Silencing of AJAP1 expression by promoter methylation activates the Wnt/β-catenin signaling pathway to promote tumor proliferation and metastasis in salivary adenoid cystic carcinoma

Liehao Jiang; Yunye Liu; Yan Pan; Zhuo Tan; Jiafeng Wang; Guowan Zheng; Chenhong Qian; Shiying Xu; Xin Zhu; Wenli Ma; Susanna Guerrini; Pedro Infante-Cossio; Jiajun Wu; Minghua Ge; Xiujun Cai

doi:10.21037/gs-23-127

Silencing of AJAP1 expression by promoter methylation activates the Wnt/β-catenin signaling pathway to promote tumor proliferation and metastasis in salivary adenoid cystic carcinoma

Gland Surg. 2023 Jun 30;12(6):834-852. doi: 10.21037/gs-23-127. Epub 2023 Jun 27.

Authors

Liehao Jiang^{1

2

3}, Yunye Liu², Yan Pan², Zhuo Tan^{2

3}, Jiafeng Wang^{2

3}, Guowan Zheng^{2

3}, Chenhong Qian⁴, Shiying Xu⁵, Xin Zhu⁶, Wenli Ma⁷, Susanna Guerrini⁸, Pedro Infante-Cossio⁹, Jiajun Wu⁷, Minghua Ge^{2

3}, Xiujun Cai^{1

10}

Affiliations

¹ Zhejiang University School of Medicine, Zhejiang University, Hangzhou, China.
² Otolaryngology & Head and Neck Center, Cancer Center, Department of Head and Neck Surgery, Zhejiang Provincial People's Hospital (Affiliated People's Hospital, Hangzhou Medical College), Hangzhou, China.
³ Key Laboratory of Endocrine Gland Diseases of Zhejiang Province, Hangzhou, China.
⁴ Department of Breast Surgery, Jiaxing Second Hospital, Jiaxing, China.
⁵ Department of Breast Armor Surgery, Huzhou First People's Hospital, Huzhou, China.
⁶ Zhejiang Cancer Research Institute, The Cancer Hospital of the University of Chinese Academy of Sciences (Zhejiang Cancer Hospital), Hangzhou, China.
⁷ Bengbu Medical College, Bengbu, China.
⁸ Unit of Diagnostic Imaging, Department of Medical Sciences, Azienda Ospedaliero-Universitaria Senese, University of Siena, Siena, Italy.
⁹ Department of Surgery, School of Medicine, University of Seville, Seville, Spain.
¹⁰ Department of General Surgery, Sir Run-Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China.

Abstract

Background: Salivary adenoid cystic carcinoma (SACC) is a unique malignant tumor of the salivary gland with poor prognosis, which is not effective with chemotherapy and targeted drugs. Therefore, it is important to explore the molecular mechanism underlying SACC invasion and metastasis to develop novel therapeutic strategies and targets in clinical research.

Methods: Real-time quantitative polymerase chain reaction (RT-qPCR) and western blot (WB) were performed to detect the expression of Adherens Junctions Associated Protein 1 (AJAP1). Methylation-specific PCR was used to evaluate the methylation of the AJAP1 promoter. AJAP1 was overexpressed or knocked down by lentivirus-mediated transfection. Kaplan-Meier analysis was conducted to create a survival curve and the log-rank test was used to analyze the overall survival (OS). The prognostic correlation was assessed using univariate and multivariate Cox regression analyses. Co-immunoprecipitation (Co-IP) was utilized to pull down the possible binding protein of AJAP1 and laser scanning confocal microscopy was applied to detect the subcellular localization of AJAP1, E-cadherin, and β-catenin. Cell viability, colony formation, wound healing, and Transwell invasion assays were performed to evaluate the function of AJAP1 in vitro. A subcutaneous xenograft assay in nude mice was performed to verify the function of AJAP1 in vivo.

Results: AJAP1 was downregulated in SACC tumors and was closely related to SACC lymph node/distant metastasis, which was an independent risk factor for SACC prognosis. Methylation-specific PCR confirmed that high methylation of the AJAP1 promoter was the main cause of its silencing. Overexpression or knockdown of AJAP1 in SACC cells could significantly inhibit or promote the proliferation, invasion, and metastasis of SACC cells, respectively, in both the in vitro and in vivo experiments. Mechanically, we found that AJAP1 binds to E-cadherin and β-catenin to form a complex in cytomembrane, reducing the nuclear translocation of β-catenin and blocking the Wingless/Integrated/β-catenin (Wnt/β-catenin) signaling pathway to play a suppressive role in cancer.

Conclusions: In conclusion, these results suggest that the downregulation of AJAP1 protein expression may play a certain role in progression and metastasis of SACC. Our study indicates that AJAP1 may be a potential prognostic molecular marker and therapeutic target for SACC.

Keywords: AJAP1; Salivary adenoid cystic carcinoma (SACC); metastasis; methylation; β-catenin.