Smurf2-Mediated Stabilization of DNA Topoisomerase Iiα Controls Genomic Integrity

Author Manuscript Published OnlineFirst on June 13, 2017; DOI: 10.1158/0008-5472.CAN-16-2828 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Smurf2-mediated stabilization of DNA topoisomerase II 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

1Laboratory of Molecular and Cellular Cancer Biology, Faculty of Medicine in the Galilee,

Bar-Ilan University, Safed, Israel

2Department of Pathology, The Galilee Medical Center, Nahariya, Israel

Running Title: The Smurf2/Topo II axis and genome stability

Keywords: Smurf2, Topo II, ubiquitination, proteasome degradation, chromatin bridges.

Financial Support: This work was supported by several grants including ICRF 00636 (M. Blank),

Marie-Curie FP-7 CIG 612816 (M. Blank), and Dayan Family Foundation award (M. Blank).

Corresponding Author: Michael Blank, Laboratory of Molecular and Cellular Cancer Biology,

Faculty of Medicine in the Galilee, Bar-Ilan University, 1311502 Safed, Israel. Phone: 972-54-222-

0547; Fax: 972-4-622-9256; E-mail: [email protected]

Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 2017 American Association for Cancer Research. Author Manuscript Published OnlineFirst on June 13, 2017; DOI: 10.1158/0008-5472.CAN-16-2828 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Abstract

DNA topoisomerase II (Topo II) ensures genomic integrity and unaltered chromosome inheritance and serves as a major target of several anticancer drugs. Topo II function is well understood, but how its expression is regulated remains unclear. Here we identify the E3 ubiquitin ligase Smurf2 as a physiological regulator of Topo II levels. Smurf2 physically interacted with

Topo II and modified its ubiquitination status to protect Topo II from the proteasomal degradation in dose- and catalytically-dependent manners. Smurf2-depleted cells exhibited a reduced ability to resolve DNA catenanes and pathological chromatin bridges formed during mitosis, a trait of Topo II-deficient cells and a hallmark of chromosome instability. Introducing

Topo IIα into Smurf2-depleted cells rescued this phenomenon. Smurf2 was a determinant of Topo

II protein levels in normal and cancer cells and tissues, and its levels affected cell sensitivity to the

Topo II-targeting drug etoposide. Our results identified Smurf2 as an essential regulator of Topo II, providing novel insights into its control and into the suggested tumor suppressor functions of

Smurf2.

Introduction

DNA topoisomerase II (Topo II is the major form of the Topo II enzyme in cycling vertebrate cells that acts to untangle chromosomal catenanes forming during the duplication of genetic material. Topo IIplays a pivotal role in chromatin organization, dynamics and unperturbed chromosome inheritance. The failure of Topo II to maintain DNA supercoiling homeostasis and properly disentangle daughter chromosomes can lead to the formation of pathological chromosome bridges, chromosomal instability (CIN), and ultimately to cancer (1–4). Furthermore, due to the high abundance of Topo II in rapidly proliferating cells and its vital roles in mitotic processes,

Topo II is a core target of several anticancer drugs (4, 5). Despite the importance of Topo II, the mechanisms governing its cellular levels remain largely unknown.

Recently, we reported that HECT-type E3 ubiquitin ligase Smurf2 operates in mammalian cells as a critical regulator of chromosome integrity, and acts to prevent the CIN, and carcinogenesis (6). We showed that genomic ablation of Smurf2 leads to accumulation of chromosomal aberrations, including Robertsonian translocations, undefined translocations and marker chromosomes. Furthermore, we demonstrated that mice knock-out for Smurf2 develop a wide spectrum of tumors in different organs and tissues. These and other studies established Smurf2 as an important regulator of genomic integrity, whose inactivation results in carcinogenesis (7).

However, the mechanisms underlying tumor suppressor functions of Smurf2 are far from understood.

Here, we report a novel mechanism by which Smurf2 is involved in genome integrity regulation–via stability regulation of Topo II and prevention of anaphase bridge formation. We also show that cellular levels of Smurf2 delineate Topo II protein levels in both mouse and human normal and cancer cells and tissues, and could determine cell sensitivity to Topo II poison etoposide.

Materials and Methods

Cell culture

Smurf2 knockout (Smurf2-/- ) mouse embryonic fibroblasts (MEFs) and wild-type cells derived from littermate control embryos were originally established in the laboratory of Dr. Ying Zhang

(National Cancer Institute, NIH), and obtained from her laboratory in 2012 (6). These cells were obtained at passages 30-35, and used between passages 40-55. Human cell lines used in this study were generously provided by Prof. Yosef Shiloh (Tel Aviv University, Israel). These cells were obtained in 2013, and originated from American Type Culture Collection (ATCC, Manassas, VA).

These cell lines were propagated, frozen and used in culture for up to 12 passages. All cell lines were maintained in high glucose DMEM medium (4,5 g/l D-Glucose, Gibco) supplemented with

10% (v/v) fetal bovine serum, 2 mM L-glutamine and 1% (v/v) Pen-Strep, and incubated at 37˚C with 5% CO2. Cell strains were selectively tested for mycoplasma at the Faculty Core Facility using a PCR-based approach. The latest date for testing of our leading cellular models, which include

U2OS wild-type cells, Smurf2 knock-out cells, Smurf2 knockdown strains, and cells expressing

Smurf2 catalytically-inactive (Cys716Gly) and wild type forms, was 01/24/2017. Cell line authentication was not conducted.

Tissue microarrays (TMAs) and Immunohistochemistry (IHC)

Human TMAs were purchased from US Biomax, Inc (Rockville, MD). Mice tissues were fixed in

4% formalin and 5µm tissue sections were prepared. IHC was conducted as previously described

(6). All comparable samples were sampled on the same slide, and all staining procedures were conducted on slides positioned horizontally. Histological evaluations and TMAs scoring were conducted by a board-certified pathologist at the Galilee Medical Center, Israel.

GST-fusion protein, pull-down assays and ubiquitination assays

GST fusion proteins were prepared from E.coli using glutathione-Sepharose beads (Amersham); purified full-length human Topo IIα was purchased from TopoGen (TG200H). An in vitro binding assay was performed as described (6). Briefly, GST or GST-Smurf2 were incubated with Topo IIα in binding buffer for 15 minutes at 37°C and GST-Smurf2 was pulled-down using Glutathione

Sepharose 4B beads (GE Healthcare). The beads were then washed four times with ice-cold binding buffer and proteins were eluted with 5X SDS sample buffer.

In vivo and in vitro ubiquitination assays were performed as previously described (6, 8) with a few modifications. In brief, for the in vivo ubiquitination assay cells were lysed with either RIPA buffer supplemented with 5 mM NEM or in 1% SDS followed by an immediate boiling of samples for 15 minutes. Following the boiling, cell lysates were equilibrated with RIPA/NEM buffer to reduce SDS concentration in the samples down to 0.1%. Cell lysates were then sonicated, FLAG-

Topo IIα was immunoprecipitated, and its ubiquitination pattern analyzed.

For the in vitro ubiquitination assay, 500 ng of Topo IIα were incubated with 250 ng of GST or GST-Smurf2, 5 µg of HA-ubiquitin protein, E1 (UBE1; 100 ng) and E2 enzyme (UbcH5c; 150 ng), and 100 mM ATP-Mg in the E3 ligase reaction buffer (BostonBiochem) for 2 h at 37°C. RIPA buffer was added to the reactions and Topo IIα was pulled down using anti-Topo IIα antibody

(Abcam) and protein G-Sepharose beads.

Topoisomerase II extraction and DNA decatenation assay

Nuclear extracts for DNA decatenation assay were prepared as described (9), with a few modifications. In brief, cell pellets were resuspended in TEMP buffer (10 mM Tris-HCl, pH 7.5, 1

mM EDTA, 4 mM MgCl2, 0.5 mM PMSF), and incubated on ice for 15 min. Subsequently,

0.6%Nonidet-P40 substitute was added, samples vortexed for 10 sec, and centrifuged (20,000g, 30 sec). The supernatants, representing the cytoplasmic fraction, were collected and saved for Western blot analysis. The remaining nuclei were then washed in ice-cold TEMP, re-suspended in a TEP

buffer (10mM TrisHCl pH 7.5, 1mM EDTA, 500 mM NaCl, 0.5 mM PMSF), vortexed at 4˚C for

30 min, and centrifuged for 20 min at 20,000g. The supernatants, representing the nuclear fraction, were collected and saved for subsequent analyses. To verify the complete extraction of Topo II from the samples, the remaining insoluble fractions were solubilized in TEP buffer using a sonication, and subsequently analyzed in immunoblots.

DNA decatenation assay was performed using nuclear extracts and kinetoplast DNA (kDNA,

TopoGEN) in a complete buffer assay. In particular, each reaction contained 50 mM TrisHCl pH 8,

150 mM NaCl, 10 mM MgCl2, 2 mM ATP, 400 ng of kinetoplast DNA, 2 µg of nuclear extract and double-distilled water to bring the final volume up to 40 µl. Reactions were incubated at 37˚C for

30 min, and stopped by adding 8 µl of 5xStop Buffer (TopoGEN). Subsequently, samples were incubated with RNase (40 µg/ml) at 37˚C for 15 min and with Proteinase K (150 µg/ml) for an additional 15 min at 37˚C. Finally, samples were separated by electrophoresis through a 1% agarose gel stained with SYBR safe DNA-stain gel (Invitrogen), and visualized in the SyngeneG:BOX.

All other methods and reagents we used in this study are detailed in Supplementary method section.

Results

Identification of Topo II as a novel interactor of Smurf2

To identify novel Smurf2 binding partners, we employed immunoprecipitation (IP) coupled with

Mass Spectrometry (MS) analyses. These analyses were conducted on Smurf2-deficient MEFs reconstituted either with a full-length FLAG-tagged Smurf2 or with an empty vector as a control.

FLAG-Smurf2 immunoprecipitates were resolved in SDS-PAGE, and protein bands were visualized using Coomassie staining (Fig. 1A). Bands specifically associated with Smurf2 immunoprecipitates were excised from the gel and submitted for identification in MS. To deduct a background, the bands were cut side-by-side from both positive (Smurf2-reconstituted cells) and control lanes

(empty vector). Using this approach, we identified Topo II as a novel Smurf2 interactor with a

high degree of reliability: Topo II-specific protein sequence was detected in 146 peptides in

Smurf2-immunoprecipitated samples vs. 0 peptides in control samples (IP from cells expressing an empty vector) (Fig. 1A).

To validate the MS results, we conducted several lines of experiments. First, we demonstrated the interactions between Smurf2 and Topo II by co-IP experiments in MEFs, the cell model that has been used to discover the Smurf2-Topo II protein interaction (Fig. 1B). Next, we show that this interaction is also preserved in human cells. IP of MYC-tagged Smurf2 from HEK-

293T cells detected endogenous Topo II in complex with Smurf2; and vice versa: IP of FLAG- tagged Topo II detected endogenous Smurf2 complexed with Topo II(Fig. 1C). Finally, using these cells we demonstrated the interaction between both endogenous Smurf2 and Topo II (Fig.

1D). Essentially, the discovered interaction between Smurf2 and Topo II appears to be direct, since we were also able to detect the interaction between purified human Topo II and Smurf2 in the tube (Fig. 1E).

Smurf2 physically interacts with Topo II during interphase

To visualize the interaction between Smurf2 and Topo IIin cells, we expressed GFP-Smurf2 in human osteosarcoma U2OS cells. These cells have a large nucleus and are commonly used in localization microscopy to detect molecular interactions, in particular in the nuclear compartment.

Following cell fixation and immunostaining with the Topo II-specific antibody, we evaluated the co-localization between GFP-Smurf2 and Topo II under the confocal microscope. The specificity of the anti-Topo IIantibody used in our studies was rigorously validated using Topo II knockdown cells. The results (Fig. 2A) show that in interphase cells Smurf2 occupies both nuclear and cytoplasmic compartments, while Topo IIexhibits exclusively nuclear localization. In the nucleus, Smurf2 showed a high degree of co-localization with Topo IIin particular with its nucleolar fraction, as evident from the images recorded in the Nomarski imaging mode.

To validate the interaction between Smurf2 and Topo II, we recorded cell images of

Smurf2/Topo II-stained cells at different focal planes through the cell volume (Z-stack analysis).

The results we obtained corroborated the interaction between Smurf2 and Topo IIand indicated that in interphase cells Smurf2 associates with Topo IIthrough the nuclear volume

(Supplementary Fig. S1).

Next, an in situ proximity ligation assay (PLA), which enables determining the protein- protein interactions directly within the cell, provided further evidence that Smurf2 and Topo II physically interact with each other (Fig. 2B and C). Immunoprecipitation studies conducted in

U2OS cells further corroborated the interaction between Smurf2 and Topo II in this cell model

(Fig. 2D).

During mitosis, Topo II is tightly associated with chromatin, and follows the chromatin movement pattern. The examination of Topo II and Smurf2 biodistribution in U2OS cells going through the unperturbed mitosis revealed that while Topo II associates with mitotic chromosomes,

Smurf2 is mainly excluded from the interactions with chromatids (Fig. 2E). Moreover, in mitotic cells Smurf2 was mostly found at the periphery of Topo II-chromatin templates, including the spindle midzone. The observed biodistribution of Smurf2 and Topo II in interphase and mitotic cells is in accordance with the previous studies, which determined the localization of each of these proteins in human cells, but independently of each other (6, 10, 11). Collectively, these findings establish Topo II as a novel Smurf2 interactor.

Smurf2 controls the steady-state levels of Topo II in different cell types, and in tissues

To gain an insight into the biological significance of the complex formation between Smurf2 and

Topo II, we first examined the levels of Topo II in Smurf2 knock-out (KO) vs. wild-type MEF cells. These cells were derived from littermate control embryos. We found that the steady-state levels of Topo II were profoundly diminished in Smurf2KO MEFs (Fig. 3A, Left panel). To

determine whether the loss of Smurf2 alone is responsible for the decrease in the cellular levels of

Topo II, we restored Smurf2 expression in KO MEFs and found that upon Smurf2 reconstitution the cellular levels of Topo II were significantly increased (Fig. 3A, Right panel). Essentially, IHC and western blot analyses conducted on the tissue samples of Smurf2-deficient vs. control mice revealed that diminished Topo II protein levels were also a characteristic of Smurf2-ablated tissues (Fig. 3B and C; Supplementary Fig. S2A and B). The levels of Topo II, which activities are mostly associated with transcription regulation (2), were comparable between Smurf2KO and control tissues.

Similar results were also obtained in different human cell models: in U2OS osteosarcoma cells, HCT116 colon carcinoma cells, and DU145 and PC-3 prostate carcinoma cells. In all these cells, decrease of Smurf2 expression levels either through acute (using siRNAs) or stable knockdown (using lentiviral-based shRNAs) reduced the steady-state levels of Topo II(Fig. 3D). These effects were monitored through the use of four different si/shRNAs: designed to target Smurf2 mRNA either at 3’UTR or its coding sequence.

Next, to validate our biochemical data at a single-cell resolution, we performed immunofluorescence staining of Topo II in Smurf2 knockdown cells. The immunofluorescence studies were conducted concomitant to the Western blot analyses performed on WCL, as well as on the fractionated samples: cytosolic, nucleoplasmic and chromatin fractions (Fig. 3E and

Supplementary Fig. S2C). The results we obtained corroborated that the steady-state levels of Topo

II in Smurf2-depleted cells are significantly diminished. Of note, the observed Topo II-staining pattern and its decreased levels in Smurf2 knock-down cells were independent of the cell fixation procedure, and monitored in both formaldehyde- and methanol/acetone-fixed cells (Supplementary

Fig. S2C).

Furthermore, using the CRISPR/Cas9-based gene-editing system, we generated

Smurf2CRISPR U2OS cell line, in which we succeeded to decrease Smurf2 cellular levels down to

10%-15% of the original level (Fig. 3F). Using these cells, we demonstrated that targeting SMURF2 for inactivation at the genome level produced in human cells results similar to both mouse Smurf2-/- cells and tissues (Fig. 3A-C, and Supplementary Fig. S2B), and to human cell lines knocked-down for Smurf2 with RNAi (Fig. 3D and E). Of note, both the levels of Topo II and DNA topoisomerase 1 (Top1), which also operates in DNA transition processes to maintain supercoiling homeostasis (12), were unaffected by Smurf2 depletion (Fig. 3F). Furthermore, we conducted bi- parametric FACS analyses on cells in which Smurf2 was depleted either through CRISPR/Cas9 gene editing or using RNAi. These analyses provided evidence that targeting of Smurf2 for inactivation does not affect cell cycle distribution, although a moderate decrease in the mitotic population of Smurf2-knockdown cells was observed (Supplementary Fig. S3).

To determine whether Smurf2 affects Topo II protein levels by modulating its gene expression, we analyzed the mRNA expression levels of Topo II in Smurf2 knock-down and control U2OS and HCT116 cells using real time qRT-PCR. The data showed that mRNA levels of

Topo II were unaffected by Smurf2 depletion (Fig. 3G).

Finally, we demonstrated that overexpression in these cells of MYC-Smurf2 increases Topo

II protein levels proportional to the amount of Smurf2 transduced to the cells (Fig. 3H). Altogether, these data reveal Smurf2 as an essential regulator of the steady-state levels of Topo II and suggest that Smurf2 regulates Topo II protein levels post-translationally.

Smurf2 regulates the stability of Topo II through the inhibition of Topo II proteasomal degradation

To investigate whether Smurf2 manages Topo II protein levels by regulating its degradation, we conducted several lines of experiments. First, we treated Smurf2-deficient and -overexpressing cells with the proteasome inhibitor MG-132 and found that the stability of Topo II is regulated through the proteasome-mediated degradation, and is under Smurf2 control (Fig. 4A and B; Supplementary

Fig. S4A and B). Following cell treatment with a proteasome inhibitor, the Topo II levels in

Smurf2-proficient cells were notably increased, yet slightly affected in Smurf2 knockdown cells

(Fig. 4A, lane 2 vs. 4). In contrast, overexpression of Smurf2 in these cells dramatically increased the levels of Topo II in both untreated and MG-132-treated cells as compared to control samples transduced with an empty vector (Fig. 4B, lanes 3 and 4 vs.1 and 2). Noteworthy, MG-132 cell treatment did not lead to further stabilization of Topo II in Smurf2 overexpressing cells. This finding suggests that high Smurf2 levels were sufficient to prevent Topo II proteasomal degradation. Finally, the co-expression of FLAG-Topo II together with MYC-Smurf2 protected

Topo II from the proteasome-mediated degradation in Smurf2 dose-dependent manner

(Supplementary Fig. S4A). Of note, we did not find significant changes in the levels of Topo IIα in chloroquine-treated vs. control cells (Supplementary Fig. S4B). Taken together, these data suggest that Smurf2 stabilizes Topo II by inhibiting its proteasomal degradation.

E3 ubiquitin ligase activities of Smurf2 are required for Topo II stability regulation

Smurf2 is a HECT-type E3 ubiquitin ligase in which active-site cysteine 716 (Cys716) is crucial for its catalytic activity. To determine whether E3 ligase functions of Smurf2 are required for Topo II stability regulation, we analyzed Topo II protein levels in U2OS cells expressing either

Smurf2WT or its catalytically-inactive form in which active-site cysteine was substituted with glycine (Cys716Gly; Smurf2CG). Cells transduced with an empty vector served as a control. The data (Fig. 4C) show that only the expression of Smurf2WT was able to increase the Topo II protein levels. Similar results were also obtained in Smurf2-ablated MEF cells reconstituted with

Smurf2WT or its E3 ligase mutant form (Fig. 4D).

Smurf2 operates as a molecular editor of the Topo II ubiquitination code

To determine whether Smurf2 is capable of ubiquitinating Topo II, we conducted in cellulo ubiquitination assay in which FLAG-Topo II was co-expressed together with HA-tagged ubiquitin

and Myc-tagged Smurf2. FLAG-Topo II was then immunoprecipitated and its ubiquitination pattern was analyzed. To perform this analysis, we used an anti-HA antibody, which specifically recognized the HA-tagged ubiquitin attached to Topo II This experimental design allowed us to identify that Topo II undergoes ubiquitination (Fig. 4E, lanes 3-5 vs. 1 and 2). This design also allowed us to discover that the addition of catalytically-active Smurf2 significantly enriched Topo

II monoubiquitination (Fig. 4E, lanes 4 vs. 3). Moreover, the results show that the substitution of

Smurf2WT with its mutant form failed to produce this ubiquitination phenomenon (Fig. 4E, lane 5 vs. 4). The data also show that in the cells, which expressed Smurf2CG, the Topo II is polyubiquitinated. The observed polyubiquitination of Topo II while evident in Smurf2CG- expressing cells, was less visible in cells transfected with an empty vector (Fig. 4E, Left panel; lane

5 vs. 3). We assumed that this finding due to the incomplete inactivation of cellular deubiquitinases

(DUBs) upon cell lysis. To clarify this point, we conducted the Topo II ubiquitination experiment again, but to deactivate the cellular DUBs more efficiently, in the second round, the cells were lysed directly in 1%SDS, followed by an immediate sample boiling. Similar to our previous findings, the addition of Smurf2WT, but not Smurf2CG or an empty vector, significantly enriched the monoubiquitinated fraction of Topo II(Fig. 4E, Right panel)In addition, we verified that the expression of catalytically-active Smurf2 switched the ubiquitination code on Topo II from poly- to monoubiquitination, as compared to samples expressing an empty vector or Smurf2CG (Fig. 4E,

Right panel; lane 4 vs. 3 and 5).

Our data indicate that the protein stability of Topo II is regulated through the proteasome- mediated degradation. This type of proteolysis relies on the formation of a particular type of ubiquitination–K48-linked polyubiquitination. To examine whether Smurf2 protects Topo II from the degradation by modulating this particular type of ubiquitination, we co-expressed FLAG-Topo

II with Smurf2 together with either wild type ubiquitin or its mutant form (K48-only ubiquitin). In this mutant ubiquitin, all the lysines except K48 were mutated to arginines. These mutations abolish

the ability of ubiquitin to form polyubiquitin chains other than through the K48-linked chain. Both wild-type and mutant forms of ubiquitin were HA-tagged. FLAG-Topo II was then immunoprecipitated and its ubiquitination pattern analyzed using anti-HA antibody. The results show that similar to the previous findings the expression of Smurf2 together with a wild type form of ubiquitin switched the Topo II ubiquitination from poly- to monoubiquitination (Fig. 4F, lane 2 vs. 1). However, when the wild type ubiquitin was substituted to K48-only ubiquitin, and expressed together with Smurf2, the K48-linked polyubiquitination of Topo II was completely abolished

(Fig. 4F, lane 4 vs. lane 3). These data suggest that Smurf2 operates as a molecular editor that modifies the Topo II ubiquitination code to protect Topo II from the degradation-promoting-K48 polyubiquitination. The data also indicate that the E3 ligase activities of Smurf2 are required for the switch.

Finally, we performed an ubiquitination reconstitution assay using purified ubiquitin- activating enzyme (E1), ubiquitin conjugase (E2), HA-ubiquitin, Smurf2 (either wild type or E3 mutant form), and human Topo II. Using this experimental settings, we demonstrate that Smurf2 ubiquitinates Topo II directly (Fig. 4G). The specificity of this reaction is demonstrated by the unique ubiquitination pattern of Topo II monitored only in the presence of Smurf2WT. In addition, we validated that the observed ubiquitination pattern belongs to Topo II and not to Smurf2 or any other components used in the ubiquitination assay (Supplementary Fig. S4C and D).

Smurf2 depletion decreases the Topo II decatenation activity, and increases the formation of anaphase bridges

Topo II is a key player in the decatenation checkpoint, and its inability to unwind chromosomal entanglements can lead to the formation of pathological chromosome bridges–one of the major sources of chromosomal translocations. Based on these findings, and our data establishing Smurf2 as a positive regulator of Topo II, we hypothesized that cells depleted of Smurf2 will exhibit a

phenotype similar to the Topo II-depleted cells. Specifically, we hypothesized that these cells will exhibit the reduced ability to untangle chromosomal catenanes, and will show the exacerbated formation of anaphase bridges.

To test this hypothesis, we first examined the Topo II decatenation activity in nuclear extracts prepared from Smurf2-knockdown and control cells using the decatenation assay. Nuclear extracts prepared from Topo II and Topo II knockdown cells were also incorporated in the analysis, and served as additional controls. The data (Fig. 5A and B, and Supplementary Fig. S5A) show that DNA decatenation was significantly reduced in Smurf2 knockdown cells, and was comparable to the cells knockdown for Topo II. In addition, using Topo II and Topo II knockdown cells, we demonstrate that Topo II decatenation assay used in our study is specific to monitor activities of Topo IIThis finding is in agreement with results previously published by

Bower et al., and showing that Topo II assay mainly measures activities of Topo II (13).

Furthermore, by assaying Topoisomerase I activity, we demonstrate that the extracts derived from

Smurf2 knockdown cells are otherwise normal, and have unaltered Top1 activity (Supplementary

Fig. S5B). The sensitivity of Top1 assay was validated using Top1 knockdown cells

(Supplementary Fig. S5C and D).

Next, we analyzed and compared the occurrence of chromosome bridges, and lagging chromosomes, in Smurf2- and Topo II-depleted cells. To this end, we employed the U2OS cell model. These cells have been previously shown to exhibit an increased DNA bridge formation after

Topo II knockdown (14). The data (Fig. 5C and D; Supplementary Fig. S5E) show that Smurf2 depletion significantly increased the formation of DNA bridges in mitotic cells (P = 0.000212, 2- test). Moreover, the incidence of DNA bridges in Smurf2-depleted mitotic cells was highly similar to the incidence of these bridges in Topo II knockdown cells (Fig. 5C). This finding suggests that

Smurf2 depletion phenocopies the Topo II depletion in the resolving of DNA catenanes.

Essentially, we demonstrated that Smurf2 inactivation had no significant effect on the occurrence of

lagging chromosomes (Fig. 5E and F). These chromosomes are formed through the mechanisms distinct from the mechanisms underlying anaphase bridge formation, and are independent of

TopoII–16

Finally, we reconstituted Smurf2CRISPR U2OS cells with mCherry-tagged Topo II (or with an empty vector as a control) and, following the generation of stable cell lines, analyzed the formation of anaphase bridges and lagging chromosomes in these cells. The data (Fig. 5G;

Supplementary Fig. S5F) show that in Smurf2-depleted cells transduced with Topo II the cellular phenotype was rescued, as evident by a significant decrease in the population of mitotic cells with chromatin bridges compared to control (P = 0.014, 2-test). The incidence of lagging chromosomes in these cells remained unaffected (Fig. 5H). Of note, decatenation assay conducted on Topo II- reconstituted cells suggested that the N-terminally tagged mCherry-Topo II is catalytically active

(Supplementary Fig. S5G); similar results were reported by Lane et al., showing that mCherry- tagged Topo II can functionally complement the loss of endogenous protein (17).

Smurf2-depletion decreases cell sensitivity to Topo II poison etoposide

The cellular levels of Topo II are an important determinant of tumor cell sensitivity to Topo II- targeting drugs, in particular to etoposide: higher Topo II levels–higher sensitivity, and vice versa

(18–21). Topo II poison etoposide acts to stabilize transient Topo II-bridged break and lead to generation of highly cytotoxic DNA damage–double strand breaks. To determine whether and how manipulations with Smurf2 cellular levels affect cell sensitivity to etoposide, we assessed the sensitivity of Smurf2CRISPR and control wild-type U2OS cells to this drug. The data (Fig. 5I) show that Smurf2CRISPR cells were more resistant to etoposide treatment than control cells. Similar results were also observed in Smurf2KO vs. WT MEF cells (Supplementary Fig. S5H).

The Smurf2/Topo II relationship is preserved in human tissues

To determine whether Smurf2 is also a relevant determinant of Topo II protein levels in human tissues, we conducted IHC analyses on two tissue microarrays (TMAs): prostate (PR1921), which included both normal (n=31) and cancer tissues (n=149); and breast TMA (BR10010) containing primary and metastatic carcinoma tissues (n=98). These TMAs were stained with anti-Topo II and anti-Smurf2 IHC-specific antibodies, and counterstained with hematoxylin. The subsequent histopathological examination coupled with the Spearman’s rank correlation analysis revealed that the expression levels of Smurf2 and Topo II are positively correlated. This correlation was statistically significant for all analyzed tissues: P < 0.001 for breast and prostate carcinoma tissues, and P < 0.05 for normal prostate tissues (Fig. 6A-D, and Supplementary Table S1).

Discussion

In this study, we identified a novel mechanism by which Smurf2, a HECT-type E3 ubiquitin ligase and recently discovered tumor suppressor, is involved in the regulation of genome integrity.

Recently, we reported that cells depleted of Smurf2 accumulate multiple chromosome abnormalities in their genome, where translocations were the most notable hallmark (6). However, the mechanisms underlying this phenomenon are far from understood.

The formation of pathological chromatin bridges is considered one of the major causes of chromosomal translocations, which are viewed as an outcome of the compromised decatenation checkpoint mediated by Topo II(16, 22). Inability of Topo II to decatenate DNA (e.g. due to its reduced cellular levels and/or activities) can lead to CIN and, ultimately, to cancer. Moreover, in established tumors, CIN can drive tumor progression by accelerating the gain of oncogenic loci and the loss of tumor suppressor loci (2, 4).

Using different human and mouse cellular models, Smurf2 targeting approaches, human tissue arrays, genetically modified systems and rescue experiments, we provided confirmatory

insights that Smurf2 acts as a key cellular factor that stabilizes Topo II and prevents the formation of pathological DNA bridges. Mechanistically, we demonstrated that Smurf2 physically interacts with Topo II and protects it from the proteasome-mediated degradation in Smurf2 dose- and catalytically-dependent manners. We showed that Smurf2 operates as a molecular switcher that modifies the Topo II ubiquitination code to reduce its degradation-promoting-K48 polyubiquitination and increase monoubiquitination. Essentially, we demonstrated that Smurf2 is capable to directly bind to and ubiquitinate Topo II. Finally, we provided evidence that Smurf2 is a relevant determinant of Topo II protein levels in mouse and human normal and cancer tissues, and that Smurf2 levels could affect the cell sensitivity to Topo II poison and anticancer drug etoposide.

Currently, there is limited information about the mechanisms that regulate Topo II protein levels and stability. Studies published to date mainly report that following cell treatment with topoisomerase II poisons, Topo II undergoes proteasome degradation, which enables repair of

DNA lesions (23–26). Our study demonstrates that Topo II undergoes proteasomal degradation also in undamaged cells, and it is under the control of Smurf2. This finding is intriguing because

Smurf2 is primarily known as a degradation-promoting E3 (7, 27). Nonetheless, our data strongly suggest that Smurf2 is an authentic positive regulator of Topo II that protects it from the proteasome-mediated proteolysis in Smurf2 dose- and E3 ligase-dependent manners. The data obtained in IHC studies conducted on tissues derived from Smurf2-deficient and wild-type mice, as well as on human TMAs (278 normal and tumor tissues), provided further support that Smurf2 is also a positive regulator of Topo II in tissues.

The functional and cellular assays conducted in this study provided evidence that Smurf2- depletion phenocopies Topo II depletion. This finding is quite intriguing especially in light of mouse phenotypes reported for Topo II and Smurf2: Topo II-deficient mice are embryonic lethal

(28), whereas Smurf2KO mice are viable and exhibit no overt developmental defect during

embryogenesis (6, 29). There are a few possible explanations that could reconcile these mouse phenotypes. First, as we demonstrated in a panel of different human and mouse cells and tissues

(Fig. 3), the depletion of Smurf2 significantly reduced, but not ablated Topo II levels. It is possible that the remaining levels of Topo II were sufficient to complete embryogenesis. Second, during embryogenesis other members of the NEDD4 E3 ligase family (nine in total) could compensate for Smurf2 deficiency. For example, mice with a targeted disruption of either the

Smurf2 or Smurf1 allele are viable and survive to adulthood (6, 29, 30), however disruption of both

Smurf2 and Smurf1 alleles leads to embryonic lethality (31). The possibility that Smurf2 does not affect the cellular levels of Topo II during early embryogenesis, where it appears to be particularly important (28), also exists.

Collectively, our findings establish a novel functional link between an E3 ubiquitin ligase

Smurf2 and Topo II, and propose a new paradigm in the stability regulation of Topo II, and genome integrity maintenance. Our data also suggest an intriguing role of Smurf2 as an E3 ligase that can protect some of the key cellular proteins from degradation; and provide a novel insight into the tumor suppressor functions of Smurf2.

Acknowledgements

We thank Meir Shamay for helpful discussions during this manuscript preparation, and Basem

Hijazi for statistical analysis. We are also grateful to Ying E. Zhang for providing Smurf2 deficient mice, and for other support.

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Figure Legends

Figure 1. Smurf2 physically interacts with Topo II. A, coomassie gel staining of Smurf2 immunopreciptates obtained from FLAG-Smurf2-reconstituted and control Smurf2-/- MEFs. The ratio (R) shows the number of peptides identified for Topo II in MS vs. control samples. B, validation of Smurf2 interactions with Topo II in Smurf2-/- MEFs reconstituted with a FLAG- tagged Smurf2. WCL, whole cell lysate. C, reciprocal co-IP analysis showing interactions between

Smurf2 and Topo II in human HEK-293T cells. D, co-IP analysis of endogenous Smurf2 and Topo

II interaction in HEK-293T cells. E, GST-pull down experiment showing direct interaction

between purified GST-Smurf2 and Topo II.

Figure 2. Smurf2 associates with Topo II in interphase cells. A, confocal images showing co- localization of GFP-Smurf2 and endogenous Topo II in the interphase nucleus of U2OS cells.

DNA was counterstained with Hoechst. Two representative images are shown in the figure. Bars, 10

µm. B, PLA assay indicating sites of direct protein-protein interaction of FLAG-Smurf2 and endogenous Topo II in U2OS cell nuclei. Bars, 20 µm. The diagram on the bottom of the figure shows the conditions under which the fluorescent signal in PLA is generated. C, quantification of

PLA (n=109). D, co-IP analysis substantiating the interaction of FLAG-Smurf2 and Topo II in

U2OS cell model. E, biodistribution of GFP-Smurf2 and Topo II in U2OS cells sequestered at different stages of mitosis. Note distinct Smurf2 and Topo II localization patterns in mitotic cells:

Topo II is localized on mitotic chromosomes; Smurf2 is mostly excluded from the chromatin templates. Bars, 10 µm.

Figure 3. Smurf2 positively regulates the steady-state levels of Topo II. A, western blot analysis of Topo II in Smurf2 wild type (WT; Smurf2+/+) and Smurf2 knockout (KO; Smurf2-/-) MEFs (left panel); and in Smurf2-/- MEFs reconstituted with either an empty FLAG vector (Smurf2-/- (E)) or with

FLAG-Smurf2 (Smurf2-/- (R)) (right panel). B, IHC staining of Topo II (brown) in spleen tissue sections prepared from WT and KO mice. The nuclei were counterstained with hematoxylin (blue).

Bars, 50 µm. 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 IIand expression in the spleen tissues. D, western blot analysis of Topo II 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 on the bottom shows the position of each siRNA and shRNA on the mRNA’s map of Smurf2. E, immunofluorescence staining of Topo II in U2OS cells showing diminished Topo II levels upon Smurf2 knockdown. The specificity of Topo II antibody was validated on siTopo II cells. Bars, 20 µm. Bottom panel shows western blot analyses of Topo II in Smurf2 knockdown cells conducted concomitantly with the immunofluorescent studies. Analyses were performed on 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 are demonstrated by using antibodies against the cytosolic α-tubulin and the chromatin component histone H2B. F, western blot analysis of Topo II, II 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 II 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

II protein levels in Smurf2 dose-dependent manner. 

Figure 4. Smurf2 regulates the stability of Topo II through the inhibition of its proteasomal degradation. A, western blot analysis of Topo II in Smurf2 knockdown U2OS cells after treatment

with MG-132 (5 M; 4 hrs). B, western blot analysis showing that Smurf2 overexpression protects

Topo II, but not Topo II from proteasome-mediated degradation in U2OS cells. C, overexpression of Smurf2WT, but not its catalytically-inactive mutant form (Smurf2CG), increases

Topo II protein levels in U2OS cells. D, western blot analysis of Topo II in Smurf2-/- MEFs reconstituted with Smurf2WT or its E3 ligase mutant form. E, western blot analysis showing that

Smurf2 modifies Topo II 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 (5mM; left panel) or in 1%SDS followed by immediate sample boiling (right panel).

F, Smurf2 reduces the K48-linked polyubiquitination of Topo II in vivo. G, western blot analysis of Topo II 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 II was pulled down from the reaction using anti-Topo II antibody. The arrow points to monoubiquitinated Topo II. Right panel shows coomassie blue staining of purified

GST and GST-Smurf2 used in this study.

Figure 5. Smurf2 depletion phenocopies Topo II depletion. A, DNA decatenation assay. B, relative decatenation activity in Smurf2 and Topo II knockdown cells. Data was quantified on two independent experiments. C, quantification of the percentage of U2OS cells with Hoechst-positive ana/telophase bridges. Bars represent means+SD (n=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. Bars, 10 µm. E, quantification of the percentage of U2OS cells with lagging chromosomes in Smurf2- and Topo II-depleted samples. Bars represent means+SD

(n=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.

Bars, 10 µm. G, the incidence of anaphase bridge formation in Smurf2CRISPR U2OS cells transduced

with either an empty vector or with mCherry-Topo II Data are represented as mean+SD (n=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.

Figure 6. The Smurf2/Topo II relationship is preserved in human tissues. A, a correlative relationship between Smurf2 and Topo II 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 =<10%; 1=10-24%; 2=25-

49%; 3=50-74%; 4=75-100%. Data are presented as counts±SD. B, representative images of IHC staining of Smurf2 and Topo II quantified in (A). L5, L11, E1 and C3 are the coordinates of the samples in the tissue array. Scale bars, 50 μm. C, quantification of the expression levels of Smurf2 and Topo II in human breast cancer TMA. D, representative images of IHC staining of Smurf2 and

Topo II in breast TMA. B8 and F8 are the coordinates of the samples in the tissue array. Bars, 50

μm.

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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