Tripartite motif containing 69 elicits ERK2-dependent EYA4 turnover to impart pancreatic tumorigenesis

Yu Jia; Hui-Yan Li; Jue Wang; Xing Chen; Lu Lou; Yan-Yan Wei; Ying Wang; Shi-Jing Mo

doi:10.7150/jca.79905

Tripartite motif containing 69 elicits ERK2-dependent EYA4 turnover to impart pancreatic tumorigenesis

J Cancer. 2023 Jan 1;14(2):200-218. doi: 10.7150/jca.79905. eCollection 2023.

Authors

Yu Jia¹, Hui-Yan Li², Jue Wang³, Xing Chen², Lu Lou², Yan-Yan Wei², Ying Wang⁴, Shi-Jing Mo²

Affiliations

¹ Cancer Research Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei, P.R. China.
² General Surgical Laboratory, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, 510080, Guangdong, P.R. China.
³ Department of Pathology, The First Affiliated Hospital, Sun Yet-Sen University, Guangzhou 510080, Guangdong, P.R. China.
⁴ Department of Surgery, Huashan Hospital, Fudan University, Shanghai 200040, P.R. China.

Abstract

Eyes absent homologue 4 (EYA4) is silenced in pancreatic ductal adenocarcinoma (PDAC) and functions as a tumor suppressor to restrain PDAC development, albeit the molecular mechanism underlying its downregulation remains enigmatic. Methods: Functional studies were determined by immunohistochemistry of PDAC samples from patients and Pdx1-Cre; LSL-Kras^G12D/+; Trp53^fl/+ (KPC) mice, three-dimensional spheroid culture, flow cytometry, MTT and subcutaneous xenograft experiments. Mechanistical studies were examined by cellular ubiquitination, cycloheximide (CHX) pulse-chase, co-immunoprecipitation, chromatin immunoprecipitation, GST-pulldown, in vitro protein kinase assay, immunofluorescence and luciferase reporter assays. Results: We screen E3 ligase that is negatively correlated with EYA4 and uncover a mutually exclusive interaction of tripartite motif containing 69 (TRIM69) with EYA4 in human PDAC. TRIM69 elicits EYA4 polyubiquitylation and turnover independent of P53 and impedes the EYA4-driven deactivation of β-catenin/ID2 cascade, fueling PDAC cell proliferation in vitro and tumor development in mice. Expression of TRIM69 is upregulated in PDAC samples from independent cohorts of patients and the Pdx1-Cre; LSL-Kras^G12D/+; Trp53^fl/+ (KPC) mice, and associated with unfavorable prognosis. Depleting TRIM69 preferentially induces lethality in the EYA4-deficient PDAC cells. We further unearth that ERK2 directly binds to the D-site of mitogen-activated protein kinase (MAPK) docking groove in EYA4 Leu512/514 and phosphorylates EYA4 at Ser37, which is instrumental for EYA4 polyubiquitylation and turnover by TRIM69. Conclusion: Our results define a previously unappreciated role of TRIM69-EYA4 axis in pancreatic tumorigenesis and underscore that targeting TRIM69 might be an effective therapeutic approach for PDAC harboring EYA4 deficiency.

Keywords: Eyes absent homologue 4; Pancreatic ductal adenocarcinoma; Tripartite motif containing 69; Turnover.