Smurf2-Mediated Stabilization of DNA Topoisomerase Iiα Controls Genomic Integrity

Published OnlineFirst June 13, 2017; DOI: 10.1158/0008-5472.CAN-16-2828 Cancer Molecular and Cellular Pathobiology Research

Smurf2-Mediated Stabilization of DNA Topoisomerase IIa Controls Genomic Integrity Andrea Emanuelli1, Aurora P. Borroni1, Liat Apel-Sarid2, Pooja A. Shah1, Dhanoop Manikoth Ayyathan1, Praveen Koganti1, Gal Levy-Cohen1, and Michael Blank1

Abstract

DNA topoisomerase IIa (Topo IIa) ensures genomic integ- pathological chromatin bridges formed during mitosis, a trait rity and unaltered chromosome inheritance and serves as a of Topo IIa–deficient cells and a hallmark of chromosome major target of several anticancer drugs. Topo IIa function is instability. Introducing Topo IIa into Smurf2-depleted cells well understood, but how its expression is regulated remains rescued this phenomenon. Smurf2 was a determinant of Topo unclear. Here, we identify the E3 ubiquitin ligase Smurf2 as a IIa protein levels in normal and cancer cells and tissues, and its physiologic regulator of Topo IIa levels. Smurf2 physically levels affected cell sensitivity to the Topo II–targeting drug interacted with Topo IIa and modified its ubiquitination status etoposide. Our results identified Smurf2 as an essential regu- to protect Topo IIa from the proteasomal degradation in dose- lator of Topo IIa, providing novel insights into its control and catalytically dependent manners. Smurf2-depleted cells and into the suggested tumor-suppressor functions of Smurf2. exhibited a reduced ability to resolve DNA catenanes and Cancer Res; 77(16); 1–11. 2017 AACR.

Introduction mice knockout for Smurf2 develop a wide spectrum of tumors in different organs and tissues. These and other studies estab- DNA topoisomerase IIa (Topo IIa) is the major form of the lished Smurf2 as an important regulator of genomic integrity, Topo II enzyme in cycling vertebrate cells that acts to untangle whose inactivation results in carcinogenesis (7). However, the chromosomal catenanes forming during the duplication of genet- mechanisms underlying tumor-suppressor functions of Smurf2 ic material. Topo IIa plays a pivotal role in chromatin organiza- are far from understood. tion, dynamics, and unperturbed chromosome inheritance. The Here, we report a novel mechanism by which Smurf2 is failure of Topo IIa to maintain DNA supercoiling homeostasis involved in genome integrity regulation—via stability regulation and properly disentangle daughter chromosomes can lead to the of Topo IIa, and prevention of anaphase bridge formation. We formation of pathological chromosome bridges, chromosomal also show that cellular levels of Smurf2 delineate Topo IIa protein instability (CIN), and ultimately to cancer (1–4). Furthermore, levels in both mouse and human normal and cancer cells and due to the high abundance of Topo IIa in rapidly proliferating tissues, and could determine cell sensitivity to Topo II poison cells and its vital roles in mitotic processes, Topo IIa is a core target etoposide. of several anticancer drugs (4, 5). Despite the importance of Topo IIa, the mechanisms governing its cellular levels remain largely unknown. Materials and Methods Recently, we reported that HECT-type E3 ubiquitin ligase Cell culture Smurf2 operates in mammalian cells as a critical regulator of Smurf2 knockout (Smurf2 / ) mouse embryonic ﬁbroblasts chromosome integrity, and acts to prevent the CIN, and carci- (MEF) and wild-type cells derived from littermate control nogenesis (6). We showed that genomic ablation of Smurf2 embryos were originally established in the laboratory of Dr. leads to accumulation of chromosomal aberrations, including Ying Zhang (National Cancer Institute,NIH,Bethesda,MD), Robertsonian translocations, undeﬁned translocations and and obtained from her laboratory in 2012 (6). These cells were marker chromosomes. Furthermore, we demonstrated that obtained at passages 30–35, and used between passages 40 and 55. Human cell lines used in this study were generously provided by Prof. Yosef Shiloh (Tel Aviv University, Tel Aviv, Israel). These cells were obtained in 2013, and originated from 1Laboratory of Molecular and Cellular Cancer Biology, Faculty of Medicine in the 2 the ATCC. These cell lines were propagated, frozen, and used in Galilee, Bar-Ilan University, Safed, Israel. Department of Pathology, The Galilee culture for up to 12 passages. All cell lines were maintained in Medical Center, Nahariya, Israel. high glucose DMEM medium (4,5 g/L D-Glucose, Gibco) sup- Note: Supplementary data for this article are available at Cancer Research plemented with 10% (v/v) FBS, 2 mmol/L L-glutamine, and 1% Online (http://cancerres.aacrjournals.org/). (v/v) penicillin–streptomycin, and incubated at 37Cwith5% Corresponding Author: Michael Blank, Bar-Ilan University, 8 Henrietta Szold, CO2. Cell strains were selectively tested for mycoplasma at the Safed 1311502, Israel. Phone: 9725-4222-0547; Fax: 972-4622-9256; E-mail: Faculty Core Facility using a PCR-based approach. The latest [email protected] date for testing of our leading cellular models, which include doi: 10.1158/0008-5472.CAN-16-2828 U2OS wild-type cells, Smurf2 knockout cells, Smurf2-knock- 2017 American Association for Cancer Research. down strains, and cells expressing Smurf2 catalytically inactive

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(Cys716Gly) and wild-type forms, was January 24, 2017. Cell extraction of Topo IIa from the samples, the remaining insol- line authentication was not conducted. uble fractions were solubilized in TEP buffer using a sonication, and subsequently analyzed in immunoblots. Tissue microarrays and IHC DNA decatenation assay was performed using nuclear extracts Human tissue microarrays (TMA) were purchased from U. and kinetoplast DNA (kDNA; TopoGEN) in a complete buffer S. Biomax, Inc. Mice tissues were fixedin4%formalinand5- assay. In particular, each reaction contained 50 mmol/L Tris HCl mm tissue sections were prepared. IHC was conducted as pH 8, 150 mmol/L NaCl, 10 mmol/L MgCl2, 2 mmol/L ATP, described previously (6). All comparable samples were sam- 400 ng of kinetoplast DNA, 2 mg of nuclear extract and double- pled on the same slide, and all staining procedures were distilled water to bring the final volume up to 40 mL. Reactions conducted on slides positioned horizontally. Histologic eva- were incubated at 37 C for 30 minutes, and stopped by adding 8 luations and TMAs scoring were conducted by a board-cer- mLof5Stop Buffer (TopoGEN). Subsequently, samples were tified pathologist at the Galilee Medical Center (Nahariya, incubated with RNase (40 mg/mL) at 37 C for 15 minutes and Israel). with Proteinase K (150 mg/mL) for an additional 15 minutes at 37C. Finally, samples were separated by electrophoresis through GST-fusion protein, pull-down assays, and ubiquitination a 1% agarose gel stained with SYBRS Safe DNA Gel Stain (Invi- assays trogen), and visualized in the SyngeneG:BOX. GST fusion proteins were prepared from E. coli using gluta- All other methods and reagents we used in this study are thione-Sepharose beads (Amersham); purified full-length detailed in Supplementary Methods Section. human Topo IIa was purchased from TopoGen (TG200H). An in vitro binding assay was performed as described previously Results (6). Briefly, GST or GST-Smurf2 were incubated with Topo IIa in binding buffer for 15 minutes at 37CandGST-Smurf2was Identification of Topo IIa as a novel interactor of Smurf2 pulled-down using Glutathione Sepharose 4B beads (GE To identify novel Smurf2-binding partners, we employed Healthcare). The beads were then washed four times with immunoprecipitation coupled with mass spectrometry (MS) ice-cold binding buffer and proteins were eluted with 5 SDS analyses. These analyses were conducted on Smurf2-deficient sample buffer. MEFs reconstituted either with a full-length FLAG-tagged In vivo and in vitro ubiquitination assays were performed as Smurf2 or with an empty vector as a control. FLAG-Smurf2 previously described (6, 8) with a few modifications. In brief, immunoprecipitates were resolved in SDS-PAGE, and protein for the in vivo ubiquitination assay cells were lysed with either bands were visualized using Coomassie staining (Fig. 1A). RIPA buffer supplemented with 5 mmol/L NEM or in 1% SDS Bands specifically associated with Smurf2 immunoprecipitates followed by an immediate boiling of samples for 15 minutes. were excised from the gel and submitted for identification in Following the boiling, cell lysates were equilibrated with MS. To deduct a background, the bands were cut side-by-side RIPA/NEM buffer to reduce SDS concentration in the samples from both positive (Smurf2-reconstituted cells) and control down to 0.1%. Cell lysates were then sonicated, FLAG-Topo lanes (empty vector). Using this approach, we identified Topo IIa was immunoprecipitated, and its ubiquitination pattern IIa as a novel Smurf2 interactor with a high degree of reliability: analyzed. Topo IIa-specific protein sequence was detected in 146 pep- For the in vitro ubiquitination assay, 500 ng of Topo IIa were tides in Smurf2-immunoprecipitated samples versus 0 peptides incubated with 250 ng of GST or GST-Smurf2, 5 mgofHA- in control samples (immunoprecipitates from cells expressing ubiquitin protein, E1 (UBE1; 100 ng) and E2 enzyme (UbcH5c; an empty vector; Fig. 1A). 150 ng), and 100 mmol/L ATP-Mg in the E3 ligase reaction To validate the MS results, we conducted several lines of buffer (Boston Biochem) for 2 hours at 37C. RIPA buffer was experiments. First, we demonstrated the interactions between added to the reactions and Topo IIa was pulled down using Smurf2 and Topo IIa by coimmunoprecipitation experiments in anti-Topo IIa antibody (Abcam) and protein G-Sepharose MEFs, the cell model that has been used to discover the Smurf2- beads. Topo IIa protein interaction (Fig. 1B). Next, we show that this interaction is also preserved in human cells. Immunoprecipita- Topoisomerase II extraction and DNA decatenation assay tion of MYC-tagged Smurf2 from HEK-293T cells detected endog- Nuclear extracts for DNA decatenation assay were prepared enous Topo IIa in complex with Smurf2, and vice versa, immu- as described previously (9), with a few modifications. In brief, noprecipitation of FLAG-tagged Topo IIa detected endogenous cell pellets were resuspended in TEMP buffer (10 mmol/L Tris- Smurf2 complexed with Topo IIa (Fig. 1C). Finally, using these HCl, pH 7.5, 1 mmol/L EDTA, 4 mmol/L MgCl2, 0.5 mmol/L cells we demonstrated the interaction between both endogenous PMSF), and incubated on ice for 15 minutes. Subsequently, Smurf2 and Topo IIa (Fig. 1D). Essentially, the discovered inter- 0.6% Nonidet-P40 substitute was added, samples vortexed for action between Smurf2 and Topo IIa appears to be direct, because 10 seconds, and centrifuged (20,000 g,30seconds).The we were also able to detect the interaction between purified supernatants, representing the cytoplasmic fraction, were col- human Topo IIa and Smurf2 in the tube (Fig. 1E). lected and saved for Western blot analysis. The remaining nuclei were then washed in ice-cold TEMP, resuspended in a Smurf2 physically interacts with Topo IIa during interphase TEP buffer (10 mmol/L Tris-HCl pH 7.5, 1 mmol/L EDTA, 500 To visualize the interaction between Smurf2 and Topo IIa in mmol/L NaCl, 0.5 mmol/L PMSF), vortexed at 4Cfor30 cells, we expressed GFP-Smurf2 in human osteosarcoma U2OS minutes, and centrifuged for 20 minutes at 20,000 g.The cells. These cells have a large nucleus and are commonly used in supernatants, representing the nuclear fraction, were collected localization microscopy to detect molecular interactions, in and saved for subsequent analyses. To verify the complete particular in the nuclear compartment. Following cell fixation

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Figure 1. Smurf2 physically interacts with Topo IIa. A, Coomassie gel staining of Smurf2 immunoprecipitates obtained from FLAG-Smurf2-reconstituted and control Smurf2/ MEFs. The ratio (R) shows the number of peptides identiﬁed for Topo IIa in MS versus control samples. B, Validation of Smurf2 interactions with Topo IIa in Smurf2/ MEFs reconstituted with a FLAG-tagged Smurf2. WCL, whole cell lysate. C, Reciprocal coimmunoprecipitation analysis showing interactions between Smurf2 and Topo IIa in human HEK-293T cells. D, Coimmunoprecipitation analysis of endogenous Smurf2 and Topo IIa interaction in HEK-293T cells. E, GST-pull down experiment showing direct interaction between puriﬁed GST-Smurf2 and Topo IIa.

and immunostaining with the Topo IIa-specificantibody,we of these proteins in human cells, but independently of each evaluated the colocalization between GFP-Smurf2 and Topo other (6, 10, 11). Collectively, these findings establish Topo IIa IIa under the confocal microscope. The specificity of the anti- as a novel Smurf2 interactor. Topo IIa antibody used in our studies was rigorously validated using Topo IIa knockdown cells. The results (Fig. 2A) show that Smurf2 controls the steady-state levels of Topo IIa in different in interphase cells Smurf2 occupies both nuclear and cyto- cell types and in tissues plasmic compartments, whereas Topo IIa exhibits exclusively To gain an insight into the biological significance of the nuclear localization. In the nucleus, Smurf2 showed a high complex formation between Smurf2 and Topo IIa,wefirst degree of colocalization with Topo IIa,inparticularwithits examined the levels of Topo IIa in Smurf2 knock-out (KO) nucleolar fraction, as evident from the images recorded in the vs. wild-type MEF cells. These cells were derived from littermate Nomarski imaging mode. control embryos. We found that the steady-state levels of Topo To validate the interaction between Smurf2 and Topo IIa,we IIa were profoundly diminished in Smurf2KO MEFs (Fig. 3A, recorded cell images of Smurf2/Topo IIa-stained cells at dif- Left). To determine whether the loss of Smurf2 alone is respon- ferent focal planes through the cell volume (Z-stack analysis). sibleforthedecreaseinthecellularlevelsofTopoIIa,we The results we obtained corroborated the interaction between restored Smurf2 expression in KO MEFs and found that upon Smurf2 and Topo IIa, and indicated that in interphase cells Smurf2 reconstitution the cellular levels of Topo IIa were Smurf2 associates with Topo IIa through the nuclear volume significantly increased (Fig. 3A, Right). Essentially, IHC and (Supplementary Fig. S1). western blot analyses conducted on the tissue samples of Next, an in situ proximity ligation assay (PLA), which enables Smurf2-deficient vs. control mice revealed that diminished determining the protein–protein interactions directly within Topo IIa protein levels were also a characteristic of Smurf2- the cell, provided further evidence that Smurf2 and Topo IIa ablated tissues (Fig. 3B and C; Supplementary Fig. S2A and physically interact with each other (Fig. 2B and C). Immuno- S2B). The levels of Topo IIb, which activities are mostly asso- precipitation studies conducted in U2OS cells further corrob- ciated with transcription regulation (2), were comparable orated the interaction between Smurf2 and Topo IIa in this cell between Smurf2KO and control tissues. model (Fig. 2D). Similar results were also obtained in different human cell During mitosis, Topo IIa is tightly associated with chroma- models: in U2OS osteosarcoma cells, HCT116 colon carcinoma tin, and follows the chromatin movement pattern. The exam- cells, and DU145 and PC-3 prostate carcinoma cells. In all these ination of Topo IIa and Smurf2 biodistribution in U2OS cells cells, decrease of Smurf2 expression levels either through acute going through the unperturbed mitosis revealed that while (using siRNAs) or stable knock-down (using lentiviral-based Topo IIa associates with mitotic chromosomes, Smurf2 is shRNAs) reduced the steady-state levels of Topo IIa (Fig. 3D). mainly excluded from the interactions with chromatids (Fig. These effects were monitored through the use of four different si/ 2E). Moreover, in mitotic cells Smurf2 was mostly found at the shRNAs: designed to target Smurf2 mRNA either at 30UTR or its periphery of Topo IIa-chromatin templates, including the coding sequence. spindle midzone. The observed biodistribution of Smurf2 and Next, to validate our biochemical data at a single-cell reso- Topo IIa in interphase and mitotic cells is in accordance with lution, we performed immunofluorescence staining of Topo IIa the previous studies, which determined the localization of each in Smurf2 knockdown cells. The immunofluorescence studies

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Figure 2. Smurf2 associates with Topo IIa in interphase cells. A, Confocal images showing colocalization of GFP-Smurf2 and endogenous Topo IIa in the interphase nucleus of U2OS cells. DNA was counterstained with Hoechst. Two representative images are shown in the figure; scale bars, 10 mm. B, PLA assay indicating sites of direct protein–protein interaction of FLAG-Smurf2 and endogenous Topo IIa in U2OS cell nuclei. Scale bars, 20 mm. The diagram at the bottom of the figure shows the conditions under which the fluorescent signal in PLA is generated. C, Quantification of PLA (n ¼ 109). D, Coimmunoprecipitation analysis substantiating the interaction of FLAG-Smurf2 and Topo IIa in U2OS cell model. E, Biodistribution of GFP-Smurf2 and Topo IIa in U2OS cells sequestered at different stages of mitosis. Note distinct Smurf2 and Topo IIa localization patterns in mitotic cells. Topo IIa is localized on mitotic chromosomes. Smurf2 is mostly excluded from the chromatin templates. Scale bars, 10 mm.

were conducted concomitant to the Western blot analyses and DNA topoisomerase 1 (Top1), which also operates in performed on WCL, as well as on the fractionated samples: DNA transition processes to maintain supercoiling homeosta- cytosolic, nucleoplasmic, and chromatin fractions (Fig. 3E; sis(12),wereunaffectedbySmurf2depletion(Fig.3F).Fur- Supplementary Fig. S2C). The results we obtained corroborated thermore, we conducted bi-parametric FACS analyses on cells that the steady-state levels of Topo IIa in Smurf2-depleted cells in which Smurf2 was depleted either through CRISPR/Cas9 are significantly diminished. Of note, the observed Topo IIa- gene editing or using RNAi. These analyses provided evidence staining pattern and its decreased levels in Smurf2 knock-down that targeting of Smurf2 for inactivation does not affect cell- cells were independent of the cell fixation procedure, and cycle distribution, although a moderate decrease in the mitotic monitored in both formaldehyde- and methanol/acetone-fixed population of Smurf2-knockdown cells was observed (Supple- cells (Supplementary Fig. S2C). mentary Fig. S3). Furthermore, using the CRISPR/Cas9-based gene-editing To determine whether Smurf2 affects Topo IIa protein levels by system, we generated Smurf2CRISPR U2OS cell line, in which modulating its gene expression, we analyzed the mRNA expres- we succeeded to decrease Smurf2 cellular levels down to 10%– sion levels of Topo IIa in Smurf2 knockdown and control U2OS 15% of the original level (Fig. 3F). Using these cells, we and HCT116 cells using real-time qRT-PCR. The data showed that demonstrated that targeting SMURF2 for inactivation at the mRNA levels of Topo IIa were unaffected by Smurf2 depletion genome level produced in human cells results similar to both (Fig. 3G). mouse Smurf2 / cellsandtissues(Fig.3A–C; Supplementary Finally, we demonstrated that overexpression in these cells of Fig. S2B), and to human cell lines knocked-down for Smurf2 MYC-Smurf2 increases Topo IIa protein levels proportional to the with RNAi (Fig. 3D and E). Of note, both the levels of Topo IIb amount of Smurf2 transduced to the cells (Fig. 3H). Altogether,

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Figure 3. Smurf2 positively regulates the steady-state levels of Topo IIa. A, Western blot analysis of Topo IIa in Smurf2 wild-type (WT; Smurf2þ/þ) and Smurf2 knockout (KO; Smurf2/) MEFs (left), and in Smurf2/ MEFs reconstituted with either an empty FLAG vector (Smurf2/(E)) or with FLAG-Smurf2 (Smurf2/(R); right). B, IHC staining of Topo IIa (brown) in spleen tissue sections prepared from WT and KO mice. The nuclei were counterstained with hematoxylin (blue). Scale bars, 50 mm. Both WT and KO tissues were sampled on the same slide and processed for IHC simultaneously. The images were acquired and processed under the equal settings. C, Western blot analysis of Topo IIa and IIb expression in the spleen tissues. D, Western blot analysis of Topo IIa in different human cell models knockdown for Smurf2. Non-silencing siRNA (NS) and shRNA directed against luciferase (Luc) were used as controls for siRNA and shRNA experiments, respectively. The diagram at the bottom shows the position of each siRNA and shRNA on the mRNA's map of Smurf2. E, Immunofluorescence staining of Topo IIa in U2OS cells showing diminished Topo IIa levels upon Smurf2 knockdown. The specificity of Topo IIa antibody was validated on siTopo IIa cells. Scale bars, 20 mm. Bottom, Western blot analyses of Topo IIa in Smurf2 knockdown cells conducted concomitantly with the immunofluorescent studies. Analyses were performed on whole cell lysate (WCL) as well as on fractionated samples, in which cytosolic (C), nucleoplasmic (N), and chromatin fractions (IF) were extracted. Protein loadings and degree of fractionations were demonstrated by using antibodies against the cytosolic a-tubulin and the chromatin component histone H2B. F, Western blot analysis of Topo IIa,IIb, and Top1 in U2OS cells, in which Smurf2 was depleted using CRISPR/Cas9-based gene editing system (Smurf2CRISPR cells). The diagram on the right shows the position of SMURF2 gene targeting and the targeting sequence. G, qRT-PCR analysis of Topo IIa mRNA levels in Smurf2 knockdown U2OS and HCT116 cell models. Data are represented as mean SD of three independent experiments, with three technical replicates for each experiment. H, Western blot analysis showing that overexpression of MYC-Smurf2 increases Topo IIa protein levels in Smurf2 dose-dependent manner. these data reveal Smurf2 as an essential regulator of the steady- with a proteasome inhibitor, the Topo IIa levels in Smurf2- state levels of Topo IIa, and suggest that Smurf2 regulates Topo IIa proficient cells were notably increased, yet slightly affected in protein levels posttranslationally. Smurf2 knockdown cells (Fig. 4A, lane 2 vs. 4). In contrast, overexpression of Smurf2 in these cells dramatically increased Smurf2 regulates the stability of Topo IIa through the the levels of Topo IIa in both untreated and MG-132–treated cells inhibition of Topo IIa proteasomal degradation as compared with control samples transduced with an empty To investigate whether Smurf2 manages Topo IIa protein levels vector (Fig. 4B, lanes 3 and 4 vs.1 and 2). Noteworthy, MG-132 by regulating its degradation, we conducted several lines of cell treatment did not lead to further stabilization of Topo IIa in experiments. First, we treated Smurf2-deficient and -overexpres- Smurf2-overexpressing cells. This finding suggests that high sing cells with the proteasome inhibitor MG-132 and found that Smurf2 levels were sufficient to prevent Topo IIa proteasomal the stability of Topo IIa is regulated through the proteasome- degradation. Finally, the coexpression of FLAG-Topo IIa together mediated degradation, and is under Smurf2 control (Fig. 4A and with MYC-Smurf2 protected Topo IIa from the proteasome- B; Supplementary Fig. S4A and S4B). Following cell treatment mediated degradation in Smurf2 dose-dependent manner

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Figure 4. Smurf2 regulates the stability of Topo IIa through the inhibition of its proteasomal degradation. A, Western blot analysis of Topo IIa in Smurf2 knockdown U2OS cells after treatment with MG-132 (5 mmol/L; 4 hours). B, Western blot analysis showing that Smurf2 overexpression protects Topo IIa, but not Topo IIb, from proteasome- mediated degradation in U2OS cells. C, Overexpression of Smurf2WT, but not its catalytically inactive mutant form (Smurf2CG), increases Topo IIa protein levels in U2OS cells. D, Western blot analysis of Topo IIa in Smurf2/ MEFs reconstituted with Smurf2WT or its E3 ligase mutant form. E, Western blot analysis showing that Smurf2 modifies Topo IIa ubiquitination in E3 ligase-dependent manner in vivo (in HEK-293T cells). Cells were lysed using either RIPA lysis buffer supplemented with the deubiquitinase inhibitor NEM (5 mmol/L; left) or in 1% SDS, followed by immediate sample boiling (right). F, Smurf2 reduces the K48- linked polyubiquitination of Topo IIa in vivo. G, Western blot analysis of Topo IIa ubiquitination by recombinant GST-Smurf2 in the presence of E1, E2, and HA-tagged ubiquitin (HA-Ub). GST-Smurf2CA (Cys716Ala) mutant protein as well as purified GST were used as controls. Topo IIa was pulled down from the reaction using anti-Topo IIa antibody. Arrow, monoubiquitinated Topo IIa. Right, Coomassie blue staining of purified GST and GST-Smurf2 used in this study. WCL, whole cell lysate.

(Supplementary Fig. S4A). Of note, we did not find significant tin and Myc-tagged Smurf2. FLAG-Topo IIa was then immuno- changes in the levels of Topo IIa in chloroquine-treated versus precipitated and its ubiquitination pattern was analyzed. To control cells (Supplementary Fig. S4B). Taken together, these data perform this analysis, we used an anti-HA antibody, which suggest that Smurf2 stabilizes Topo IIa by inhibiting its protea- specifically recognized the HA-tagged ubiquitin attached to Topo somal degradation. IIa. This experimental design allowed us to identify that Topo IIa undergoes ubiquitination (Fig. 4E, lanes 3–5 vs. 1 and 2). This E3 ubiquitin ligase activities of Smurf2 are required for Topo IIa design also allowed us to discover that the addition of catalytically stability regulation active Smurf2 significantly enriched Topo IIa monoubiquitina- Smurf2 is a HECT-type E3 ubiquitin ligase in which active-site tion (Fig. 4E, lanes 4 vs. 3). Moreover, the results show that the cysteine 716 (Cys716) is crucial for its catalytic activity. To substitution of Smurf2WT with its mutant form failed to produce determine whether E3 ligase functions of Smurf2 are required this ubiquitination phenomenon (Fig. 4E, lane 5 vs. 4). The data for Topo IIa stability regulation, we analyzed Topo IIa protein also show that in the cells, which expressed Smurf2CG, the Topo levels in U2OS cells expressing either Smurf2WT or its catalytically IIa is polyubiquitinated. The observed polyubiquitination of inactive form, in which active-site cysteine was substituted with Topo IIa, whereas evident in Smurf2CG-expressing cells, was less glycine (Cys716Gly; Smurf2CG). Cells transduced with an empty visible in cells transfected with an empty vector (Fig. 4E, left; lane 5 vector served as a control. The data (Fig. 4C) show that only the vs. 3). We assumed that this finding due to the incomplete expression of Smurf2WT was able to increase the Topo IIa protein inactivation of cellular deubiquitinases upon cell lysis. To clarify levels. Similar results were also obtained in Smurf2-ablated MEF this point, we conducted the Topo IIa ubiquitination experiment cells reconstituted with Smurf2WT or its E3 ligase mutant form again, but to deactivate the cellular deubiquitinases more effi- (Fig. 4D). ciently, in the second round, the cells were lysed directly in 1% SDS, followed by an immediate sample boiling. Similar to our Smurf2 operates as a molecular editor of the Topo IIa previous findings, the addition of Smurf2WT, but not Smurf2CG ubiquitination code or an empty vector, significantly enriched the monoubiquitinated To determine whether Smurf2 is capable of ubiquitinating fraction of Topo IIa (Fig. 4E, right). In addition, we verified that Topo IIa, we conducted in cellulo ubiquitination assay in which the expression of catalytically active Smurf2 switched the ubiqui- FLAG-Topo IIa was coexpressed together with HA-tagged ubiquitination code on Topo IIa from poly- to monoubiquitination, as

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compared with samples expressing an empty vector or Smurf2CG IIa. In addition, using Topo IIa and Topo IIb knockdown cells, we (Fig. 4E, right; lane 4 vs. 3 and 5). demonstrate that Topo II decatenation assay used in our study is Our data indicate that the protein stability of Topo IIa is specific to monitor activities of Topo IIa. This finding is in regulated through the proteasome-mediated degradation. This agreement with results previously published by Bower and col- type of proteolysis relies on the formation of a particular type leagues (13), and showing that Topo II assay mainly measures of ubiquitination–K48–linked polyubiquitination. To exam- activities of Topo IIa. Furthermore, by assaying Topoisomerase I ine whether Smurf2 protects Topo IIa from the degradation by activity, we demonstrate that the extracts derived from Smurf2- modulating this particular type of ubiquitination, we coex- knockdown cells are otherwise normal, and have unaltered Top1 pressed FLAG-Topo IIa with Smurf2 together with either wild- activity (Supplementary Fig. S5B). The sensitivity of Top1 assay type ubiquitin or its mutant form (K48-only ubiquitin). In was validated using Top1 knockdown cells (Supplementary Fig. this mutant ubiquitin, all the lysines except K48 were mutated S5C and S5D). to arginines. These mutations abolish the ability of ubiquitin Next, we analyzed and compared the occurrence of chromo- to form polyubiquitin chains other than through the K48- some bridges, and lagging chromosomes, in Smurf2- and Topo linked chain. Both wild-type and mutant forms of ubiquitin IIa-depletedcells.Tothisend,weusedtheU2OScellmodel. were HA-tagged. FLAG-Topo IIa was then immunoprecipi- These cells have been previously shown to exhibit an increased tated and its ubiquitination pattern analyzed using anti-HA DNA bridge formation after Topo IIa knockdown (14). The antibody. The results show that similar to the previous find- data (Fig. 5C and D; Supplementary Fig. S5E) show that Smurf2 ings the expression of Smurf2 together with a wild-type form depletion significantly increased the formation of DNA bridges of ubiquitin switched the Topo IIa ubiquitination from poly- in mitotic cells (P ¼ 0.000212, c2 test). Moreover, the incidence to monoubiquitination (Fig. 4F, lane 2 vs. 1). However, when of DNA bridges in Smurf2-depleted mitotic cells was highly the wild-type ubiquitin was substituted to K48-only ubiquitin, similar to the incidence of these bridges in Topo IIa knock- and expressed together with Smurf2, the K48-linked polyubi- down cells (Fig. 5C). This finding suggests that Smurf2 deple- quitination of Topo IIa was completely abolished (Fig. 4F, tion phenocopies the Topo IIa depletionintheresolvingof lane 4 vs. lane 3). These data suggestthatSmurf2operatesasa DNA catenanes. Essentially, we demonstrated that Smurf2 molecular editor that modifies the Topo IIa ubiquitination inactivation had no significant effect on the occurrence of code to protect Topo IIa from the degradation-promoting– lagging chromosomes (Fig. 5E and F). These chromosomes are K48 polyubiquitination. The data also indicate that the E3 formed through the mechanisms distinct from the mechanisms ligase activities of Smurf2 are required for the switch. underlying anaphase bridge formation, and are independent of Finally, we performed a ubiquitination reconstitution assay Topo IIa (14–16). using purified ubiquitin-activating enzyme (E1), ubiquitin con- Finally, we reconstituted Smurf2CRISPR U2OS cells with jugase (E2), HA-ubiquitin, Smurf2 (either wild-type or E3 mutant mCherry-tagged Topo IIa (or with an empty vector as a control) form), and human Topo IIa. Using these experimental settings, and, following the generation of stable cell lines, analyzed the we demonstrate that Smurf2 ubiquitinates Topo IIa directly (Fig. formation of anaphase bridges and lagging chromosomes in these 4G). The specificity of this reaction is demonstrated by the unique cells. The data (Fig. 5G; Supplementary Fig. S5F) show that in ubiquitination pattern of Topo IIa monitored only in the pres- Smurf2-depleted cells transduced with Topo IIa the cellular ence of Smurf2WT. In addition, we validated that the observed phenotype was rescued, as evident by a significant decrease in ubiquitination pattern belongs to Topo IIa, and not to Smurf2 or the population of mitotic cells with chromatin bridges compared any other components used in the ubiquitination assay (Supple- with control (P ¼ 0.014, c2 test). The incidence of lagging mentary Fig. S4C and S4D). chromosomes in these cells remained unaffected (Fig. 5H). Of note, decatenation assay conducted on Topo IIa-reconstituted Smurf2 depletion decreases the Topo II decatenation activity cells suggested that the N-terminally tagged mCherry-Topo IIa is and increases the formation of anaphase bridges catalytically active (Supplementary Fig. S5G); similar results were Topo IIa is a key player in the decatenation checkpoint, and reported by Lane and colleagues (17), showing that mCherry- its inability to unwind chromosomal entanglements can lead to tagged Topo IIa can functionally complement the loss of endog- the formation of pathological chromosome bridges—one of enous protein. the major sources of chromosomal translocations. On the basis of these findings, and our data establishing Smurf2 as a positive Smurf2-depletion decreases cell sensitivity to Topo II poison regulator of Topo IIa, we hypothesized that cells depleted of etoposide Smurf2 will exhibit a phenotype similar to the Topo IIa- The cellular levels of Topo IIa are an important determinant depleted cells. Specifi cally, we hypothesized that these cells of tumor cell sensitivity to Topo II-targeting drugs, in particular will exhibit the reduced ability to untangle chromosomal to etoposide: higher Topo IIa levels, higher sensitivity, and vice catenanes, and will show the exacerbated formation of ana- versa (18–21). Topo II poison etoposide acts to stabilize tran- phase bridges. sientTopoII-bridgedbreakandleadtogenerationofhighly To test this hypothesis, we first examined the Topo II decatena- cytotoxic DNA damage–double-strand breaks. To determine tion activity in nuclear extracts prepared from Smurf2-knockdown whether and how manipulations with Smurf2 cellular levels and control cells using the decatenation assay. Nuclear extracts affect cell sensitivity to etoposide, we assessed the sensitivity of prepared from Topo IIa and Topo IIb knockdown cells were also Smurf2CRISPR and control wild-type U2OS cells to this drug. The incorporated in the analysis, and served as additional controls. data (Fig. 5I) show that Smurf2CRISPR cellsweremoreresistant The data (Fig. 5A and B; Supplementary Fig. S5A) show that DNA to etoposide treatment than control cells. Similar results were decatenation was significantly reduced in Smurf2-knockdown also observed in Smurf2KO versus WT MEF cells (Supplemen- cells, and was comparable with the cells knockdown for Topo tary Fig. S5H).

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Figure 5. Smurf2 depletion phenocopies Topo IIa depletion. A, DNA decatenation assay. B, Relative decatenation activity in Smurf2 and Topo IIa knockdown cells. Data were quantified on two independent experiments. C, Quantification of the percentage of U2OS cells with Hoechst-positive ana/telophase bridges. Scale bars, means SD (n ¼ 3). At least 100 ana/telophase cells were scored per experiment. D, Representative confocal images showing the formation of anaphase bridges in Smurf2CRISPR U2OS cells. Scale bars, 10 mm. E, Quantification of the percentage of U2OS cells with lagging chromosomes in Smurf2- and Topo IIa-depleted samples. Scale bars, means SD (n ¼ 3). A minimum 100 ana/telophase cells were scored per experiment. F, Representative confocal images showing the occurrence of lagging chromosomes in both Smurf2CRISPR and in control cells. Scale bars, 10 mm. G, The incidence of anaphase bridge formation in Smurf2CRISPR U2OS cells transduced with either an empty vector or with mCherry-Topo IIa. Data are represented as mean SD (n ¼ 3). H, Quantification of the occurrence of lagging chromosomes in cells described in G. I, XTT assay showing decreased sensitivity of Smurf2CRISPR to etoposide treatment. Graphs represent the average values of three independent experiments performed in hexaplicates. , P < 0.01; , P < 0.001. ns, nonsignificant.

The Smurf2/Topo IIa relationship is preserved in Discussion human tissues In this study, we identifiedanovelmechanismbywhich To determine whether Smurf2 is also a relevant determi- Smurf2, an HECT-type E3 ubiquitin ligase and recently discov- nant of Topo IIa protein levels in human tissues, we con- ered tumor suppressor, is involved in the regulation of genome ducted IHC analyses on two TMAs: prostate (PR1921), which integrity. Recently, we reported that cells depleted of Smurf2 included both normal (n ¼ 31) and cancer tissues (n ¼ 149); accumulate multiple chromosome abnormalities in their and breast TMA (BR10010) containing primary and meta- genome, where translocations were the most notable hallmark static carcinoma tissues (n ¼ 98). These TMAs were stained (6). However, the mechanisms underlying this phenomenon with anti-Topo IIa and anti–Smurf2 IHC-specific antibodies, are far from understood. and counterstained with hematoxylin. The subsequent histo- The formation of pathological chromatin bridges is consid- pathologic examination coupled with the Spearman's ered one of the major causes of chromosomal translocations, rank correlation analysis revealed that the expression levels which are viewed as an outcome of the compromised decate- of Smurf2 and Topo IIa are positively correlated. This nation checkpoint mediated by Topo IIa (16, 22). Inability of correlation was statistically significant for all analyzed tis- Topo IIa to decatenate DNA (e.g., due to its reduced cellular sues: P < 0.001 for breast and prostate carcinoma tissues, and levels and/or activities) can lead to CIN and, ultimately, to P < 0.05 for normal prostate tissues (Fig. 6A–D; Supplemen- cancer. Moreover, in established tumors, CIN can drive tumor tary Table S1).

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Figure 6. The Smurf2/Topo IIa relationship is preserved in human tissues. A, A correlative relationship between Smurf2 and Topo IIa protein levels in human prostate TMA. These tissues were sampled on the same slide and processed for IHC simultaneously. The intensity and the percentages of positive cells were scored using the following system: 0 ¼ <10%; 1 ¼ 10%–24%; 2 ¼ 25%–49%; 3 ¼ 50%–74%; 4 ¼ 75%–100%. Data are presented as counts SD. B, Representative images of IHC staining of Smurf2 and Topo IIa quantified in A. L5, L11, E1, and C3 are the coordinates of the samples in the tissue array. Scale bars, 50 mm. C, Quantification of the expression levels of Smurf2 and Topo IIa in human breast cancer TMA. D, Representative images of IHC staining of Smurf2 and Topo IIa in breast TMA. B8 and F8 are the coordinates of the samples in the tissue array. Scale bars, 50 mm. progression by accelerating the gain of oncogenic loci and the also in undamaged cells, and it is under the control of Smurf2. loss of tumor-suppressor loci (2, 4). This finding is intriguing because Smurf2 is primarily known as a Using different human and mouse cellular models, Smurf2- degradation promoting E3 (7, 27). Nonetheless, our data strongly targeting approaches, human tissue arrays, genetically modified suggest that Smurf2 is an authentic positive regulator of Topo IIa systems and rescue experiments, we provided confirmatory that protects it from the proteasome-mediated proteolysis in insights that Smurf2 acts as a key cellular factor that stabilizes Smurf2 dose- and E3 ligase-dependent manners. The data Topo IIa and prevents the formation of pathological DNA obtained in IHC studies conducted on tissues derived from bridges. Mechanistically, we demonstrated that Smurf2 physically Smurf2-deficient and wild-type mice, as well as on human TMAs interacts with Topo IIa and protects it from the proteasome- (278 normal and tumor tissues), provided further support that mediated degradation in Smurf2 dose- and catalytically depen- Smurf2 is also a positive regulator of Topo IIa in tissues. dent manners. We showed that Smurf2 operates as a molecular The functional and cellular assays conducted in this study switcher that modifies the Topo IIa ubiquitination code to reduce provided evidence that Smurf2-depletion phenocopies Topo IIa its degradation-promoting K48 polyubiquitination and increase depletion. This finding is quite intriguing especially in light of monoubiquitination. Essentially, we demonstrated that Smurf2 is mouse phenotypes reported for Topo IIa and Smurf2: Topo IIa- capable to directly bind to and ubiquitinate Topo IIa. Finally, we deficient mice are embryonic lethal (28), whereas Smurf2KO mice provided evidence that Smurf2 is a relevant determinant of Topo are viable and exhibit no overt developmental defect during IIa protein levels in mouse and human normal and cancer tissues, embryogenesis (6, 29). There are a few possible explanations that and that Smurf2 levels could affect the cell sensitivity to Topo II could reconcile these mouse phenotypes. First, as we demonstrat- poison and anticancer drug etoposide. ed in a panel of different human and mouse cells and tissues Currently, there is limited information about the mechanisms (Fig. 3), the depletion of Smurf2 significantly reduced, but not that regulate Topo IIa protein levels and stability. Studies pub- ablated Topo IIa levels. It is possible that the remaining levels of lished to date mainly report that following cell treatment with Topo IIa were sufficient to complete embryogenesis. Second, topoisomerase II poisons, Topo IIa undergoes proteasome deg- during embryogenesis other members of the NEDD4 E3 ligase radation, which enables repair of DNA lesions (23–26). Our study family (nine in total) could compensate for Smurf2 deficiency. demonstrates that Topo IIa undergoes proteasomal degradation For example, mice with a targeted disruption of either the Smurf2

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or Smurf1 allele are viable and survive to adulthood (6, 29, 30); Writing, review, and/or revision of the manuscript: M. Blank however, disruption of both Smurf2 and Smurf1 alleles leads to Administrative, technical, or material support (i.e., reporting or organizing embryonic lethality (31). The possibility that Smurf2 does not data, constructing databases): A. Emanuelli, A.P. Borroni, P.A. Shah, a D. Manikoth Ayyathan, G. Levy-Cohen affect the cellular levels of Topo II during early embryogenesis, Study supervision: M. Blank where it appears to be particularly important (28), also exists. Other (carried out most of the experimental studies in this article): Collectively, our ﬁndings establish a novel functional link A. Emanuelli between an E3 ubiquitin ligase Smurf2 and Topo IIa, and propose Other (generation of cell lines): P. Koganti a new paradigm in the stability regulation of Topo IIa, and genome integrity maintenance. Our data also suggest an intrigu- Acknowledgments ing role of Smurf2 as an E3 ligase that can protect some of the key We thank Meir Shamay for helpful discussions during this article preparation cellular proteins from degradation, and provide a novel insight and Basem Hijazi for statistical analysis. We are also grateful to Ying E. Zhang for into the tumor suppressor functions of Smurf2. providing Smurf2-deﬁcient mice and for other support.

Disclosure of Potential Conﬂicts of Interest Grant Support No potential conﬂicts of interest were disclosed. This work was supported by several grants, including ICRF 00636, Marie-Curie FP-7 CIG 612816, and Dayan Family Foundation award to Authors' Contributions M. Blank. Conception and design: M. Blank The costs of publication of this article were defrayed in part by the payment of Development of methodology: P. Koganti, G. Levy-Cohen page charges. This article must therefore be hereby marked advertisement in Acquisition of data (provided animals, acquired and managed patients, accordance with 18 U.S.C. Section 1734 solely to indicate this fact. provided facilities, etc.): A. Emanuelli Analysis and interpretation of data (e.g., statistical analysis, biostatistics, Received October 20, 2016; revised December 13, 2016; accepted June 9, computational analysis): L. Apel-Sarid, M. Blank 2017; published OnlineFirst June 13, 2017.

References 1. Grue P, Grasser A, Sehested M, Jensen PB, Uhse A, Straub T, et al. 15. Thompson SL, Compton DA. Chromosome missegregation in human cells Essential mitotic functions of DNA topoisomerase IIa are not adopted arises through specific types of kinetochore—microtubule attachment by topoisomerase IIb in human H69 cells. J Biol Chem 1998;273: errors. Proc Natl Acad Sci U S A 2011;108:17974–8. 33660–6. 16. Ganem NJ, Pellman D. Linking abnormal mitosis to the acquisition of 2. Wang JC. Cellular roles of DNA topoisomerases: a molecular perspective. DNA damage. J Cell Biol 2012;199:871–81. Nat Rev Mol Cell Biol 2002;3:430–40. 17. Lane AB, Gimenez-Abian JF, Clarke DJ. A novel chromatin tether domain 3. Farr CJ, Antoniou-Kourounioti M, Mimmack ML, Volkov A, Porter controls topoisomerase IIa dynamics and mitotic chromosome formation. ACG. The a isoform of topoisomerase II is required for hypercompac- J Cell Biol 2013;203:471–86. tion of mitotic chromosomes in human cells. Nucleic Acids Res 2014; 18. Zhou Z, Zwelling LA, Ganapathi R, Kleinerman ES. Enhanced etoposide 42:4414–26. sensitivity following adenovirus-mediated human topoisomerase IIalpha 4. Chen T, Sun Y, Ji P, Kopetz S, Zhang W. Topoisomerase IIa in chromosome gene transfer is independent of topoisomerase IIbeta. Br J Cancer 2001; instability and personalized cancer therapy. Oncogene 2015;34:4019–31. 85:747–51. 5. Nitiss JL. Targeting DNA topoisomerase II in cancer chemotherapy. Nat Rev 19. Burgess DJ, Doles J, Zender L, Xue W, Ma B, McCombie WR, et al. Cancer 2009;9:338–50. Topoisomerase levels determine chemotherapy response in vitro and in 6. Blank M, Tang Y, Yamashita M, Burkett SS, Cheng SY, Zhang YE. A vivo. Proc Natl Acad Sci U S A 2008;105:9053–58. tumor suppressor function of Smurf2 associated with controlling 20. Erriquez J, Becco P, Olivero M, Ponzone R, Maggiorotto F, Ferrero A, et al. chromatin landscape and genome stability through RNF20. Nat Med TOP2A gene copy gain predicts response of epithelial ovarian cancers to 2012;18:227–34. pegylated liposomal doxorubicin: TOP2A as marker of response to PLD in 7. Zou X, Levy-Cohen G, Blank M. Molecular functions of NEDD4 E3 ovarian cancer. Gynecol Oncol 2015;138:627–33. ubiquitin ligases in cancer. Biochim Biophys Acta 2015;1856:91–106. 21. Kirk JS, Schaarschuch K, Dalimov Z, Lasorsa E, Ku S, Ramakrishnan S, et al. 8. Levy-Cohen G, Blank M. Functional analysis of protein ubiquitination. Top2a identifies and provides epigenetic rationale for novel combination Anal Biochem 2015;484:37–9. therapeutic strategies for aggressive prostate cancer. Oncotarget 2015;6: 9. Congdon LM, Pourpak A, Escalante AM, Dorr RT, Landowski TH. Protea- 3136–46. somal inhibition stabilizes topoisomerase IIa protein and reverses resis- 22. Germann SM, Schramke V, Pedersen RT, Gallina I, Eckert-Boulet N, tance to the topoisomerase II poison ethonafide (AMP-53, 6-ethoxyazo- Oestergaard VH, et al. TopBP1/Dpb11 binds DNA anaphase bridges to nafide). Biochem Pharmacol 2008;75:883–90. prevent genome instability. J Cell Biol 2014;204:45–59. 10. Osmundson EC, Ray D, Moore FE, Gao Q, Thomsen GH, Kiyokawa H. The 23. Nayak MS, Yang JM, Hait WN. Effect of a single nucleotide polymor- HECT E3 ligase Smurf2 is required for Mad2-dependent spindle assembly phism in the murine double minute 2 promoter (SNP309) on the checkpoint. J Cell Biol 2008;183:267–77. sensitivity to topoisomerase II-targeting drugs. Cancer Res 2007;67: 11. Christensen MO, Larsen MK, Barthelmes HU, Hock R, Andersen CL, 5831–9. Kjeldsen E, et al. Dynamics of human DNA topoisomerases IIalpha and 24. Congdon LM, Pourpak A, Escalante AM, Dorr RT, Landowski TH. Protea- IIbeta in living cells. J Cell Biol 2002;157:31–44. somal inhibition stabilizes topoisomerase IIalpha protein and reverses 12. Li M, Liu Y. Topoisomerase I in Human Disease Pathogenesis and Treat- resistance to the topoisomerase II poison ethonafide (AMP-53, 6-ethox- ments. Genomics Proteomics Bioinformatics 2016;14:166–71. yazonafide). Biochem Pharmacol 2008;75:883–90. 13. Bower JJ, Karaca GF, Zhou Y, Simpson DA, Cordeiro-Stone M, 25. Alchanati I, Teicher C, Cohen G, Shemesh V, Barr HM, Nakache P, et al. The Kaufmann WK. Topoisomerase IIalpha maintains genomic stability E3 ubiquitin-ligase Bmi1/Ring1A controls the proteasomal degradation of through decatenation G(2) checkpoint signaling. Oncogene 2010;29: Top2alpha cleavage complex—a potentially new drug target. PLoS ONE 4787–99. 2009;4:e8104. 14. Broderick R, Nieminuszczy J, Blackford AN, Winczura A, Niedzwiedz W. 26. GaoR,SchellenbergMJ,HuangSY,AbdelmalakM,MarchandC,Nitiss TOPBP1 recruits TOP2A to ultra-fine anaphase bridges to aid in their KC, et al. Proteolytic degradation of topoisomerase II (Top2) enables resolution. Nat Commun 2015;6:6572. the processing of Top2*DNA and Top2*RNA covalent complexes by

OF10 Cancer Res; 77(16) August 15, 2017 Cancer Research

The Smurf2/Topo IIa Axis and Genome Stability

tyrosyl-DNA-phosphodiesterase 2 (TDP2). J Biol Chem 2014;289: 29. Tang LY, Yamashita M, Coussens NP, Tang Y, Wang X, Li C, et al. Ablation of 17960–9. Smurf2 reveals an inhibition in TGF-b signalling through multiple mono- 27. David D, Nair SA, Pillai MR. Smurf E3 ubiquitin ligases at the cross roads ubiquitination of Smad3. EMBO J 2011;30:4777–89. of oncogenesis and tumor suppression. Biochim Biophys Acta 2013;1835: 30. Yamashita M, Ying SX, Zhang GM, Li C, Cheng SY, Deng CX, et al. Ubiquitin 119–28. ligase Smurf1 controls osteoblast activity and bone homeostasis by target- 28. Akimitsu N, Adachi N, Hirai H, Hossain MS, Hamamoto H, Kobayashi M, ing MEKK2 for degradation. Cell 2005;121:101–13. et al. Enforced cytokinesis without complete nuclear division in embryonic 31. Narimatsu M, Bose R, Pye M, Zhang L, Miller B, Ching P, et al. cells depleting the activity of DNA topoisomerase IIa. Genes Cells Regulation of planar cell polarity by Smurf ubiquitin ligases. Cell 2003;8:393–402. 2009;137:295–307.

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Smurf2-Mediated Stabilization of DNA Topoisomerase IIα Controls Genomic Integrity

Andrea Emanuelli, Aurora P. Borroni, Liat Apel-Sarid, et al.

Cancer Res Published OnlineFirst June 13, 2017.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-16-2828

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