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. Ó2017 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 À À 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- Ó2017 American Association for Cancer Research. down strains, and cells expressing Smurf2 catalytically inactive www.aacrjournals.org OF1 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2017 American Association for Cancer Research. Published OnlineFirst June 13, 2017; DOI: 10.1158/0008-5472.CAN-16-2828 Emanuelli et al. (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- mLof5ÂStop 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 5Â 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