Aleksandra Toloczko1,2,5, Fusheng Guo1,5, Hiu-Fung Yuen1, Qing Wen3, Stephen A. Wood4, Yan Shan Ong1, Pei Yi Chan1, Asfa Alli Shaik1, Jayantha Gunaratne1, Mark J. Dunne2, Wanjin Hong1,6 and Siew Wee Chan1,6
1 Institute of Molecular and Cell Biology, Agency for Science, Technology and Research (A*STAR), Singapore, Republic of Singapore
2 ISchool of Medical Sciences, Faculty of Biology, Medicine and Health, University of Manchester, United Kingdom
3 ICentre for Public Health and Centre for Cancer Research & Cell Biology, School of Medicine, Dentistry and Biomedical Sciences, Queen’s University Belfast, Belfast, United Kingdom
4 Eskitis Institute for Drug Discovery, Griffith University, Queensland, Australia
5 These authors contributed equally to this work
6 Corresponding authors: email@example.com (Siew Wee Chan), firstname.lastname@example.org (Wanjin Hong).
Published in Cancer Research, 77(18):4921-4933 on September 15, 2017
The core LATS kinases of the Hippo tumor suppressor pathway phosphorylate and inhibit the downstream transcriptional co-activators YAP and TAZ, which are implicated in various cancers. Recent studies have identified various E3 ubiquitin ligases that negatively regulate the Hippo pathway via ubiquitination, yet few deubiquitinating enzymes (DUB) have been implicated. In this study, we report the DUB USP9X is an important regulator of the core kinases of this pathway. USP9X interacted strongly with LATS kinase and to a lesser extent with WW45, KIBRA, and Angiomotin, and LATS co-migrated exclusively with USP9X during gel filtration chromatography analysis. Knockdown of USP9X significantly downregulated and destabilized LATS and resulted in enhanced nuclear translocation of YAP and TAZ, accompanied with activation of their target genes. In the absence of USP9X, cells exhibited an epithelial-to-mesenchymal transition phenotype, acquired anchorage-independent growth in soft agar, and led to enlarged, disorganized, three-dimensional acini. YAP/TAZ target gene activation in response to USP9X knockdown was suppressed by knockdown of YAP, TAZ, and TEAD2. Deletion of USP9X in mouse embryonic fibroblasts resulted in significant downregulation of LATS. Furthermore, USP9X protein expression correlated positively with LATS but negatively with YAP/TAZ in pancreatic cancer tissues as well as pancreatic and breast cancer cell lines. Overall, these results strongly indicate that USP9X potentiates LATS kinase to suppress tumor growth.
Figure legend: (A) Knockdown of USP9X (USP9X-KD) in MCF10A cells resulted in epithelial-mesenchymal transition (EMT). (B) Three-dimensional culture of USP9X-KD cells in matrigel. USP9X-KD acini have enlarged and disorganized morphology. (C) Immunohistochemical (IHC) staining of USP9X and LATS in normal and pancreatic cancer tissue arrays. USP9X and LATS protein expression were predominantly negative in pancreatic cancer tissues. (D) USP9X and LATS were expressed weakly as opposed to strong YAP/TAZ expression in tissue sections derived from pancreatic cancer patients.
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