ORIGINAL ARTICLE KLHL39 suppresses colon cancer metastasis by blocking KLHL20-mediated PML and DAPK ubiquitination HY Chen 1,2 , JY Hu 2,3,6 , TH Chen 2,3,6 , YC Lin 2 , X Liu 1 , MY Lin 2 , YD Lang 4 , Y Yen 1 and RH Chen 2,3,5 Cullin 3 (Cul3)-family ubiquitin ligases use the BTB-domain-containing proteins for the recruitment of substrates, but the regulation of this family of ubiquitin ligases has not been completely understood. KLHL20 is a BTB-family protein and targets tumor suppressor promyelocytic leukemia protein (PML) and death-associated protein kinase (DAPK) to its kelch-repeat domain for ubiquitination and degradation. Here, we show that another BTB-kelch protein KLHL39 is recruited to the substrate-binding domain of KLHL20 but is not a substrate of Cul3–KLHL20 complex. Interestingly, KLHL39 does not bind Cul3 because of the absence of certain conserved residues in the BTB domain. Instead, KLHL39 blocks KLHL20-mediated ubiquitination of PML and DAPK by disrupting the binding of these substrates to KLHL20 as well as the binding of KLHL20 to Cul3. Through the two mechanisms, KLHL39 increases the stability of PML and DAPK. In human colon cancers, downregulations of KLHL39, PML and DAPK are associated with metastatic progression. Furthermore, preclinical data indicate that KLHL39 promotes colon cancer migration, invasion and survival in vitro and metastasis in vivo through a PML- and DAPK-dependent mechanism. Our study identifies KLHL39 as a negative regulator of Cul3-KLHL20 ubiquitin ligase and reveals a role of KLHL39-mediated PML and DAPK stabilization in colon cancer metastasis. Oncogene (2015) 34, 5141–5151; doi:10.1038/onc.2014.435; published online 26 January 2015 INTRODUCTION Metastasis is responsible for most cancer mortality. A sequence of discrete steps is required for disseminating primary cancer cells to colonize distant sites. 1,2 At each step, metastatic cells face multiple obstacles that are overcome with molecular alterations of specific metastasis-related genes. Understanding the molecular mecha- nisms that mediate metastatic progression would be essential for the treatment of this devastating disease. Protein degradation through polyubiquitination has funda- mental roles in many physiological and pathological processes, including cancer metastasis. A critical step in the ubiquitination process involves the transfer of ubiquitin moiety to the substrate by ubiquitin ligases. The Cullin–RING complexes comprise the largest known class of ubiquitin ligases, in which Cullin serves as a scaffold for linking two functional modules: the catalytic RING- finger protein Roc1 or Roc2 that recruits E2, and a substrate- binding molecule that brings substrate within the proximity to the catalytic module. 3 Mammalian genomes encode for seven Cullin proteins (Cullin 1, 2, 3, 4a, 4b, 5 and 7). The Cullin 3 (Clu3)-based E3 ligases exploit BTB (Bric-a-brac/Tramtrack/Broad complex) domain-containing proteins as the substrate adaptor to bridge Cul3 and substrate. 4,5 While the BTB domain is responsible for Cul3 binding, many BTB-domain proteins contain additional protein interaction domains for substrate recruitment. In humans, the kelch-repeat domain is the most prevalent substrate-binding domain and 95 BTB-kelch proteins are found in human genome. 6 However, it is unclear whether all of these BTB-kelch proteins are engaged in functional Cul3 E3 ligase complex, and the physio- logical roles of many BTB-kelch proteins are poorly characterized. Furthermore, the mechanism for regulating Cul3-family E3 ligase remains incompletely understood. The BTB-kelch protein KLHL20 was identified in our laboratory as a substrate adaptor of Cul3 E3 ligase complex. 7 This E3 ligase complex catalyzes the ubiquitination of two tumor suppressor proteins, death-associated protein kinase (DAPK) and promyelo- cytic leukemia protein (PML), thereby promoting their proteolysis. 7,8 DAPK is a well-known suppressor of metastasis and its expression is downregulated in many types of metastatic cancers. 9,10 Furthermore, preclinical data indicate that DAPK elicits multiple mechanisms to inhibit metastatic progression of cancers, such as increase of the susceptibility of tumor cells to death signals, regulation of cell–matrix adhesion and cell motility and modulation of tumor microenvironments. 11 As to PML, it is a pleiotropic tumor suppressor protein 12,13 and is downregulated in many types of cancers. 14 A recent study revealed its inhibitory role in cell migration by suppressing integrin β1 expression. 15 In addition, PML inhibits epithelial–mesenchymal transition in the hypoxic cells, 8 an important step in tumor metastasis. Consistent with these functions, PML downregulation is associated with advanced stages of certain tumors. 14,16 As KLHL20 targets both DAPK and PML for ubiquitin-dependent degradation, it likely has a promoting role in cancer metastasis. KLHL39, also known as IVNS1ABP, NS1-BP or Nd1, possesses domain architecture similar to KLHL20, that is, a BTB domain in its N terminus and six kelch repeats in the C terminus. KLHL39 was identified as a human protein interacting with the nonstructural NS1 protein of the influenza A virus. 17 A recent study revealed that KLHL39 forms a complex with heterogeneous nuclear 1 Graduate Institute of Cancer Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan; 2 Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan; 3 Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan; 4 Institute of Biomedical Science, Academia Sinica, Taipei, Taiwan and 5 Institute of Biochemical Sciences, College of Life Science, National Taiwan University, Taipei, Taiwan. Correspondence: Dr R-H Chen, Institute of Biological Chemistry, Academia Sinica, 128 Academia Road, Section II, Nankang, Taipei 115, Taiwan, Taiwan. E-mail: rhchen@gate.sinica.edu.tw 6 These authors contribute equally to this work. Received 30 June 2014; revised 23 October 2014; accepted 25 November 2014; published online 26 January 2015 Oncogene (2015) 34, 5141 – 5151 © 2015 Macmillan Publishers Limited All rights reserved 0950-9232/15 www.nature.com/onc