The non-canonical ubiquitin activating enzyme UBA6 suppresses epithelial-mesenchymal transition of mammary epithelial cells

// Xianpeng Liu 1 , Limin Sun 1 , Demirkan B. Gursel 2 , Chonghui Cheng 3, 7, 9 , Sui Huang 4, 7 , Alfred W. Rademaker 5, 7 , Seema A. Khan 6, 7 , Jun Yin 8 and Hiroaki Kiyokawa 1, 7 1 Department of Pharmacology, Northwestern University, Chicago, Illinois 60611, USA 2 Department of Pathology, Northwestern University, Chicago, Illinois 60611, USA 3 Division of Hematology/Oncology, Northwestern University, Chicago, Illinois 60611, USA 4 Department of Cell and Molecular Biology, Northwestern University, Chicago, Illinois 60611, USA 5 Department of Preventive Medicine, Northwestern University, Chicago, Illinois 60611, USA 6 Department of Surgery, Northwestern University, Chicago, Illinois 60611, USA 7 Robert H. Lurie Comprehensive Cancer Center, Northwestern University, Chicago, Illinois 60611, USA 8 Department of Chemistry, Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, Georgia 30303, USA 9 Current/Present address: Lester and Sue Smith Breast Center, Baylor College of Medicine, Houston, TX 77030, USA Correspondence to: Xianpeng Liu, email: Xianpeng.liu@northwestern.edu Hiroaki Kiyokawa, email: Kiyokawa@northwestern.edu Keywords: ubiquitination, breast cancer, EMT, Rho GTPase, cell cycle Received: May 18, 2017 Accepted: August 17, 2017 Published: September 15, 2017 ABSTRACT Ubiquitination plays critical roles in the regulation of oncoproteins and tumor suppressors during carcinogenesis. The two ubiquitin activating enzymes (E1) in human genome, UBA1 and UBA6, initiate ubiquitination by ATP-dependent activation of ubiquitin. Recent evidence suggests that UBA1 and UBA6 play partially overlapped yet distinct roles in controlling the proteome. Here we demonstrate that ubiquitination pathways initiated specifically by UBA6 set a suppressive barrier against critical steps of mammary carcinogenesis such as loss of polarity, anoikis resistance and epithelial-mesenchymal transition (EMT). Mammary epithelial MCF-10A cells expressing shRNA against UBA6 fail in establishing cell cycle arrest in response to detachment from extracellular matrix, confluency with fully engaged cell-cell contact or growth factor deprivation. Moreover, UBA6-deficient MCF-10A cells undergo spontaneous EMT under growth factor deprivation and exhibit accelerated kinetics of TGF-β-induced EMT. The Rho-GTPase CDC42 is one of the specific targets of UBA6-initiated ubiquitination and plays a key role in the function of UBA6 in controlling epithelial homeostasis, since a CDC42 inhibitor, ML141, rescues UBA6-deficient cells from the EMT phenotype. Immunohistochemical analysis of human breast cancer tissues demonstrates that 38% of invasive carcinomas express low or undetectable expression of UBA6, suggesting that downregulation of this non-canonical E1 plays a role in breast cancer development.