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Cellular and Molecular Biology

Aberrant PJA2-CHRM3 signaling creates a therapeutic vulnerability in gastric tumor

British Journal of Cancer volume 133, pages 1122–1136 (2025)Cite this article

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Abstract

Background

Vagal innervation plays a pivotal role in gastric tumorigenesis and tumor progression. However, the upstream signaling regulating acetylcholine receptors (AChRs) and its contribution to carcinogenesis remains largely elusive.

Methods

We constructed Trp53−/−; Cdh1−/− mouse gastric organoids to recapitulate the morphological and functional characteristics of diffuse-type gastric cancer (DGC) for FDA-approved drug (1464 compounds) screening. We investigated the effects of AChR inhibitors in diffuse-type patient-derived organoids (PDOs) by examining IC50 and xenograft tumorigenesis. Mass spectrometry and Co-immunoprecipitation was used to identify the interaction between cholinergic receptor muscarinic 3 (CHRM3) and PJA2. Ubiquitination and degradation assays were used to explore the regulation of CHRM3 by PJA2. 120 human gastric cancer specimens and GEO database were used to explore the clinical relevance of PJA2-CHRM3 signaling.

Results

mAChR inhibitors were identified as the most effective at suppressing Trp53−/−; Cdh1−/− organoids. Consistently, the viability of PDOs with aberrant expression of CHRM3 can be significantly inhibited by oxybutynin hydrochloride (OXY) and nortriptyline hydrochloride (NOR). Mechanistically, the RING E3 ligase PJA2 ubiquitinates and degrades CHRM3, subsequently suppressing downstream TGFβ-pSMAD3 signaling and tumor cell progression. Clinically, low PJA2 expression was correlated with high CHRM3, p-SMAD3 and choline acetyltransferase (ChAT) expression and predicted poor outcomes. Biologically, PJA2 but not its catalytically dead ΔRING mutant could suppress PDOs with aberrant CHRM3 signaling. PJA2 depletion enhanced tumor metastasis of gastric cancer cells, subsequently reversed by OXY and NOR treatment.

Conclusions

This previously unknown PJA2-CHRM3 signaling axis provides further understanding of cholinergic innervation as well as identifies a new therapeutic vulnerability in DGC.

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Fig. 1: mAChR inhibitors effectively suppress the vitality of Trp53−/−; Cdh1−/− organoids.
Fig. 2: OXY and NOR significantly inhibits PDOs both in vitro and in vivo.
Fig. 3: OXY and NOR suppresses TGFβ signaling pathway via CHRM3.
Fig. 4: PJA2 negatively regulates the stability of CHRM3.
Fig. 5: PJA2 inhibits gastric cancer via the CHRM3-TGFβ signaling pathway.
Fig. 6: The clinical relevance of PJA2-CHRM3 signaling in gastric cancer.
Fig. 7: Aberrant PJA2-CHRM3-TGFβ signaling and treatment strategies for diffuse-type gastric tumor innervation.

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Data availability

The results displayed here are partially based on data generated by The Cancer Genome Atlas (TCGA) Research Network (https://www.cancer.gov/tcga), Asian Cancer Research Group (ACRG) database (https://consortiapedia.fastercures.org/consortia/acrg/) and Chinese Glioma Genome Atlas (CGGA) database (http://www.cgga.org.cn/). The publicly available dataset reused in this study, including Kumar et al.’ dataset (11 NT and 29 PT samples), was retrieved from GSE183904 (PubMed ID: 34642171). Kaplan–Meier Plotter (https://kmplot.com/analysis/) were used for survival analysis of patients with gastric cancer by survival curves tests. The RNA-Seq data has been uploaded to the Sequence Read Archive (SRA), BioProject ID: PRJNA1073869. All the data supporting the findings of this study are available from the corresponding authors upon reasonable request.

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Acknowledgements

We thank Dr. Rongkui Luo (Department of Pathology, Gastric Cancer Center, Zhongshan Hospital, Fudan University, Shanghai, China) for his kind help in evaluation, IHC staining, and Lauren classification of tissue microarrays. This work was supported by the National Natural Science Foundation of China to LYH (82472666, 82272994 and 82073098), and the Interdisciplinary Program of Shanghai Jiao Tong University (YG2022QN086 and YG2025QNB47).

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Author notes

  1. These authors contributed equally: Lingmeng Li, Jingyi Yin.

Authors and Affiliations

  1. Key Laboratory of Systems Biomedicine (Ministry of Education) and Collaborative Innovation Center of Systems Biomedicine, Shanghai Center for Systems Biomedicine, Shanghai Jiao Tong University, Shanghai, China

    Lingmeng Li, Jingyi Yin, Ling Zhang, Yinan Niu & Liyu Huang

  2. Department of Gastrointestinal Surgery, Gastric cancer center, Zhongshan Hospital, Fudan University, Shanghai, China

    Wenshuai Liu, Leihao Wang, Junjie Zhao, Yuan Gu, Zhaoming Wang & Xuefei Wang

  3. Department of General Surgery, Zhongshan Hospital, Fudan University, Shanghai, China

    Wenshuai Liu, Leihao Wang, Junjie Zhao, Yuan Gu, Zhaoming Wang & Xuefei Wang

  4. Department of General Surgery, Zhongshan Hospital (Xiamen), Fudan University, Shanghai, China

    Junjie Zhao & Xuefei Wang

  5. Laboratory animal center, Instrumental analysis center, Shanghai Jiao Tong University, Shanghai, China

    Dongli Liang

  6. D1 Medical Technology Company, Nanxiang Precision Medicine Industrial Park, Shanghai, China

    Guoxiang Fu & Guoqiang Hua

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Contributions

LYH designed and supervised the study; LML and JYY performed most of the experiments; LZ, WSL, YNN, LHW and GXF helped some biochemical experiments and data curation; DLL helped some animal experiments; LYH, LML and JYY wrote the manuscript; JJZ, YG, ZMW, GQH and XFW were responsible for writing–review and editing. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Liyu Huang.

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The authors declare no competing interests.

Ethics approval and consent to participate

The animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) of Shanghai Jiao Tong University (Approval Number: 20221035). This study, involving human participants, was approved by the Ethics Committee of Fudan University (Approval Number: B2021-449R) and was carried out in accordance with the ethical standards stipulated in the 1964 Declaration of Helsinki. Each patient receives written informed consent.

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Li, L., Yin, J., Zhang, L. et al. Aberrant PJA2-CHRM3 signaling creates a therapeutic vulnerability in gastric tumor. Br J Cancer 133, 1122–1136 (2025). https://doi.org/10.1038/s41416-025-03145-8

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