2015. 161(4): p. 803–16. https://doi.org/10.1016/j.cell.2015.04.012. https://www.ncbi.nlm.nih.gov/pubmed/25913192.
Banh, R.S., et al., Neurons Release Serine to Support mRNA Translation in Pancreatic Cancer. Cell, 2020. https://doi.org/10.1016/j.cell.2020.10.016. https://www.ncbi.nlm.nih.gov/pubmed/33142117.
Pundavela, J., et al., Nerve fibers infiltrate the tumor microenvironment and are associated with nerve growth factor production and lymph node invasion in breast cancer. Mol Oncol, 2015. 9(8): p. 1626–35. https://doi.org/10.1016/j.molonc.2015.05.001. https://www.ncbi.nlm.nih.gov/pubmed/26009480.
Zahalka, A.H., et al., Adrenergic nerves activate an angio-metabolic switch in prostate cancer. Science, 2017. 358(6361): p. 321–326. https://doi.org/10.1126/science.aah5072.
Wang, Z., et al., Acetylcholine promotes the self-renewal and immune escape of CD133 + thyroid cancer cells through activation of CD133-Akt pathway. Cancer Lett, 2020. 471: p. 116–124. https://doi.org/10.1016/j.canlet.2019.12.009. https://www.ncbi.nlm.nih.gov/pubmed/31830559.
Tibensky, M. and B. Mravec, Role of the parasympathetic nervous system in cancer initiation and progression. Clin Transl Oncol, 2021. 23(4): p. 669–681. https://doi.org/10.1007/s12094-020-02465-w.
Zhao, C., et al., Denervation suppresses gastric tumorigenesis. Science translational medicine, 2014. 6(250): p. 250ra115. https://doi.org/10.1126/scitranslmed.3009569.
Raufman, J.P., et al., Muscarinic receptor subtype-3 gene ablation and scopolamine butylbromide treatment attenuate small intestinal neoplasia in Apcmin/+ mice. Carcinogenesis, 2011. 32(9): p. 1396–402. https://doi.org/10.1093/carcin/bgr118.
Griffin, N., et al., Clinicopathological Significance of Nerves in Esophageal Cancer. Am J Pathol, 2020. 190(9): p. 1921–1930. https://doi.org/10.1016/j.ajpath.2020.05.012.
Wang, N., et al., Autocrine Activation of CHRM3 Promotes Prostate Cancer Growth and Castration Resistance via CaM/CaMKK-Mediated Phosphorylation of Akt. Clin Cancer Res, 2015. 21(20): p. 4676–85. https://doi.org/10.1158/1078-0432.Ccr-14-3163.
Sleigh, J.N., G.A. Weir, and G. Schiavo, A simple, step-by-step dissection protocol for the rapid isolation of mouse dorsal root ganglia. BMC Res Notes, 2016. 9: p. 82. https://doi.org/10.1186/s13104-016-1915-8.
Kamiya, A., et al., Genetic manipulation of autonomic nerve fiber innervation and activity and its effect on breast cancer progression. Nat Neurosci, 2019. 22(8): p. 1289–1305. https://doi.org/10.1038/s41593-019-0430-3.
Arvidsson, U., et al., Vesicular acetylcholine transporter (VAChT) protein: a novel and unique marker for cholinergic neurons in the central and peripheral nervous systems. J Comp Neurol, 1997. 378(4): p. 454–67.
Bernardini, N., et al., Detection of basal and potassium-evoked acetylcholine release from embryonic DRG explants. J Neurochem, 2004. 88(6): p. 1533–9. https://doi.org/10.1046/j.1471-4159.2003.02292.x.
Corsetti, V., et al., Expression of Cholinergic Markers and Characterization of Splice Variants during Ontogenesis of Rat Dorsal Root Ganglia Neurons. Int J Mol Sci, 2021. 22(11). https://doi.org/10.3390/ijms22115499.
Jing, M., et al., An optimized acetylcholine sensor for monitoring in vivo cholinergic activity. Nat Methods, 2020. 17(11): p. 1139–1146. https://doi.org/10.1038/s41592-020-0953-2.
Greaves, D. and Y. Calle, Epithelial Mesenchymal Transition (EMT) and Associated Invasive Adhesions in Solid and Haematological Tumours. Cells, 2022. 11(4). https://doi.org/10.3390/cells11040649.
Chen, J., et al., Acetylcholine receptors: Key players in cancer development. Surg Oncol, 2019. 31: p. 46–53. https://doi.org/10.1016/j.suronc.2019.09.003.
von Rosenvinge, E.C., et al., Bedside to bench: role of muscarinic receptor activation in ultrarapid growth of colorectal cancer in a patient with pheochromocytoma. Mayo Clin Proc, 2013. 88(11): p. 1340–6. https://doi.org/10.1016/j.mayocp.2013.06.023.
Goto, Y., et al., Muscarinic receptors promote castration-resistant growth of prostate cancer through a FAK-YAP signaling axis. Oncogene, 2020. 39(20): p. 4014–4027. https://doi.org/10.1038/s41388-020-1272-x.
Tolaymat, M., et al., The Role of M3 Muscarinic Receptor Ligand-Induced Kinase Signaling in Colon Cancer Progression. Cancers (Basel), 2019. 11(3). https://doi.org/10.3390/cancers11030308.
Arteaga, C.L. and J.A. Engelman, ERBB receptors: from oncogene discovery to basic science to mechanism-based cancer therapeutics. Cancer Cell, 2014. 25(3): p. 282–303. https://doi.org/10.1016/j.ccr.2014.02.025.
Sabbah, D.A., R. Hajjo, and K. Sweidan, Review on Epidermal Growth Factor Receptor (EGFR) Structure, Signaling Pathways, Interactions, and Recent Updates of EGFR Inhibitors. Curr Top Med Chem, 2020. 20(10): p. 815–834. https://doi.org/10.2174/1568026620666200303123102.
Sierra, J.C., et al., Epidermal growth factor receptor inhibition downregulates Helicobacter pylori-induced epithelial inflammatory responses, DNA damage and gastric carcinogenesis. Gut, 2018. 67(7): p. 1247–1260. https://doi.org/10.1136/gutjnl-2016-312888.
da Cunha Santos, G., F.A. Shepherd, and M.S. Tsao, EGFR mutations and lung cancer. Annu Rev Pathol, 2011. 6: p. 49–69. https://doi.org/10.1146/annurev-pathol-011110-130206.
Sheng, Q. and J. Liu, The therapeutic potential of targeting the EGFR family in epithelial ovarian cancer. Br J Cancer, 2011. 104(8): p. 1241–5. https://doi.org/10.1038/bjc.2011.62.
Partecke, L.I., et al., Subdiaphragmatic vagotomy promotes tumor growth and reduces survival via TNFα in a murine pancreatic cancer model. Oncotarget, 2017. 8(14): p. 22501–22512. https://doi.org/10.18632/oncotarget.15019.
Renz, B.W., et al., Cholinergic Signaling via Muscarinic Receptors Directly and Indirectly Suppresses Pancreatic Tumorigenesis and Cancer Stemness. Cancer Discov, 2018. 8(11): p. 1458–1473. https://doi.org/10.1158/2159-8290.Cd-18-0046.
Pastushenko, I. and C. Blanpain, EMT Transition States during Tumor Progression and Metastasis. Trends Cell Biol, 2019. 29(3): p. 212–226. https://doi.org/10.1016/j.tcb.2018.12.001.
Qureshi, R., H. Arora, and M.A. Rizvi, EMT in cervical cancer: its role in tumour progression and response to therapy. Cancer Lett, 2015. 356(2 Pt B): p. 321 – 31. https://doi.org/10.1016/j.canlet.2014.09.021.
Russo, P., et al., Cholinergic receptors as target for cancer therapy in a systems medicine perspective. Curr Mol Med, 2014. 14(9): p. 1126–38. https://doi.org/10.2174/1566524014666141015152601.
Wang, L., et al., Muscarinic acetylcholine receptor 3 mediates vagus nerve-induced gastric cancer. Oncogenesis, 2018. 7(11): p. 88. https://doi.org/10.1038/s41389-018-0099-6.
Revesz, D., et al., Effects of vagus nerve stimulation on rat hippocampal progenitor proliferation. Exp Neurol, 2008. 214(2): p. 259 – 65. https://doi.org/10.1016/j.expneurol.2008.08.012.
Erin, N., et al., Capsaicin-mediated denervation of sensory neurons promotes mammary tumor metastasis to lung and heart. Anticancer Res, 2004. 24(2b): p. 1003–9.
Erin, N., et al., Vagotomy enhances experimental metastases of 4THMpc breast cancer cells and alters substance P level. Regul Pept, 2008. 151(1–3): p. 35–42. https://doi.org/10.1016/j.regpep.2008.03.012.

Table. 1 Relationship between parasympathetic nerve density and tumor characteristics in patients with ESCC

Features	No . of patients	Nerve density		p value
Features	No . of patients	high	low	p value
All patients	76	38	38
Age (years)				0.622
＜65	52	27	25
≥65	24	11	13
Gender				0.105
Male	58	26	32
Female	18	12	6
Grade				0.358
1	29	15	14
2	45	23	22
3	2	0	2
T infiltrate				0.446
T1	2	0	2
T2	10	5	5
T3	58	29	29
T4	6	4	2
Lymphatic metastasis (N)				0.049*
N0	62	27	35
N1	12	9	3
N2	2	2	0
Metastasis (M)				0.159
M0	46	20	26
M1	30	18	12
TNM stage				0.019*
Ⅰ	3	0	3
Ⅱ	54	24	30
Ⅲ	19	14	5

(*p < 0.05)

No competing interests reported.

Cholinergic signals promote esophageal squamous cell carcinoma metastasis by activating EGFR/AKT signaling pathway

Status:

Version 3

Abstract

Figures

Introduction

Materials And Methods

Results

Discussion

Declarations

References

Tables

Additional Declarations

Supplementary Files

Status:

Version 3