Deoxycholic Acid Drives ATP4A-Dependent Growth and Migration of Vocal Fold Leukoplakia Epithelial Cells via TGR5/JAK2/STAT3 Signaling

Jia-Yu Zhang; Tian-Jiao Bi; Qin-Ying Wang; Jiang-Tao Zhong; Yang-Yang Bao; Zi-Ming Fu; Zhe Chen; Heng-Chao Chen; Zai-Zai Cao; Shui-Hong Zhou

doi:10.1016/j.jvoice.2025.11.012

Deoxycholic Acid Drives ATP4A-Dependent Growth and Migration of Vocal Fold Leukoplakia Epithelial Cells via TGR5/JAK2/STAT3 Signaling

J Voice. 2025 Nov 29:S0892-1997(25)00490-4. doi: 10.1016/j.jvoice.2025.11.012. Online ahead of print.

Authors

Jia-Yu Zhang¹, Tian-Jiao Bi², Qin-Ying Wang³, Jiang-Tao Zhong⁴, Yang-Yang Bao⁵, Zi-Ming Fu⁶, Zhe Chen⁷, Heng-Chao Chen⁸, Zai-Zai Cao⁹, Shui-Hong Zhou¹⁰

Affiliations

¹ Department of Otolaryngology, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003, Zhejiang, China; Department of Otolaryngology, Shaoxing Yuecheng People's Hospital, Shaoxing City 312000, Zhejiang Province, China. Electronic address: 460270380@qq.com.
² Department of Otolaryngology, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003, Zhejiang, China; Zhejiang Provincial Clinical Research Center for Head & Neck Cancer, Hangzhou 310014, China. Electronic address: btj674013426@qq.com.
³ Department of Otolaryngology, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003, Zhejiang, China; Zhejiang Provincial Clinical Research Center for Head & Neck Cancer, Hangzhou 310014, China. Electronic address: huxl324@163.com.
⁴ Department of Otolaryngology, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003, Zhejiang, China; Zhejiang Provincial Clinical Research Center for Head & Neck Cancer, Hangzhou 310014, China. Electronic address: 724318981@qq.com.
⁵ Department of Otolaryngology, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003, Zhejiang, China; Zhejiang Provincial Clinical Research Center for Head & Neck Cancer, Hangzhou 310014, China. Electronic address: bao4300383@yeah.net.
⁶ Department of Otolaryngology, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003, Zhejiang, China; Zhejiang Provincial Clinical Research Center for Head & Neck Cancer, Hangzhou 310014, China. Electronic address: 929526150@qq.com.
⁷ Department of Otolaryngology, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003, Zhejiang, China; Zhejiang Provincial Clinical Research Center for Head & Neck Cancer, Hangzhou 310014, China. Electronic address: 153187129@qq.com.
⁸ Department of Otolaryngology, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003, Zhejiang, China; Zhejiang Provincial Clinical Research Center for Head & Neck Cancer, Hangzhou 310014, China. Electronic address: Chenhengchao@126.com.
⁹ Department of Otolaryngology, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003, Zhejiang, China; Zhejiang Provincial Clinical Research Center for Head & Neck Cancer, Hangzhou 310014, China. Electronic address: 352093177@qq.com.
¹⁰ Department of Otolaryngology, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003, Zhejiang, China; Zhejiang Provincial Clinical Research Center for Head & Neck Cancer, Hangzhou 310014, China. Electronic address: 1190051@zju.edu.cn.

PMID: 41320587
DOI: 10.1016/j.jvoice.2025.11.012

Abstract

Purpose: Vocal fold leukoplakia (VFL) is the most common precancerous lesion of laryngeal carcinoma, with variable malignant potential. The pathogenesis of VFL remains incompletely understood. Increasing evidence indicates that laryngopharyngeal reflux, especially bile acid reflux, may drive epithelial transformation.

Methods: ATP4A and ATP4B expression were assessed by immunohistochemistry in 58 VFL and 15 laryngeal squamous cell carcinoma (LSCC) specimens. Primary VFL epithelial cells were treated with graded concentrations of deoxycholic acid (DCA, 50-400 μM). Small interfering RNAs targeting ATP4A and TGR5, as well as the JAK2/STAT3 inhibitor AG490, were employed to delineate signaling mechanisms. Proliferation and migration were measured by cell counting kit (CCK)-8 and Transwell assays, respectively, while mRNA and protein levels were analyzed using quantitative real-time-PCR and Western blotting.

Results: ATP4A positivity was significantly higher in LSCC than in VFL (47.57 ± 15.75% vs 33.47 ± 21.22%, (P = 0.018), whereas ATP4B showed no significant difference. In vitro, DCA at 0-200 μM dose-dependently increased proliferation, migration, ATP4A expression, and TGR5/JAK2/STAT3 phosphorylation, while higher concentrations (300-400 μM) suppressed cell activity. ATP4A knockdown reversed DCA-induced proliferation and migration. Similarly, TGR5 silencing or AG490 treatment abrogated DCA-mediated upregulation of ATP4A and functional enhancement.

Conclusions: DCA activates the TGR5/JAK2/STAT3 axis to upregulate ATP4A, thereby promoting VFL epithelial cell proliferation and migration. These findings provide novel mechanistic insight linking bile acid reflux to VFL progression and identify TGR5/ATP4A as potential therapeutic targets.

Keywords: ATP4A; Bile acids; JAK2/STAT3 pathway; Laryngopharyngeal reflux; TGR5; Vocal fold leukoplakia.