an atm/trim37/nemo axis counteracts genotoxicity by ...molecular cell biology an atm/trim37/nemo...

Molecular Cell Biology

An ATM/TRIM37/NEMO Axis CounteractsGenotoxicity by Activating Nuclear-to-Cytoplasmic NF-kB SignalingGeyan Wu1,2, Libing Song3, Jinrong Zhu1,2, Yameng Hu1,2, Lixue Cao1,2,Zhanyao Tan1,2, Shuxia Zhang1,4, Ziwen Li1,2, and Jun Li1,2

Abstract

Blocking genotoxic stress-induced NF-kB activationwould substantially enhance the anticancer efficiency ofgenotoxic chemotherapy. Unlike the well-established classicalNF-kB pathway, the genotoxic agents-induced "nuclear-to-cytoplasmic" NF-kB pathway is initiated from the nucleusand transferred to the cytoplasm. However, the mechanismlinking nuclear DNA damage signaling to cytoplasmic IKKactivation remains unclear. Here, we report that TRIM37, anovel E3 ligase, plays a vital role in genotoxic activation ofNF-kB via monoubiquitination of NEMO at K309 in thenucleus, consequently resulting in nuclear export of NEMOand IKK/NF-kBactivation.Clinically, TRIM37 levels correlatedpositively with levels of activated NF-kB and expression ofBcl-xl and XIAP in esophageal cancer specimens, which alsoassociated positively with clinical stage and tumor-node-metastasis classification and associated inversely with overalland relapse-free survival in patients with esophageal cancer.Overexpression of TRIM37 conferred resistance to the DNA-

damaging anticancer drug cisplatin in vitro and in vivothrough activation of the NF-kB pathway. Genotoxicstress-activated ATM kinase directly interacted with andphosphorylated TRIM37 in the cytoplasm, which inducedtranslocation of TRIM37 into the nucleus, where it formed acomplex with NEMO and TRAF6 via a TRAF6-binding motif(TBM). Importantly, blocking the ATM/TRIM37/NEMO axisvia cell-penetrating TAT-TBM peptide abrogated genotoxicagent-induced NEMO monoubiquitination and NF-kBactivity, resulting in hypersensitivity of cancer cells to gen-otoxic drugs. Collectively, our results unveil a pivotal rolefor TRIM37 in genotoxic stress and shed light on mechan-isms of inducible chemotherapy resistance in cancer.

Significance: In response to genotoxic stress, TRIM37 acti-vates NF-kB signaling via monoubiquitination of NEMO,which subsequently promotes cisplatin chemoresistance andtumor relapse in cancer.CancerRes; 78(22); 6399–412.�2018AACR.

IntroductionChromosomal integrity of all living organisms is endlessly

jeopardized by genotoxic stress generated from endogenous met-abolic sources, such as reactive oxygen species (ROS), and envi-ronmental resources, such as ionizing radiation and genotoxicchemicals (1–4). Unrepaired or inappropriately repaired DNAdamage consequently attribute to genetic variation, apoptosis,aging, degenerative diseases, inflammation, and cancer (2–5).

Meanwhile, cells have evolved a precisely controlled network ofDNA damage response (DDR) to respond to genotoxic stresses,includes sensing of damaged DNA, activation of cell cycle check-points, assembly of DNA repair machineries, and transactivationof DNA damage-responsive gene expression. For instance, ATMkinase and PARP-1 act as sensor proteins to detect DNA lesionsand modify a variety of proteins, which initiate DNA repair andcell-cycle checkpoint control (6, 7).

On the other hand, the NF-kB signaling pathway was emergedas a vital mediator for cellular responses to genotoxic threats viainducing survival genes that allow cells to repair damaged DNAand promote survival (8, 9). However, unlike thewell-establishedclassical NF-kB pathway in which signals are initiated fromcell surface receptors and transduced from the cytoplasm tothe nucleus (10, 11), genotoxic agents triggered the "nuclear-to-cytoplasmic" NF-kB pathway that is initiated from the nucleusand transferred to the cytoplasm (8, 9). In response to DNAdamage signals, PARP-1 is rapidly recruited to DNA damage sitesand induces auto-poly(ADP-ribosyl)ation (PARylation), whichassembles NEMO, PIASy, ATM, PIDD, and LRP16 into a nucle-oplasmic signalosome (12–16). Furthermore, signalosome for-mation induces sumoylation of NEMO at K277/K309 in a PIDD/PARP1/PIASy-dependent manner and ATM-dependent phos-phorylation of NEMO at S85 (12–17). Then phosphorylated-NEMO is monoubiquitinated and exported from the nucleustogether with ATM and ELKS, which form a complex with IKKcatalytic subunits in the cytoplasm, consequently resulting in

1Key Laboratory of Liver Disease of Guangdong Province, The Third AffiliatedHospital, Sun Yat-sen University, Guangzhou, P.R. China. 2Department of bio-chemistry, Zhongshan school of medicine, Sun Yat-sen University, Guangzhou,P.R. China. 3State Key Laboratory of Oncology in South China, CollaborativeInnovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center,Guangzhou, P.R. China. 4Key Laboratory of Protein Modification and Degrada-tion, School of Basic Medical Sciences, Affiliated Cancer Hospital &Institute ofGuangzhou Medical University, Guangzhou Medical University, Guangzhou, P.R.China.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

G. Wu, L. Song, and J. Zhu contributed equally to this article.

Corresponding Author: Jun Li, Sun Yat-sen University, 74 Zhongshan Road II,Guangzhou, Guangdong 510080, P.R. China; Phone: 86-20-87335828;Fax: 86-20-87335828; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-18-2063

�2018 American Association for Cancer Research.

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activation of IKK/NF-kB signaling (18). Therefore, nuclearmono-ubiquitin of NEMO,which is essential for its nuclear export, is thekey step in genotoxic agent-triggered "nuclear-to-cytoplasmic"NF-kB signaling. However, the E3 ligase responsible for NEMOnuclear monoubiquitination remains unknown.

Tripartite motif containing 37 (TRIM37) is a newly identifiedE3 ubiquitin ligase that comprises a tripartite motif (TRIM,RING-B-box-coiled-coil) domain, TRAF domain (TD), andpolyacidic domain (19–21). It has been recently reported thatTRIM37 plays vital roles in various biological processesdepending on TRIM domain-dependent E3 ligase activity, suchas promotion of peroxisomal matrix protein import via directmonoubiquitination of PEX5 at K464 and silencing of geneexpression through monoubiquitination of histone H2A (22,23). In this study, we unveiled a novel function of TRIM37 inregulating nuclear-to-cytoplasmic NF-kB signaling. We foundthat, upon genotoxic stimulation, TRIM37 rapidly translocatedinto the nucleus where it interacted directly with TRAF6 tocatalyze monoubiquitination of NEMO at K309. BlockingTRIM37/TRAF6 interaction using a cell-penetrating TAT-TDpeptide abrogated NEMO monoubiquitination-dependentNF-kB signaling, resulting in hypersensitivity of esophagealcancer cells to DNA-damaging chemotherapeutics. Hence ourstudy reveals a crucial role of TRIM37 in genotoxic stress-induced NF-kB activation and sheds light on mechanisms ofinducible chemotherapy resistance in esophageal cancer.

Materials and MethodsEthics statement

Informed consent was signed by all patients, and the investi-gation has been conducted in accordance with the ethical stan-dards according to the Declaration of Helsinki, national andinternational guidelines, which has also been approved by theauthors' Institutional Review Board.

Tissue specimens and patient informationAll of the patients received standardized platinum-based che-

motherapy. Informed consent was obtained from all patients andapprovals from Sun Yat-sen University Cancer Center Institution-al Research Ethics Committee were obtained for this study. A totalof 441 paraffin-embedded, archived esophageal cancer specimensand freshly collected 1 esophageal cancer-adjacent normal tissueand 9 esophageal cancer tissues were histopathologically andclinically diagnosed at Sun Yat-sen University Cancer Center(Guangdong, China) between 2005 and 2016. The clinical infor-mation of the samples is shown in Supplementary Tables S1 to S3.Detailed description provided in Supplementary methods.

CellsPrimary cultures of normal esophageal epithelial cells were

established from fresh specimens of the adjacent noncancer-ous esophageal tissue, which was over 5 cm from the canceroustissue, according previously report (24). The Eca109 cells werekindly provided by Professors Tsao SW (The University ofHong Kong) and grown in DMEM medium (Invitrogen) sup-plemented with 10% FBS (HyClone). All the cell lines beentested for Mycoplasma contamination. All cell lines wereauthenticated by short tandem repeat (STR) fingerprinting atMedicine Lab of Forensic Medicine Department of Sun Yat-SenUniversity.

Plasmids, virus constructs, and retroviral infection of targetcells

Human TRIM37, NEMO, and TRAF6 were amplified by PCRand cloned into the pSin-EF2 vector. Fragments of the humanTRIM37 and TRAF6-coding sequence were amplified by PCRand cloned into the pSin-EF2 vector. The indicated mutantswere created using primers and a Stratagene Mutagenesis Kitaccording to the protocol recommended by the manufacturer.pNF-kB-luc and control plasmids (Clontech) was used toquantitatively examine NF-kB activity. Transfection of siRNAsor plasmids was performed using the Lipofectamine 3000reagent (Invitrogen) according to the manufacturer's instruc-tion. Retroviral production and infection were performed asdescribed previously (25). Stable cell lines expressing indicatedgenes were selected for 10 days with 0.5 mg/mL puromycin48 hours after infection. The primers used were listed inSupplementary Table S4.

ImmunohistochemistryIHC analysis was performed to study altered protein expression

in441humanesophageal cancer tissues accordingprevious report(26). Paraffin-embedded tissues were analyzed using IHC withanti-TRIM37 antibody (Abcam; 1:200), anti-NF-kB p65 antibody(Abcam; 1:200), anti-Bcl-XL antibody (Cell Signaling; 1:100),anti-XIAP (1007-1008) antibody (Proteintech; 1:100). All theantibodies used in this study has been listed in SupplementaryTable S5. The degree of immunostaining of formalin-fixed, par-affin-embedded sections were reviewed and scored separately bytwo independent pathologists uninformed of the histopathologicfeatures and patient data of the samples. The scores were deter-mined by combining the proportion of positively-stained tumorcells and the intensity of staining. The scores given by the twoindependent pathologists were combined into a mean score forfurther comparative evaluation. Tumor cell proportions werescored as follows: 0, no positive tumor cells; 1, <10% positivetumor cells; 2, 10% to 35% positive tumor cells; 3, 35% to 75%positive tumor cells; 4, >75% positive tumor cells. Stainingintensity was graded according to the following standard: 1, nostaining; 2, weak staining (light yellow); 3, moderate staining(yellow brown); 4, strong staining (brown). The staining index(SI) was calculated as the product of the staining intensity scoreand the proportion of positive tumor cells. Using this method ofassessment, we evaluated protein expression in benign esophage-al epithelia and malignant lesions by determining the SI, withpossible scores of 0, 2, 3, 4, 6, 8, 9, 12, and 16. Samples with an SI� 8 were determined as high expression and samples with an SI <8 were determined as low expression. Cutoff values were deter-mined on the basis of a measure of heterogeneity using the log-rank test with respect to OS.

Coimmunoprecipitation assayCells grown in 100-mm culture dishes were lysed using 500 mL

of lysis buffer [25 mmol/L HEPES (pH 7.4), 150 mmol/L NaCl,1% NP-40, 1 mmol/L EDTA, 2% glycerol, 1 mmol/L phenyl-methylsulfonyl fluoride (PMSF)]. After being maintained on icefor 30minutes, the lysateswere clarified bymicrocentrifugation at12,000 rpm for 10 minutes. To preclear the supernatants, thelysates were incubated with 20 mL of agarose beads (Calbiochem)for 1 hours with rotation at 4�C. After centrifugation at 2,000 rpmfor 1 minutes, the supernatants were incubated with 20 mL ofantibody-cross-linked protein G-agarose beads overnight at 4�C.

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The agarose beads were then washed six times with wash buffer[25 mmol/L HEPES (pH 7.4), 150 mmol/L NaCl, 0.5% NP-40,1 mmol/L EDTA, 2% glycerol, 1 mmol/L PMSF]. After removingall the liquid, the pelleted beadswere resuspended in 30 mL of 1Mglycine (pH 3), after which, 10 mL of 4� sample buffer was added,the samples were denatured, and the sample components wereelectrophoretically separated on SDS-PAGE for immunoblotanalysis.

Stochastic optical reconstruction microscopyEca109 cells seeded on No. 118 mm round coverslips were

washed twice with PBS, fixed with 4% PFA in PBS for 1 hour,permeabilized with the PBST for 10 minutes, and blocked with4% BSA in PBS for 1 hour. The cells were then incubated withprimary antibodies for 3 hours and with Alexa Fluor 647 conju-gated goat anti-rabbit IgG (1:200 dilution) and pre-adsorbedAlexa Fluor 568 conjugated goat anti-mouse IgG (1:200 dilution;Abcam, ab175733) antibodies for 1.5 hours. The sample was keptin PBS until imaging.

Generation and preparation of TAT-37/TBM peptidesPeptides were synthesized by the ChinaPeptides using standard

HOBt/Fmoc chemistry and purified by reverse phase HPLC to>95% purity. The final amino acid compositions were verifiedutilizing amino acid analysis andMALDI TOFmass spectrometry.The cell permeation sequence used was previously demonstratedto have 10-fold increased intracellular concentration comparedwith the native TAT sequence from HIV-1 (27). We designed anegative control peptide by changing DFEVGE to AFAVGA. Theamino acid sequences of these peptides are: TAT-Ctrl:RKKRRORNRRRAFAVGA; TAT-TBM: RKKRRORNRRRDFEVGE.

Xenografted tumor models, IHC, and H&E stainingIn the subcutaneous patient-derived xenografts (PDX) tumor

model, freshly isolated clinical esophageal cancer patienttissues were subdivided into 2 to 3 mm3 pieces, coated withMatrigel (BD Biosciences) and media in a 1:1 ratio, andembedded within the subcutaneous space underneath the skinof female NOD/Shi-scid/IL-2Rgnull (NOG) mouse (6–8 weeksold; CREA Japan Inc.). Tumor growth was monitored by mea-suring the tumor diameters, and the tumor volume was calcu-lated using the equation (L � W2)/2. When the tumor becamepalpable, mice were intratumoral treated with cisplatin (CDDP;5 mg/kg) and intratumoral injection of TAT-control peptide orTAT-TRIM37/TBM peptide three times per week (as per cycle)for up to 6 weeks. At the end of treatment, the mice weresacrificed and the tumors were excised and weighed, andconfirmed by histology.

In the subcutaneous tumor model, the indicated luciferaseexpressing cells (1� 106) were injected subcutaneously into nudemice. When the luminescence signal reached 2 � 107 p/sec/cm2/sr, mice were intratumoral treated with CDDP (5 mg/kg) threetimes per week (as per cycle) for up to 6 weeks. Mice weresacrificed when moribund as determined by an observerblinded to the treatment, and tumors were excised and paraffin-embedded. Serial 4.0 mm sections were cut and subjected to IHCand hemotoxylin and eosin (H&E) staining. After deparaffiniza-tion, sections were H&E-stained with Mayer's hematoxylin solu-tion, or IHC-stained using antibodies of NF-kB p65 (1:100;Abcam), or stained with TUNEL (In Situ Cell Death DetectionKit, TMR red; Roche Applied Science), and counterstained with

phalloidin (Alexa Fluor 488; Invitrogen) and DAPI (Sigma-Aldrich) according to manufacturer's protocols. The images werecaptured using the AxioVision Rel.4.6 computerized image anal-ysis system (Carl Zeiss). Themice used in this study were sacrificedwhen the volume of control tumors reached to 1.5-cmdiameter orthe mice become moribund. All of the animal procedures wereapproved by the Sun Yat-sen University Animal Care Committee.

Statistical analysisAll statistical analyses were carried out using SPSS130.0 statis-

tical software. A chi-squared test was used to analyze the rela-tionship between TRIM37 expression and the clinicopathologiccharacteristics. Survival curves were plotted using Kaplan–Meiermethod and compared by log-rank test. Survival data were eval-uated by univariate and multivariate Cox regression analyses. P <0.05 was considered statistically significant.

ResultsTRIM37 promotes genotoxic stress-induced NF-kB activation

To identify potential nuclear E3 enzyme for NEMO monou-biquitination, affinity purification and mass spectrometry (MS)was conducted using nuclear extracts from etoposide-treatedEca-109 esophageal cancer cells. In addition to previouslyreported interacting proteins, such as TRAF6, ATM, and PIASy(12–17), we found that E3 ligase TRIM37 may also be a potentnuclear NEMO-interacting protein (Fig. 1A). Prominently, com-pared with control cells, etoposide-induced NF-kB activation wasrapidly elevated in TRIM37-overexpressing cells but decreased inTRIM37�/� cells (Fig. 1B and C), suggesting that TRIM37 played avital role in etoposide-induced NF-kB activation. This hypothesiswas further confirmed by multiple assays, in which overexpres-sion of TRIM37 significantly increased, but knockout or knock-down of TRIM37 decreased the IKK activity, the expression of NF-kB-regulated antiapoptotic genes Bcl-XL and XIAP, and theNF-kBtranscriptional activity in the etoposide-treated cells (Fig. 1C;Supplementary Fig. S1A and S1B). Importantly, the elevatedNF-kB activity induced by irradiation, camptothecin, or CDDPwas also drastically decreased in TRIM37�/� cells (SupplementaryFig. S1C and S1D), demonstrating that TRIM37 contributed togenotoxic stress-induced NF-kB activation.

Clinical relevance of TRIM37/NF-kB signaling in humanesophageal cancer

Statistical analyses revealed that TRIM37 levels were positivelycorrelated with level of activated NF-kB and expression of Bcl-xland XIAP in 441 paraffin-embedded and nine freshly collectedesophageal cancer specimens (Fig. 1D and E). IHC stainingshowed that TRIM37 protein was slightly expressed in normalesophageal tissues but showed markedly higher expression inesophageal cancer and was further elevated in relapsed esoph-ageal cancer (Fig. 1F). Furthermore, we found that TRIM37expression was positively associated with the clinical stage (P ¼0.001), tumor-node-metastasis classification (P ¼ 0.026; P ¼0.004; P ¼ 0.039) and inversely associated with overall andrelapse-free survival in patients with esophageal cancer (Supple-mentary Table S1–S3 and Fig. 1G). Importantly, Kaplan–Meierplotter analysis revealed that expression of TRIM37 were signif-icant correlated with shorter overall and relapse-free survival inpatients with breast cancer, ovarian cancer, or liver cancer (Sup-plementary Fig. S1E). These results indicate that TRIM37

TRIM37 Activates NF-kB Signaling via NEMO Monoubiquitination

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Figure 1.

TRIM37 promotes genotoxic stress-induced NF-kB activation. A, Immunoprecipitation assay was performed in nuclear extracts from etoposide (Etop; 10 mmol/L,2 hours)-treated NE1/Flag-NEMO cells using anti-Flag affinity agarose, followed by mass-spectrometric peptide sequencing. E3 ligase TRIM37 was identifiedas one of the proteins present in the precipitate.B andC,NF-kBDNA-binding activity by EMSA and IKK kinase activity by in vitro kinase activity assaywere examinedin NE1 cells transfected with 0, 0.5, 1.5, and 5.0 mg of a Flag-tagged TRIM37 plasmid (B) or in Eca-109/TRIM37�/� cells, followed by the treatment with etoposide (10mmol/L, 2 hours) or TNFa (10 ng/mL, 15 minutes; C).D,Analysis of expression (left) and correlation (right) of TRIM37with Bcl-XL and XIAPmRNA expression, as wellas NF-kBDNA-binding activity in one freshly collected esophageal cancer-adjacent sample and nine esophageal cancer samples. Each bar represents themean� SDof three independent experiments. E, TRIM37 levels associated with nuclear NF-kB p65, Bcl-XL, or XIAP expression in 441 primary human esophageal cancerspecimens. Left, two representative specimens with low and high levels of TRIM37 expression are shown. Original magnification, �200. Right, percentages ofspecimens showing low or high TRIM37 expression relative to the level of nuclear NF-kB p65, Bcl-XL, or XIAP. Scale bars, 50 mm. F, IHC staining of TRIM37 in normalesophageal tissue, nonrelapsed, and relapsed esophageal cancer tissues. Scale bars, 50 mm. G, Kaplan–Meier curves of overall survival (left) and relapse-freesurvival (right) of patients with esophageal cancer with low versus high expression of TRIM37 (n ¼ 441; P < 0.001, log-rank test).

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overexpression may contribute to development and progressionof multiple types of human cancer.

TRIM37 confers resistance to CDDP in vitro and in vivoNF-kB activation usually induces various antiapoptotic genes

that allow cells to survive in the presence of DNA-damaging drugs(8, 9). We therefore examined the effect of TRIM37 on resistanceto CDDP, a DNA-damaging drug that is commonly used inanticancer therapies. As shown in Fig. 2A and B, overexpressingTRIM37 decreased the CDDP-induced apoptotic death andincreased the colony formation. The same results were alsoobtained using an in vivo tumor model in which TRIM37/tumors

exhibited remarkable resistance to CDDP therapy, as indicated byrapid tumor progression, lower proportion of apoptotic cells, andincreased NF-kB activation, consequently resulting in shortersurvival of tumor-bearing mice (Fig. 2C–F). In contrast, CDDPtreatment led to significant remission of TRIM37�/�/tumors(Fig. 2C–F). These results demonstrate that TRIM37 overexpres-sion confers resistance to CDDP via NF-kB activation.

TRIM37 induces NEMO monoubiquitination at residue K309Although we observed no alterations of etoposide-induced

sumoylation and phosphorylation of NEMO between TRIM37-dysregulated and control cells, the expression of etoposide-induced

Figure 2.

TRIM37 overexpression confers esophageal cancer cells resistance to CDDP in vitro and in vivo.A, FACS analysis of Annexin V staining (left) and quantification (right)of indicated cells treated with vehicle or CDDP (5 mmol/L) at 24 hours. B, Representative images (left) and quantification (right) of colony number of theindicated cells. C, Xenograft model in nude mice. Representative images of tumor-bearing mice (left) and tumor volumes were examined on the indicated days(right; n ¼ 6/group). D, EMSA assay of NF-kB activity in the indicated tumors (n ¼ 6/group). OCT-1/DNA-binding complex served as a control. a-Tubulinwas used as a loading control. E, H&E and IHC staining of nuclear NF-kB p65 and TUNEL-positive cells in the indicated tumors (n ¼ 6/group), Scale bars, 50 mm.F, Kaplan–Meier survival of the indicated mice (n ¼ 6/group). Each bar in A, B, and E represents the mean � SD of three independent experiments.� , P < 0.05; �� , P < 0.01; �� , P < 0.001.






Figure 3.

TRIM37 promotes NEMOmonoubiquitination at residue K309. A, The whole cell extracts prepared from the indicated cells treated with etoposide (Etop; 10 mmol/L,2 hours), then immunoblot analysis of expression of p-ATM, total ATM, sumo-NEMO, p-NEMO (S85), immunoprecipitated NEMO, and ubiquitinated NEMO.a-Tubulin was used as a loading control. B, Immunoblot analysis of expression of immunoprecipitated NEMO, IKKb, cIAP1, TAK1, ATM, and PIASy in vector- andmyc-TRIM37–transfected cells, followed by treatment with etoposide (10 mmol/L, 2 hours). (Continued on the following page.)

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monoubiquitinated NEMO was rapidly increased in TRIM37-transduced cells but decreased in TRIM37�/� cells (Fig. 3A;Supplementary Fig. S2A). The promotive effect of TRIM37 onetoposide-induced NEMO monoubiquitination was drasticallyabrogated by silencing of ATM or PIASy but remained in IKKb-,cIAP1-, or TAK1-silenced cells (Fig. 3B). These results suggest thatTRIM37 may participate in NEMO monoubiquitination.

MS analysis revealed that Lys309 was the TRIM37-monoubi-quitinated residue (Fig. 3C). Accordingly, overexpressing TRIM37in the etoposide-treated NE1/NEMO�/� cells had no impact onthe monoubiquitinated level of Lys309 NEMO mutant (K309A)but significantly increased the monoubiquitinated level ofNEMO/WT and other NEMO lysine mutants (K277A, K285A,andK399A; Fig. 3D).Meanwhile, TRIM37overexpression inNE1/NEMO�/� cells dramatically enhanced the recovery effect ofNEMO/WT, but not NEMO/K309A, on the genotoxic stress-inducedDNA-binding and transcriptional activities of NF-kB andIKK activity (Fig. 3E and F; Supplementary Fig. S2B–S2D). Theseresults demonstrate that TRIM37-mediated NEMOmonoubiqui-tination at K309 is vital for genotoxic NF-kB activation.

TRIM37 promotes nuclear export of NEMOIn agreement with previous findings that monoubiquitination

of NEMO is crucial for its nuclear export (15), we found thatoverexpressing TRIM37 dramatically decreased the nuclearexpression of NEMO/WT but had no impact on nuclear level ofNEMO/K309A in etoposide-treated cells (Fig 3G), suggesting thatTRIM37 promoted nuclear export of NEMO. This hypothesiswas further supported by the observation that the duration ofetoposide-induced nuclear NEMO in TRIM37-transduced cellswasmuch shorter than that in control cells, while TRIM37�/� cellsshowed sustained nuclear NEMO signal (Fig. 3H; SupplementaryFig. S2E). However, the effect of TRIM37 on nuclear export ofNEMO was abolished by NEMO/K309A (Fig. 3H). Consistentwith previous report (28), calcium chelation prevented the cyto-plasmic expression ofmonoubiquitinatedNEMO in the TRIM37-transduced cells (Fig. 3I and J), which provided further evidencethat TRIM37 mediated monoubiqutination of NEMO in thenucleus. Remarkably, the etoposide-induced association ofNEMO with Ran, an export receptor and the cargo protein fornuclear export of NEMO (29), was increased in TRIM37-trans-duced cells but was nearly undetectable in TRIM37�/� cells, andno interaction of Ran and NEMO/K309A was observed (Fig. 3K).

These results demonstrated that TRIM37-mediatedmonoubiqui-tination of NEMO promotes nuclear export of NEMO.

Genotoxic stress induces nuclear translocation of TRIM37Coimmunoprecipitation (Co-IP) assays revealed the genotoxic

stress-induced TRIM37/NEMO interaction only occurred in thenucleus but not in the cytoplasm (Fig. 4A–C; Supplementary Fig.S3A and S3B). We then asked whether genotoxic stress couldinduce translocation of TRIM37 into nuclear whereas TRIM37interacted with and monoubiquitinatd NEMO. Interestingly, thenuclear expression of TRIM37 dramatically elevated at 30 minafter genotoxic stress (Fig. 4D and E; Supplementary Fig. S3C).However, TRIM37 containing amutant nuclear localization signalprevented genotoxic stress-induced nuclear translocation ofTRIM37 and interaction with NEMO, as well as TRIM37-inducedgenotoxic NF-kB activation and NEMO monoubiquitination(Supplementary Fig. S3D–S3G).

TRAF6 is required for TRIM37-mediated NEMOmonoubiquitination

Far-Western blot analysis that TRIM37 could not interactdirectly withNEMO, suggesting that TRIM37-mediated genotoxicNEMO monoubiquitination required other protein(s). By indi-vidually silencing all identified nuclear NEMO-interacting pro-teins in Figure 1A,we found that silencing TRAF6 in the etoposide-treated cells almost entirely abrogated genotoxic stress-inducedTRIM37/NEMO interaction but knocking down NEMO did notreduce thebinding affinity of TRIM37 for TRAF6, and that ablatingTRIM37 had no obvious impact on NEMO/TRAF6 association,indicating that TRAF6 is required for formation of a TRIM37/TRAF6/NEMO complex (Fig. 4F). Consistently, the nuclear levelof TRAF6 was also drastically elevated at 30 min after genotoxicstress (Supplementary Fig. S4A). The direct nuclear interaction ofTRIM37 and TRAF6 was confirmed by far-western blot andSTORM analyses (Fig. 4G and H). Interestingly, although thezoom-in STORM image showed TRAF6 (red) stained in closeproximity with TRIM7(green) both in the cytoplasm (middle,top) and nucleus (middle, bottom), 3D render revealed thatTRAF6 interacted directly with TRIM37 in the nucleus (right,bottom) but not in the cytoplasm (right, top; Fig. 4H). Theseresults provided further evidence that TRIM37 forms complexwith TRAF6 in the nucleus upon genotoxic treatment. Consis-tently, silencing TRAF6 abolished the effect of TRIM37 on

(Continued.) Total cell lysates were probed with anti-Flag antibody and a-tubulin was used as a loading ycontrol. C,MS spectrum of an ubiquitinated NEMO peptideshown in the myc-TRIM37/Flag-NEMO cotransduced cells, followed by treatment with etoposide (10 mmol/L, 2 hours). Fragment ions are indicated. D, Immunoblotanalysis of expression of immunoprecipitated NEMO in etoposide-treated control or myc-TRIM37-overexpressing in NE1/NEMO�/� cells transfected with plasmidsencoding Flag-NEMO/WT or the indicated Flag-NEMO/mutant. Total cell lysates were probed with anti-Flag antibody and a-tubulin was used as a loadingcontrol. E, NF-kB DNA-binding and IKK kinase activities were examined in vector-, Flag-NEMO/WT-, or Flag-NEMO/K309A–transduced NEMO�/� cellscotransfectedwith vector ormyc-TRIM37. Thirty-six hours after transfection, cellswere stimulatedwith etoposide (10mmol/L, 2 hours) and analyzed for anti-Flag andanti-NEMO antibodies. a-Tubulin was used as a loading control. F, NF-kB DNA-binding and IKK kinase activities were examined in the indicated cells2 hours after treated with ionizing radiation (IR; 10 Gy), camptothecin (CPT; 10 mmol/L), or CDDP (5 mmol/L). Total cell lysates were probed with anti-Flag antibodyand a-tubulin was used as a loading control. G, Immunoblot analysis of expression of TRIM37/wt and TRIM37/mutant in nuclear extracts from indicated etoposide-treated NEMO�/� cells (10 mmol/L, 2 hours). b-Actin was used as a cytoplasmic loading control and lamin B1 was used as a nuclear loading control. H,Representative pictures (left) and quantification (right) of nuclear Flag-NEMO/WT or Flag-NEMO/K309A in the indicated cells treated with etoposide for theindicated times. Each bar represents themean� SDof three independent experiments. � ,P <0.05; �� ,P<0.01; �� ,P<0.001. Scale bars, 10mm. I, Immunoblot analysisof expression of monoubiqutinated-NEMO in the cytoplasm (CE) and nucleus (NE) of indicated cells, preincubated either with DMSO or BAPTA (20 mmol/L)for 30minutes and further treated with etoposide (10 mmol/L, 2 hours). b-Actin was used as a cytoplasmic loading control and lamin B1 was used as a nuclear loadingcontrol. J and K, Immunoprecipitation assay was performed using anti-NEMO antibody in nuclear extracts of NE-1/V and NE-1/TRIM37 cells or Eca-109/Ctrland Eca-109/TRIM37�/� cells (J), or using anti-NEMO antibody in nuclear extracts of Flag-NEMO/WT- or Flag-NEMO/K309A-transduced NEMO�/� cells (K), whichwere preincubated with BAPTA (20 mmol/L, 30 minutes) and then treated with etoposide (10 mmol/L, 2 hours), and then analyzed by immunoblot withanti-Ran antibody.






Figure 4.

TRAF6 is required for TRIM37-mediatedNEMOmonoubiquitination.A, Immunoprecipitation assayswereperformed in cells transfectedwithvector- ormyc-TRIM37prior tothe etoposide (Etop) exposure (10 mmol/L, 2 hours) or TNFa treatment (10 ng/mL, 15 minutes) using anti-Flag antibody and immunoblot was analyzed using anti-NEMO and anti-IKKb antibodies.B, Immunoprecipitation/immunoblot analyseswere performed in cells treatedwith etoposide (10 mmol/L, 2 hours) or TNFa (10 ng/mL, 15minutes) using anti-TRIM37 or anti-NEMO antibodies. C, Immunoprecipitation/immunoblot analyses were performed in fractionated cytoplasmic (C) and nuclear (N)extracts frometoposide-treatedcells (10mmol/L, 2 hours) using anti-TRIM37andanti-NEMOantibodies.D, Immunoblotanalysis of TRIM37expression in the nucleus extractfrom etoposide-treated cells at the indicated time. Lamin B1 was used as a nuclear loading control. E, Representative images of TRIM37 immunostained with anti-TRIM37antibody (left) and the expression of TRIM37 in subcellular fractions (right) of the Eca-109 cells treated with or without etoposide (10 mmol/L, 30 minutes). Scale bars,10 mm. F, Co-IP assays were performed in the indicated cells using anti-TRIM37 or anti-NEMO antibodies. Left, depletion of TRAF6 impaired the interaction betweenTRIM37 and NEMO. Middle, depletion of TRIM37 did not affect the interaction between NEMO and TRAF6. Right, depletion of NEMO did not alter the interaction betweenTRIM37 and TRAF6. G, Far-Western blotting analysis was performed using IgG or TRIM37 antibody-immunoprecipitated proteins and detected using anti-TRAF6antibody and then reblotted with anti-TRIM37 antibody. Recombinant GST-TRAF6 was used as a control. H, The interaction of TRAF6 and TRIM37 was examinedusing STORMcaptured inwide shot (left; scale bars, 8mm), further zoomed-in (middle; scale bars, 100 nm) and 3D-rendered (right). I,NF-kBDNA-binding activity byEMSAand expression of monoubiqutinated NEMO by immunoblot analysis were examined in vector/cells or TRIM37/cells pretransfected with control or TRAF6-siRNAand then analyzed 2 hours after treatment with ionizing radiation (10 Gy), camptothecin (CPT; 10 mmol/L), or CDDP (5 mmol/L). J, Immunoprecipitation assays wereperformed using anti-TRIM37 antibody in the etoposide-treated cells at the indicated times and analyzed by immunoblot with anti-NEMO and anti-TRAF6 antibodies.K, Immunoprecipitation assay was performed using anti-TRIM37 antibody in etoposide-treated cells (10 mmol/L, 2 hours) transfected with the indicated doses of TRAF6and analyzed by immunoblot with anti-NEMO antibody. L, NF-kB DNA-binding activity by EMSA and immunoblot analysis was performed with indicated cells treatedwith etoposide (10 mmol/L, 2 hours). M, In vitro ubiquitination assay was performed with purified recombinant GST-NEMO, GST-NEMO(85A), or GST-NEMO/K309Athat was incubated with His-TRIM37 (10, 50, or 100 ng) in a reaction mixture containing 2 mmol/L ATP, 1 mg His-ubiquitin, 50 ng E1 (UBE1), and 100 ng E2 (His-UBCH5B)with or without 1 mg TRAF6 or 20 mL p-ATM for 60 minutes at 37�C. Blots were probed with anti-NEMO, anti-ub, anti-TRAF6, anti-p-ATM, and anti-TRIM37 antibodies.


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genotoxic stress-induced NEMOmonoubiquitination and NF-kBactivity (Fig. 4I; Supplementary Fig. S4B). Moreover, co-IP assaysrevealed that with increasing degrees of genotoxic stress, moreTRIM37 was found complexed with TRAF6 and NEMO, and thatTRAF6 induced-interaction between TRIM37 and NEMOwas in aTRAF6 dose-dependent manner (Fig. 4J and K; SupplementaryFig. S4C). Importantly, overexpressing TRAF6-C70A mutant orS13A/T330A failed to recover the level of NEMO monoubiqui-tination in TRAF6-silencing cells treated with etoposide, suggest-ing that nuclear translocation andE3activity of TRAF6 contributesto TRIM37-mediated NEMO ubiquitination (Fig. 4L). In vitroprotein binding and ubiquitination assays further demonstratedthat TRAF6 was essential for TRIM37-mediated NEMO mono-ubiquitination via direct interaction with TRIM37 (Fig. 4M).Notably, NEMO S85A mutant also dramatically abrogated theeffect of TRIM37 on NEMOmonoubiquitination, indicating thatATM-mediated S85 phosphorylation of NEMOwas early event ofits monoubiquitination (Fig. 4M).

ATM-mediated TRIM37 phosphorylation and nucleartranslocation

TRIM37 was previously reported to be localized in peroxi-somal membranes (19–23) and ATM kinase was also found tobe recruited by PEX5 to peroxisomal membranes (30, 31).Consistently, immunofluorescence and subcellular fraction-ation assays revealed that TRIM37 and ATM were colocalizedin peroxisomal membranes (Fig. 5A and B). Interestingly,etoposide-induced TRIM37 nuclear translocation was drastical-ly prevented by an inhibitor of ATM (Fig. 5C), suggesting thatATM was involved in TRIM37 nuclear translocation upongenotoxic stress. Meanwhile, we found that genotoxic stressinduced the phosphorylation of TRIM37 at 196TQ/801SQmotifs (Fig. 5D) and mutating TRIM37 TQ/SQ sites T196 andS801 to alanine reduced genotoxic stress-induced phosphory-lation and nuclear translocation of TRIM37 (Fig. 5E). More-over, we observed that that the genotoxic stress-induced ATM/TRIM37 complex only formed in the cytoplasm but not in thenucleus (Fig. 5F and G). Co-IP assays using ATM and seriallytruncated TRIM37 fragments demonstrated that ATM interactedwith the TD of TRIM37 (Fig. 5H). Importantly, treatmentwith antioxidants not only dramatically prevented genotoxicstress-induced TRIM37 nuclear translocation but also reducedgenotoxic stress-induced ATM/TRIM37 interaction and NEMOmonoubiquitination (Fig. 5I–L). Therefore, oxidative stressmay be also involved in ATM-mediated phosphorylation andnuclear translocation of TRIM37 and NEMO monoubiquitina-tion upon genotoxic stress.

TRAF6-binding motif in TRIM37 is required for the TRAF6interaction

Co-IP assays using serially truncated TRIM37 and TRAF6 frag-ments demonstrated that the TD was the interaction region ofTRIM37 and TRAF6 (Fig. 6A and B). The TD of TRIM37 contains asequence (DFEVGE; residues 366-371) with homology to a con-sensus TRAF6-binding motif (TBM), PXEXX (aromatic/acidicresidue; ref. 32). Interestingly, a TRIM37 mutant containing aTBMdeletion (TRIM37/DTBM) lost the capacity to directly bind toTRAF6, resulting in resistance to genotoxic stress-induced mono-ubiquitination and nuclear export of NEMO, as well as NF-kBactivation (Fig. 6C–F; Supplementary Fig. S5A and B). Consis-tently, immunoprecipitation assays using anti-Ran antibody

showed that TRIM37/DTBM and TRIM37/RF-mu abrogated theinteraction between NEMO and Ran (Fig. 6G).

TRIM domain was essential for TRIM37-mediated NEMOmonoubiquitination

To further determine whether the E3 ligase activity of TRIM37was essential for NEMO monoubiquitination, a TRIM37 deriva-tive (TRIM37/RF-mu) bearing a point mutation in a conservedcysteine residue in the RING fingermotif (C18R), which interfereswith catalytic activity, was constructed. Although TRIM37/RF-mucould still form a complex with TRAF6/NEMO upon etoposidetreatment, overexpressing TRIM37/RF-mu in the TRIM37�/� cellscould not recover the genotoxic stress-induced monoubiquiti-nation and nuclear export of NEMO and NF-kB activation(Fig. 6C–G; Supplementary Fig. S5A and S5B), demonstratingthat TRIM37-mediated NEMO monoubiquitination is TRIMdomain-dependent.

TAT-TRIM37/TBM peptide promotes genotoxic agent-inducedtumor regression

The cell-penetrating HIV-1 transactivator of transcription(TAT)-peptide has been widely used as an anticancer moleculardelivery system due to its high solubility and penetrability (33,34). We then examined the inhibitory effect of TAT-conjugatedTRIM37/TBM (TAT-TBM) peptide on genotoxic NF-kB activationand cancer progression. As shown in Fig. 7A and B, treatment withTAT-TBM peptide dramatically reduced genotoxic stress-inducedTRIM37/TRAF6/NEMO complex formation, IKK/NF-kB activa-tion, and NEMO monoubiquitination in the cells with higherTRIM37 expression, consequently resulting in enhanced effect ofCDDP on cell death as indicated by increased caspase 3 positivecells and decreased colony formation (Fig. 7C and D). Further-more, a PDX model, using two freshly collected clinical esoph-ageal cancer tissues, showed that cotreatment with CDDP andTAT-TBM peptide resulted in significant remission of esophagealcancer tumor volume andmass (T#6: 34mg vs. 657mg; T#9: 65.8mg vs. 983mg) comparedwith control tumors (Fig. 7E), as well ashigher percentage of TUNELþ cells and reduced genotoxic NF-kBactivity (Fig. 7F and G), in comparison with CDDP treatmentalone. Taken together, these results further support thenotion thatTRIM37 overexpression enhanced genotoxic stress-induced NF-kB activation, consequently resulting in chemoresistance andpoorer clinical outcomes in human cancer.

DiscussionUnlike activation of the classical NF-kB signaling pathway that

displays a fast response to typical signals initiated fromcell surfacereceptors, "atypical" NF-kB activators, such as DNA damage oroxygen stress, trigger a slow NF-kB signal (with peak activitiesreached after 2–4 hours; refs. 8, 35). These delayed kinetics werepreviously thought to be at least partially attributable to the timerequired to transfer the nuclear damage signal to the cytoplasmicIKK complex. Multiple recent studies have provided insightsthat genotoxic threats triggering NF-kB activation requiremolecular trafficking between the nucleus and cytoplasm. Forinstance, genotoxic stress-induced nuclear translocation of IKKunbound-NEMO is the key event for DNA damage-dependentIKK/NF-kB signaling (36). Meanwhile, genotoxic stress-inducednuclear translocation of PIDD resulted in augmentation ofsumoylation and ubiquitination of NEMO (14). However,






Figure 5.

ATM-mediated TRIM37 phosphorylation and nuclear translocation. A, Colocalization of ATM and TRIM37 on peroxisomes in Eca-109 cells as analyzed byimmunofluorescence. Scale bars, 10 mm (left), 2.5 mm (right). B, Peroxisomal fractionation/immunoblot analysis of expression of ATM, TRIM37, catalase, andPMP70. WCE, whole cell extracts; PO, peroxisome. C, Indicated cells were exposed to ATM inhibitor KU-55933 (10 mmol/L) or DMSO for 1 hour before addition ofetoposide (Etop; 10 mmol/L, 2 hours) and then analyzed for subcellular fractionation/immunoblot analysis of TRIM37 expression. b-Actin was used as a cytoplasmicloading control and lamin B1 was used as a nuclear loading control.D, Immunoprecipitation assays using anti-Flag antibodywere performed in TRIM37/wt- and TRIM37/mutant-transfected cells treated with etoposide (10 mmol/L, 2 hours) with or without ATM inhibitor KU-55933 (10 mmol/L, 1 hour) pretreatment, and analyzed byimmunoblot with anti-pTQ/SQ antibody. E, Immunoblot analysis of expression of TRIM37/wt- and TRIM37/mutant in nuclear/cytoplasmic extracts form the cellstreated with or without etoposide (10 mmol/L, 2 hours). b-Actin was used as a cytoplasmic loading control and lamin B1 was used as a nuclear loading control. F,Immunoprecipitation/immunoblot analyses were performed in fractionated cytoplasmic (C) and nuclear (N) extracts from etoposide-treated cells (10 mmol/L, 2 hours)using anti-TRIM37 and anti-ATM antibodies. Scale bars, 8 mm (left), 100 nm (middle). G, Immunofluorescence analysis revealed that TRIM37 and ATM interactedin the cytoplasm. H, Left, schematic illustration of wild-type and truncated TRIM37. Right, immunoprecipitation assays were performed using anti-Flag antibody inetoposide-treated cells transfected with Flag-tagged TRIM37 or the indicated Flag-tagged TRIM37 mutants, and immunoblot analyzed with anti-Flag, ATM, and p-ATMantibodies. I–J, Immunoblot (I) and immunofluorescence (J) analyses of TRIM37 expression in cells treated with etoposide (10 mmol/L, 2 hours) with or withoutpyrrolidinedithiocarbamatepretreatment (PDTC;20mmol/L, 1 hour).b-Actinwasusedasa cytoplasmic loading control and laminB1wasusedasanuclear loadingcontrol.Scale bars, 10 mm. K, Immunoblot analysis of expression of ATM-immunoprecipitated TRIM37 in cells treated with the indicated agents. L, Immunoprecipitation/immunoblot analyses of monoubiquitinated NEMO expression and EMSA of NF-kB DNA-binding activity in cells treated with the indicated agents.


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Figure 6.

TRAF6-binding motif of TRIM37 is required for TRIM37/TRAF6 interaction. A, Left, schematic illustration of wild-type and truncated TRAF6. Right, co-IP assayswere performed using anti-Flag antibody in etoposide-treated HEK293T cells (10 mmol/L, 2 hours) after transfection with Flag-tagged TRIM37 or the indicatedmyc-tagged TRAF6 mutants and analyzed by immunoblot with anti-Flag and anti-myc antibodies. B, Co-IP assays were performed using anti-myc antibodyin etoposide-treated HEK293T cells (10 mmol/L, 2 hours) pretransfected with myc-tagged TRAF6 or the indicated Flag-tagged TRIM37 mutants for 36 hours andanalyzed by immunoblot with anti-Flag and anti-myc antibodies. C, Left, schematic illustration of TRIM37mutants TRIM37/DTBM and TRIM37/RF-mu. Bottom, co-IPassays were performed in etoposide-treated HEK293T cells (10 mmol/L, 2 hours) pretransfected with myc-TRIM37/wt, myc-TRIM37/DTBM, or myc-TRIM37/RF-mu,and analyzed by immunoblot with anti-Flag, anti-NEMO, and anti-TRAF6 antibodies. D, Far-Western analysis was performed using immunoprecipitatedmyc-TRIM37/wt, myc-TRIM37/DTBM, or myc-TRIM37/RF-mu, which were gel-purified, transferred to a membrane, and incubated with recombinant TRAF6, thendetected using anti-TRAF6 antibody and then reblotted with anti-TRIM37 antibody. Recombinant GST-TRAF6 was used as a control. E, NF-kB DNA-bindingand IKK activities (left) and expression ofmonoubiquitinated NEMOwere examined in etoposide-treated cells (10 mmol/L, 2 hours) pretransfectedwithmyc-TRIM37,myc-TRIM37/DTBM, or myc-TRIM37/RF-mu. BCL-XL and XIAP expression was analyzed at 6 hours in the indicated cells treated with etoposide (Etop; 10 mmol/L).GST-IkBa or a-tubulin was used as loading control. F, Representative pictures (top) and quantification (bottom) of nuclear Flag-NEMO/WT in the indicatedcells treated with etoposide for the indicated times. Each bar represents the mean � SD of three independent experiments. � , P < 0.05, ��, P < 0.01, �� , P < 0.001.Scale bars, 10 mm. G, Immunoprecipitation assays using anti-Ran antibody were performed in TRIM37/wt-, TRIM37/DTBM-, or TRIM37/RF-mu-transfectedcells, which were preincubated with BAPTA (20 mmol/L, 30 minutes), and then treated with etoposide (10 mmol/L, 2 hours) and analyzed by immunoblot withthe indicated antibody.






Figure 7.

TAT-TRIM37/TBM peptide augments genotoxic stress-induced tumor regression. A, Co-IP assays using anti-TRIM37 antibody were performed in NE1/TRIM37 (left)and Eca-109 (right) cells preincubated with TAT-Ctrl or TAT-37/TBM peptide for 2 h then further treated with CDDP (5 mmol/L, 2 hours), and analyzed byimmunoblot with anti-NEMO and anti-TRAF6 antibodies.B,NF-kBDNA-binding and IKK activities and expression ofmonoubiquitinated NEMO in the indicated cellspreincubated with TAT-Ctrl or TAT-37/TBM peptide for 2 hours then further treated with etoposide (Etop; 10 mmol/L, 2 hours) or CDDP (5 mmol/L, 2 hours). Theexpression of BCL-XL and XIAP was examined at 6 hours after indicated treatment. OCT-1 DNA-binding complex served as a DNA-binding control and GST-IkBa ora-tubulin was used as loading control. C, Representative pictures (left) and quantification (right) of activated caspase-3þ-cells in the indicated cells preincubatedwith TAT-Ctrl or TAT-37/TBM peptide for 2 hours, then further treated with or without CDDP (5 mmol/L, 24 hours). Scale bars, 50 mm. D, Representativepictures (left) and quantification (right) of colony numbers of indicated cells as determined by an anchorage-independent growth assay. E, Representativeimages of tumors from the PDX model cotreated with CDDP (5 mg/kg, three times per week for up to 6 weeks) plus TAT-Ctrl or TAT-37/TBM peptide (top);tumor volumes were examined on the indicated days (bottom). Detailed information for T#6 and T#9 is shown in Fig. 1D. F, NF-kB DNA-binding activity andexpression of monoubiquitinated NEMO and activated caspase-3 were examined in the indicated tumors. OCT-1 DNA-binding complex served as a DNA-bindingcontrol and a-tubulin was used as a loading control. G, IHC staining of nuclear NF-kB p65 and TUNEL-positive cells in the indicated tumors. Each bar inD and G represents the mean � SD of three independent experiments. � , P < 0.05; �� , P < 0.01; �� , P < 0.001. Scale bars, 50 mm.


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monoubiquitinated NEMO, which complexes with ATM andELKS, was exported from the nucleus and was shown to beessential for cytoplasmic IKK activation (18). Herein, we foundthat in response to genotoxic stress, it took nearly 30 minutes forperoxisomal E3 ligase TRIM37 to translocate into the nucleuswhere it associated TRAF6 and NEMO, which resulted in NEMOmonoubiquitination at K309. Therefore, the DNA damage-triggered slow NF-kB signal may be caused by TRIM37 nucleartranslocation. Importantly, we demonstrated that ATM kinase, asensor of DNA damage, played an important role in genotoxicstress-induced nuclear translocation of TRIM37 via direct physi-cal interaction with and phosphorylation of TRIM37. Interesting-ly, two ATM-mediated phosphorylation sites are present inTRIM37, in which T196Q is within the peroxisomal targetingsignal of TRIM37, and S801Q is in proximity to the nuclearlocalization signal of TRIM37. These results suggested thatATM-mediated phosphorylation may lead to dissociation ofTRIM37 from the peroxisome and nuclear transition via exposureof the TRIM37 nuclear localization signal. Although it has beenreported that genotoxic stress-activated nuclear ATM translocatedinto cytosolic and membrane fractions within 10 minutes (36),we found that antioxidants prevented genotoxic stress-inducedATM/TRIM37 interaction and TRIM37 nuclear translocation,suggesting that oxidative stress-activated ATM, instead of DNAdamage-activated ATM, contributed to phosphorylation ofTRIM37. This was consistent with previous reports that oxidativestress could induce activation of peroxisomal ATM kinase (30,31). Therefore, our study provided a novel mechanism and rolefor ATM in genotoxic stress-induced NF-kB activation.

The TRIM37 gene on chromosome 17q22–23 was originallyfound tobe frequentlymutated in patientswithmulibrey nanism,a disease with dramatic growth impairment in several organs (19,20). Further studies demonstrated that the roles of TRIM37 invarious biological processes depend on TRIM domain-dependentE3 ligase activity. For instance, TRIM37 is involved in peroxisomalmatrix protein import via monoubiquitination of PEX5 (22).Enforced expression of a TRIM37 mutant that lacks E3 ligaseactivity could not prevent the TRIM37 depletion-resulted super-numerary centrosomal-component foci (37). Although multiplestudies reported that TRIM37 is mainly distributed in the cyto-plasm, such as in peroxisomes (20–22), Bhatnagar and colleaguesfound that TRIM37 associatedwith PRCcomplex in the nucleus toestablish a repressive chromatin structure (23), suggesting thatTRIM37 could translocate into the nucleus. Herein, we found thatin response to DNA damage, ATM-mediated phosphorylation ofTRIM37 led to its rapid translocation into the nucleus, where itforms a TRIM37/TRAF6/NEMO complex that catalyzes NEMOmonoubiquitination, ultimately leading toNF-kB-mediated anti-

apoptotic transcription. Treatment with a cell-penetrating TAT-TBMpeptide, which blocked interaction of TRIM37/TRAF6, abro-gated genotoxic stress-induced NEMO monoubiquitination andNF-kB activation, resulting in hypersensitivity of cancer cells togenotoxic chemotherapy. Therefore, our findings not only reveal acrucial role of TRIM37 in genotoxic stress-induced NF-kB activa-tion, but also have important translational implications for themechanistic understanding of therapeutic TRIM37 inhibitors thatcan potentate the effect of chemotherapeutic drugs or ionizingradiation in cancer therapy.

In conclusion, preventing genotoxic stress-induced NF-kB acti-vation, which results in development of chemotherapy resistance,will be beneficial for a large group of patients with cancer. Herein,we demonstrated that genotoxic stress-induced E3 ligase TRIM37contributed to NEMO monoubiquitination and genotoxic IKK/NF-kB activation, consequently leading to genotoxic stress resis-tance of esophageal cancer. Therefore, further investigation intothe role of TRIM37 in resistance of chemotherapy-induced geno-toxic stress will not only provide valuable insights to betterunderstand imitation and progression of cancers but also mayeventually lead to the development of novel therapeutic strategiesfor treatment of human cancers.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: L. Song, J. LiDevelopment of methodology: G. Wu, J. Zhu,Analysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): G. Wu, J. Zhu, Y. Hu, L. Cao, Z. Tan, S. Zhang, Z. Li,Writing, review, and/or revision of the manuscript: L. Song, J. LiStudy supervision: J. Li

AcknowledgmentsThis work was supported by Natural Science Foundation of China

[No. 81830082, 91740119, 91529301, and 81621004 (all to J. Li);91740118, 81773106, and 81530082 (all to L. Song)]; Guangzhou Scienceand Technology Plan Projects (201803010098 to J. Li); Guangdong NaturalScience Foundation (2018B030311009 to J. Li; 2016A030308002 toL. Song); The Fundamental Research Funds for the Central Universities(No. 17ykjc02 to J. Li).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received July 5, 2018; revised August 28, 2018; accepted September 21, 2018;published first September 25, 2018.

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Wu et al.




2018;78:6399-6412. Published OnlineFirst September 25, 2018.Cancer Res Geyan Wu, Libing Song, Jinrong Zhu, et al.

B SignalingκActivating Nuclear-to-Cytoplasmic NF-An ATM/TRIM37/NEMO Axis Counteracts Genotoxicity by

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