KLHL39 Suppresses Colon Cancer Metastasis by Blocking KLHL20-Mediated PML and DAPK Ubiquitination

ORIGINAL ARTICLE KLHL39 suppresses colon cancer metastasis by blocking KLHL20-mediated PML and DAPK ubiquitination

HY Chen1,2,JYHu2,3,6, TH Chen2,3,6, YC Lin2, X Liu1, MY Lin2, YD Lang4,YYen1 and RH Chen2,3,5

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 Furthermore, the mechanism for regulating Cul3-family E3 ligase Metastasis is responsible for most cancer mortality. A sequence of remains incompletely understood. discrete steps is required for disseminating primary cancer cells to The BTB-kelch protein KLHL20 was identified in our laboratory 7 colonize distant sites.1,2 At each step, metastatic cells face multiple as a substrate adaptor of Cul3 E3 ligase complex. This E3 ligase obstacles that are overcome with molecular alterations of specific complex catalyzes the ubiquitination of two tumor suppressor metastasis-related genes. Understanding the molecular mecha- proteins, death-associated protein kinase (DAPK) and promyelo- nisms that mediate metastatic progression would be essential for cytic leukemia protein (PML), thereby promoting their 7,8 the treatment of this devastating disease. proteolysis. DAPK is a well-known suppressor of metastasis Protein degradation through polyubiquitination has funda- and its expression is downregulated in many types of metastatic mental roles in many physiological and pathological processes, cancers.9,10 Furthermore, preclinical data indicate that DAPK elicits including cancer metastasis. A critical step in the ubiquitination multiple mechanisms to inhibit metastatic progression of cancers, process involves the transfer of ubiquitin moiety to the substrate such as increase of the susceptibility of tumor cells to death by ubiquitin ligases. The Cullin–RING complexes comprise the signals, regulation of cell–matrix adhesion and cell motility and largest known class of ubiquitin ligases, in which Cullin serves as a modulation of tumor microenvironments.11 As to PML, it is a scaffold for linking two functional modules: the catalytic RING- pleiotropic tumor suppressor protein12,13 and is downregulated in finger protein Roc1 or Roc2 that recruits E2, and a substrate- many types of cancers.14 A recent study revealed its inhibitory role binding molecule that brings substrate within the proximity to the in cell migration by suppressing integrin β1 expression.15 In catalytic module.3 Mammalian genomes encode for seven Cullin addition, PML inhibits epithelial–mesenchymal transition in the proteins (Cullin 1, 2, 3, 4a, 4b, 5 and 7). The Cullin 3 (Clu3)-based E3 hypoxic cells,8 an important step in tumor metastasis. Consistent ligases exploit BTB (Bric-a-brac/Tramtrack/Broad complex) with these functions, PML downregulation is associated with domain-containing proteins as the substrate adaptor to bridge advanced stages of certain tumors.14,16 As KLHL20 targets both Cul3 and substrate.4,5 While the BTB domain is responsible for Cul3 DAPK and PML for ubiquitin-dependent degradation, it likely has a binding, many BTB-domain proteins contain additional protein promoting role in cancer metastasis. interaction domains for substrate recruitment. In humans, the KLHL39, also known as IVNS1ABP, NS1-BP or Nd1, possesses kelch-repeat domain is the most prevalent substrate-binding domain architecture similar to KLHL20, that is, a BTB domain in its domain and 95 BTB-kelch proteins are found in human genome.6 N terminus and six kelch repeats in the C terminus. KLHL39 was However, it is unclear whether all of these BTB-kelch proteins are identified as a human protein interacting with the nonstructural engaged in functional Cul3 E3 ligase complex, and the physio- NS1 protein of the influenza A virus.17 A recent study revealed logical roles of many BTB-kelch proteins are poorly characterized. that KLHL39 forms a complex with heterogeneous nuclear

1Graduate Institute of Cancer Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan; 2Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan; 3Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan; 4Institute of Biomedical Science, Academia Sinica, Taipei, Taiwan and 5Institute 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: [email protected] 6These authors contribute equally to this work. Received 30 June 2014; revised 23 October 2014; accepted 25 November 2014; published online 26 January 2015 KLHL39 suppresses metastasis through PML and DAPK HY Chen et al 5142 ribonucleoproteins to regulate the alternative splicing of influenza from baculovirus and then incubated together in the presence of A virus M1 mRNA, thereby facilitating the generation of viral M2 anti-Flag M2 agarose beads. The beads pull down both Flag- protein.18 Consistent with its role in pre-mRNA splicing, KLHL39 is KLHL39 and Myc-KLHL20, demonstrating their direct interaction found in the nucleus and enriched in the nuclear speckles (Figure 1d). Taken together, these data identified KLHL39 as a containing spliceosome assembly factor SC35 and this localization binding protein of KLHL20. is disrupted upon viral infection.17 KLHL39 is also colocalized and 19 physically associated with actin in the cytoplasm. A previous KLHL39 is not a substrate of Cul3-KLHL20 ubiquitin ligase and study with a murine model of metastasis revealed a down- does not interact with Cul3 regulation of murine homolog of KLHL39 in metastatic nodules The identification of a direct interaction between two BTB-kelch compared with the primary tumors. Consistent with this finding, proteins raised several possible functions for KLHL39. As KLHL39 KLHL39 overexpression in murine colon cancer and melanoma cell interacts with the kelch-repeat domain of KLHL20, we first lines suppresses migration and invasion in vitro and pulmonary 20 investigated whether KLHL39 is a substrate of Cul3-KLHL20 and liver metastasis in vivo. These observations support a metastasis suppressive role for KLHL39. However, whether a ubiquitin ligase. Overexpression of Roc1-Cul3-KLHL20 E3 ligase similar effect could be observed with human KLHL39 and the did not increase the ubiquitination level of KLHL39, even though underlying mechanism of this effect have not been explored. the ubiquitination of PML, a substrate of this E3 ligase, was In this study, we show that KLHL39 interacts with KLHL20 and elevated (Supplementary Figure 1A). In the reciprocal experiment, functions as an inhibitor of Cul3-KLHL20 ubiquitin ligase. Through KLHL20 depletion reduced the ubiquitination of PML but not KLHL39 (Supplementary Figure 1B). Consistent with these findings, this function, KLHL39 prevents ubiquitin-mediated proteolysis of overexpression or knockdown of KLHL20 did not alter the steady- PML and DAPK. We provide preclinical and clinical evidence for state level of KLHL39, whereas the level of PML was significantly the contribution of PML and DAPK stabilization to the metastasis- reduced or elevated in the same experiment, respectively suppression function of KLHL39. (Supplementary Figures 1C and D). Thus, KLHL39 is not a substrate of Cul3-KLHL20 E3 ligase. 4 RESULTS Viewing the dimeric structure of Cul3 raises the second possibility that the interaction of KLHL39 with KLHL20 leads to KLHL39 interacts with KLHL20 the formation of a Cul3 E3 ligase complex containing both KLHL39 In an effort to identify the regulator or effector of KLHL20, we and KLHL20 as the substrate adaptors. To evaluate the formation performed a yeast two-hybrid screen with its kelch-repeat domain of such hybrid complex, we first examined whether KLHL39 as the bait. Several positive clones identified encoded for the interacts with Cul3 via its BTB domain. To our surprise, KLHL39 did C-terminal portion of KLHL39. Reciprocal co-immunoprecipitation not co-precipitate with Cul3 from lysates of co-transfected cells, analysis on 293T cells transfected with Flag-KLHL39 and Myc- whereas Roc1 readily co-precipitated with Cul3 (Supplementary KLHL20 revealed a physical association between the two proteins Figure 2A). Furthermore, in vitro binding assay using purified (Figures 1a and b). Furthermore, domain mapping analysis KLHL39 and Cul3 failed to detect their interaction, even though indicated that the kelch-repeat domains of the two proteins, the interaction between KLHL39 and KLHL20 was observed in the rather than the BTB domains, were responsible for their same experiment (Supplementary Figure 2B). To explore the interaction. In addition, interaction of endogenous KLHL39 with underlying mechanism for the inability of KLHL39 to bind Cul3, we endogenous KLHL20 was detected by co-immunoprecipitation performed a sequence alignment using the BTB domains of (Figure 1c). To demonstrate a direct interaction between the two KLHL39 and several known Cul3 substrate adaptors. This analysis proteins, Myc-KLHL20 and Flag-KLHL39 were purified separately revealed that KLHL39 carries a phenylalanine at amino acid 75 and

Figure 1. KLHL39 interacts with KLHL20. (a) Immunoprecipitation analysis of the interaction between Flag-KLHL39 and full-length (FL) or various deletion mutants of Myc-KLHL20 in transfected 293T cells. (b) Immunoprecipitation analysis of the interaction between Myc-KLHL20 and FL or various truncated mutants of KLHL39 in transfected 293T cells. (c) Immunoprecipitation analysis of the interaction between endogenous KLHL20 and endogenous KLHL39 in HeLa cells. (d) In vitro binding assay for puriﬁed KLHL20 and KLHL39. Myc-KLHL20 and Flag- KLHL39 were separately puriﬁed from baculovirus by anti-myc and anti-Flag antibody, incubated together with anti-Flag M2 beads, and analyzed by western blot.

Oncogene (2015) 5141 – 5151 © 2015 Macmillan Publishers Limited KLHL39 suppresses metastasis through PML and DAPK HY Chen et al 5143 a methionine at 114, whereas other BTB proteins possess leucine (Figure 4b). Consequently, the steady-state levels of endogenous or isoleucine at these two positions (Supplementary Figure 2C). PML and DAPK were both decreased in KLHL39-depleted cells Importantly, a previous study indicated that two equivalent (Figure 4c). These results point to a physiological role of KLHL39 in residues in MEL-26 (i.e., amino acids 245 and 285) are critical for regulating KLHL20-dependent ubiquitination and degradation of Cul3 interaction.21 Thus, the inability of KLHL39 to bind Cul3 may PML and DAPK. be because of the presence of these two nonconserved residues. In line with this notion, replacement of either of the two residues Downregulations of KLHL39, DAPK and PML correlate with with leucine resulted in a detectable interaction with Cul3 metastatic progression of colon cancers (Supplementary Figure 2D). Our findings indicate that KLHL39 is Having identified an inhibitory role of KLHL39 in KLHL20-mediated not a substrate adaptor of Cul3-family ubiquitin ligase owing to ubiquitination and degradation of PML and DAPK, we sought to the lack of certain conserved residues. examine the biological significance of this regulation. DAPK is a well-known metastasis suppressor,11 whereas the function of PML KLHL39 interferes with both substrate and Cul3 binding to KLHL20 in blocking migration and invasion has also been reported.15 On the basis of the finding that KLHL39 binds KLHL20 but not Interestingly, a previous study implicated a suppressive function of Cul3, together with the inability of KLHL20 to promote KLHL39 KLHL39 in metastasis.20 Accordingly, data derived from two degradation, we speculated that KLHL39 acts as a pseudosub- independent sets of microarray analysis revealed the association strate to block substrate recruitment to the Cul3–KLHL20 complex. of KLHL39 downregulation with shorter metastasis-free survival of Consistent with this hypothesis, KLHL39 overexpression decreased colon cancer patients (Supplementary Figure 3). We therefore the binding of endogenous PML to KLHL20 (Figure 2a). To evaluated the expression of KLHL39, PML and DAPK in this type of demonstrate a direct role of KLHL39 in blocking the binding of cancers. Immunohistochemistry (IHC) analysis on a cohort of colon PML to KLHL20, we performed an in vitro binding assay. To this cancer specimens revealed that low expression of KLHL39, DAPK end, we separately purified GST-KLHL20, Flag-KLHL39 and Flag- or PML each correlated with higher tumor grade, lymph node PML-I from baculovirus. By incubating these proteins together, we metastasis and distant metastasis (Figures 5a and b). Furthermore, revealed that KLHL39 induced a dose-dependent reduction of KLHL39 expression correlated with the expression of both DAPK KLHL20 binding to PML-I, which was accompanied with an and PML (Figure 5c), supporting the existence of KLHL39- increased binding of KLHL20 to KLHL39 (Figure 2b). Next, we dependent DAPK and PML regulation in this cohort. To further sought to extend this finding to another KLHL20 substrate, DAPK. investigate the association of KLHL39–DAPK/PML axis with tumor Similarly, KLHL39 interfered with the binding of DAPK to KLHL20 metastasis, we investigated the expression of these three proteins both in vivo and in vitro (Figures 2c and d). These results indicate in primary tumors and their lymph node metastases in another that KLHL39 inhibits the interaction of KLHL20 with its substrates, cohort of colon cancer patients. Low expression of each KLHL39, DAPK and PML. Unexpectedly, we also observed that KLHL39 DAPK and PML was more frequently observed in lymph node overexpression compromised the formation of Cul3-KLHL20 metastases than primary tumors (Figures 5d and e). Collectively, complex in vivo (Figure 2e). This effect of KLHL39 was these clinical data support the association of KLHL39-DAPK/PML recapitulated using in vitro binding assay (Figure 2f, upper panel). pathway with metastatic progression of colon cancers. Furthermore, the kelch-repeat deletion mutant of KLHL39 (KLHL39Δkelch), which could not bind KLHL20, did not affect KLHL39 suppresses colon cancer cell migration, invasion and the interaction between Cul3 and KLHL20 (Figure 2f, lower panel). resistance to anoikis through PML and DAPK Taken together, our study indicates that KLHL39 not only blocks – KLHL20 binding to its substrates but also interferes with the The demonstration of a clinical relevance for the KLHL39 DAPK/ formation of KLHL20–Cul3 complex. PML axis to the suppression of colon cancer metastasis prompted us to investigate the functions of this pathway in several metastasis-related characters using cell-based assays. We first KLHL39 blocks KLHL20-induced ubiquitination and degradation of expressed KLHL39 in a highly metastatic colon cancer cell line, PML and DAPK HCT-116. As expected, KLHL39 overexpression increased the Given that KLHL39 compromises the interaction of KLHL20 with abundance of both DAPK and PML, which was reversed by DAPK both the substrates and Cul3, we reasoned that it should block the shRNA and PML shRNA, respectively (Figure 6a). When tested for ubiquitination and degradation of KLHL20 substrates. Consistent cell migration and invasion activities, we observed that KLHL39 with this notion, overexpression of KLHL39 blocked PML-I significantly suppressed HCT-116 cell migration and invasion. ubiquitination induced by Roc1-Cul3-KLHL20 E3 ligase complex, More importantly, these functions were partially reversed by DAPK and this effect of KLHL39 was abolished by deletion of the kelch- or PML knockdown and almost completely abrogated by repeat domain (Figure 3a). Similar effect was observed with combined depletion of DAPK and PML (Figure 6b). Resistance to another KLHL20 substrate, DAPK (Figure 3b). Consequently, the anoikis (i.e., apoptosis induced by cell detachment from extra- KLHL20-induced downregulation of both PML and DAPK was cellular matrix) is a trait of metastatic cells. As DAPK is a mediator Δ abrogated by KLHL39 but not KLHL39 kelch mutant (Figures 3c of anoikis22 and PML promotes various types of cell death,12,13 we fi and d). These ndings indicate that KLHL39 blocks KLHL20- evaluated the role of KLHL39–DAPK/PML axis in anoikis. Remark- induced substrate ubiquitination and degradation through its ably, KLHL39 overexpression increased the anoikis susceptibility of interaction with KLHL20. HCT-116 cells, which was again partially reversed by PML or DAPK knockdown and more significantly reversed by combined knock- KLHL39 knockdown promotes the ubiquitination and degradation down of PML and DAPK (Figure 6c). In a reciprocal set of of PML and DAPK experiments, we depleted KLHL39 in a less metastatic colon To investigate the effect of endogenous KLHL39 on the regulation cancer cell line, HT-29. KLHL39 knockdown resulted in the of KLHL20 substrates, we established HeLa cells stably expressing downregulation of PML and DAPK, increased cell migration/ either of the two KLHL39 short hairpin RNAs (shRNAs) (Figure 4a). invasion and decreased anoikis susceptibility (Figures 6d, e and f). Consistent with the inhibitory role of KLHL39 in KLHL20- Importantly, these biological effects of KLHL39 knockdown were dependent ubiquitination and degradation events, KLHL39 knock- all attenuated by overexpression of DAPK or PML-IV. Thus, our down elevated PML ubiquitination in KLHL20-overexpressing cells study indicates that KLHL39 is capable of suppressing multiple (Figure 4a). A similar effect was observed for DAPK ubiquitination metastasis-related functions through DAPK and PML.

Figure 2. KLHL39 inhibits the recruitment of PML and DAPK to KLHL20 and the binding of Cul3 to KLHL20. (a) Immunoprecipitation analysis of the interaction between KLHL20 and endogenous PML in 293T cells transfected with indicated constructs. (b) GST pull-down analysis for in vitro interactions of KLHL20 with PML-I and KLHL39. GST-KLHL20 was purified from baculovirus by glutathione-sepharose beads and incubated with Flag-PML-I in the presence of increasing concentrations of Flag-KLHL39 separately purified from baculovirus. The bound proteins were analyzed by western blot. (c) Immunoprecipitation analysis of the interaction between KLHL20 and DAPK (or KLHL39) in 293T cells transfected with indicated constructs. (d) GST pull-down analysis for in vitro interactions of KLHL20 with DAPK and KLHL39 as in (b). (e) Immunoprecipitation analysis of the interaction between KLHL20 and Cul3 in 293T cells transfected with indicated constructs. (f) GST pull- down analysis for the recruitment of KLHL20 to Cul3 in the presence or absence of KLHL39. All proteins were separately purified from baculovirus. For each panel, the intensities of the PML (a and b), DAPK (c and d), Cul3 (e) and KLHL20 (f) bands were quantified, normalized with the control protein in the precipitates and indicated on the bottom.

To corroborate that the effects of KLHL39 are resulted from its DAPK and PML expression, cell migration, invasion and anoikis in antagonizing the functions of KLHL20, we overexpressed KLHL39 KLHL20-depleted HT-29 cells (Supplementary Figures 4D–F). Thus, in HCT-116 cells stably expressing KLHL20 shRNA. Remarkably, the effects of KLHL39 on regulating metastasis-related traits are KLHL39 could no longer regulate the expression of DAPK dependent on KLHL20. and PML in KLHL20-depleted cells (Supplementary Figure 4A). As a consequence, KLHL39 overexpression failed to affect cell migration, invasion and anoikis in KLHL20-depleted cells KLHL39 suppresses tumor metastasis through PML and DAPK (Supplementary Figures 4B and C). In a reciprocal set of We next determined the effect of KLHL39–DAPK/PML axis on experiments, we showed that KLHL39 shRNA failed to regulate tumor metastasis in vivo by using the experimental metastasis

Figure 3. Overexpression of KLHL39 reduces KLHL20-induced ubiquitination and degradation of PML and DAPK. (a and b) Analysis of PML-I (a) and DAPK (b) ubiquitination in 293T cells transfected with indicated constructs by anti-Flag immunoprecipitation, followed by western blot with anti-ubiquitin antibody. (c and d) Western blot analysis of endogenous PML and DAPK expression in 293T cells (c) and PC-3 cells (d) transfected with indicated constructs. model. Injection of HCT-116 cells into the circulation of nude mice confirmed the data obtained from bioluminescence analysis led to the formation of lung metastases as early as 3 weeks. (Figures 7b and c). Thus, our findings support that KLHL39- Bioluminescence image detected the persistent growth of induced stabilization of DAPK and PML has a suppressive role in metastatic lung tumors (Figure 7a). In sharp contrast, the colon cancer metastasis. HCT-116 cells carrying overexpressed KLHL39 failed to develop lung metastases even at 5 weeks after injection (Figure 7a). The antimetastasis function of KLHL39 was partially reversed by DISCUSSION depletion of either DAPK or PML. Examination of lungs harvested In this study, we identified KLHL39 as a binding protein of KLHL20 at 5 weeks after injection for the presence of metastatic nodules and the kelch domains of these two proteins mediate their

Figure 4. KLHL39 depletion enhances KLHL20-dependent ubiquitination to destabilize PML and DAPK. (a and b) Analysis of PML-I (a) and DAPK (b) ubiquitination in HeLa cells stably expressing control or KLHL39 shRNAs and transfected with indicated constructs. (c) Western blot analysis of endogenous PML and DAPK expression in HeLa cells stably expressing control or KLHL39 shRNAs.

interaction. Unexpectedly, KLHL39 is neither a substrate of Cul3– ubiquitination and degradation induced by KLHL39 downregula- KLHL20 complex nor a substrate adaptor of Cul3 ubiquitin ligase. tion is associated with colon cancer metastatic progression. Our Instead, KLHL39 interferes with the interaction of KLHL20 with its findings also identify a new mechanism for PML degradation in substrates and blocks the assembly of Cul3–KLHL20 complex. As a human cancers. consequence, KLHL39 functions as a negative regulator of Cul3- Our previous studies revealed a tumor-promoting role of KLHL20 ubiquitin ligase to prevent the degradation of KLHL20 KLHL20 by its induction of ubiquitination and degradation of substrates DAPK and PML. We further explored the biological two tumor suppressor proteins, DAPK and PML. In this study, we significance of KLHL39-mediated stabilization of DAPK and PML further identify the same family protein, KLHL39, as a negative and demonstrated that the KLHL39–DAPK/PML axis suppresses regulator of KLHL20. This finding not only discovers a hierarchy in tumor cell migration, invasion and resistance to anoikis in vitro the BTB-kelch-family proteins but also suggests a role of the ratio and tumor metastasis in vivo. Consistent with these preclinical of KLHL39 to KLHL20 in determining the fate of tumor cells. data, examination of human colon cancer patient specimens Importantly, KLHL20 is an HIF-1 target and is therefore highly revealed that downregulation of each KLHL39, DAPK or PML induced by hypoxic tumor microenvironment.8 In response to correlates with metastasis incidence. Furthermore, by examining hypoxia, KLHL20 substrate PML is rapidly degraded,8 presumably tumors taken from the primary and metastatic sites of the same due to a high KLHL20/KLHL39 ratio. The upstream signal that patients, we found that these three proteins are expressed regulates the expression of KLHL39 in tumors remains elusive and significantly less in metastatic sites. Consistent with these findings, it would be interesting to determine how KLHL39 is down- low expression of each of these three proteins is associated with regulated in metastatic colon cancers. shorter metastasis-free period and overall survival. Thus, our study To date, several mechanisms are reported to regulate the identifies KLHL39 as a negative regulator of Cul3-KLHL20 E3 ligase activity of Cul3-family ubiquitin ligases, such as dimerization, and a suppressor of tumor metastasis by stabilizing KLHL20 neddylation and posttranslational modifications of the substrate substrates DAPK and PML (Figure 7d). adaptors.4 In addition, pseudosubstrate that binds substrate DAPK is frequently downregulated in many types of cancers, adaptor to interfere with the recruitment of genuine substrates including metastatic tumors.10 Although hypermethylation of the has emerged as a new mechanism. For instance, several DAPK promoter is a major mechanism for DAPK downregulation in pseudosubstrates are identified for Cul3-Keap1 ubiquitin ligase, tumors, conflicting observations have been reported for the such as p62,31 PALB2,32 WTX33 and DPP3.34 These pseudosub- frequency of DAPK promoter methylation.23–25 Our previous study strates can each displace Nrf2 from the Keap1–Nrf2 complex, identified an miRNA-dependent mechanism for DAPK down- thereby preventing the ubiquitination and degradation of Nrf2. regulation in metastatic colon cancer.26 In this study, our findings Similarly, we identify KLHL39 as a pseudosubstrate of Cul3-KLHL20 of KLHL39-mediated DAPK stabilization and its association with E3 ligase, as it inhibits substrate binding to KLHL20. However, colon cancer metastatic progression indicate that DAPK can also unlike those Keap1 pseudosubstrates, KLHL39 not only blocks be regulated at posttranslational level in human cancers. Similar to substrate–KLHL20 interaction but also impairs the formation of DAPK, PML is frequently downregulated in various human Cul3–KLHL20 complex. It is unclear how KLHL39 inhibits the cancers.14 Evidence has emerged that proteasomal degradation binding of KLHL20 to Cul3. One possibility is that binding KLHL39 is the major cause of PML downregulation in tumors.8,27–30 to KLHL20 can induce a long-range conformational change to alter Consistent with this notion, our study indicates that aberrant PML the architecture of KLHL20 BTB domain. Regardless of the

Figure 5. Downregulations of KLHL39, DAPK and PML are associated with colon cancer metastasis. (a and d) Representative IHC data for KLHL39, DAPK and PML expression in a stage I and III colon cancer specimen (a) and a pair of specimens of primary colon cancer and its lymph node metastasis (d). Bars, 10 μm. (b) The KLHL39, DAPK and PML expression proﬁles with relation to tumor stages and metastasis. (c) The association of KLHL39 expression with DAPK and PML expression in colon cancer specimens. (e) The expression proﬁles of KLHL39, DAPK and PML in primary colon cancer and lymph node metastatic lesion. For (b, c and e), the P-values are determined by Fisher’s exact test.

Figure 6. KLHL39–PML/DAPK axis inhibits migration, invasion and promotes anoikis of colon cancer cells. (a) Western blot analysis of DAPK and PML expression in HCT-116 cells stably expressing indicated constructs and/or shRNAs. (b and c) Migration, invasion and anoikis assays of HCT-116 derivatives as in (a). (d) Western blot analysis of HT-29 cells stably expressing indicated constructs and/or shRNAs. (e and f) Migration, invasion and anoikis assays of HT-29 derivatives as in (d). In (b, c, e and f), data shown are mean ± s.d., **Po0.005, ***Po0.0005 and n = 3.

underlying mechanism, our study uncovers a novel regulatory to determine whether KLHL39 can act on other types of cancers mechanism for Cul3-family E3 ligases. and whether KLHL39 can regulate other BTB-family proteins. Although Cul3-family ubiquitin ligases exploit exclusively BTB- domain-containing proteins as substrate adaptors, structural and bioinformatic analyses predict that BTB proteins do not all function MATERIALS AND METHODS as Cul3 adaptors. A helical structure at the C terminus of BTB Plasmids domain, named 3-box domain, is involved in forming tight complex Plasmids encoding HA-ubiquitin, myc-Cul3, myc-Roc1, Flag-PML-I, with Cul3 and a number of BTB proteins lacks 3-box domain.35 Flag-PML-IV were described previously.8 Plasmids encoding Flag-DAPK, Although KLHL39 contains an intact 3-box domain, it lacks at least myc-KLHL20, myc-KLHL20 kelch and myc-KLHL20 BTB were described.7 two conserved residues in the BTB domain. KLHL39 carries a Phe at KLHL39 cDNA was amplified by reverse transcription–PCR and subcloned position 75 and a Met at position 114, whereas many BTB proteins to pRK5F, pBybe-Flag and pVL1393-Flag. Various KLHL39 deletion mutants that bind Cul3 possess Leu or Ile at the two positions. Notably, were generated by PCR or ligation-mediated PCR. Phe75 and M114 are predicted to localize in the β4/α4 loop and the α5/α6 loop of the BTB domain, respectively. Recent structural Antibodies and reagents analyses of the complex between Cul3 N-terminal domain and BTB- Anti-KLHL20 antibody was described previously.8 Other antibodies used in BACK domain of SPOP, KLHL3 or KLHL11 revealed that both β4/α4 this study were obtained as follows: anti-DAPK (D2178; Sigma, St. Louis, MO, loop and the α5/α6 loop of the BTB domain are within the regions USA), anti-Flag (M2; Sigma), anti-GFP (sc-9996; Santa Cruz, Dallas, TX, USA), that contribute to Cul3 binding.36–38 Thus, the presence of anti-c-myc (9E10; Santa Cruz), anti-KLHL39 (ab101278 for western blot/ nonconserved residues in these two regions are likely responsible ab127566 for IHC; Abcam, Cambridge, MA, USA), anti-Roc1 (ab2977; Abcam), for the inability of KLHL39 to form a complex with Cul3. In line with anti-Cul3 (2759; Cell Signaling, Danvers, MA, USA), anti-ubiquitin (3933; Cell Signaling), anti-α-tubulin (Millipore, Billerica, MA, USA), anti-GAPDH this notion, we show that replacement of either residue with a fi (Epitomics, Burlingame, CA, USA), anti-actin (GTX110564; GeneTex, Hsinchu, conserved Leu residue increases the af nity of KLHL39 to Cul3. Taiwan) and anti-PML (NB100-59787 for western blot; Novus, Littleton, CO, Thus, KLHL39 is likely evolved to diverge from a Cul3 substrate USA). Human plasma fibronectin was obtained from Millipore. MG132 was adaptor to an inhibitor of a particular Cul3–adaptor complex. It obtained from Calbiochem (Billerica, MA, USA). would be interesting to determine whether other BTB-domain proteins can function as an inhibitor of Cul3-family ubiquitin ligases. Cell cultures and transfection In summary, our study identifies a new regulatory mechanism HeLa, 293T, 293FT, PC-3, HCT-116, HT-29 and SF21 cells were obtained from the for Cul3-family E3 ligase. The BTB-kelch protein KLHL39 does not American Type Culture Collection (ATCC, Manassas, VA, USA). HeLa, 293T and serve as a substrate adaptor of Cul3 but functions as an inhibitor 293FT cells were cultured in Dulbecco's modified Eagle's medium containing of Cul3-KLHL20 E3 ligase. Through this mechanism, KLHL39 10% fetal calf serum. PC-3 cells were cultured in Dulbecco's modified Eagle's stabilizes Cul3-KLHL20 substrates DAPK and PML, thereby medium high glucose medium supplemented with 10% fetal calf serum, contributing to colon cancer metastasis. It would be interesting nonessential amino acid, 1 mM sodium pyruvate and 20 mM L-glutamine.

Figure 7. KLHL39 suppresses tumor metastasis through DAPK and PML stabilization. (a) Bioluminescence analysis and quantiﬁcation of lung metastasis derived from injection of indicated HCT-116 derivatives (as in Figure 6a) into the circulation of nude mice. Representative images at week 4 (left) and the kinetic of metastasis at indicated time points (right) after injection are shown. (b) Lung metastasis and histological analysis of the lung at 5 weeks after injection. Nodules are indicated by arrowheads. Bar, 1 mm (top) and 10 μm (bottom). (c) Number of metastatic nodules in the lung at 5 weeks after injection. (d) Model for KLHL39-induced stabilization of PML and DAPK in suppressing tumor metastasis. Numerical data for panels a and c are mean ± s.d., n = 6, *Po0.05, **Po0.005 and ***Po0.0005.

HCT-116 and HT-29 cells were cultured in RPMI containing 10% fetal calf of polybrene and infected cells were selected by 2 μg/ml puromycin, serum. SF21 cells were cultured in TNM-FH insect medium (AppliChem, 10 μg/ml blasticidin, 800 μg/ml G418 and/or 100 μg/ml hygromycin. Darmstadt, Germany) containing 10% fetal calf serum. Transfection was performed with Lipofectamine 2000 Reagent (Invitrogen, Carlsbad, CA, USA). Immunoprecipitation and in vitro binding assay Cell extract was prepared with RIPA lysis buffer containing 50 mM Tris RNA interference (pH 8.0), 0.15 M NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% sodium Lentivirus-based shRNA constructs were obtained from National RNAi Core dodecylsulfate, 1 mM phenylmethyl sulfonyl fluoride, 1 μg/ml aprotinin and 39 Facility, Taipei, Taiwan. Lentivirus-based constructs for expressing shRNAs 1 μg/ml leupeptin. Immunoprecipitation was performed as described. For targeting KLHL20, DAPK or PML were described previously.7,8 The 21 target in vitro binding analysis, recombinant Flag-KLHL39, Flag-PML-I, Flag-DAPK sequences for KLHL39 shRNAs are: KLHL39 shRNA no. 1, 5′-CCTCTCAAACTA and myc-KLHL20 were purified from baculovirus using anti-Flag agarose ACAGGCTTA-3′ and KLHL39 shRNA no. 2, 5′-CTGCATCTCTTACCGAAATTT-3′. beads and c-myc AD agarose beads (Santa Cruz), followed by elution with Flag and myc peptide, respectively. GST-Cul3–Roc1 complex was purified from SF21 by glutathione beads. The beads were incubated with purified Establishment of stable cell lines Flag-PML-I for 40 min in binding buffer containing 20 mM Tris (pH 7.5), Stable cell lines were generated by lentivirus transduction as described 150 mM NaCl, 1% NP-40, 0.1% sodium dodecylsulfate and 1% sodium previously.8 For infection, the viral stock was supplemented with 8 μg/ml deoxycholate. The bound proteins were analyzed by western blot.

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Supplementary Information accompanies this paper on the Oncogene website (http://www.nature.com/onc)