Naturally Occurring Isothiocyanates Exert Anticancer ... · The molecular mechanisms underlying the anticancer proper-ties of isothiocyanates are under debate (1). Isothiocyanates

Therapeutics, Targets, and Chemical Biology

Naturally Occurring Isothiocyanates ExertAnticancer Effects by Inhibiting DeubiquitinatingEnzymesAnn P. Lawson1, Marcus J.C. Long2, Rory T. Coffey1,3, Yu Qian4, Eranthie Weerapana4,Farid El Oualid5, and Lizbeth Hedstrom1,6

Abstract

The anticancer properties of cruciferous vegetables are wellknown and attributed to an abundance of isothiocyanates suchas benzyl isothiocyanate (BITC) and phenethyl isothiocyanate(PEITC). While many potential targets of isothiocyanates havebeen proposed, a full understanding of the mechanisms under-lying their anticancer activity has remained elusive.Herewe reportthat BITC and PEITC effectively inhibit deubiquitinating enzymes(DUB), including the enzymes USP9x and UCH37, which areassociated with tumorigenesis, at physiologically relevant con-centrations and time scales. USP9x protects the antiapoptoticprotein Mcl-1 from degradation, and cells dependent on Mcl-1were especially sensitive to BITC and PEITC. These isothiocya-nates increased Mcl-1 ubiquitination and either isothiocyanatetreatment, or RNAi-mediated silencing of USP9x decreased Mcl-1levels, consistent with the notion that USP9x is a primary target of

isothiocyanate activity. These isothiocyanates also increased ubi-quitination of the oncogenic fusion protein Bcr-Abl, resulting indegradation under low isothiocyanate concentrations and aggre-gation under high isothiocyanate concentrations. USP9x inhibi-tion paralleled the decrease in Bcr-Abl levels induced by isothio-cyanate treatment, and USP9x silencing was sufficient to decreaseBcr-Abl levels, further suggesting that Bcr-Abl is aUSP9x substrate.Overall, our findings suggest that USP9x targeting is critical to themechanism underpinning the well-established anticancer activityof isothiocyanate. We propose that the isothiocyanate-inducedinhibition of DUBs may also explain how isothiocyanates affectinflammatory and DNA repair processes, thus offering a unifyingtheme in understanding the function and useful application ofisothiocyanates to treat cancer as well as a variety of otherpathologic conditions. Cancer Res; 75(23); 5130–42. �2015 AACR.

IntroductionThe dietary consumption of cabbage, broccoli, and other cru-

ciferous vegetables is associated with a decreased risk of cancer(1–3). The chemoprotective properties of these vegetables areattributed to isothiocyanates such as benzyl isothiocyanate(BITC), phenethyl isothiocyanate (PEITC), and sulforaphane(Fig. 1A; refs. 1, 2). Plasma concentrations of isothiocyanates canreach 0.25 mmol/L from a single serving of broccoli, and intra-cellular concentrations can be 200-fold higher due to concentra-tive processes (3, 4). Numerous studies demonstrate that thesecompounds have antiproliferative activity against tumors in both

cell culture and animal models (2, 3), and PEITC has enteredclinical trials for lung and oral cancers (3). Isothiocyanates induceapoptosis in many cancer cell lines, and exposure to BITC orPEITC for only 3 hours inhibits cell growthwith EC50 values of 1.8to 17 mmol/L (5). Sulforaphane also inhibits growth under theseconditions, although the values of EC50 are typically much higher(50 mmol/L). Isothiocyanates perturb many cellular processes,including DNA repair (3, 6), autophagy (2), the inflammatoryresponse (1), and the antioxidant response (1, 2). Isothiocyanatesalso modulate the activity of several oncogenic proteins. Forexample, both PEITC and BITC reduce the levels of the antiapop-totic proteinMcl-1 in leukemia cells (7–9), and PEITC induces theknockdown of Bcr-Abl kinase, the oncogenic fusion protein thatcauses chronic myeloid leukemia (10).

The molecular mechanisms underlying the anticancer proper-ties of isothiocyanates are under debate (1). Isothiocyanates areelectrophiles that form reversible adducts with small-moleculethiols such as glutathione (Fig. 1B; ref. 3). Amines can form stableadducts with isothiocyanates, although this reaction is not facileat neutral pH. Depletion of glutathione, and resulting generationof reactive oxygen species (ROS), is an appealing mechanismfor the anticancer activities of isothiocyanates (11). However,L-butathionine sulfoximine depletes glutathione and inducesROS to greater extents than PEITC yet does not induce apoptosis(12). This finding discredits glutathione depletion/ROS produc-tion as the mechanism of anticancer activity. Isothiocyanates canalso modify proteins at thiol and amine residues. At least 30proteins have been reported to be potential isothiocyanate targets,

1DepartmentofBiology,BrandeisUniversity,Waltham,Massachusetts.2Graduate Program in Biochemistry and Biophysics, Brandeis Univer-sity, Waltham, Massachusetts. 3Graduate Program in Molecular andCellular Biology, Brandeis University, Waltham, Massachusetts.4Department of Chemistry, Merkert Center, Boston College, ChestnutHill, Massachusetts. 5UbiQ, Amsterdam, the Netherlands. 6Depart-ment of Chemistry, Brandeis University,Waltham, Massachusetts.

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

A.P. Lawson and M.J.C. Long contributed equally to this article.

Corresponding Author: Lizbeth Hedstrom, MS009, Brandeis University, 415South Street, Waltham, MA 02453. Phone: 781-736-2333; Fax: 781-736-2349;E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-15-1544

�2015 American Association for Cancer Research.

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including P450s, glutathione reductase, thioredoxin reductase,mutant p53, migration-inhibitory factor, protein phosphatases,and tubulin (1), but the functional consequences of isothio-cyanate modification are usually unknown and the correlationwith cellular phenotypes uncertain. Moreover, the reversiblenature of isothiocyanate–thiol adducts suggests that Cys-mod-ified proteins were unlikely to be identified in previous experi-ments. Therefore, the wide array of potential isothiocyanatetargets does not satisfactorily explain the pleiotropic cellulareffects of isothiocyanates.

Catalytic cysteine residues are generally very nucleophillic andreact readily with electrophiles, so enzymes with catalytic cysteineresidues are good candidates for isothiocyanate targets. Cysteineproteases are particularly attractive candidates for isothiocyanateinhibition because the thiol adduct resembles the thioester inter-mediate of peptide hydrolysis (Fig. 1B and C). The C¼S bond islonger and more electronegative than C¼O and thus may resem-ble the transition state for peptide hydrolysis, potentially provid-ing additional binding energy. Isothiocyanates areweak reversibleinhibitors of papain (13), the prototypical cysteine protease frompapaya, but the effects of isothiocyanates on other cysteineproteases have not been investigated. Papain is distantly relatedto deubiquitinating enzymes (DUB), the hydrolases that removeubiquitin from target proteins and disassemble ubiquitin chains.DUBs regulate many important physiologic processes, includingprotein degradation, DNA repair, autophagy, and protein traf-ficking (6, 14) and are potential targets for the treatment of manydiseases, including cancer (15), neurodegeneration, inflamma-tion, and infection (14). We recognized that many of the phe-notypes associated with dietary isothiocyanates are also observedwhen cells are treated with DUB inhibitors. Therefore, we hypoth-esized that DUBs might be targets of isothiocyanates.

Here, we report that both BITC and PEITC inhibit USP9X andUCH37 and other DUBs at physiologically relevant concentrationsand time scales. DUB inhibition provides a molecular mechanismfor the anticancer properties of dietary isothiocyanates.

Materials and MethodsDetailed methods are included in the Supplementary Material.

MaterialsAll chemicals and reagents were from Sigma Aldrich unless

otherwise stated. Solvents (except DMSO) were from Fisher.Other reagents used in this study: G5 isopeptidase inhibitor1 (50-230-7928, Calbiochem); PEITC (Acros Organics); bor-tezomib (Millennium Pharmaceuticals); Mini-Complete andPhosSTOP inhibitory cocktails (Roche Applied Science); bor-tezomib (LC laboratories); Alamar Blue (Invitrogen); USP9x,USP7(catalytic domain), UCH-L3, Ubiquitin-AMC, Suc-Leu-Leu-Val-Tyr-AMC, RAP80 UIM Domains Agarose AM-120, and20S human proteasome (Boston Biochem); normal goat IgGSC-2028 (Santa Cruz); TAMRA-ubiquitin propargylamide andCy5-ubiquitin vinyl methyl ester (UbiQ); HA-ubiquitin vinyl-sulfone and HA-ubiquitin vinyl methyl ester were synthesizedusing standard methods previously described (16). The plas-mid encoding the HA-1-75Ub-Intein-chitin–binding domainfusion protein was a gift from Prof. H. Ploegh (WhiteheadInstitute, Cambridge, MA). BaF3 and BaF3/p210 cells wereprovided by Nathaniel Gray (Harvard University, Cambridge,MA).

AntibodiesThe following antibodies were used: anti-K48-linked ubiqui-

tin, clone APU2 and anti-K63-linked ubiquitin, clone APU3(Millipore); anti-PARP 9542, anti-cAbl 2862, anti-a-tubulin2156, anti-Mcl-1 D35A5, anti-Flag 2368 (Cell Signaling Tech-nologies); anti-actin clone AC-40 A3853, anti-GAPDH cloneG9295, and anti-HA, Clone 3F10 (Roche); anti-ubiquitin, clone6C1.17 (BD Pharmingen); anti-HSP70, anti-USP7, anti-UCH37,anti-USP24, and anti-USP9x (all rabbit monoclonal; Abcam);and horseradish peroxidase (HRP)-conjugated secondary anti-bodies (Abcam).

Tissue culture assays and preparation of cell lysatesB16/F10 andMCF7 cells were purchased fromATCC. BaF3 and

BaF3/p210 cells were provided by Dr. Nathaniel Gray (HarvardMedical School, Boston, MA; obtained 2013), K562 cells wereprovided by Jeffrey Strovel (Avalon Pharmaceuticals; obtainedin 2011), HeLa cells were provided by Benjamin F. Cravatt

Figure 1.Proposed mechanism of DUB inhibition. A, structures of naturally occurring isothiocyanates. B, proposed mechanism of isothiocyanate inhibition.C, mechanism of DUB catalysis.

Naturally Occurring Isothiocyanates Inhibit DUBs

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(The Scripps Research Institute; obtained in 2010), NIH/3T3 cellswere provided by Dr. Rubio Ren (Brandeis University, Waltham,MA; obtained in 2012), and COS1 were provided by Dr. DanielOprian (Brandeis University; obtained in 2010). Cell lines wereauthenticated (9-Marker STR May 2015). The genetic profiles ofK562, MCF-7, HeLa, BaF3, BaF3/p210, and NIH/3T3 cells wereidentical to reported genetic profiles. COS1 cells were confirmedto be of African greenmonkey in origin and free of all interspeciescontamination.

Cells were cultured in DMEM (HeLa, COS1, NIH/3T3, B16-F10, and MCF-7) or RPMI (BaF3, BaF3/p210 and K562) supple-mented with 10% heat-inactivated FBS (DBS was used forNIH/3T3 cells), 1� GlutaMAX, and 1% penicillin/streptomycinat 37�C in a 5%CO2 humidified atmosphere. BaF3 cells were alsosupplemented with 1 ng/mL recombinant mouse IL3 (rmIL3,R&D Systems).

For lysate preparation, nonadherent cells were harvested bycentrifugation, resuspended in 10 mmol/L HEPES (pH 7.9),5 mmol/L MgCl2, 140 mmol/L KCl, 1% NP-40, protease, andPhosphatase InhibitorCocktail II, lysedusing3� freeze thawcycles,and clarified by centrifugation.Whole-cell lysates were prepared byadding 0.1%SDS to the cells togetherwith supernatant followedbysonication. Protein was analyzed by Western blotting (protein,antibody dilution): K48-linked ubiquitin (6 mg, 1:9,000 antibodydilution; 30–40 mg, 1:20,000), PARP (30–40 mg, 1:1,500), K63-linked ubiquitin (30–40 mg, 1:1,500), Mcl-1 (30–40 mg, 1:1,000),FLAG(30–40mg,1:6,000), cAbl (30–40mg,1:1,000), andubiquitin(10–20 mg, 1:14,000). Signals were normalized to actin (1:10,000for 6 mg lysate; 1:30,000 for 30–40 mg lysate), a-tubulin (1:8,000),or GAPDH (1:35,000).

UbiquitinG76V-GFP assayCOS1 cells were transfected with an expression plasmid for

ubiquitinG76V-GFP (plasmid 11941 from Addgene, from thelaboratory of Nico Dantuma) using Mirus 2020 (Madison, WI).FACS was carried out on a Beckman FACS Calibur. All data wereanalyzed using FlowJo V10, from TreeStar.

20S proteasome assayProteasome activity wasmeasured bymonitoring the hydrolysis

of Suc-Leu-Leu-Val-Tyr-AMC in 50mmol/L potassium phosphate,pH 7.6, 50mmol/L NaCl, 1 mmol/L dithiothreitol (DTT) at 25�C.The K0.5 value for human 20S proteasome was experimentallydetermined to be 12 � 2 mmol/L (Hill coefficient ¼ 2), in goodagreement with literature values (17). The apparent K0.5 of protea-some activity of rabbit reticulocyte lysate was 28� 3 mmol/L (Hillcoefficient ¼ 1.3).

Activity profiling of reactive cysteinesHeLa cell lysates were treated with either DMSO or PEITC

(20 mmol/L), followed by 100 mmol/L of IA-alkyne. Clickchemistry and peptide analysis was performed as describedpreviously (18, 19).

DUB activity profilingCell lysate was treated with isothiocyanate or 1% DMSO

control and then treated with HA-UbVS, HA-UbVME, Cy5-Ub-VME, or TAMRA-Ub-PA. Aliquots were removed and immediatelyquenched in 2� DTT loading buffer and frozen until analyzed.BaF3/p210 cells were treated with isothiocyanate or 0.1% DMSO

control, harvested, and washed two times with ice-cold PBS. Cellpellets were then lysed with glass beads in ice-cold 75 mmol/LK2HPO4, pH 7.5, 150 mmol/L NaCl, and 250 mmol/L sucrose.The clarified supernatant was incubated with Cy5-UbVME (250nmol/L) for 5 minutes at 37�C. Aliquots were quenched andtreated as above. Western blot analysis was carried out usingstandard methods. In-gel fluorescent scans were obtained using aGE Typhoon scanner.

Recombinant DUB assaysThe hydrolysis of Ub-AMC was measured by monitoring the

production of AMC at 37�C in a black 96-well plate. Assay buffercontained 50 mmol/L HEPES, pH 7.6, 100 mmol/L NaCl, 0.75mmol/L BME. The fluorescence intensities were quantified withthe appropriate AMC standard curves.

Proliferation assaysMeasured using the Alamar Blue Method as described in the

Supplementary Methods.

ImmunoprecipitationsFor Mcl-1 immunoprecipitations, COS1 cells were transiently

transfected with an expression plasmid for 3� FLAG-taggedmouse Mcl-1 (Addgene plasmid 32978, from the lab of JosephOpferman) using Mirus 2020. 3� Flag–Mcl-1 was immunopre-cipitated with anti-Flag M2 magnetic beads and eluted with500 mg/mL 3�Flag peptide.

For the isolation of polyubiquitinated proteins, BaF3/p210cells were incubated with 5 mmol/L BITC or PEITC or with 0.1%DMSO for 1 hour at 37�C. K562 cells were incubated with5 mmol/L BITC or PEITC or with 0.1% DMSO for 2 hours at37�C. Poly-K63–linked ubiquitinated proteins were enrichedwith RAP80-UIM agarose (25 mL resuspended slurry). ForBcr-Abl immunoprecipitation, the PEITC-, BITC- (5 mmol/L), orDMSO-treated cell lysates were adjusted to the same proteinconcentration (1 mg/mL) and precleared with protein G (1 hourat 4�C). The cleared lysate was incubated with anti-cAbl (1 mL/100 mL lysate) overnight at 4�C. cAbl was immunoprecipitatedwith protein G beads.

Cell transfection and RNA interferenceBaF3/p210 and K562 cells were transfected with siRNAs using

the Amaxa Nucleofector II (Amaxa). NIH/3T3/p210 cells weretransfected using Dharmafect 1 (GE Dharmacon). PredesignedON-TARGET plus siRNA pools (nontargeting and targetingUSP9x) were obtained from Dharmacon. The following siRNApools (Ms or Hu, target sequence) were used:

Ms si-USP9x no. 09, 50-CAGCAAAACUGUUCGUCAA-30;Ms si-USP9x no. 10, 50-GGGCUAACGAUCUCAUUUA-30;Ms si-USP9x no. 11, 50-GCUAAUGUGUAAAUGGCAA-30;Ms si-USP9x no. 12, 50-GAUGAGGCUUCAAGAUAUA-30;Hu si-USP9x no. 06, 50-AGAAAUCGCUGGUAUAAAU -30;Hu si-USP9x no. 07, 50-ACACGAUGCUUUAGAAUUU -30;Hu si-USP9x no. 08, 50-GUACGACGAUGUAUUCUCA -30;Hu si-USP9x no. 09, 50-GAAAUAACUUCCUACCGAA -30;si-nontargeting no. 1, 50-UGGUUUACAUGUCGACUAA-30

si-nontargeting no. 2, 50-UGGUUUACAUGUUGUGUGA-30

si-nontargeting no. 3, 50-UGGUUUACAUGUUUUCUGA-30

si-nontargeting no. 4, 50-UGGUUUACAUGUUUUCCUA-30

Lawson et al.

Cancer Res; 75(23) December 1, 2015 Cancer Research5132




Retroviral transductionMSCV-p210-IRES-GFP vector obtained from the Ren labora-

tory (Brandeis University; ref. 20) was used to produce retroviralpseudovirus. NIH/3T3 cells were transduced with virus GFP–expressing NIH/3T3 cells collected on a FACS Aria FlowCytometer.

ResultsIsothiocyanates increase the high-molecular-weight ubiquitinpool

DUB inhibition can be revealed by the accumulation of high-molecular-weight ubiquitinated proteins (HMW-Ub). Inhibitors

of the proteasome and P97 can also cause the accumulation ofK48-linked ubiquitination, but as yet only DUB inhibitors areknown to cause the accumulation of K63-linked ubiquitination.We examined the effects of BITC and PEITC on the HMW-Ub inthe pro-B-cell line BaF3/p210, which expresses Bcr-Abl kinase.Both BITC and PEITC caused the accumulation of K48-linkedHMW-Ub (Fig. 2A and B) while sulforaphane had no effect.Increases in K48-linked Ub could be observed within 4 hours at7 mmol/L BITC or PEITC and reached 2- to 4-fold at 15 mmol/L.Similar results were obtained in BaF3 cells (SupplementaryFig. S1A and S1B). Both isothiocyanates also caused a 9- to 18-fold increase in K63-linkedUb (Fig. 2C andD and SupplementaryFig. S1D). Treatment with the proteasome inhibitor bortezomib

Figure 2.Naturally occurring isothiocyanates cause HMW-Ub accumulation in cells but do not inhibit the proteasome. A, BaF3/p210 cells were treated with PEITC or BITC for4 hours. Whole-cell lysates were analyzed by SDS-PAGE and probed for K48-linked Ub. Actin is shown as a loading control. Data are representative of twoindependent experiments and reproduced independently in the parent BaF3 cell line. B, densitometric quantification of the blot shown in A. C, BaF3/p210 cells wereincubated with BITC or PEITC for 4 hours. Whole-cell lysates were analyzed by SDS-PAGE and immunoblotted as indicated. Data are representative of twoindependent experiments and reproduced independently in the parent BaF3 cells. D, quantification of immunoblot shown in C. E, BaF3/p210 cells were treated withBITC (15 mmol/L), BITC (15 mmol/L) combined with bortezomib (20 nmol/L), or bortezomib (20 nmol/L) alone for 4 hours. Whole-cell lysates were analyzed bySDS-PAGE and Western blotting. "Control" denotes 0.1% DMSO vehicle only. Actin is shown as a loading control. Data are representative of two independentexperiments. F, COS1 cells transiently expressing UbG76V-GFP were treated for 8 hours with PEITC (12 mmol/L), BITC (12 mmol/L), sulforaphane (SFN; 12 mmol/L),USP9x inhibitor WP1130 (5 mmol/L), the proteasome inhibitor bortezomib (Bort, 4 mmol/L), or 0.1% DMSO (vehicle control). GFP was quantified by flowcytometry. Data presented are the mean � SD of quadruplicate samples from three independent experiments. Significance was determined using theStudent t test.






also increased the levels of K48-linked Ub but had no effect onK63-linked Ub (Fig. 2E). Thus, the accumulation of both K48 andK63-linked HMW-Ub strongly suggests that BITC and PEITCinhibit DUBs.

BITC and PEITC do not inhibit the flux through the ubiquitinproteasome system

The accumulation of K48-linked ubiquitin suggests that theanticancer effects of Isothiocyanates might arise from inhibitionof flux through the ubiquitin–proteasome system, much like theanticancer effects of the proteasome inhibitor bortezomib. There-fore, we used the UbG76V-GFP assay to monitor 26S proteasomeactivity and flux through the ubiquitin–proteasome system in livecells (21, 22). This reporter protein consists of a ubiquitin linkedto the N-terminus of GFP. The G76V mutation prevents thecleavage of ubiquitin from GFP, leading to its degradation bythe 26S proteasome (22). When COS1 cells expressing UbG76V-GFPwere treated with the proteasome inhibitor bortezomib, GFPfluorescence increased 1.5-fold, consistent with reports fromother laboratories (e.g., ref. 23; Fig. 2F). In contrast, BITC andPEITC did not increase GFP levels, indicating that proteasomeactivity was not inhibited. Similarly, the DUB inhibitor WP1130also did not cause an increase in GFP levels (Fig. 2F). Isothiocya-nates did not inhibit purified 20S proteasome or proteasomeactivity in rabbit reticulocyte lysate, contrary to a previous report(Supplementary Fig. S2A–S2C; ref. 24). Collectively, these obser-vations strongly suggest that the isothiocyanate-induced accumu-lation of HMW-Ub results from DUB inhibition.

PEITC does not perturb the global cysteine reactomeThemethods used to identify isothiocyanate targets in previous

reports were unlikely to detect modifications of cysteine residuesinDUBsorother proteins. Therefore,weused competitive cysteinereactivity profiling with an iodoacetamide-alkyne (IA-alkyne)probe (100 mmol/L) to more thoroughly investigate the effectsof isothiocyanates on the global cysteine reactome of HELA cells(18). More than 1,000 IA-alkyne–labeled cysteine residues wereidentified in HELA cells usingmass spectrometric analysis. Unfor-tunately, only two of these cysteine residues belonged to DUBs(UCHL1 and otubain 1), alluding to the poor affinity ofIA-alkyne toward the active-site cysteines in DUBs, as well as thelow abundance of DUBs relative to other cysteine-containingproteins in HELA lysates. Despite low coverage of DUBs, thiscysteine profiling experiment provides a measure of the generalpromiscuityof PEITCacrosshighly reactive cysteine residues in theproteome. Treatment of HELA lysates with PEITC (20 mmol/L)significantly inhibited the labeling of only 14 of 1,400 profiledcysteines in at least one of two independent experiments (inhi-bition � 67%; Supplementary Dataset S1), although in no casewas labeling inhibited inboth experiments. Seventyof theprofiledcysteine residues are found in enzymeactive sitesormetal-bindingsites, including six dehydrogenases and six cysteine proteases(Supplementary Dataset S2). PEITC blocked the labeling of onlyone of these, mitochondrial phosphoenolpyruvate carboxykinase(Supplementary Fig. S3). These experiments demonstrate thatPEITC is not a promiscuous cysteine-modifying agent.

BITC and PEITC inhibit USP9x and UCH37We turned to competitive activity profiling to identify which

DUBs are inhibited by BITC and PEITC. In addition to the

commonly used HA-tagged ubiquitin vinyl sulfone (HA-UbVS)andHA-tagged ubiquitin vinylmethyl ester (HA-UbVME; refs. 25,26), we also used the more sensitive fluorogenic probesCy5-UbVME (26) and TAMRA-ubiquitin propargylamide(TAMRA-UbPA; refs. 27, 28). These irreversible DUB inhibitorslabel 10 to 30 DUBs in cell lysates with varying repertoiresdepending on the probe and experimental conditions (25). Thetreatment of cell lysates with HA-UbVS produced the character-istic pattern of protein bands at 300, 150 to 100, 45, and 36 kDa,generally ascribed toUSP9x/USP24 (292 kDa),USP19 (146kDa),USP7/8 (128 and 127 kDa, respectively), USP28/15 (122 and 112kDa, respectively), UCH37 (38 kDa), UCH-L3 (26 kDa), andUCH-L1 (25 kDa; Supplementary Fig. S4A; ref. 25). Similarresults, although with varying band intensities, were obtainedwith HA-UbVME, TAMRA-UbPA, and Cy5-UbVME (Supplemen-tary Fig. S4B–S4D). As expected, WP1130 and isopeptidaseinhibitor G5 reduced the labeling of several DUBs (e.g., bandsbetween 120 and 300 kDa), validating the experimental method(Supplementary Fig. S4A; refs. 25, 29).

BITC and PEITC inhibited the labeling of several DUBs inlysates prepared from cells pretreated with isothiocyanates (Fig.3A). When lysate was incubated with either BITC or PEITC andthen diluted into a buffer containing HA-UbVS, the labelingincreased with time, demonstrating that isothiocyanate inhibi-tion was slowly reversible as expected (Fig. 3B). Labeled DUBswith molecular weights at 300, 135, and 45 kDa were among themost susceptible to isothiocyanates. These bands likely corre-sponded to three DUBs that are potential therapeutic targets forcancer: USP9x, USP7, and UCH37 (Fig. 3A, Supplementary Fig.S4A–S4D). The identities of USP9x and UCH37 were confirmedby observing the molecular weight shift upon labeling withimmunoblotting (Fig. 3C and D and Supplementary Fig. S4Eand S4F). USP24, a close relative of USP9x (16), was also a targetof BITC and PEITC (Fig. 3E). However, immunoblotting revealedthat the 128-kDa isothiocyanate target was not USP7 (Fig. 3C).

The IC50 values for the inhibition of USP9x labeling were 27�6 mmol/L and 15� 3 mmol/L for BITC and PEITC, respectively, incell lysates (Supplementary Fig. S5A and S5B). The values of IC50

for the inhibition of UCH37 labeling were 22 � 4 mmol/L and13 � 1 mmol/L for BITC and PEITC, respectively (SupplementaryFig. S5C). The values for USP24with IC50 values of 56� 5 mmol/Land 28� 5 mmol/L for BITC and PEITC, respectively (Fig. 3E andSupplementary Fig. S5D).

BITC and PEITC inhibit USP9x and UCH37 in vitroThe inhibition of USP9x was confirmed by assaying purified

recombinant enzyme. PEITC inhibited labeling of recombinantUSP9x with Cy5-UbVME with an IC50 value of 20� 2 mmol/L, ingood agreement with the lysate assays (Supplementary Fig. S6Aand S6B). Both BITC and PEITC were slow binding competitiveinhibitors of recombinant USP9x, with values of Ki ¼ 25� 1 and23 � 2 mmol/L, respectively (Supplementary Fig. S6C–S6E). Thevalues of kon decreased with increasing substrate concentration,indicating competitive inhibition (Supplementary Fig. S6F).

UCH37 (also known as UCHL5) is a component of the 19Sregulatory particle and the INO80 chromatin remodeling com-plex. The 19S regulatory particle activates UCH37, whereasUCH37 is inactive in the INO80 complex (30). Therefore, weassayed the DUB activity of UCH37 in the 19S regulatory particleusing activity profiling. Both PEITC and BITC inhibited UCH37with values of EC50 of 36 � 5 mmol/L and 31 � 6 mmol/L, in

Lawson et al.





reasonable agreement with the lysate assays (Fig. 3F and Supple-mentary Fig. S5E). No inhibition of recombinant UCHL3 or thecatalytic domain of USP7 was observed, confirming the lysateassays (Supplementary Fig. S6A and S6B).

USP9x inhibition provides a molecular mechanism for theantileukemic effects of isothiocyanates

Both USP9x and UCH37 are overexpressed in many cancers(30–33), so inhibition of these DUBs provides an attractive

mechanism for the anticancer effects of isothiocyanates (30,31, 33). USP9x is known to protect the antiapoptotic proteinMcl-1 from ubiquitination and degradation (33). Intriguingly,isothiocyanates decrease Mcl-1 levels in several cell lines (7–9),and similar decreases in Mcl-1 levels are observed when USP9xis either inhibited or silenced (32–34). These observationssuggest that the USP9x may be a primary target for BITC andPEITC. In contrast, the role of UCH37 in cancer is not under-stood and could involve either or both the 19S regulatory

Figure 3.Naturally occurring isothiocyanate inhibit USP9x and UCH37 in cell lysates and in living cells. A, BaF3/p210 cells were incubated with PEITC or BITC for 4 hours.The cells were harvested, washed, resuspended in lysis buffer, and lysed with glass beads. After centrifugation, the clarified supernatant was normalized(0.6 mg/mL), then Cy5-UbVME (250 nmol/L) was added and the mixture incubated at 37�C for 5 minutes and analyzed by SDS-PAGE. In-gel fluorescent scan wasobtained on Typhoon scanner. Data are representative of three independent experiments. � , DUB assignments that were verified by immunoblotting. In caseswherethe bands were not confirmed by immunoblotting, DUBs were identified on the basis of accepted molecular weight. B, A BaF3 cell lysate (12 mg/mL) wasincubated with PEITC or BITC (250 mmol/L) or with DMSO vehicle (1% final concentration) and then diluted 20-fold into buffer containing HA-UbVS (1.5 mmol/L).Aliquots were removed at the indicated times resolved by SDS-PAGE and immunoblotted with anti-HA. �, no compound. C, BaF3 cell lysate was incubated withPEITC or BITC, treated with HA-UbVS, resolved on a 6% PAGE, and immunoblotted with anti-USP9x and anti-USP7. �, vehicle control. D, BaF3 cell lysate(1.5 mg/mL) was incubated with PEITC, BITC, or sulforaphane (SFN; 50 mmol/L) or with G5 or WP1130 (10 mmol/L) for 10 minutes prior to addition of HA-UbVME(1.5 mmol/L). After 20 minutes, aliquots were analyzed by immunoblotting with anti-UCH37 antibody. Ratio represents UCH37-UbVME conjugate per totalUCH37. Data are representative of two independent experiments. �, indicates no compound, vehicle only. E, BaF3 cell lysate (1.5 mg/mL) was incubatedwith PEITC or BITC, treated with HA-UbVS (1.5 mmol/L) and analyzed by immunoblotting with anti-USP24. Data are representative of two independent experiments.F, 19S regulatory particles (19S RP, 30 nmol/L)were treatedwith PEITC or BITC for 30minutes atwhich time Cy5-UbVME (300 nmol/L)was added. After 20minutes,aliquots were quenched in loading buffer and resolved by SDS-PAGE. In-gel scans were obtained on a GE Typhoon scanner.






particle and the INO80 complex or recently characterizedcomplexes with SMAD proteins (30). Which of these complexespromote cancer, and whether the deubiquitinating activity ofUCH37 is required, is not understood.

If the anticancer effects of BITC and PEITC arise from theinhibition of USP9x, thenMcl-1–dependent cells should bemoresensitive to isothiocyanates than other cell lines. Therefore, wetested the isothiocyanate sensitivity of various cell lines (Fig. 4Aand B). BaF3, BaF3/p210, and K562 cells are all dependent onMcl-1 (35, 36), and these cells are the most sensitive to BITC andPEITC, with EC50 values of 1 to 3 mmol/L (Fig. 4A and B). Incontrast, MCF7, NIH3T3, and COS1 cells are 8- to 40-fold lesssensitive to BITC and PEITC, as expected given that these cells donot rely on Mcl-1. Importantly, MCF7 cells rely on the Mdm2,which is protected from ubiquitination and degradation by USP7(14). As shown above, BITC and PEITC do not inhibit USP7,which explains the resistance of these cells.

USP9x inhibition occurs early in the isothiocyanate anticancerprogram

We further examined the effects of isothiocyanates onBaF3/p210 cells to determine whether the USP9x inhibition isan initiating event in their anticancer program. USP9x levelsremained stable upon treatment with BITC (Supplementary Fig.S5F). USP9x inhibition was evident after treatment with either

BITC or PEITC for 4 hours (Fig. 4C and D). The values of IC50 forUSP9x inhibition in cells were 4� 1 mmol/L and 7� 2 mmol/L forBITC and PEITC, respectively. These values are smaller than thoseobtained in lysates (see above). However, cells are known toconcentrate isothiocyanates by factors of hundreds (4), which canaccount for this higher potency. Importantly, the inhibition ofUSP9x in cells is similar to the inhibition of cell proliferation andviability.

As in other cell types, Mcl-1 exists as two major bands inBaF3 cells (37). The fast-mobility (FM) isoform is an N-terminally truncated product formed from the full-length orslow-mobility (SM) isoform. The fast-mobility isoform hasattenuated antiapoptotic activity as well as a significantly longerhalf-life (37). A corresponding decrease in total Mcl-1 was alsoobserved when BaF3/p210 cells were treated with BITC andPEITC (Fig. 5A–C). In contrast to this isothiocyanate-induceddecrease in Mcl-1, treatment with the proteasome inhibitorsMG-132 and bortezomib increased Mcl-1 levels, as observed byothers (Fig. 5B–D; refs. 38–40). Bortezomib also induced theappearance of a lower-molecular-weight caspase cleavage Mcl-1fragment thatwasnotobservedwith either isothiocyanate (Fig. 5D;ref. 41). MG-132 rescued Mcl-1 levels in isothiocyanate-treatedcells, as expected if inhibition of USP9x increased ubiquitinationand degradation (Fig. 5B and C). Importantly, BITC also decreasedthe levels of Mcl-1 in the presence of the translation inhibitor

Figure 4.Mcl-1–dependent cells are more sensitive to BITC and PEITC. A, cells were incubated with BITC for 48 hours. With the exception of K562 cells, viability wasmeasured using the Alamar Blue method. Viability of K562 cells was measured with Promega CellTiterGlo assay. Data are presented as mean � SD of at leasttriplicate samples. B, as in A, except cells were incubated with PEITC for 48 hours. C, lysates were prepared from BaF3/p210 cells treated with PEITC orBITC for 4 hours. Samples were treated with Cy5-UbVME (250 nmol/L) and resolved by SDS-PAGE. Data presented are the mean� SD of at least two independentexperiments. D, a representative in-gel scan for the experiment described in C.

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cycloheximide, further indicating that BITC promotes the degra-dation of Mcl-1 (Supplementary Fig. S7A–S7C). Similar resultswere obtained when BaF3 and K562 cells were treated with iso-thiocyanates (Supplementary Fig. S7D–S7F).

The inhibition of USP9x should increase the ubiquitination ofMcl-1 (33). COS1 cells expressing Flag-Mcl-1 were treated withisothiocyanates for 2 hours and Flag-Mcl-1 was isolated byimmunoprecipitation. Both BITC and PEITC, as well as WP1130,

Figure 5.Isothiocyanates promote the ubiquitination of Mcl-1. A, BaF3/p210 cells were incubated with PEITC or BITC for 4 hours. Whole-cell lysates were analyzedby SDS-PAGE andWestern blotting. 0 mmol/L indicates 0.1% DMSO vehicle only. Data are representative of four independent experiments. B, BaF3/p210 cells weretreated with BITC (15 mmol/L), BITC (15 mmol/L) combined with MG-132 (20 mmol/L), or MG-132 (20 mmol/L) alone for 4 hours. Whole-cell lysates were analyzed bySDS-PAGE and immunoblotted as indicated. Control denotes 0.1% DMSO vehicle only. Actin is shown as a loading control. Data are representative of twoindependent experiments. C, quantification of B, mean � range of two experiments analyzed using the Student t test. D, BaF3/p210 cells were treated withBITC (15 mmol/L), BITC (15 mmol/L) combined with bortezomib (20 nmol/L), or bortezomib (20 nmol/L) alone for 4 hours. Whole-cell lysates were analyzed bySDS-PAGE and Western blotting. Control denotes 0.1% DMSO vehicle only. Actin is shown as a loading control. Data are representative of two independentexperiments. E, COS1 cells transiently transfected with Flag-MCL1 were treated with PEITC (15 mmol/L), PEITC (15 mmol/L) together with bortezomib (300 nmol/L),bortezomib alone (300 nmol/L), BITC (15 mmol/L), BITC (15 mmol/L) together with bortezomib (300 nmol/L), or with WP1130 (5 mmol/L) for 2 hours.Flag-MCL1 was immunopreciptated and blotted for K48-linked Ub and Flag. Control, DMSO vehicle only. �, indicates immunoprecipitation (IP) of untransfectedcells as an additional control. Data are representative of three independent experiments by two researchers.






increased the levels of ubiquitinated Flag-Mcl-1 in a concentra-tion-dependent manner (Fig. 5E and Supplementary Fig. S8).Bortezomib alone also increased Flag-Mcl-1 ubiquitination, andthe addition of bortezomib further increased ubiquitinated Flag-Mcl-1 in isothiocyanate-treated cells. These data indicate that theisothiocyanate-induced decrease in Mcl-1 results from increasedubiquitination, as expected when USP9x is inhibited. Theseobservations provide additional evidence that the mechanism ofisothiocyanates is distinct from proteasome inhibition or trans-lation inhibition and suggest that USP9x inhibition can accountfor the anticancer activity of isothiocyanates in leukemia cells.

Isothiocyanates increase ubiquitination and degradation ofBcr-Abl

PEITC has been reported to cause the knockdown of Bcr-Ablkinase, the oncogenic fusion protein that causes chronic mye-logenous leukemia (10). Both BaF3/p210 and K562 cellsdepend on Bcr-Abl, so the isothiocyanate sensitivity of thesecells could derive from the depletion of Bcr-Abl as well asMcl-1. We hypothesized that DUB inhibition could also beresponsible for this isothiocyanate effect. Intriguingly, USP9xhas been implicated in the regulation of Bcr-Abl (34, 42).Therefore we examined the correlation of Bcr-Abl ubiquitina-tion, PARP cleavage, and USP9x inhibition.

Both BITC and PEITC decreased the levels of total Bcr-Abl inBaF3/p210 cells (Fig. 6A–C and Supplementary Fig. S9A). Bcr-Ablknockdown was essentially complete after only 2-hour treatmentwith BITC (5 mmol/L), whereas PARP cleavage was not observeduntil 6 hours (Fig. 6D). Likewise, Bcr-Abl was reduced to 20%of its initial level within 2 hours after treatment with PEITC(5 mmol/L), and PARP cleavage was observed at 4 hours (Fig.6D). No aggregation of Bcr-Abl kinase was observed at lowisothiocyanate concentrations (5 mmol/L), although aggregationwas evident at high isothiocyanate concentrations (30 mmol/L;Supplementary Fig. S9B–S9D).

Two complimentary approaches were used to determinewhether isothiocyanate treatment increases ubiquitination ofBcr-Abl. First, Bcr-Abl was immunoprecipitated from lysates ofBaF3/p210 cells. Significantly, more ubiquitination was observedin Bcr-Abl isolated from isothiocyanate-treated cells than untreat-ed cells (Fig. 6E and Supplementary Fig. S9E). Bcr-Abl ubiquitina-tion predominantly involves K63 linkages, so we used RAP80-UIM–conjugated agarose resin to isolate K63-linked ubiquiti-nated proteins (34, 43). More polyubiquitinated Bcr-Abl wasrecovered from isothiocyanate-treated BaF3/p210 cells than fromuntreated cells (Fig. 6F and Supplementary Fig. S9F and S9G). Asimilar increase in Bcr-Abl ubiquitination was observed whenK562 cells were treated with BITC and PEITC (Supplementary Fig.S10). Collectively, these observations demonstrate that isothio-cyanate treatment increases the ubiquitination and degradation/aggregation of Bcr-Abl, strongly suggesting that the antileukemiaprogram of isothiocyanates results from DUB inhibition. Impor-tantly, USP9x inhibition occurred on the same time scale asBcr-Abl knockdown at low isothiocyanate concentrations (Fig.6Cand Supplementary Fig. S7E). Thus,USP9x inhibition could beresponsible for the increased ubiquitination of Bcr-Abl.

USP9x knockdown mimics the action of isothiocyanatesTo further elucidate the role of USP9x inhibition in the knock-

down of Mcl-1 and Bcr-Abl kinase, we used siRNA to decrease thelevel of USP9x in K562 cells. Optimal silencing occurred 24 hours

posttransfection in these rapidly proliferating cells, resulting in a70% decrease of USP9x (Fig. 7A and B and Supplementary Fig.S11A and S11B). The level of Mcl-1 decreased by approximately30% in the USP9x silenced cells, as reported by others (Fig. 7B;refs. 33, 34). Bcr-Abl was also reduced by 30% in USP9x silencedcells. No insoluble Bcr-Abl aggregates were observed (Supple-mentary Fig. S11C). Mcl-1 and Bcr-Abl were also depleted whenUSP9x was silenced in BaF3/p210 cells (Fig. 7C and D andSupplementary Fig. 11D). Moreover, USP9x silencing resulted insignificantly increased cell death in BaF3/p210 cells (Supplemen-tary Fig. S11E), suggesting that the isothiocyanate-induced inhi-bition of USP9x can, at least partially, account for the reducedviability and increased cell death observed upon isothiocyanatetreatment. USP9x knockdown sensitizes cells to isothiocyanates,as expected if USP9x was the primary target (Fig. 7E). However,electroporated cells appeared to be less sensitive to isothiocya-nates (Fig. 7E vs. Supplementary Fig. S11F). We suggest twoexplanations for this observation: first, electroporation causessome cell death, which increased background and decreased assaysensitivity; second, viability was measured after a short isothio-cyanate treatment (6 vs. 24 or 48 hours) to keep in the range ofoptimal siRNA-induced Bcr-Abl knockdown. Knockdown of Bcr-Abl was also observed when USP9x expression was silenced inNIH/3T3 cells stably transfected with the Bcr-Abl gene (3T3/p210cells; Supplementary Fig. S11G). Thus, experiments in three celllines, with RNAi targeting USP9x from both human and mousecells lines, confirm the role of USP9x in maintaining the levels ofBcr-Abl.

DiscussionThe anticancer effects of BITC and PEITC are well established.

Although isothiocyanates were generally believed to depleteglutathione, inducing ROS, this mechanism has recently beendiscredited (11, 12). We recognized that many of the effectsattributed to isothiocyanates are also properties of DUB inhibi-tors. Our data demonstrate that BITC and PEITC inhibit at leasttwoDUBs that are potential anticancer targets, UCH37andUSP9x(30–32). Moreover, a strong correlation exists between elevatedUSP9x levels and poor prognosis (31). While the role of UCH37in cancer is not well understood, USP9x protects the antiapopto-tic/prosurvival protein Mcl-1 from ubiquitination and depletion(32, 33). Thus, the inhibition of USP9x can account for theisothiocyanate-induced decrease in Mcl-1. Leukemia cells aredependent on Mcl-1 and especially sensitive to isothiocyanates.However, Mcl-1 levels are elevated in many cancers, so theinhibition of USP9x provides a molecular mechanism for thebroader anticarcinogenic activity of BITC and PEITC.

Isothiocyanates also increase the ubiquitination of Bcr-Abl,causing depletion via degradation at low concentrations andaggregation at high concentrations. A partial knockdown ofUSP9x also depletes Bcr-Abl. The simplest explanation for thisobservation is that Bcr-Abl is a substrate for USP9x, although wecannot rule out the involvement of other DUBs. The DUB inhib-itor WP1130 also increases the ubiquitination and aggregation ofBcr-Abl, although this work was unable to demonstrate thatUSP9x inhibition accounted for these effects (34).

In addition to inducing growth arrest and apoptosis in cancercells, isothiocyanates perturb the inflammatory response (1) andDNA repair (3, 6). DUBs also regulate these processes. At leasteight DUBs are involved in inflammation (CYLD, A20, Cezanne,

Lawson et al.





USP21, OTULIN, OTUD5, MCPIP1, and USP9x; refs. 44, 45).Eight DUBs have also been implicated in DNA repair (OTUB1,USP1, USP3, USP11, USP16, USP47, BRCC36, and POH1).Given the multitude of physiologic processes that are regulatedby ubiquitination (6), DUB inhibition is likely to be themolecular mechanism underlying the pleiotropic effects ofdietary isothiocyanates. It is important to note that our studyfocused on just two structurally similar isothiocyanates and twoDUB targets. Other DUB targets remain to be identified. Morestructurally diverse isothiocyanates such as sulphoraphane

could inhibit different cysteine proteases or even other enzymeswith cysteine nucleophiles.

DUBS, like other cysteine proteases, are challenging targets fordrug discovery because potent inhibition usually requires thepresence of electrophillic "warheads" that can react nonspecifi-cally with other proteins (46). Many DUB inhibitors fall into thiscategory. For example, G5 (47) and b-AP15 (48) are highlyreactive dienones, which can cause cross-linking. WP1130 (29)is an activated enone that has off-target effects (49). Only ahandful of selective DUB inhibitors have emerged, including the

Figure 6.DUB inhibition is an early event in isothiocyanate-induced knockdown of Bcr-Abl. A, whole-cell lysates from BaF3/p210 cells treated with PEITC or BITC for 4 hourswere resolved by SDS-PAGE and immunoblotted as indicated. Data are representative of three independent experiments. B, lysates from BaF3/p210 cellstreated with PEITC (5 mmol/L) were resolved by SDS-PAGE and immunoblotted as indicated. Data are representative of two independent experiments. C,quantification of blots shown in B and in Supplementary Fig. S9A. Data are presented as mean� range of two independent experiments. D, quantification of blotsshown in B and in Supplementary Fig. S9A. Data are presented as mean � range of two independent experiments. E, BaF3/p210 cells were treated with 5 mmol/LPEITC or BITC for 1 hour. Bcr-Abl was immunoprecipitated from lysates (1 mg/mL) with anti-cAbl antibody. Actin is shown as a loading control. Control, DMSO,vehicle only. Data are representative of two independent experiments. F, BaF3/p210 cells were treated with 5 mmol/L PEITC or BITC for 1 hour. K63-linkedpolyubiquitinated proteins were isolated from lysates (1 mg/mL) using RAP80-UIM agarose. The presence of ubiquitinated Bcr-Abl is indicated with the bracket.Data are representative of two independent experiments and were repeated using K562 cells (see Supplementary Fig. S10C and S10D).






Figure 7.siRNA-mediated silencing of USP9x decreases levels of Mcl-1 and Bcr-Abl. A, K562 cells (1.5� 106 cells)were transfected with nontargeting siRNA (Scr., 380 nmol/L)or with USP9x siRNA (380 nmol/L) using the Amaxa Nucleofector II (Program T-016). "MOCK" indicates cells that were subject to electroporation with no siRNA.Cells were harvested 24 hours posttransfection and immunoblotted as indicated. Data are representative of more than seven independent experiments.B, K562 cells (1.5 � 106 cells) were transfected with nontargeting siRNA (Scr., 380 nmol/L) or with USP9x siRNA (380 nmol/L) as in A, harvested 24 hoursposttransfection, and analyzed by SDS-PAGE and immunoblot. Data presented are the mean � SD of three independent experiments; two were performed induplicate and were analyzed using the Student t test. Representative blot shown in A. C and D, BaF3/p210 cells (2� 106 cells) were transfected with nontargetingsiRNA (Scr., 1.5 mmol/L) or with USP9x siRNA (1.5 mmol/L or 380 nmol/L) using the Amaxa Nucleofector II (Program X-001). "MOCK" indicates cells that weresubject to electroporation with no siRNA.�. no treatment control. Forty-eight hours after transfection, cell lysates were analyzed by SDS-PAGE and immunoblottedas indicated. a-Tubulin is shown as a loading control. Mcl-1 protein level was determined by normalizing the top Mcl-1 band (long form with antiapoptoticfunction) to the loading control. Data are representative of twoexperiments. E, BaF3/p210 cells (2� 106 cells)were transfectedwith nontargeting siRNA (Scr) orwithUSP9x siRNA. Forty-eight hours posttransfection, cells were treated with BITC or PEITC (100 mmol/L) for 6 hours and then analyzed with CellTiterGlo.

Lawson et al.





USP14-specific DUB inhibitor IU1, the USP7-specific inhibitorsP5091 andHBX 19,818, and the USP1 inhibitor ML323 (50). Thereversible nature of isothiocyanate adducts and the relatively lowintrinsic toxicity of this function offer a promising new avenue forthe design of inhibitors for DUBs and other cysteine proteases.Since more than 120 isothiocyanate are available from bothdietary and other natural sources, these compounds form a richresource for lead compounds, drugdiscovery, and functional fooddesign.

Disclosure of Potential Conflicts of InterestF. El Oualid is a guest scientist (starting August 12, 2015) at the Nether-

lands Cancer Institute; has ownership interest in Shares in UbiQ (Bio)company; and gave expert testimony for Netherlands Cancer Institute (untilAugust 11, 2015). No potential conflicts of interest were disclosed by theother authors.

Authors' ContributionsConception and design: A.P. Lawson, M.J.C. Long, R.T. Coffey, L. HedstromDevelopment of methodology: A.P. Lawson, M.J.C. Long, R.T. CoffeyAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): A.P. Lawson, M.J.C. Long, R.T. Coffey, Y. Qian,E. Weerapana

Analysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): A.P. Lawson, M.J.C. Long, R.T. Coffey, Y. Qian,E. Weerapana, L. HedstromWriting, review, and/or revision of the manuscript: A.P. Lawson, M.J.C. Long,R.T. Coffey, F. El Oualid, L. HedstromAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): A.P. Lawson, M.J.C. Long, Y. Qian, F. El OualidStudy supervision: L. Hedstrom

AcknowledgmentsThe authors thank Arifa Ahsan for the TOC graphic, the Ploegh laboratory for

the ubiquitin intein plasmid, and Nelson Lau and Yuliya Sytnikova for assis-tance with the knockdown experiments.

Grant SupportThis study was funded by the NIH (grant GM100921 to L. Hedstrom) and

a Sprout Grant from Brandeis University (M.J.C. Long). M.J.C. Long wassupported by a Howard Hughes Medical Institute International StudentResearch Fellowship.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received June 8, 2015; revised August 24, 2015; accepted August 31, 2015;published OnlineFirst November 5, 2015.

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Lawson et al.




2015;75:5130-5142. Published OnlineFirst November 5, 2015.Cancer Res Ann P. Lawson, Marcus J.C. Long, Rory T. Coffey, et al. Inhibiting Deubiquitinating EnzymesNaturally Occurring Isothiocyanates Exert Anticancer Effects by

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