AbstractMetastasis is the main cause of death in cancer patients. TGF-尾 is pro-metastatic for malignant cancer cells. Here we report a loss-of-function screen in mice with metastasis as readout and identify OTUD1 as a metastasis-repressing factor. OTUD1-silenced cancer cells show mesenchymal and stem-cell-like characteristics. Further investigation reveals that OTUD1 directly deubiquitinates the TGF-尾 pathway inhibitor SMAD7 and prevents its degradation. Moreover, OTUD1 cleaves Lysine 33-linked poly-ubiquitin chains of SMAD7 Lysine 220, which exposes聽the SMAD7 PY motif, enabling SMURF2 binding and subsequent聽T尾RI turnover聽at the cell surface. Importantly, OTUD1 is lost in multiple types of human cancers and loss of OTUD1 increases metastasis in intracardial xenograft and orthotopic transplantation models, and correlates with poor prognosis among breast cancer patients. High levels of OTUD1 inhibit聽cancer stemness and shut off metastasis. Thus, OTUD1 represses breast cancer metastasis by mitigating TGF-尾-induced pro-oncogenic responses via deubiquitination of聽SMAD7. IntroductionMetastatic disease is largely incurable because of its systemic nature and the resistance of disseminated tumor cells to existing therapeutic agents1. To colonize distant organs, circulating tumor cells must overcome many obstacles, including surviving in circulation, infiltrating distant tissues, evading immune defenses, adapting to supportive niches, surviving as latent tumor-initiating seeds, and eventually breaking out to replace the host tissue2. Metastasis is a highly inefficient process and the mechanisms are poorly understood. TGF-尾 signaling is one of the most important pathways involved in all these metastatic processes3,4,5. In many late-stage tumors, TGF-尾 signaling is redirected from suppressing cell proliferation and instead found to activate epithelial-to-mesenchymal transition (EMT), a cellular program that promotes cancer cell intravasation and confers cancer stem cells traits associated with high-grade malignancy6,7,8.TGF-尾 signals via specific complexes of type 袉 (T尾RI) and type II Ser/Thr kinase receptors. The activated TGF-尾 type I receptor induces SMAD2/3 phosphorylation; phosphorylated SMAD2/3 forms hetero-oligomers with SMAD4, which accumulate in the nucleus to regulate the expression of target genes9. SMAD7 functions as an inhibitory SMAD by recruiting the E3-ubiquitin ligase SMURF2 to T尾RI and mitigating TGF-尾 signaling10,11,12. Various E3 ligases, including ARKADIA and RNF12 can potentiate TGF-尾 signaling by targeting SMAD7 for poly-ubiquitination and degradation13,14,15,16.Recently, we developed an in vivo screen in mice that enables the isolation of genetic entities involved in activation of breast cancer metastasis. Here, the results of one such screen using a DUB shRNA library is presented. The top hit, termed OTU domain-containing protein 1 (OTUD1), was found to inhibit breast cancer stem cell traits and metastasis. We also elucidate the underlying mechanism and show that OTUD1 empowers SMAD7 to inhibit TGF-尾 signaling in breast cancer metastasis.ResultsGenetic聽screen identified OTUD1 as a metastasis聽suppressorWe designed a loss-of-function screen in mice to identify deubiquitinating enzymes (DUBs) that antagonize metastasis (Fig.聽1a; Supplementary Fig.聽1a) and applied it to early passage MDA-MB-231 cells, which still show epithelial-like morphology and exhibit relatively low metastatic ability. We used a shRNA library targeting 74 DUBs, in which each DUB is covered by 4鈥? independent short hairpins with at least two of them validated (Supplementary Data聽1). Instead of making a pool of shRNA virus, we produced up to 371 distinct shRNA lentiviruses in HEK293T cells and individually introduced them into early passage MDA-MB-231 cells. After puromycin selection for three days, this gave rise to 371 stable cell lines. We used an equal amount of cells from each cell line (10鈥壝椻€?03 cells per shRNA stable cell line) and mixed them for nude mice intracardial injection (Supplementary Fig.聽1a). Within 4 weeks, the mixed shRNA stable cells produced a total of seven strong metastatic nodules in multiple mice (3 from 30 mice shown in Fig.聽1b); some of the other mice developed weak micrometastasis (6 from 30 mice shown in Fig.聽1b). In contrast, cells infected with empty vector did not produce macroscopic lesions upon injection in 30 mice after 4 weeks (Fig.聽1b). This screening strategy can thus be used to identify essential DUBs that suppress metastasis.Fig. 1An in vivo genetic screen identifies OTUD1 as potent suppressor of breast cancer metastasis. a, b Flow chart and figures of the in vivo screen identifying DUBs that inhibit breast cancer metastasis. Low metastatic MDA-MB-231-Luciferase/GFP breast cancer cells were infected with lentiviruses expressing DUB shRNAs and intracardially injected into nude mice. The mice were monitored for 4 weeks by in vivo bioluminescent imaging (BLI) and the early metastatic nodules were isolated and the corresponding shRNAs were identified by sequencing. See Supplementary Fig.聽1a for details. c Immunoblot (IB) analysis of OTUD1 shRNA and sgRNA-mediated knockdown and knockout in MDA-MB-231 (BM) cells. d IB analysis of MDA-MB-231 (BM) cells stably expressing doxycycline (dox)-inducible OTUD1 and treated with the indicated doses of Dox. e Left panel: BLI of three representative mice injected with MDA-MB-231 control cells or cells deficient in OTUD1, Images were taken 4 weeks after injection. Two independent sgRNAs were used to generate OTUD1 knockout cells. Right upper panel: BLI signals of all mice in each experimental group at week 5. Right lower panel: The percentage of metastasis-free mice in each experimental group followed in time. f MDA-MB-231 cells expressing DOX-inducible OTUD1-wt/CA was intracardially injected in mice. DOX was administrated 21 days after inoculation of the cells. Metastasis was analyzed by BLI. Left panel: BLI of representative mice from each group at indicated days. Right upper panel: BLI signal of each experimental group followed in time. Right lower panel: The percentage of metastasis-free mice in each experimental group followed in time. g Oncomine database summary for OTUD1 expression in multiple cancers (left). Box plots of OTUD1 expression levels in invasive ductal breast carcinoma (IDBC) and lung adenocarcinoma (LA) compared with normal tissue (right). h Kaplan鈥揗eier analysis of relapse-free survival of patients in publicly available breast cancer datasets (NKI 295). i BRCA1 target genes are enriched in OTUD1-high versus OTUD1-low expressing patients shown by pre-ranked gene-set enrichment analysis (GSEA). *p鈥?lt;鈥?.05, **p鈥?lt;鈥?.01 and ***p鈥?lt;鈥?.001 (two-tailed Student鈥瞫 t-test (e, f), Log-rank test (h) or two-way analysis of variance (ANOVA) (e, f))Full size image After examination and reintroduction in the early passage MDA-MB-231 cells, two of the seven shRNAs isolated from individual lesions promoted lung metastasis without affecting primary tumor growth. We focused on these two because both shRNAs target OTUD1 (Fig.聽1b), an OTU domain family member with a largely unexploited function in cancer. OTUD1 was reported to be differentially expressed in thyroid cancer and to deubiquitinate and stabilize p5317,18. To consolidate our observations, we generated OTUD1 knockout cell lines of the highly bone-metastatic MDA-MB-231 (BM) cells19,20 by two independent guide RNAs of CRISPR/Cas9 technology (Fig.聽1c). Mice injected with these two OTUD1 knockout clones developed larger bone metastases and had significantly shorter bone metastases-free survival periods (Fig.聽1e), corroborating a strong anti-metastatic activity of endogenous OTUD1. To directly implicate OTUD1 in this process, we used a doxycycline-regulated promoter to initiate its expression in MDA-MB-231 (BM) cells 3 weeks after intracardial injection (Fig.聽1d, f). Although cell transplantation induced bone metastasis to similar levels around day 21 post-injection, expression of OTUD1 wild-type (wt) beginning at day 21 significantly suppressed metastasis around day 35, which also led to increased mice survival (Fig.聽1f). Thus, induction of OTUD1 causes inhibition of metastatic outgrowth, which is not observed with its deubiquitinating enzyme inactive form CA (carrying a point mutation in one of the key cysteines of the catalytic domain), directly implicating OTUD1 as a critical DUB in metastasis suppression.Given the role of OTUD1 in the regulation of metastasis, we investigated the possibility that OTUD1 might be a relevant factor in late-stage cancer. Oncomine expression analysis revealed that OTUD1 mRNA levels are frequently downregulated in human cancers21,22,23,24,25,26 (Fig.聽1g; Supplementary Fig.聽1b). Using the NKI295 breast cancer database27, we observed that low OTUD1 expression is associated with poor prognosis of distant metastasis-free survival in patients (Fig.聽1h). This feature is apparently not limited to breast cancer; in The Cancer Genome Atlas (TCGA) database28, bladder urothelial patients with lower OTUD1 expression had a shorter disease-free life expectancy than those with higher OTUD1 expression (Supplementary Fig.聽2a), suggesting that OTUD1 inhibits metastatic relapse in patients. Finally, hypothesizing that OTUD1 has a signaling function, we compared NKI295 tumor microarray data聽of 54 OTUD1-high expressing patients and 64 OTUD1-low expressing patients and applied it to Gene Set Enrichment Analysis (GSEA)29. Intriguingly, we observed that the 70 core targets of tumor suppressor Breast Cancer 1 (BRCA1) are all significantly enriched in OTUD1-high patients (Fig.聽1i; Supplementary Fig.聽2b and Supplementary Data聽2), strongly indicating that OTUD1 could support BRCA1 function. These results are consistent with a role for OTUD1 in restricting metastasis of human breast cancer.OTUD1 inhibits cancer stem cell traitsWe next examined the role of OTUD1 in breast cancer cell phenotypic behavior. Depletion of endogenous OTUD1 potentiated the capacity of RAS transformed MCF10A cells to form tumor organoids in 3D Matrigel but did not inhibit tumor cell survival and proliferation under standard 2D culture conditions (Fig.聽2a, b). Conversely, lentiviral-mediated expression of wt OTUD1, but not the CA mutant, significantly reduced the number of tumor-like colonies (Fig.聽2c). To investigate the ability of OTUD1 to regulate cancer stem cell activity, we performed mammosphere assays30. Although silencing of endogenous OTUD1 strongly increased the capacity of MCF10A (RAS) cells to form tumor spheres, ectopic expression of OTUD1-wt, but not the CA mutant form, greatly reduced this (Fig.聽2d鈥?a data-track=\"click\" data-track-label=\"link\" data-track-action=\"figure anchor\" href=\"/articles/s41467-017-02029-7#Fig2\">f). Consistent results in both tumor organoids and mammosphere assays were obtained when using mouse 4T1 mammary carcinoma cells (Supplementary Fig.聽2c, d). These results indicate that OTUD1 inhibits stemness of breast cancer cells.Fig. 2OTUD1 inhibits cancer stem cell traits. a, b Control and OTUD1-silenced MCF10A-RAS cells were cultured in 3D Matrigel. Representative wells a (scale bar, 2 mm)聽and mean number of organoids (卤SE) per 200 cells from triplicate samples b. c Mean number of organoids (卤SE) per 200 cells from triplicate samples of control and OTUD1-wt/CA overexpressed MCF10A-RAS cells cultured in 3D Matrigel. d, e Control and OTUD1-silenced MCF10A-RAS cells were analyzed in a tumor sphere assay. The pictures show representative images of tumor spheres聽(d, scale bar, 500聽渭m), and the graph shows the number of tumor spheres per 5鈥壝椻€?03 cells seeded e. f Mean number of tumor spheres per 5鈥壝椻€?03 cells from triplicate samples of control and OTUD1-wt/CA overexpressing MCF10A-RAS cells. g Control and OTUD1-shRNA silenced MCF10A-RAS cells were subcutaneous injected into nude mice at the indicated numbers. Mean of tumor volumes at week 5. h Tumor latency at indicated cell number of cells described in g. i Control and OTUD1-silenced MCF10A-RAS cells were subjected to qPCR analysis. j Control MCF10A-RAS cells or cells stably expressing OTUD1-wt or OTUD1-CA were subcutaneous injected into nude mice at the indicated numbers. Mean of tumor volumes at week 5. k Tumor latency at indicated cell number described in j. l Control MCF10A-RAS cells or cells stably expressing OTUD1-wt or OTUD1-CA were subjected to qPCR analysis. Error bars, mean鈥壜扁€塖D. *p鈥?lt;鈥?.05, **p鈥?lt;鈥?.01, and *** p鈥?lt;鈥?.001 (two-tailed Student鈥瞫 t-test b鈥?b>l)Full size image We subsequently examined the mitigating effect of OTUD1 on cancer stemness in vivo employing subcutaneous injections. Inactivation of OTUD1 promoted tumor incidence and tumor growth and shortened latency after injection of limiting numbers of MCF10A-RAS cells in mice (Fig.聽2g, h). Statistical analysis confirmed that silencing of OTUD1 increases the frequency of tumor-initiating cells (Supplementary Table聽1). Conversely, in the same limited cell number injection assay we observed that ectopic expression of OTUD1-wt, but not the CA mutant, inhibited tumor incidence and growth and prolonged latency after injection of 102, 103, 104 but not 105 cells (Fig.聽2j, k). In line with this, the transcription of the pluripotency factors NANOG, SOX2, OCT4, and TAZ were significantly elevated in OTUD1-depleted tumors and severely inhibited in tumors expressing wild-type OTUD1 (Fig.聽2i, l). These results suggest that OTUD1 reduces the manifestation of cancer stem cell traits.OTUD1 causes suppression of TGF-尾 downstream signals and EMTStem cell traits of breast carcinoma are associated with EMT, which requires interplay of multiple tumor-promoting pathways including TGF-尾8. In breast cancer cells, TGF-尾 treatment strongly聽promoted tumor organoids and tumor spheres formation; and these effects were inhibited by T尾RI kinase inhibitor SB431542 (Supplementary Fig.聽3a, b), demonstrating that TGF-尾 signaling indeed play a positive role in supporting stem-cell-like properties. TGF-尾 signals via its downstream activation of SMAD2 and SMAD36, which can be measured with ARE-Luc and CAGA-Luc transcriptional reporters, respectively31,32. Among more than 70 tested DUB cDNAs, OTUD1 was identified as one of a few potent DUBs that antagonized both TGF-尾 induced ARE-Luc and CAGA-Luc reporters (Fig.聽3a). OTUD1-CA did not inhibit TGF-尾 signaling (Fig.聽3b), suggesting that DUB activity is essential. Knockdown assays demonstrated that loss of OTUD1 is required for a proper TGF-尾-induced transcriptional response (Fig.聽3c). Ectopic expression of OTUD1-wt, but not OTUD1-CA, mitigated the magnitude and duration of TGF-尾-induced SMAD2 phosphorylation and SMAD2鈥揝MAD4 complex formation in MCF10A-RAS cells (Fig.聽3d). Depletion of OTUD1 showed the opposite effect (Fig.聽3e). These results indicate that OTUD1 is a potent antagonist of TGF-尾/SMAD signaling.Fig. 3OTUD1 causes suppression of TGF-尾 downstream signals and EMT. a Diagram of DUB cDNA screen data in HEK293T cells in which DUBs that activate TGF-尾-induced SMAD3/SMAD4-dependent CAGA12-Luc transcriptional reporter and TGF-尾-induced SMAD2/SMAD4-dependent ARE-Luc are indicated. The x-axe is the ARE-luciferase activity and y-axe is the CAGA-luciferase activity, shown as Log_2 value of ligand-induced fold changes. b, c Effect of ectopic OTUD1 wild-type (OTUD1-wt) and OTUD1-CA mutant (b) or OTUD1knockdown (sh-OTUD1 #1 and #2) (c) on CAGA12-Luc transcriptional response induced by TGF-尾 in HEK293T cells. Co.vec, empty vector; Co.sh, non-targeting shRNA. d, e IB of total cell lysate and anti-SMAD2/3 immunoprecipitates derived from control and MCF10A-RAS cells stably depleted for OTUD1 (d) or stably expressing Myc-OTUD1-wt/C320A (e) and treated with TGF-尾 (5鈥塶g/ml) as indicated. SMAD2/3-associated SMAD4 was analyzed by IB. 5% total cell lysate was loaded as input and were analyzed for P-SMAD2, OTUD1, SMAD2/3, and SMAD4. f Immunofluorescence and 4, 6-diamidino-2-phenylindole (DAPI) staining of control and HaCaT cells stably depleted of OTUD1 and treated with TGF-尾 (2.5 ng/ml) for 72鈥塰. Scale bar, 50鈥壩糾. g IB of cell lysates derived from MCF10A(RAS) cells stably expressed with control empty vector (Vec), or OTUD1-wt/CA constructs and treated with TGF-尾 (2.5 ng/ml) and SB431542 (10鈥壩糓) for 72鈥塰. h Heat聽map of TGF-尾 regulated genes in control cells (Co.sh) or MCF10A (RAS) cells stably depleted for OTUD1 with two independent shRNA (#1 and #2) and treated with or without TGF-尾 (2.5 ng/ml) for 8鈥塰. i qRT-PCR analysis of TGF-尾 target genes Snail, COL1A1, and SERPINE1 in control and OTUD1 stably depleted MCF10A (RAS) cells treated with TGF-尾 (2.5 ng/ml) for 8鈥塰. Values and error bars represent the means鈥壜扁€塖D of triplicates and are representative of at least two independent experiments. j qRT-PCR analysis of MDA-MB-231 (BM) cells infected with control of OTUD1-wt-expressing virus for 3 days (left panel) or 12 days (right panel). *p鈥?lt;鈥?.05, **p鈥?lt;鈥?.01 and ***p鈥?lt;鈥?.001 (two-tailed Student鈥瞫 t-test b鈥?b>j)Full size image Typical features of stemness-associated EMT include upregulation of N-cadherin, fibronectin, smooth muscle actin and vimentin, and downregulation of E-cadherin. Upon depletion of OTUD1, epithelial HaCaT keratinocytes gained mesenchymal features as analyzed by confocal-microscopy (Fig.聽3f). This latter effect is inhibited by the treatment of SB431542, a selective T尾R袉 kinase inhibitor, suggesting that the autocrine TGF-尾 is sufficient to promote EMT in OTUD1-depleted cells. In breast cancer cells, silencing OTUD1 promoted TGF-尾-induced changes in EMT-marker expression, whereas ectopic expression of OTUD1-wt, but not OTUD1-CA, had the reverse effect (Fig.聽3g; Supplementary Fig.聽3a). In line with this, PCR array analyses confirmed that loss of OTUD1 led to several molecular features of mesenchymal cells, including the upregulation of key transcriptional inducers such as TWISTs, SNAILs, ZEBs, and other EMT-related targets such as COL1A1, SERPINE1 (Fig.聽3h, i; Supplementary Data聽3); We next analyzed the influence of OTUD1 overexpression on well-established EMT-related makers genes at 3 days (immediate response) and 12 days (delayed response) post-transduction of OTUD1 expressing lentiviruses in breast cancer MDA-MB-231 (BM) cells (Fig.聽3j). As expected, OTUD1 overexpression was associated with an increased expression level of the epithelial maker CDH1 and decreased expression levels of the mesenchymal markers CDH2, FN1, and VIM (Fig.聽3j). Reduced expression of several EMT-related transcription factors, including SNAIL, SLUG, ZEB1, and ZEB2, was observed at 12 days post-OTUD1 overexpression (Fig.聽3j), showing a reversal of the mesenchymal phenotype of MDA-MB 231 (BM) cells. Besides, all of these genes were suppressed by an increase of OTUD1-wt, but not by OTUD1-CA, either at聽the basal or TGF-尾-induced level (Supplementary Fig.聽3b; Supplementary Data聽3). Taken together, these results indicate that OTUD1 is a critical and selective inhibitor of TGF-尾/SMAD signaling and EMT.OTUD1 associates with SMAD7 and deubiquitinates SMAD7 in vitroWe next investigated the molecular mechanism by which OTUD1 inhibits TGF-尾/SMAD signaling. The inhibitory effect of OTUD1 on TGF-尾-induced SMAD2 phosphorylation (Fig.聽3d) indicates that OTUD1 acts on the聽receptor/SMAD level. As OTUD1鈥檚 DUB activity is required, targeting of an inhibitor of TGF-尾 signaling, such as inhibitory SMAD7, appeared most likely. Ectopic expression of OTUD1 could suppress TGF-尾 signaling in control cells but barely showed effect in SMAD7-deficient cells; also knockdown of OTUD1 could promote TGF-尾 signaling in control cells but not in SMAD7-deficient cells (Fig.聽4a; Supplementary Fig.聽4a), suggesting that聽inhibitory SMAD7 might be the major target of OTUD1 in the TGF-尾 pathway. To consolidate this observation in vivo, we generated a聽SMAD7 knockout cell line of early passage MDA-MB-231 cells using聽CRISPR/Cas9 technology (Supplementary Fig.聽4a). Mice injected with this聽SMAD7 knockout clone developed聽more rapid and stronger metastasis and had significantly shorter metastasis-free survival periods (Fig.聽4b). Moreover, ectopic expression of OTUD1 significantly inhibited metastasis in control cells but did not show significant inhibitory effect in the SMAD7 knockout cells. This demonstrates that the anti-metastatic activity of OTUD1 is largely mediated via regulation of聽SMAD7. Consistent with this notion, in vitro purified SMAD7 protein was found to associate directly with OTUD1 (Fig.聽4c). In cells, Myc-tagged OTUD1 strongly and specifically co-precipitated with SMAD7 (Fig.聽4d). Moreover, endogenous OTUD1 was found to interact with SMAD7 in breast cancer cells (Fig.聽4e).Fig. 4OTUD1 interacts with and deubiquitinates SMAD7 in vitro. a Effect of OTUD1-wt and CA mutant (left panel) or sh-OTUD1 #1 (right panel) on CAGA12-Luc transcriptional response induced by TGF-尾 (2.5 ng/ml) for 16鈥塰 in HEK293T cells. Co., control sgRNA. b Left panel: BLI of three representative mice injected with MDA-MB-231 control cells or cells deficient in SMAD7 (sg-SMAD7) that ectopically expressed with control vector (Co.vec) or OTUD1, images were taken 6 weeks after injection. Middle panel: BLI signals of all mice in each experimental group at week 6. Right panel: the percentage of metastasis-free mice in each experimental group followed in time. c Purified SMAD7 and OTUD1-wt/CA interaction in vitro. Prokaryotic purified SMAD7 and eukaryotic purified OTUD1 proteins were incubated and immunoprecipitated with anti-OTUD1 (left panel) or with anti-SMAD7 (right panel) antibodies. Immunoprecipitates were then immunoblotted for OTUD1 and SMAD7. d IB analysis of total cell lysate (TCL) and immunoprecipitates derived from HEK293T cells transfected with Myc-OTUD1 and Flag-SMAD1-7 constructs as indicated. e IB of anti-SMAD7 immunoprecipitate derived from HEK293T cells. Association between OTUD1 and SMAD7 was examined. 5% total cell lysates was loaded as input. f鈥?b>h OTUD1 deubiquitinates poly-ubiquitination of SMAD7 in vitro. Flag-SMAD7 and HA-Ub were transfected in HEK293T cells. Poly-ubiquitinated SMAD7 was then immunoprecipitated with anti-Flag M2 beads and incubated with purified OTUD1-wt/CA protein as indicated time. Lysates were analyzed by IB with anti-HA-Ub, anti-SMAD7, and anti-OTUD1 antibodies f. Flag-SMAD7 and His-Ub were transfected into HEK293T cells. Subsequently, poly-ubiquitinated SMAD7 from the cell lysate was pulled down by Nickle beads and incubated with purified OTUD1-wt/CA for 60鈥塵ins. Lysates were analyzed by IB with anti-SMAD7 and anti-OTUD1 antibodies, asterisk indicates free SMAD7 g. His-SMAD7 was first incubated with E1, E2 (UbcH6), GST-RNF12, and ubiquitin in vitro for 3鈥塰 at 37鈥壜癈. The poly-ubiquitinated His-SMAD7 was immunoprecipitated by anti-SMAD7 antibody then incubated with purified OTUD1-wt/CA for 60鈥塵ins. The assay mixtures were analyzed by anti-Ub, anti-SMAD7 and anti-OTUD1 antibodies (h). *p鈥?lt;鈥?.05, **p鈥?lt;鈥?.01, and ***p鈥?lt;鈥?.001 (two-tailed Student鈥瞫 t-test a, b or two-way analysis of variance (ANOVA) b)Full size image Although OTUD1-wt-associated SMAD7 was not ubiquitinated, OTUD1-CA mutant-associated SMAD7 was highly ubiquitinated, with the SMAD7 poly-ubiquitination band presenting most prominently (Supplementary Fig.聽4b). We then examined whether OTUD1 serves as a DUB for SMAD7. Firstly, Flag-tagged SMAD7 proteins were affinity purified, and their ubiquitination pattern was visualized by immunoblotting for HA-ubiquitin: poly-ubiquitination appeared as a major modification of SMAD7. To demonstrate that OTUD1 can directly deubiquitinate SMAD7, we performed in vitro deubiquitination assays. Purified OTUD1-wt, but not OTUD1-CA, removed poly-ubiquitin chains from SMAD7; when an optimal concentration of OTUD1 was applied for this assay, more than 50% of the poly-ubiquitylated SMAD7 was cleaved within 20鈥塵in (Fig.聽4f). When poly-ubiquitinated SMAD7 was purified with nickel beads from His-Ubiquitin expressing cells and applied to an in vitro deubiquitination assay, the poly-ubiquitin chains were deubiquitinated by OTUD1-wt. This results in a concomitant accumulation of free SMAD7 (Fig.聽4g), indicating that poly-ubiquitinated SMAD7 is a substrate of OTUD1. Previously, we have identified RNF12 as an E3 ligase for SMAD713. Purified Gluthathion S-tranferease (GST)-RNF12 wt, but not GST-RNF12 CA mutant (the essential catalytic sites in C3HC4 motif of RNF12 RING domain were mutated), greatly promoted SMAD7 poly-ubiquitination13. Upon聽incubation of聽His-SMAD7 with RNF12 in vitro, the poly-ubiquitin chains of SMAD7 were cleaved by OTUD1-wt (Fig.聽4h). This confirms that OTUD1 deubiquitinates SMAD7 in vitro.OTUD1 deubiquitinates SMAD7 and sustains SMAD7 stabilityIn cells we found overexpression of OTUD1-wt, but not OTUD1-CA, to deubiquitinate SMAD7 poly-ubiquitination including the Lysine 48 chain conjugation (as revealed by K48-linkage specific antibody) either in the absence or presence of the proteasome inhibitor MG132 (Fig.聽5a). RNF12 promoted poly-ubiquitination of SMAD7 in control cells or OTUD1-CA-expressing cells, but not in OTUD1-wt-expressing cells (Fig.聽5b). Depletion of endogenous OTUD1 promoted SMAD7 poly-ubiquitination both at聽the basal and at聽the RNF12-induced level (Fig.聽5c).Fig. 5OTUD1 deubiquitinates SMAD7 in vivo and sustains SMAD7 stability. a IB of total cell lysate (TCL) and immunoprecipitates derived from HEK293T cells transfected with HA-Ub, Flag-SMAD7, Myc-OTUD1-wt/CA and treated with control DMSO or MG132 (5鈥壩糓 for 4鈥塰) as indicated. b, c IB of TCL and immunoprecipitates derived from HEK293T stably expressing HA-Ub and transfected with Flag-SMAD7, Myc-RNF12, and Myc-OTUD1-wt/CA (b) or depleted for OTUD1 with shRNA (#1 and #2) c as indicated. Poly-ubiquitinated SMAD7 was immunoprecipitated with anti-Flag M2 beads and analyzed by IB with anti-HA-Ub antibody. d IB of TCL and immunoprecipitates derived from OTUD1 +/+ and OTUD1 鈭?i>/鈭?/sup> HEK293T cells stably expressing HA-Ub and restored with or without Myc-OTUD1 expression plasmid. Endogenous poly-ubiquitinated SMAD7 was immunoprecipitated with anti-SMAD7 (S7) antibody and immunoblotted with anti-HA-Ub antibodies. e [35S]-methionine labeling and pulse-chase studies of SMAD7 in control (Co.sh) and OTUD1-depleted PC3 (#1 and #2). The amount of immunoprecipitated labeled protein after the chase was expressed as the percentage of that at the beginning of the chase (time 0) and shown in the right panel. Results are shown as means鈥壜扁€塖D of two independent sets of experiments in duplicate. f IB of lysates derived from PC3 cells stably expressing empty vector (Co.vec), Myc-OTUD1-wt or OTUD1-CA and treated with cycloheximide (CHX, 20鈥壩糶/ml) at the indicated time points. Actin was analyzed as an internal loading control. Quantification of the band intensities was shown in the right panel. Band intensity was normalized to the t鈥?鈥? controls. Results are shown as means鈥壜扁€塖D of three independent sets of experiments. *p鈥?lt;鈥?.05 (two-tailed Student鈥瞫 t-test e, f)Full size image Using CRISPR/Cas9 technology, we generated OTUD1 knockout HEK293T cells. When endogenous SMAD7 was precipitated in these cells, we observed an accumulation of SMAD7 poly-ubiquitination in OTUD1 鈭?i>/鈭?/sup> cells compared with OTUD1 +/+ cells. This effect was reversed when OTUD1 expression was restored (Fig.聽5d). To investigate whether OTUD1 affects SMAD7 turnover, we measured SMAD7 protein stability. Pulse-chase labeling experiments showed that the endogenous SMAD7 displayed a severely impaired half-life in OTUD1-depleted cells compared to wild-type cells (Fig.聽5e). Ectopic expression of OTUD1-wt, but not OTUD1-CA, prolonged the half-life of endogenous SMAD7 (Supplementary Fig.聽4c). In line with this, the turnover rate of SMAD7, as measured by cycloheximide (CHX) treatment, was reduced by OTUD1 but enhanced in OTUD1-depleted cells (Fig.聽5f; Supplementary Fig.聽4d). Taken together, endogenous OTUD1 contributes to SMAD7 stability through its deubiquitination.OTUD1 cleaves K33-poly-ubiquitin chain on SMAD7 Lysine 220By mass spectrometry, we found that SMAD7 was ubiquitinated on Lysine 220 (Fig.聽6a; Supplementary Data聽4), in close proximity to the unique poly-proline-tyrosine 鈥淧PPY鈥?motif that mediates the recruitment SMURF2 to SMAD733 (Fig.聽6b). Conjugation of ubiquitin on this site might therefore affect the ability of SMAD7 to interact with SMURF2. To determine the type of ubiquitin linkage on this residue, we co-transfected SMAD7 and OTUD1 with individual ubiquitin expression constructs that can only form ubiquitin linkages at one lysine (for example, K27- indicates every lysine except K27 is changed to Arginine). As shown (Fig.聽6c), SMAD7 was primarily conjugated with K27-, K33-, and K48- ubiquitin chains and all of those linkage could be antagonized by OTUD1. Mutation of Lysine 220 to Arginine (K220R) on SMAD7 completely abolished its K33-poly-ubiquitination but had no obvious effect on K27- or K48-poly-ubiquitination (Fig.聽6d), suggesting that K220 is the major site specifically responsible for K33-poly-ubiquitination on SMAD7.Fig. 6OTUD1 cleaves K33-poly-ubiquitin chain on SMAD7 Lysine 220. a K220 ubiquitination of SMAD7 identified by mass spectrometry. Right: Comassie staining of Flag-SMAD7 precipitations. b Sequence alignment of the PY motif and identified K220 ubiquitination site in SMAD7 orthologs of different species. c Immunoblot (IB) of total cell lysate (TCL) and immunoprecipitates derived from Flag-SMAD7 overexpressing HEK293T cells transfected with K6-, K11-, K27-, K29-, K33-, K48-, and K63-linked HA-Ub constructs and Myc-OTUD1 as indicated. d IB of TCL and immunoprecipitation derived from HEK293T cells transfected with wt, K27-, K33-, K48-linked HA-Ub, together with Flag-SMAD7-wt or K220R mutant as indicated. e Upper panel: the model of WW domain of E3-ubiquitin ligase Smurf2 binding to PY loop (residue 203-220) of SMAD7. Middle and lower panels: the model of mono-ubiquitin (PDB ID: 2XK5) conjugation to PY loop (residue 203-220) at residue Lys220. Both WW domain (green) and PY loop (red) were shown in ribbon, ubiquitin was shown in gray surface, and the key residues involved in domain interaction are drawn in stick representations. f IB of TCL and anti-SMAD7 immunoprecipitants derived from K33-linked HA-Ub expressing HEK293T cells depleted of OTUD1. g IB of TCL and Nickle-pull down precipitants derived from K33-linked His-Ub expressing HEK293T cells transfected with Flag-SMAD7-wt/ K220R and depleted of OTUD1 as indicated. h IB of input and anti-Flag immunoprecipitates derived from HEK293T cells transfected with Flag-SMAD7-wt or K220R mutant as indicated. i IB of input and anti-Flag immunoprecipitates derived from HEK293T cells transfected with Flag-SMAD7 and depleted of OTUD1 as indicated. j CAGA12-Luc transcriptional response of HEK293T cells transfected with empty vector (Co.vec), SMAD7-wt or K220R at different doses as indicated and treated with TGF-尾 (0.5 ng/ml) overnight. k IB of biotinylated cell surface T尾RI in HeLa cells stably transfected with empty vector (Co. vec), SMAD7-wt or K220R mutant expression plasmids and treated with TGF-尾 (5鈥塶g/ml) for indicated time points. Quantification of the band intensities is shown in the lower panel. Band intensity was normalized to the t鈥?鈥? controls. Results are shown as means鈥壜扁€塖D of three independent sets of experiments. l Working model of OTUD1-mediated SMAD7 deubiquitinationFull size image The NMR structure of the聽SMAD7 PY motif region in complex with the聽SMURF2 WW3 domain revealed that the residues C-terminal to the PY motif (PY-tail), including D217, are involved in direct WW3 domain binding33. K220 locates in a flexible region right after the PY motif in SMAD7. Structure modeling suggested that a covalent conjugated ubiquitin molecule on K220 is likely to occupy聽the interaction space between the PY-tail and the 尾1-strand and 尾1鈥撐? loop of the WW3 domain (Fig.聽6e, middle and lower panels), thereby聽interfering with SMURF2 binding. Considering the compact conformation of K33-linked poly-ubiquitin chain34, a poly-ubiquitin chain has higher possibility to obstruct the聽SMURF2 interaction. This suggest that K33-linked poly-ubiquitination on K220 might block SMAD7鈥揝MURF2 binding. Thus, OTUD1-mediated removal of K33-linked poly-ubiquitination on K220 is likely needed for the SMAD7 PY motif to recruit SMURF2. We next set to confirm this hypothesis with experiments. In OTUD1-depleted cells, we precipitated endogenous SMAD7 and observed accumulated K33-linked poly-ubiquitination (Fig.聽6f). Moreover, OTUD1-loss accumulated K33-linked poly-ubiquitination was only observed with SMAD7-wt protein and not with SMAD7 K220R (Fig.聽6g). These results corroborate that OTUD1 is a DUB that cleaves K33-linked poly-ubiquitination of SMAD7 K220. We then compared SMAD7-wt and SMAD7 K220R and indeed found SMURF2 to strongly associate with SMAD7 K220R mutant (Fig.聽6h). In line with this, knockdown of OTUD1, which results in increased K33-poly-ubiquitin conjugation on SMAD7 K220, reduced the ability of SMURF2 to interact with SMAD7 (Fig.聽6i). This effect of the SMAD7 K220 mutation was reflected in a direct readout of TGF-尾 signaling as the K220R mutant is more potent in suppressing the TGF-尾/SMAD-induced CAGA-Luc reporter (Fig.聽6j).Upon activation of TGF-尾 signaling, SMAD7 binds T尾RI as part of a negative feedback response10,11, and recruits SMURF2 to promote T尾RI turnover at the plasma membrane12. Given our results that OTUD1 enables SMAD7/SMURF2 function, we examined whether OTUD1 misexpression can regulate T尾RI levels at the cell surface, the location where intracellular signaling is initiated. When OTUD1 was depleted, biotin-labeled cell surface T尾RI receptor displayed a prolonged half-life (Supplementary Fig.聽5a). In line with this finding, ectopic expression of OTUD1-wt, but not the OTUD1-CA mutation, led to accelerated T尾RI degradation (Supplementary Fig.聽5b). Moreover, SMAD7 K220R showed stronger capacity than SMAD7-wt to shorten the half-life of T尾RI at the cell surface (Fig.聽6k). Together, these findings indicate that OTUD1 is a DUB that removes K33-poly-ubiquitination on SMAD7 K220, which promotes SMAD7鈥檚 inhibitory role on antagonizing T尾RI (Fig.聽6l).OTUD1 inhibits metastasis in transplantable mouse modelsTo verify our MDA-MB-231 xenograft results we investigated the effect of OTUD1 on lung metastasis using allograft models. Luciferase-labeled control or OTUD1-depleted 4T07 cells were injected intravenously into nude mice and subjected to bioluminescent imaging (BLI). OTUD1-depleted cells exhibited significantly increased lung metastasis abilities around two weeks (Fig.聽7a; Supplementary Fig.聽6a). Continued BLI monitoring revealed a further enhancement of metastatic outgrowth in the lungs of animals injected with OTUD1-depleted cells (Fig.聽7a), and histological analyses indicated a significant increase in the number of metastatic lesions and also in the average surface areas produced by OTUD1-depleted cells when compared with the control cells (Fig.聽7b, c; Supplementary Fig.聽6b). Taken together, these analyses show that loss of OTUD1 strongly promotes breast cancer lung metastasis.Fig. 7OTUD1 inhibits lung metastasis in transplantable mouse models of breast cancer metastasis. a鈥?b>d Control (Co.) or OTUD1-silenced (sh#1 and sh#2) 4T07 cells were tail vein-injected into nude mice. Lung metastasis was measured by BLI. Normalized photon flux at the indicated times left (卤SD) and representative images (right) are shown; p-values; Student鈥檚 t-test (a). Lung metastasis nodules in each group were calculated (卤SD) and the representative images of bright view (c, upper panel; scale bar, 2鈥塵m) and bioluminescent view (c, lower panel; scale bar, 2鈥塵m) are shown. Representative HE and IHC staining pictures of lung sections of control and one shRNA-depleted OTUD1 group (d), scale bars, 1鈥塵m and 20鈥壩糾 for lung haematoxylin and eosin (H E) and SMAD7 immunohistochemistry images, respectively). e鈥?b>i Control (Co.) 4T1 cells or cells stably expressing OTUD1-wt or CA were tail vein-injected into nude mice. Lung metastasis was measured by BLI. Normalized photon flux (卤SD) at the indicated times are shown in the upper panel and representative images in the lower; p values; Student鈥檚 t-test e. Lung metastasis nodules (卤SD) f and average lung lesion surface area (卤SD) g (arbitrary units based on pixel quantification from digital images) in each group were calculated and the representative images of bioluminescent view (h, scale bar, 2鈥塵m) are shown. Representative HE and IHC staining pictures of lung sections of every experimental group (i, Scale bars, 1鈥塵m and 20鈥壩糾 for lung haematoxylin and eosin (H E) and SMAD7 immunohistochemistry images, respectively) are shown. j鈥?b>m Control (Co.) or OTUD1-depleted 4T1 cells were orthotropic injected in syngeneic BALB/c mice. Primary tumor formation in mice after orthotropic injection of each group at day 21 after implantation. Normalized BLI signals (卤SD) (Left) and representative BLI signals of primary tumor. Number of lung metastasis nodules (k) and average lung lesion surface area (卤SD) (l) (arbitrary units based on pixel quantification from digital images). Representative bioluminescent view of isolated lung (m, left, scale bar, 2鈥塵m) and HE staining of lung section of each group (m, right, scale bar, 20鈥壩糾). *p鈥?lt;鈥?.05 and **p鈥?lt;鈥?.01 (two-tailed Student鈥瞫 t-test a鈥?b>l)Full size image Considering the importance of the immune system in lung metastasis, we extended our analysis to an immunocompetent mouse model of lung metastasis. We manipulated OTUD1 expression with either ectopic expression or knockdown strategies in the 4T1 murine breast cancer cell line (Supplementary Fig.聽6c) and examined their ability to influence metastasis in vivo. Stable cells that ectopically expressed OTUD1-wt or OTUD1-CA were introduced into nude mice through tail vein injection; mice were subjected to BLI. Compared to the control cells, lung metastasis was reduced in OTUD1-wt-expressing cells, but not in the OTUD1-CA-expressing cells (Fig.聽7e鈥?a data-track=\"click\" data-track-label=\"link\" data-track-action=\"figure anchor\" href=\"/articles/s41467-017-02029-7#Fig7\">g). It was clear that both the number and the average lesion surface of the lung metastasis nodules are significantly reduced in OTUD1-wt group (Fig.聽7f鈥搃). Interestingly, we also observed an increase in SMAD7 expression in OTUD1-wt when compared with control or OTUD1-CA tumors (Fig.聽7i). To mimic the pathological condition of tumorigenesis and metastasis, we then injected 4T1 cells under the nipple of BALB/c mice, which allows us to examine both primary tumor growth and spontaneous lung metastasis. Here OTUD1-depleted 4T1 cells showed increased spontaneous lung metastasis without affecting primary tumor growth (Fig.聽7j鈥?a data-track=\"click\" data-track-label=\"link\" data-track-action=\"figure anchor\" href=\"/articles/s41467-017-02029-7#Fig7\">m; Supplementary Fig.聽6d). qRT-PCR analysis indicated upregulation of cancer stem cell markers and EMT genes in OTUD1-depleted 4T1 lung-metastatic nodules when compared with control group (Supplementary Fig.聽6e), again suggesting that OTUD1 functions to oppose cancer stemness and EMT-related gene-expression programs. Therefore, our studies suggest that OTUD1 inhibits lung metastasis in transplantable mouse models.Loss of OTUD1 gene copies correlates with poor prognosisTo determine the clinical relevance of the above findings in advanced human cancers, we first analyzed the TCGA database and found that the OTUD1 gene is lost or even deleted in many tumor types as revealed by GISTIC analysis35, the total percentage is more than 50% in glioblastoma, melanoma and lung cancer (Fig.聽8a; Supplementary Table聽2). Expression of OTUD1 showed positive correlation with its gene copy number in breast cancer. Meanwhile, we observed that the very rare breast cancer patients with gain or amplification of OTUD1 have co-occurrence with p53 expression and mutual exclusivity with expression of PIK3CA and AKT1 (Fig.聽8b; Supplementary Fig.聽6f). Combined with our previous analysis, these observations strongly favor the notion that OTUD1 is a metastasis suppressor.Fig. 8OTUD1 correlates with SMAD7 in human specimens and the loss of OTUD1 gene copies in human cancer correlates with poor prognosis. a Graph showing OTUD1 gene copy loss or deletion in different tumor types. Data were obtained from the TCGA database65, 66. Detailed values are listed in Supplementary Table聽2. b Volcano plot showing that the rare breast cancer patients with gain or amplification of OTUD1 have co-occurrence with p53 expression and mutual exclusivity with expression of PIK3CA and AKT1. Data were obtained from the TCGA database65, 66. c Analysis of BRCA1, BRCA2, and OTUD1 mRNA level in p53 wt- and p53 null- MCF10A cells expressed with or without wild-type HRAS (GSE81593 dataset). d qPCR analysis of OTUD1 mRNA level in MCF7, MDA-MB-231 (early passage), MDA-MB-231 bone-metastatic (BM) cells that ectopically express HRASV12 or control vector. Values and error bars represent the mean鈥壜扁€塖D of triplicates and are representative of at least two independent experiments. e Heat map of mRNA level of listed genes in MCF10A cells that stably express control or HRAS with depletion of OTUD1 (sh-OTUD1), as indicated. f鈥?b>h Immunohistochemistry analysis of OTUD1 and SMAD7 in breast cancer tissue microarrays; representative pictures of each antibody staining are shown (f). Percentage of specimens displaying low or high SMAD7 expression compared to the expression levels of OTUD1 (g). Scatterplot showing the positive correlation between OTUD1 and SMAD7 expression in patients; Pearson鈥檚 coefficient tests were performed to assess statistical significance h. i Kaplan鈥揗eier curves showing that the metastasis-free survival of individuals was positively correlated with OTUD1 expression. j Kaplan鈥揗eier curves showing that the metastasis-free survival of individuals was positively correlated with OTUD1 expression in lymph node positive patient (Left) and has no correlation with OTUD1 in lymph node negative patients (Right). *p鈥?lt;鈥?.05 and ***p鈥?lt;鈥?.001 (two-tailed Student鈥瞫 t-test (d, g), Pearson鈥檚 coefficient tests (h) or Log-rank test (i, j))Full size image In addition, we investigated whether OTUD1 transcription is downregulated in cancer. Similar as the BRCA1 and BRCA2 tumor suppressors, expression of OTUD1 was significantly suppressed by wild-type HRAS in p53 null MCF10A cells, but not in the control MCF10A cells (p53-wt), indicating that gain of oncogenic RAS together with loss of p53 tumor suppressor could lead to decreased OTUD1 function (GSE81593) (Fig.聽8c). Consistent with the notion that activation of HRAS inhibits OTUD1 expression in p53 mutant cells, we found that upon ectopic expression of constitutively active HRASV12, OTUD1 is downregulated in p53 mutant MDA-MB-231 (both the early passage and the bone-metastatic (BM) cell lines) but not in p53 wt MCF7 cells (Fig.聽8d), TGF-尾 and HRAS collaborate to promote EMT, invasion and cancer stem traits in breast cancer cells36. In line with this, depletion of OTUD1 enhanced the effect of HRAS in MCF10A cells (Fig.聽8e; Supplementary Data聽3).From surgical resections, we collected 100 patient-derived samples of breast cancer for tissue microarray analysis. The immunohistochemical (IHC) analysis of OTUD1 and SMAD7 levels revealed a statistically significant positive correlation (Fig.聽8f鈥?a data-track=\"click\" data-track-label=\"link\" data-track-action=\"figure anchor\" href=\"/articles/s41467-017-02029-7#Fig8\">h). In a large public clinical microarray database of human breast tumors, we found a trend towards good prognosis for OTUD1-high patients (Fig.聽8i). Low OTUD1 correlates with poor survival in patients with lymph node invasion/metastasis. But for the patients without lymph node signal, OTUD1 failed to distinguish prognosis (Fig.聽8j), suggesting a more significant role of OTUD1 in antagonizing metastatic tumors. These findings are in support of our hypothesis that OTUD1 shuts off breast cancer metastasis by deubiquitinating SMAD7.DiscussionMetastasis is a process in which cancer cells relocalize to another organ. To reach distant organs, circulating tumor cells (CTCs) that have intravasated from the primary tumor must overcome many obstacles through mechanisms that are not well understood37. Recent findings have just started to define the extracellular cues, phenotypic properties, hosting niches, and signaling pathways that support the survival, self-renewal, dormancy, and reactivation of cancer cells that initiate metastasis: metastatic stem cells (MSC)38. By dissecting the biology of this multistep process, vulnerabilities are being exposed that could be exploited to prevent metastasis. To enable search of factors that favor metastatic outgrowth, we developed an in vivo screening system in mice to select for genetic determinants that promote metastasis. Our loss of function screen shares some similarity to other screens that have recently been reported using shRNA- or a CRISPR/Cas9-based approaches to identify modulators of metastasis using intravenous injection or subqutaneous transplant delivery of tumor cells, respectively39,40. In this study, we screened a shRNA library that covers human DUB family members that inhibit metastasis and use an intracardial administration route of tumor cells. From our screen, we have identified that loss of OTUD1 enables breast cancer cells to undergo EMT and gain cancer stem cell traits thereby driving metastatic spread to distant organs, such as bone and lung. OTUD1 was subsequently identified as a potent negative regulator of the TGF-尾 signaling pathway, a strong inducer of EMT and cancer cell stemness.DUBs antagonize the action of E3-ubiquitin ligases, of which the latter mediate the Ub modification of certain target proteins. DUBs remove Ub post-translational modifications of proteins,thereby regulating聽virtually all Ub-dependent processes41. Both DUBs and E3 ligases are emerging classes of enzymatic drug targets42,43. Although K48-linked Ub chains generally serve as a proteasomal degradation signal41, K63-linked chains are nondegradative and, for example, activate protein kinase cascades44. Lys11 linkages constitute an alternative degradation signal used during cell-cycle progression45. Met1-linked chains cooperate with K63 linkages in NF-魏B signaling46. But the cellular roles of the remaining types (K6, K27, K29, and K33) are elusive47.The deubiquitinase OTUD1 was found to enable the antagonistic role of SMAD7 on TGF-尾 signaling. SMAD7 recruits SMURF2 to the activated T尾RI at the cell surface to promote its ubiquitination and degradation. Moreover, SMAD7 itself is ubiquitinated by RNF12 and ARKADIA for proteasomal degradation. OTUD1 displayed two layers of regulation and specificity on SMAD7 poly-ubiquitination: OTUD1 removes K48-chain thereby inhibiting SMAD7 degradation. Moreover, OTUD1 cleaves the聽K33-chain on SMAD7 K220 residue thereby聽exposing the SMAD7聽PY-tail binding pocket for interaction with the聽WW domain of聽SMURF2. By doing so, OTUD1 enables SMAD7 function thereby antagonizing TGF-尾/SMAD signaling. Our finding is consistent with the previous finding that OTUD1 is capable of cleaving multiple types of聽ubiquitin linkage48. Of note, the聽K27-chain strongly conjugates on SMAD7 and is also cleaved by OTUD1, suggesting a 鈥渢hird鈥?layer of SMAD7 regulation by OTUD1 that requires further investigation. By generating SMAD7-deficient clone of the early passage MDA-MB-231 cells, we found that聽the anti-metastatic role of OTUD1 is dependent on endogenous SMAD7 expression. Although OTUD1 could not show significant inhibition of metastasis in SMAD7-deficient cells, our results could not exclude the possibility that OTUD1 might also target other substrates. Although OTUD1-wt can completely cleave K48-linked poly-ubiquitin chains from SMAD7 in DMSO-treated cells, this effect is not observed in the MG132-treated cells, suggesting that there might be certain DUBs present at the proteasome that have a role in deubiquitinating SMAD7.As mentioned the K33-linked ubiquitin linkage is an atypical one. Here we show by structural modeling and follow-up biochemical and cell biological experiments that K33-linked poly-ubiquitination of SMAD7 might shield the PY interaction motif, and thereby interferes with protein鈥損rotein interaction. We validated this hypothesis by showing that mutation of the SMAD7 K220 site could completely abolish SMAD7 K33-linked poly-ubiquitination (Fig.聽6d) and sharply increased SMAD7鈥檚 binding affinity with SMURF2 (Fig.聽6h). As far as we know, such聽a mechanism of action has not been reported before for K33-linked ubiquitination. In contrast to our study, others have聽reported that K33-linked ubiquitination聽can actually mediate the recruitment of regulatory proteins49,50,51, or that聽such chains compete with degradative K48-linked ubiquitination52. Of note, monoubiquitination of SMAD4 interferes with the interaction with phosphorylated SMAD2 and thereby inhibits TGF-尾 signaling53.Another aspect that deserves discussion is the broad and frequent loss of OTUD1 expression in human cancers. We observed that some breast cancers express aberrantly low concentrations of OTUD1 as a result of OTUD1 gene loss or even gene deletion and that the loss of OTUD1 expression in patient correlates with a poor prognosis. Gain of RAS activity and loss of p53 expression are often the characteristics of advanced tumors. Gain of HRAS expression and the loss of p53 signaling have been shown to collaborate with each other in promoting the loss of OTUD1 expression in breast cancer cells. Importantly, OTUD1 gene loss is also present in many other types of cancers, such as glioblastoma and lung cancers, indicating that the tumor suppressing role of OTUD1 may聽not聽be limited to breast cancer. OTUD1 is targeted for gene loss in around 50% of lung tumors (Fig.聽8a). Patients with lung carcinoma and low OTUD1 gene expression have shorter lifetimes than those with lung cancer but high OTUD1 expression (Supplementary Fig.聽6g)54. Moreover, OTUD1 correlated with the SMAD7 expression in our breast cancer tissue microarray analysis, further corroborating that OTUD1 has an essential role in the control of SMAD7 stability and activity. Moreover, in both xenograft and orthotopic mouse cancer models, loss of OTUD1 expression is closely associated with breast cancer metastasis.Combined, the in vitro and in vivo evidence in this study elucidates critical functions of OTUD1 in the termination of TGF-尾/SMAD-induced metastatic activation, indicating that OTUD1 restricts the EMT and cancer stem cell traits during metastasis. Moreover, as an in vivo deubiquitinase of SMAD7, OTUD1 represents a novel and critical mechanism that controls intrinsic SMAD7 activity and thereby TGF-尾/SMAD signaling.MethodsCell cultureHEK293T, HeLa, HaCaT, MCF7, 4T1, 4T07, MDA-MB-231 cell lines were originally from ATCC and cultured in Dulbecco鈥檚 modified Eagle鈥檚 medium (DMEM) supplemented with 10% FBS and 100鈥塙/ml penicillin鈥搒treptomycin. MCF10A (MI) and MCF10A-RAS (MII) cell lines were obtained from Dr. Fred Miller (Barbara Ann Karmanos Cancer Institute, Detroit, USA) and cultured in DMEM/F12 supplemented with 5% horse serum, 20 ng/ml epidermal growth factor (EGF), 0.5鈥壜礸/ml hydrocortisone, 100 ng/ml cholera toxin, and 100 U/ml penicillin鈥搒treptomycin. All the cells are free of mycoplasma contamination.Lentiviral transduction and generation of stable cell linesLentiviruses were produced by transfecting HEK293T cells with shRNA-targeting plasmids and the helper plasmids pCMV-VSVG, pMDLg-RRE (gag/pol), and pRSV-REV. The cell supernatants were harvested 48鈥塰 after transfection and were either used to infect cells or stored at 鈭?0鈥壜癈.To obtain stable cell lines, cells were infected at low confluence (20%) for 24鈥塰 with lentiviral supernatants diluted 1:1 with normal culture medium in the presence of 5 ng/ml of polybrene (Sigma). Forty-eight hours after infection, cells were placed under puromycin selection for one week and then passaged before use. Puromycin was used at 2鈥壜礸/ml to maintain MDA-MB-231 cells, MCF10A-RAS, 4T1 and 4T07 cells. Lentiviral shRNAs were obtained from Sigma (MISSION庐 shRNA). Typically, five shRNAs were identified and tested, and the most effective two shRNAs were used for the experiments. We used TRCN0000350761 (#1) and TRCN0000350762 (#2) for human OTUD1 knockdown and designed two primer pairs for mouse OTUD1 knockdown as indicated below:Primer1-Forward: CCGGCAGATGCTGAATGT GAATATACTCGAGTATATTCACATTCA GCATCTGTTTTTG; Primer1-Reverse: AATTCAAAAACAGATG CTGAATGTGAATATACTC GAGTATATTCACATTCA GCATCTG;Primer2-Forward: CCGGGCTCAGCAAT GGACACTATGACT CGAGTCATAGTGTCCATTG CTGAGCTTTTTG;Primer2-Reverse: AATTCAAAAAGC TCAGCAATGGACACTATGACTCG AGTCATAGTGTCCATTGCTGAGC.Transcription reporter assayHEK293T cells were seeded in 24-well plates and transfected with the indicated plasmids using calcium phosphate. Luciferase activity was measured with a PerkinElmer luminometer. The internal transfection control renilla, was used to normalize luciferase activity. Each experiment was performed in triplicate, and the data represent the mean鈥壜扁€塖D of three independent experiments.Immunoprecipitation and immunoblottingCells were lysed with 1鈥塵l lysis buffer (20鈥塵M Tris-HCl pH 7.4, 2鈥塵M EDTA, 25鈥塵M NaF, and 1% Triton X-100) containing protease inhibitors (Sigma) for 10鈥塵in at 4鈥壜癈. After centrifugation at 12鈥壝椻€?03脳g for 15鈥塵in, the protein concentrations were measured, and equal amounts of lysate were used for immunoprecipitation. Immunoprecipitation was performed with anti-FLAG M2 beads (Sigma, A2220) for 1鈥塰 at 4鈥壜癈 or with different antibodies and protein A-Sepharose (GE Healthcare BioSciences AB) for 3鈥塰 at 4鈥壜癈. Thereafter, the precipitants were washed three times with washing buffer (50鈥塵M Tris-HCl pH 8.0, 150鈥塵M NaCl, 1% Nonidet P-40, 0.5% sodium-deoxycholate, and 0.1% SDS), and the immune complexes were eluted with sample buffer containing 1% SDS for 5鈥塵in at 95鈥壜癈. The immunoprecipitated proteins were then separated by SDS-PAGE. Immunoblot (IB) analysis was performed with specific antibodies and secondary anti-mouse or anti-rabbit antibodies conjugated to horseradish peroxidase (Amersham Biosciences). Visualization was achieved with chemiluminescence.For the analysis of cell surface receptors, the proteins at the cell surface were biotinylated for 40鈥塵in at 4oC and then incubated at 37掳C for the indicated times. The biotinylated cell surface receptors were precipitated with streptavidin beads and analyzed by immunoblotting. All the uncropped and unmodified scans of western blot data were shown as Supplementary Figs.聽7鈥?a data-track=\"click\" data-track-label=\"link\" data-track-action=\"supplementary material anchor\" href=\"/articles/s41467-017-02029-7#MOESM2\">9 in the Supplementary Information.In vivo screening and mouse metastasis assayUp to 371 shRNA lentivirus (and also control shRNA) were produced in HEK293T cells. Virus were then introduced into MDA-MB-231-GFP/Luc cells individually. Forty-eight hours after infection, cells were placed under puromycin selection for another 3 days. Control and 371 shRNA stable cells were then harvested and mixed into single-cell suspensions (1鈥壝椻€?05/100鈥壩糽 PBS) then were inoculated into the left heart ventricle of 5-weeks-old female BALB/c nude mice (n鈥?鈥?0) according to the method described by Arguello et al55. Development of metastasis was monitored weekly by bioluminescent reporter imaging. Early-onset metastasis nodules were isolated and genomic DNA was purified for sequencing.聽 According聽to standard protocol (TIANamp Genomic DNA Kit, Cat. #DP304-03) the metastatic cells were harvested in lysis buffer GA containing RNaseA and then聽incubated with Proteinase K. After removing the RNA and protein, the genomic DNA was purified by TIANamp Spin Column CB3 followed by聽extensive wash with buffer PW. Details can be found in: http://tiangen.com/asset/imsupload/up0875005001348194139.pdf. No randomization and blinding was used for mice experiments.OTUD1 knockout by CRISPR/Cas9sgRNA design and cloning was performed according to the Feng聽Zhang lab general cloning protocols56,57. OTUD1 sgRNAs oligos and SMAD7 sgRNA oligos were designed based on the target site sequence (20鈥塨p) and are flanked on the 3鈥?end by a 3鈥塨p NGG PAM sequence. Using the Cas9 target design tools (http://www.genome-engineering.org), we designed two sgRNAs for each target:OTUD1 sg1 forward: 5鈥?CACCGAGGAGGAGCAG CCGGGGACG-3鈥?OTUD1 sg1 reverse: 5鈥?AAACCGTCCCCGGCTG CTCCTCCTC-3鈥?OTUD1 sg2 forward: 5鈥?CACCGGTCCACAGGACGATGTGCAG-3鈥?OTUD1 sg2 reverse: 5鈥?AAACCTGCACATCGTCCTGTGGACC-3鈥?SMAD7 sg forward: 5鈥?CACCCCAAACGATCTGCGCTCGTCG-3鈥? SMAD7 sg reverse: 5鈥?AAACGACGAGCGCAGATCGTTTGGC-3鈥?The sgRNAs were cloned into the lentiCRISPRv2 vector (Addgene). For lentivirus production, cloned lentiCRISPRv2 plasmids were co-transfected into HEK293T cells with the packaging plasmids pVSVg (AddGene 8454) and psPAX2 (AddGene 12260). Lentivirus were harvested and MDA-MB-231 cells were infected with two sgRNA mixtures for OTUD1. Forty-eight hours after infection, cells were placed under puromycin selection for 1 week and the single-cell-derived clones were picked, expanded and knockout of OTUD1 was verified by IB analysis.GSEAWe used GSEA v2.0 to perform GSEA on various functional and/or characteristic gene signatures. Gene sets were obtained from the MSigDB database v3.0 (September 2010 release). Statistical significance was assessed by comparing the enrichment score to enrichment results generated from 1,000 random permutations of the gene set to obtain p values (nominal p value). Data from NKI 295, a well-annotated human breast cancer database, were analyzed for the enriched gene signature, and OTUD1-high (OTUD1鈥夆墺鈥?.1, n鈥?鈥?4) versus OTUD1-low (OTUD1鈥夆墹鈥夆垝0.1, n鈥?鈥?4) samples were compared.Primers and reagentsThe DNA primer sequences that were used to detect target gene expression by qRT-PCR are listed in Supplementary Data聽5. Doxycycline-inducible pCW-Myc-OTUD1-wt/CA, pLv-puro-Myc-OTUD1-wt/CA and pCDNA3.1-Flag-OTUD1-wt/CA constructs were cloned and verified by DNA sequencing. MG132 was purchased from Sigma. Rhodamine phalloidin was purchased from Molecular Probe, Invitrogen. The antibodies used for immunoprecipitation, immunoblotting, and immunofluorescence were the following: N-cadherin 1:5,000 (IB; Clone 32, BD Biosciences), fibronectin 1:1,000 (IB; Sigma), vimentin 1:1,000 (IB; # 5741 Cell Signaling) and E-cadherin 1:10,000 (IB; BD 610181), 1:500 (IF; BD 610181), c-Myc 1:1,000 (IB; a-14, sc-789, Santa Cruz Biotechnology), HA 1:1,000 (IB; Y-11, sc-805, Santa Cruz Biotechnology), HA 1:10,000 (IB; 12CA5, in-house), Flag 1:10,000 (IB; M2, Sigma), Ub 1:1,000 (IB; P4D1, Santa Cruz Biotechnology), OTUD1 1:1,000 (IB; Abcam 122481), SMAD7 1:1,000(IB; sc-7004;Santa Cruz, this antibody was validated to recognize only SMAD7, not SMAD6), RNF12 1:1,000 (ab57230, Abcam and M0), SMAD4 1:1,000 (IB; B8, Santa Cruz), SMAD2/3 1:2,500 (IB); 1:500 (IP; 610842 BD), phospho-SMAD2 1:5,000 (IB; # 3101, Cell Signaling); Actin 1:10,000 (A5441, Sigma).Real-time RT-PCR聽(qRT-PCR)Total RNAs were prepared using the NucleoSpin庐 RNA II kit (BIOK脡, Netherlands). A total of 1鈥壩糶 of RNA was reverse-transcribed using the RevertAid鈩?/sup> First Strand cDNA Synthesis Kit (Fermentas). Real-time PCR was conducted with SYBR Green (Applied Bioscience) using a StepOne Plus real-time PCR system (Applied Bioscience). All target gene-expression levels were normalized to GAPDH.Immunohistochemical staining and evaluationPrimary antibodies to OTUD1 (1:50; Abcam 122481), SMAD7 (Specificity were tested to read endogenous SMAD7 at 1:200; sc-7004, Santa Cruz Biotechnology) were used for immunohistochemical staining of formalin-fixed paraffin-embedded microarrays of breast cancer tissues (obtained from US Biomax (BC081116c)), according to previously described staining protocols58. The quantification of staining was expressed as an H score. The H score was determined by the formula 3鈥壝椻€塼he percentage of strongly staining cells鈥?鈥?鈥壝椻€塼he percentage of moderately staining cells鈥?鈥塼he percentage of weakly staining cells, yielding a range of 0 to 300. Scoring was performed blindly by three independent investigators.Nickel pulldown assayCells were transfected with His-Ub plasmid and followed by nickel bead purification and immunoblot analysis59. For His-SMAD7 purification, the washed cell pellet was resuspended in lysis buffer (6鈥塎 guanidine-HCl, 0.1鈥塎 Na2HPO4/NaH2PO4, and 10鈥塵M imidazole). After sonication and a freeze-thaw step, the supernatant of the cell lysate was incubated with Talon beads (BD Biosciences) in the presence of 20鈥塵M imidazole. Beads were washed four times with lysis buffer lacking imidazole. Purified proteins were eluted in lysis buffer containing 200鈥塵M imidazole.Protein purificationSMAD7 protein and RNF12 proteins were generated in E. coli as His-tagged and GST fusion protein, respectively, as previously described13. For purification of OTUD1-wt/CA protein from mammalian cells, OTUD1-wt/CA expression plasmids were transfected in HEK293T cells and immunoprecipitated overnight with 伪-Flag-M2 resin (Sigma), followed by elution with Flag peptide (Sigma, 1鈥塵g/ml in 50鈥塵M HEPES (pH 7.5), 100鈥塵M NaCl, 0.1% NP40, 5% glycerol).In vitro binding assayFor in vitro protein鈥損rotein interactions, purified and/or recombinant proteins were diluted in binding buffer (25鈥塵M HEPES [pH 7.5], 100鈥塵M KCl, 2鈥塵M MgCl2, 0.1% NP40, 5% glycerol), immunoprecipitated with anti-OTUD1 or anti-SMAD7 for 3鈥塰 and protein A-Sepharose beads for 1鈥塰, and washed three times with the same buffer. Lysates were then analyzed by IB for coprecipitating proteins.In vitro deubiquitination of SMAD7In vitro ubiquitin assays were carried out by incubating ubiquitin-activating enzyme E1 (Ube1 40鈥塶M), Ubiquitin (8鈥壜礛), UbcH6 (0.7鈥壜礛), 500 ng His-SMAD7, 500 ng GST-RNF12, and 500 ng OTUD1-wt/CA proteins at 37 掳C for 3鈥塰 in reaction buffer, 50鈥塵M Tris-HCl (pH 7.5), 5鈥塵M MgCl2, 1鈥塵M DTT, 2鈥塵M ATP with proteases inhibitor. 9鈥壜祃 of the 10鈥壜祃 reaction system were diluted with RIPA buffer followed by immunoprecipitation and anti-Ubiquitin immunoblotting. 1鈥壜祃 of the 10鈥壜祃 reaction system was loaded as input control. In Fig.聽4f, Poly-HA-ubiquitinated Flag-SMAD7 substrate was prepared by transiently cotransfecting HEK293T cells with Flag-SMAD7 and HA-ubiquitin. Thirty-six h later, cells were treated with MG132 (5鈥壩糓) for 4鈥塰 and then the Poly-HA-ubiquitinated Flag-SMAD7 was purified in denatured conditions. To assay the ability of OTUD1 protein to deubiquitinate SMAD7 in vitro, purified OTUD1 protein was incubated with purified Poly-HA-ubiquitinated Flag-SMAD7 at 37 掳C for indicated time. Reaction was terminated by SDS sample buffer followed by a 2鈥塵in heat denaturation at 95 掳C. Reaction products were detected by IB analysis. For the experiment shown in Fig.聽4g, Poly-His-ubiquitinated Flag-SMAD7 was purified by nickel beads pulldown.In vivo ubiquitination assayAs previously described60, HEK293T cells were transfected with indicated constructs and treated with or without MG132 (5鈥壩糓) for 4鈥塰 before harvesting. 48鈥塰 after transfection, cells were washed with PBS and lysed in two pellet volumes of RIPA buffer (20鈥塵M NAP, pH 7.4, 150鈥塵M NaCl, 1% Triton, 0.5% Sodium-deoxycholate, and 1% SDS) supplemented with protease inhibitors and 10 mM N-Ethylmaleimide (NEM). Lysates were sonicated, boiled at 95掳C for 5鈥塵in, diluted with RIPA buffer containing 0.1% SDS, then centrifuged at 4掳C (16鈥壝椻€?03脳g for 15鈥塵in). The supernatant was incubated with specific antibody and protein A-Sepharose for 3鈥塰 at 4鈥壜癈. After extensive washing, bound proteins were eluted with 2脳SDS sample buffer and separated on a SDS-PAGE, followed by IB analysis.Tumor sphere assaysAs described61, single-cell suspensions of MCF10A (RAS) cells (1鈥壝椻€?03 cells/ml) were plated on ultra-low attachment plates and cultured in phenol red-free DMEM/F12 (Gibco Paisley, UK; 21041) containing B27 supplement (no vitamin A; Invitrogen, Paisley, UK; 12587) and rEGF (20 ng/ml; Sigma Aldrich Poole, UK; E-9644). Tumor spheres were visualized using a phase contrast microscope, photographed, and counted.3D matrigel assaysCells were cultured in growth-factor-reduced reconstituted basement membrane (Matrigel, Corning) as described previously62. Images were taken with an inverted microscope.Mice metastasis modelsMice experiments were approved by the Committee for Animal Welfare in Zhejiang University. Mice were purchased from animal husbandry center of the Shanghai Institute of Cell Biology, Academia Sinica, Shanghai, China. For the intracardial injection assay, five-weeks-old female BALB/c nude were anesthetized with isofluorane and single-cell suspension of MDA-MB-231/luc cells (1鈥壝椻€?05/100鈥壩糽 PBS) were inoculated into the left heart ventricle according to the method described by Arguello et al55. Bioluminescent imaging was used to verify successful injection and to monitor the metastatic outgrowth. After 6 weeks, all mice were killed and metastatic lesions were confirmed by histological analysis. For the doxycycline-inducible metastatic reactivation, MDA-MB-231 cells were infected with pCW-Myc-OTUD1-wt or CA mutant and intracardial inoculated into 5-weeks-old female BALB/c nude mice. Doxycycline was administered to mice at day 21 after injection of the cancer cells, through the diet (625鈥塵g/kg of food) as well as by intraperitoneal injection (25鈥塵g/kg of body weight; three times a week). For tail vein injection, single-cell suspensions of 4T1-Luc or 4T07-Luc cells (1鈥壝椻€?05/100鈥壩糽 PBS) cells were injected into the tail vein of five-weeks-old nude mice or female syngeneic BALB/c mice. Development of metastasis was monitored weekly by bioluminescent reporter imaging. After 6 weeks, all mice were killed and lung metastases were dissected. Mouse nipple implantation was based on a previously published method63. Female BALB/c mice were anesthetized and used for this assay. Overall, 1鈥壝椻€?05 4T1-Luc cells were injected through the nipple area into the mammary fat pad. At 21 days after injection, luciferin was injected and the primary tumors were analyzed, then the mice were euthanized and analyzed for acquisition of secondary tumor(s). All primary/metastatic tumors were detected by BLI with the IVIS 100 (Caliper Life Sciences, Hopkinton, MA, USA). The BLI signal intensity was quantified as the sum of photons within a region of interest given as the total flux (photons/s). No randomization and blinding was used for mice experiments.Pulse-chaseAs previously described13, cells were plated in 6-well plates, starved for 3鈥塰 in Met/Cys-free medium and pulsed for 40鈥塵in with 200鈥壩糃i/ml of 35S labeled Met/Cys. After two washes, the cells were chased in medium supplemented with cold (unlabeled) Met and Cys (100鈥壩糶/ml) before harvesting. Endogenous proteins were collected from the extracts by immunoprecipitation, which were thereafter resolved by SDS-PAGE and visualized by autoradiography. Each experiment was performed in duplicate.Mass spectrometryAs previously described64, SDS-PAGE gels were minimally stained with Coomassie brilliant blue, cut into six molecular weight ranges based on heavy chain IgG bands, and digested with trypsin. Immunocomplexes were identified on a Thermo Fisher LTQ (majority) or Velos-Orbitrap mass spectrometer. Spectral data were then searched against the human protein RefSeq database in BioWorks or the Proteome Discoverer Suites using either SeQuest (for LTQ data) or Mascot (Orbitrap data) software. The IP/MS results were transferred into a FileMaker-based relational database generated in-house, where protein identification numbers (protein GIs) were converted to Gene ID identifiers according to the NCBI 鈥済ene accession鈥?table.Structural modelingThe NMR structure of WW domain binding to PY loop (residue 203鈥?17) was obtained from Protein Data Bank (PDB ID: 2DJY). 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Cancer Discov. 2, 401鈥?04 (2012).Article聽 PubMed聽Google Scholar聽 Download referencesAcknowledgementsWe are grateful to Martijn Rabelink, Erik Meulmeester, Alfred Vertegaal, Joost Schimmel, and David Baker for providing reagents and for valuable discussions. We thank A.G. Jochemsen for early passage MDA-MB-231 cells. We thank Midory Thorikay for expert technical assistance. This work was supported by a special program from Ministry of Science and Technology of China (2016YFA0502500 to L.Z.), the Chinese National Natural Science Funds (31571460 to F.Z., 31471315 and 31671457 to L.Z.), PCSIRT (IRT1075 to X.G.), Jiangsu National Science Foundation (BK20150354 to F.Z.), the key R D projects of Zhejiang province (2015C03045)聽and Zhejiang outstanding youth fund (LR14C070001 to L.Z. and聽LR15C060001 to J.J.).Author informationAuthor notesZhengkui Zhang, Yao Fan and Feng Xie contributed equally to this work.AffiliationsLife Sciences Institute and Innovation Center for Cell Signalling Network, Zhejiang University, Hangzhou, 310058, Zhejiang, ChinaZhengkui Zhang,聽Yao Fan,聽Hang Zhou,聽Ke Jin,聽Pengfei Fang,聽Junling Jia,聽Jin Jin,聽Sheng Ye聽 聽Long ZhangInstitutes of Biology and Medical Science, Soochow University, 215123, Suzhou, ChinaFeng Xie聽 聽Fangfang ZhouState Key Laboratory for Diagnostic and Treatment of Infectious Diseases, The First Affiliated Hospital, School of Medicine, Zhejiang University, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Disease, 310000, Hangzhou, ChinaLi Shao聽 聽Min ZhengState Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, 100039, Beijing, ChinaWenhao Shi聽 聽Chen DingNational Center for Protein Sciences (The PHOENIX Center, Beijing), 102206, Beijing, ChinaWenhao Shi聽 聽Chen DingState Key Laboratory of Genetic Engineering and Collaborative Innovation Center for Genetics and Development, School of Life Sciences, Institute of Biomedical Sciences, Fudan University, 200433, Shanghai, ChinaWenhao Shi聽 聽Chen DingDepartment of Pharmaceutical Chemistry and the Cardiovascular Research Institute, University of California San Francisco, San Francisco, CA, 94158, USABing YangDepartment of Molecular Cell Biology and Cancer Genomics Centre Netherlands, Leiden University Medical Center, Postbus 9600, 2300 RC, Leiden, The NetherlandsHans van Dam聽 聽Peter ten DijkeDepartment of Cancer Biology, Metastasis Research Center, University of Texas MD Anderson Cancer Center, Houston, TX, 77054, USAXiaofeng ZhengTsinghua University, 100084, Beijing, ChinaXiaohua YanAuthorsZhengkui ZhangView author publicationsYou can also search for this author in PubMed聽Google ScholarYao FanView author publicationsYou can also search for this author in PubMed聽Google ScholarFeng XieView author publicationsYou can also search for this author in PubMed聽Google ScholarHang ZhouView author publicationsYou can also search for this author in PubMed聽Google ScholarKe JinView author publicationsYou can also search for this author in PubMed聽Google ScholarLi ShaoView author publicationsYou can also search for this author in PubMed聽Google ScholarWenhao ShiView author publicationsYou can also search for this author in PubMed聽Google ScholarPengfei FangView author publicationsYou can also search for this author in PubMed聽Google ScholarBing YangView author publicationsYou can also search for this author in PubMed聽Google ScholarHans van DamView author publicationsYou can also search for this author in PubMed聽Google ScholarPeter ten DijkeView author publicationsYou can also search for this author in PubMed聽Google ScholarXiaofeng ZhengView author publicationsYou can also search for this author in PubMed聽Google ScholarXiaohua YanView author publicationsYou can also search for this author in PubMed聽Google ScholarJunling JiaView author publicationsYou can also search for this author in PubMed聽Google ScholarMin ZhengView author publicationsYou can also search for this author in PubMed聽Google ScholarJin JinView author publicationsYou can also search for this author in PubMed聽Google ScholarChen DingView author publicationsYou can also search for this author in PubMed聽Google ScholarSheng YeView author publicationsYou can also search for this author in PubMed聽Google ScholarFangfang ZhouView author publicationsYou can also search for this author in PubMed聽Google ScholarLong ZhangView author publicationsYou can also search for this author in PubMed聽Google ScholarContributionsZ.Z.,聽F.X., Y.F., K.J., L.S., W.S., H.v.D., P.F, B.Y. and X.Z. performed the experiments; H.Z. and S.Y. performed the structure analysis, Z.Z.,聽H.v.D., X.Y., M.Z., J.Jia, J.Jin, and C.D. created the figures; F.Z., P.t.D. and L.Z. wrote the paper.Corresponding authorCorrespondence to Long Zhang.Ethics declarations Competing interests The authors declare no competing financial interests. 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