The anticancer properties of cruciferous vegetables are well known and attributed to an abundance of isothiocyanates such as benzyl isothiocyanate (BITC) and phenethyl isothiocyanate (PEITC). While many potential targets of isothiocyanates have been proposed, a full understanding of the mechanisms underlying their anticancer activity has remained elusive. Here we report that BITC and PEITC effectively inhibit deubiquitinating enzymes (DUB), including the enzymes USP9x and UCH37, which are associated with tumorigenesis, at physiologically relevant concentrations and time scales. USP9x protects the antiapoptotic protein Mcl-1 from degradation, and cells dependent on Mcl-1 were especially sensitive to BITC and PEITC. These isothiocyanates increased Mcl-1 ubiquitination and either isothiocyanate treatment, or RNAi-mediated silencing of USP9x decreased Mcl-1 levels, consistent with the notion that USP9x is a primary target of isothiocyanate activity. These isothiocyanates also increased ubiquitination of the oncogenic fusion protein Bcr-Abl, resulting in degradation under low isothiocyanate concentrations and aggregation under high isothiocyanate concentrations. USP9x inhibition paralleled the decrease in Bcr-Abl levels induced by isothiocyanate treatment, and USP9x silencing was sufficient to decrease Bcr-Abl levels, further suggesting that Bcr-Abl is a USP9x substrate. Overall, our findings suggest that USP9x targeting is critical to the mechanism underpinning the well-established anticancer activity of isothiocyanate. We propose that the isothiocyanate-induced inhibition of DUBs may also explain how isothiocyanates affect inflammatory and DNA repair processes, thus offering a unifying theme in understanding the function and useful application of isothiocyanates to treat cancer as well as a variety of other pathologic conditions. Cancer Res; 75(23); 5130–42. ©2015 AACR.
The dietary consumption of cabbage, broccoli, and other cruciferous vegetables is associated with a decreased risk of cancer (1–3). The chemoprotective properties of these vegetables are attributed to isothiocyanates such as benzyl isothiocyanate (BITC), phenethyl isothiocyanate (PEITC), and sulforaphane (Fig. 1A; refs. 1, 2). Plasma concentrations of isothiocyanates can reach 0.25 μmol/L from a single serving of broccoli, and intracellular concentrations can be 200-fold higher due to concentrative processes (3, 4). Numerous studies demonstrate that these compounds have antiproliferative activity against tumors in both cell culture and animal models (2, 3), and PEITC has entered clinical trials for lung and oral cancers (3). Isothiocyanates induce apoptosis in many cancer cell lines, and exposure to BITC or PEITC for only 3 hours inhibits cell growth with EC50 values of 1.8 to 17 μmol/L (5). Sulforaphane also inhibits growth under these conditions, although the values of EC50 are typically much higher (50 μmol/L). Isothiocyanates perturb many cellular processes, including DNA repair (3, 6), autophagy (2), the inflammatory response (1), and the antioxidant response (1, 2). Isothiocyanates also modulate the activity of several oncogenic proteins. For example, both PEITC and BITC reduce the levels of the antiapoptotic protein Mcl-1 in leukemia cells (7–9), and PEITC induces the knockdown of Bcr-Abl kinase, the oncogenic fusion protein that causes chronic myeloid leukemia (10).
The molecular mechanisms underlying the anticancer properties of isothiocyanates are under debate (1). Isothiocyanates are electrophiles that form reversible adducts with small-molecule thiols such as glutathione (Fig. 1B; ref. 3). Amines can form stable adducts with isothiocyanates, although this reaction is not facile at neutral pH. Depletion of glutathione, and resulting generation of reactive oxygen species (ROS), is an appealing mechanism for the anticancer activities of isothiocyanates (11). However, l-butathionine sulfoximine depletes glutathione and induces ROS to greater extents than PEITC yet does not induce apoptosis (12). This finding discredits glutathione depletion/ROS production as the mechanism of anticancer activity. Isothiocyanates can also modify proteins at thiol and amine residues. At least 30 proteins have been reported to be potential isothiocyanate targets, including P450s, glutathione reductase, thioredoxin reductase, mutant p53, migration-inhibitory factor, protein phosphatases, and tubulin (1), but the functional consequences of isothiocyanate modification are usually unknown and the correlation with cellular phenotypes uncertain. Moreover, the reversible nature of isothiocyanate–thiol adducts suggests that Cys-modified proteins were unlikely to be identified in previous experiments. Therefore, the wide array of potential isothiocyanate targets does not satisfactorily explain the pleiotropic cellular effects of isothiocyanates.
Catalytic cysteine residues are generally very nucleophillic and react readily with electrophiles, so enzymes with catalytic cysteine residues are good candidates for isothiocyanate targets. Cysteine proteases are particularly attractive candidates for isothiocyanate inhibition because the thiol adduct resembles the thioester intermediate of peptide hydrolysis (Fig. 1B and C). The C=S bond is longer and more electronegative than C=O and thus may resemble the transition state for peptide hydrolysis, potentially providing additional binding energy. Isothiocyanates are weak reversible inhibitors of papain (13), the prototypical cysteine protease from papaya, but the effects of isothiocyanates on other cysteine proteases have not been investigated. Papain is distantly related to deubiquitinating enzymes (DUB), the hydrolases that remove ubiquitin from target proteins and disassemble ubiquitin chains. DUBs regulate many important physiologic processes, including protein degradation, DNA repair, autophagy, and protein trafficking (6, 14) and are potential targets for the treatment of many diseases, including cancer (15), neurodegeneration, inflammation, and infection (14). We recognized that many of the phenotypes associated with dietary isothiocyanates are also observed when cells are treated with DUB inhibitors. Therefore, we hypothesized that DUBs might be targets of isothiocyanates.
Here, we report that both BITC and PEITC inhibit USP9X and UCH37 and other DUBs at physiologically relevant concentrations and time scales. DUB inhibition provides a molecular mechanism for the anticancer properties of dietary isothiocyanates.
Materials and Methods
Detailed methods are included in the Supplementary Material.
All chemicals and reagents were from Sigma Aldrich unless otherwise stated. Solvents (except DMSO) were from Fisher. Other reagents used in this study: G5 isopeptidase inhibitor 1 (50-230-7928, Calbiochem); PEITC (Acros Organics); bortezomib (Millennium Pharmaceuticals); Mini-Complete and PhosSTOP inhibitory cocktails (Roche Applied Science); bortezomib (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, and 20S human proteasome (Boston Biochem); normal goat IgG SC-2028 (Santa Cruz); TAMRA-ubiquitin propargylamide and Cy5-ubiquitin vinyl methyl ester (UbiQ); HA-ubiquitin vinylsulfone and HA-ubiquitin vinyl methyl ester were synthesized using standard methods previously described (16). The plasmid encoding the HA-1-75Ub-Intein-chitin–binding domain fusion protein was a gift from Prof. H. Ploegh (Whitehead Institute, Cambridge, MA). BaF3 and BaF3/p210 cells were provided by Nathaniel Gray (Harvard University, Cambridge, MA).
The following antibodies were used: anti-K48-linked ubiquitin, clone APU2 and anti-K63-linked ubiquitin, clone APU3 (Millipore); anti-PARP 9542, anti-cAbl 2862, anti-α-tubulin 2156, anti-Mcl-1 D35A5, anti-Flag 2368 (Cell Signaling Technologies); anti-actin clone AC-40 A3853, anti-GAPDH clone G9295, and anti-HA, Clone 3F10 (Roche); anti-ubiquitin, clone 6C1.17 (BD Pharmingen); anti-HSP70, anti-USP7, anti-UCH37, anti-USP24, and anti-USP9x (all rabbit monoclonal; Abcam); and horseradish peroxidase (HRP)-conjugated secondary antibodies (Abcam).
Tissue culture assays and preparation of cell lysates
B16/F10 and MCF7 cells were purchased from ATCC. BaF3 and BaF3/p210 cells were provided by Dr. Nathaniel Gray (Harvard Medical School, Boston, MA; obtained 2013), K562 cells were provided by Jeffrey Strovel (Avalon Pharmaceuticals; obtained in 2011), HeLa cells were provided by Benjamin F. Cravatt (The Scripps Research Institute; obtained in 2010), NIH/3T3 cells were provided by Dr. Rubio Ren (Brandeis University, Waltham, MA; obtained in 2012), and COS1 were provided by Dr. Daniel Oprian (Brandeis University; obtained in 2010). Cell lines were authenticated (9-Marker STR May 2015). The genetic profiles of K562, MCF-7, HeLa, BaF3, BaF3/p210, and NIH/3T3 cells were identical to reported genetic profiles. COS1 cells were confirmed to be of African green monkey in origin and free of all interspecies contamination.
Cells were cultured in DMEM (HeLa, COS1, NIH/3T3, B16-F10, and MCF-7) or RPMI (BaF3, BaF3/p210 and K562) supplemented with 10% heat-inactivated FBS (DBS was used for NIH/3T3 cells), 1× GlutaMAX, and 1% penicillin/streptomycin at 37°C in a 5% CO2 humidified atmosphere. BaF3 cells were also supplemented with 1 ng/mL recombinant mouse IL3 (rmIL3, R&D Systems).
For lysate preparation, nonadherent cells were harvested by centrifugation, resuspended in 10 mmol/L HEPES (pH 7.9), 5 mmol/L MgCl2, 140 mmol/L KCl, 1% NP-40, protease, and Phosphatase Inhibitor Cocktail II, lysed using 3× freeze thaw cycles, and clarified by centrifugation. Whole-cell lysates were prepared by adding 0.1% SDS to the cells together with supernatant followed by sonication. Protein was analyzed by Western blotting (protein, antibody dilution): K48-linked ubiquitin (6 μg, 1:9,000 antibody dilution; 30–40 μg, 1:20,000), PARP (30–40 μg, 1:1,500), K63-linked ubiquitin (30–40 μg, 1:1,500), Mcl-1 (30–40 μg, 1:1,000), FLAG (30–40 μg, 1:6,000), cAbl (30–40 μg, 1:1,000), and ubiquitin (10–20 μg, 1:14,000). Signals were normalized to actin (1:10,000 for 6 μg lysate; 1:30,000 for 30–40 μg lysate), α-tubulin (1:8,000), or GAPDH (1:35,000).
COS1 cells were transfected with an expression plasmid for ubiquitinG76V-GFP (plasmid 11941 from Addgene, from the laboratory of Nico Dantuma) using Mirus 2020 (Madison, WI). FACS was carried out on a Beckman FACS Calibur. All data were analyzed using FlowJo V10, from TreeStar.
20S proteasome assay
Proteasome activity was measured by monitoring the hydrolysis of Suc-Leu-Leu-Val-Tyr-AMC in 50 mmol/L potassium phosphate, pH 7.6, 50 mmol/L NaCl, 1 mmol/L dithiothreitol (DTT) at 25°C. The K0.5 value for human 20S proteasome was experimentally determined to be 12 ± 2 μmol/L (Hill coefficient = 2), in good agreement with literature values (17). The apparent K0.5 of proteasome activity of rabbit reticulocyte lysate was 28 ± 3 μmol/L (Hill coefficient = 1.3).
Activity profiling of reactive cysteines
HeLa cell lysates were treated with either DMSO or PEITC (20 μmol/L), followed by 100 μmol/L of IA-alkyne. Click chemistry and peptide analysis was performed as described previously (18, 19).
DUB activity profiling
Cell 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 immediately quenched 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. Cell pellets were then lysed with glass beads in ice-cold 75 mmol/L K2HPO4, pH 7.5, 150 mmol/L NaCl, and 250 mmol/L sucrose. The clarified supernatant was incubated with Cy5-UbVME (250 nmol/L) for 5 minutes at 37°C. Aliquots were quenched and treated as above. Western blot analysis was carried out using standard methods. In-gel fluorescent scans were obtained using a GE Typhoon scanner.
Recombinant DUB assays
The hydrolysis of Ub-AMC was measured by monitoring the production of AMC at 37°C in a black 96-well plate. Assay buffer contained 50 mmol/L HEPES, pH 7.6, 100 mmol/L NaCl, 0.75 mmol/L BME. The fluorescence intensities were quantified with the appropriate AMC standard curves.
Measured using the Alamar Blue Method as described in the Supplementary Methods.
For Mcl-1 immunoprecipitations, COS1 cells were transiently transfected with an expression plasmid for 3× FLAG-tagged mouse Mcl-1 (Addgene plasmid 32978, from the lab of Joseph Opferman) using Mirus 2020. 3× Flag–Mcl-1 was immunoprecipitated with anti-Flag M2 magnetic beads and eluted with 500 μg/mL 3×Flag peptide.
For the isolation of polyubiquitinated proteins, BaF3/p210 cells were incubated with 5 μmol/L BITC or PEITC or with 0.1% DMSO for 1 hour at 37°C. K562 cells were incubated with 5 μmol/L BITC or PEITC or with 0.1% DMSO for 2 hours at 37°C. Poly-K63–linked ubiquitinated proteins were enriched with RAP80-UIM agarose (25 μL resuspended slurry). For Bcr-Abl immunoprecipitation, the PEITC-, BITC- (5 μmol/L), or DMSO-treated cell lysates were adjusted to the same protein concentration (1 mg/mL) and precleared with protein G (1 hour at 4°C). The cleared lysate was incubated with anti-cAbl (1 μL/100 μL lysate) overnight at 4°C. cAbl was immunoprecipitated with protein G beads.
Cell transfection and RNA interference
BaF3/p210 and K562 cells were transfected with siRNAs using the Amaxa Nucleofector II (Amaxa). NIH/3T3/p210 cells were transfected using Dharmafect 1 (GE Dharmacon). Predesigned ON-TARGET plus siRNA pools (nontargeting and targeting USP9x) were obtained from Dharmacon. The following siRNA pools (Ms or Hu, target sequence) were used:
Ms si-USP9x no. 09, 5′-CAGCAAAACUGUUCGUCAA-3′;
Ms si-USP9x no. 10, 5′-GGGCUAACGAUCUCAUUUA-3′;
Ms si-USP9x no. 11, 5′-GCUAAUGUGUAAAUGGCAA-3′;
Ms si-USP9x no. 12, 5′-GAUGAGGCUUCAAGAUAUA-3′;
Hu si-USP9x no. 06, 5′-AGAAAUCGCUGGUAUAAAU -3′;
Hu si-USP9x no. 07, 5′-ACACGAUGCUUUAGAAUUU -3′;
Hu si-USP9x no. 08, 5′-GUACGACGAUGUAUUCUCA -3′;
Hu si-USP9x no. 09, 5′-GAAAUAACUUCCUACCGAA -3′;
si-nontargeting no. 1, 5′-UGGUUUACAUGUCGACUAA-3′
si-nontargeting no. 2, 5′-UGGUUUACAUGUUGUGUGA-3′
si-nontargeting no. 3, 5′-UGGUUUACAUGUUUUCUGA-3′
si-nontargeting no. 4, 5′-UGGUUUACAUGUUUUCCUA-3′
MSCV-p210-IRES-GFP vector obtained from the Ren laboratory (Brandeis University; ref. 20) was used to produce retroviral pseudovirus. NIH/3T3 cells were transduced with virus GFP–expressing NIH/3T3 cells collected on a FACS Aria Flow Cytometer.
Isothiocyanates increase the high-molecular-weight ubiquitin pool
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 of K48-linked ubiquitination, but as yet only DUB inhibitors are known to cause the accumulation of K63-linked ubiquitination. We examined the effects of BITC and PEITC on the HMW-Ub in the pro-B-cell line BaF3/p210, which expresses Bcr-Abl kinase. Both BITC and PEITC caused the accumulation of K48-linked HMW-Ub (Fig. 2A and B) while sulforaphane had no effect. Increases in K48-linked Ub could be observed within 4 hours at 7 μmol/L BITC or PEITC and reached 2- to 4-fold at 15 μmol/L. Similar results were obtained in BaF3 cells (Supplementary Fig. S1A and S1B). Both isothiocyanates also caused a 9- to 18-fold increase in K63-linked Ub (Fig. 2C and D and Supplementary Fig. S1D). Treatment with the proteasome inhibitor bortezomib also increased the levels of K48-linked Ub but had no effect on K63-linked Ub (Fig. 2E). Thus, the accumulation of both K48 and K63-linked HMW-Ub strongly suggests that BITC and PEITC inhibit DUBs.
BITC and PEITC do not inhibit the flux through the ubiquitin proteasome system
The accumulation of K48-linked ubiquitin suggests that the anticancer effects of Isothiocyanates might arise from inhibition of flux through the ubiquitin–proteasome system, much like the anticancer effects of the proteasome inhibitor bortezomib. Therefore, we used the UbG76V-GFP assay to monitor 26S proteasome activity and flux through the ubiquitin–proteasome system in live cells (21, 22). This reporter protein consists of a ubiquitin linked to the N-terminus of GFP. The G76V mutation prevents the cleavage of ubiquitin from GFP, leading to its degradation by the 26S proteasome (22). When COS1 cells expressing UbG76V-GFP were treated with the proteasome inhibitor bortezomib, GFP fluorescence increased 1.5-fold, consistent with reports from other laboratories (e.g., ref. 23; Fig. 2F). In contrast, BITC and PEITC did not increase GFP levels, indicating that proteasome activity was not inhibited. Similarly, the DUB inhibitor WP1130 also did not cause an increase in GFP levels (Fig. 2F). Isothiocyanates did not inhibit purified 20S proteasome or proteasome activity in rabbit reticulocyte lysate, contrary to a previous report (Supplementary Fig. S2A–S2C; ref. 24). Collectively, these observations strongly suggest that the isothiocyanate-induced accumulation of HMW-Ub results from DUB inhibition.
PEITC does not perturb the global cysteine reactome
The methods used to identify isothiocyanate targets in previous reports were unlikely to detect modifications of cysteine residues in DUBs or other proteins. Therefore, we used competitive cysteine reactivity profiling with an iodoacetamide-alkyne (IA-alkyne) probe (100 μmol/L) to more thoroughly investigate the effects of isothiocyanates on the global cysteine reactome of HELA cells (18). More than 1,000 IA-alkyne–labeled cysteine residues were identified in HELA cells using mass spectrometric analysis. Unfortunately, only two of these cysteine residues belonged to DUBs (UCHL1 and otubain 1), alluding to the poor affinity of IA-alkyne toward the active-site cysteines in DUBs, as well as the low abundance of DUBs relative to other cysteine-containing proteins in HELA lysates. Despite low coverage of DUBs, this cysteine profiling experiment provides a measure of the general promiscuity of PEITC across highly reactive cysteine residues in the proteome. Treatment of HELA lysates with PEITC (20 μmol/L) significantly inhibited the labeling of only 14 of 1,400 profiled cysteines in at least one of two independent experiments (inhibition ≥ 67%; Supplementary Dataset S1), although in no case was labeling inhibited in both experiments. Seventy of the profiled cysteine residues are found in enzyme active sites or metal-binding sites, including six dehydrogenases and six cysteine proteases (Supplementary Dataset S2). PEITC blocked the labeling of only one of these, mitochondrial phosphoenolpyruvate carboxykinase (Supplementary Fig. S3). These experiments demonstrate that PEITC is not a promiscuous cysteine-modifying agent.
BITC and PEITC inhibit USP9x and UCH37
We 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) and HA-tagged ubiquitin vinyl methyl ester (HA-UbVME; refs. 25, 26), we also used the more sensitive fluorogenic probes Cy5-UbVME (26) and TAMRA-ubiquitin propargylamide (TAMRA-UbPA; refs. 27, 28). These irreversible DUB inhibitors label 10 to 30 DUBs in cell lysates with varying repertoires depending on the probe and experimental conditions (25). The treatment of cell lysates with HA-UbVS produced the characteristic pattern of protein bands at 300, 150 to 100, 45, and 36 kDa, generally ascribed to USP9x/USP24 (292 kDa), USP19 (146 kDa), USP7/8 (128 and 127 kDa, respectively), USP28/15 (122 and 112 kDa, respectively), UCH37 (38 kDa), UCH-L3 (26 kDa), and UCH-L1 (25 kDa; Supplementary Fig. S4A; ref. 25). Similar results, although with varying band intensities, were obtained with HA-UbVME, TAMRA-UbPA, and Cy5-UbVME (Supplementary Fig. S4B–S4D). As expected, WP1130 and isopeptidase inhibitor G5 reduced the labeling of several DUBs (e.g., bands between 120 and 300 kDa), validating the experimental method (Supplementary Fig. S4A; refs. 25, 29).
BITC and PEITC inhibited the labeling of several DUBs in lysates prepared from cells pretreated with isothiocyanates (Fig. 3A). When lysate was incubated with either BITC or PEITC and then diluted into a buffer containing HA-UbVS, the labeling increased with time, demonstrating that isothiocyanate inhibition was slowly reversible as expected (Fig. 3B). Labeled DUBs with molecular weights at 300, 135, and 45 kDa were among the most susceptible to isothiocyanates. These bands likely corresponded to three DUBs that are potential therapeutic targets for cancer: USP9x, USP7, and UCH37 (Fig. 3A, Supplementary Fig. S4A–S4D). The identities of USP9x and UCH37 were confirmed by observing the molecular weight shift upon labeling with immunoblotting (Fig. 3C and D and Supplementary Fig. S4E and S4F). USP24, a close relative of USP9x (16), was also a target of BITC and PEITC (Fig. 3E). However, immunoblotting revealed that the 128-kDa isothiocyanate target was not USP7 (Fig. 3C).
The IC50 values for the inhibition of USP9x labeling were 27 ± 6 μmol/L and 15 ± 3 μmol/L for BITC and PEITC, respectively, in cell lysates (Supplementary Fig. S5A and S5B). The values of IC50 for the inhibition of UCH37 labeling were 22 ± 4 μmol/L and 13 ± 1 μmol/L for BITC and PEITC, respectively (Supplementary Fig. S5C). The values for USP24 with IC50 values of 56 ± 5 μmol/L and 28 ± 5 μmol/L for BITC and PEITC, respectively (Fig. 3E and Supplementary Fig. S5D).
BITC and PEITC inhibit USP9x and UCH37 in vitro
The inhibition of USP9x was confirmed by assaying purified recombinant enzyme. PEITC inhibited labeling of recombinant USP9x with Cy5-UbVME with an IC50 value of 20 ± 2 μmol/L, in good agreement with the lysate assays (Supplementary Fig. S6A and S6B). Both BITC and PEITC were slow binding competitive inhibitors of recombinant USP9x, with values of Ki = 25 ± 1 and 23 ± 2 μmol/L, respectively (Supplementary Fig. S6C–S6E). The values of kon decreased with increasing substrate concentration, indicating competitive inhibition (Supplementary Fig. S6F).
UCH37 (also known as UCHL5) is a component of the 19S regulatory particle and the INO80 chromatin remodeling complex. The 19S regulatory particle activates UCH37, whereas UCH37 is inactive in the INO80 complex (30). Therefore, we assayed the DUB activity of UCH37 in the 19S regulatory particle using activity profiling. Both PEITC and BITC inhibited UCH37 with values of EC50 of 36 ± 5 μmol/L and 31 ± 6 μmol/L, in reasonable agreement with the lysate assays (Fig. 3F and Supplementary Fig. S5E). No inhibition of recombinant UCHL3 or the catalytic domain of USP7 was observed, confirming the lysate assays (Supplementary Fig. S6A and S6B).
USP9x inhibition provides a molecular mechanism for the antileukemic 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 protein Mcl-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 USP9x is either inhibited or silenced (32–34). These observations suggest that the USP9x may be a primary target for BITC and PEITC. In contrast, the role of UCH37 in cancer is not understood and could involve either or both the 19S regulatory particle and the INO80 complex or recently characterized complexes with SMAD proteins (30). Which of these complexes promote cancer, and whether the deubiquitinating activity of UCH37 is required, is not understood.
If the anticancer effects of BITC and PEITC arise from the inhibition of USP9x, then Mcl-1–dependent cells should be more sensitive to isothiocyanates than other cell lines. Therefore, we tested the isothiocyanate sensitivity of various cell lines (Fig. 4A and B). BaF3, BaF3/p210, and K562 cells are all dependent on Mcl-1 (35, 36), and these cells are the most sensitive to BITC and PEITC, with EC50 values of 1 to 3 μmol/L (Fig. 4A and B). In contrast, MCF7, NIH3T3, and COS1 cells are 8- to 40-fold less sensitive to BITC and PEITC, as expected given that these cells do not 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 anticancer program
We further examined the effects of isothiocyanates on BaF3/p210 cells to determine whether the USP9x inhibition is an initiating event in their anticancer program. USP9x levels remained 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 for USP9x inhibition in cells were 4 ± 1 μmol/L and 7 ± 2 μmol/L for BITC and PEITC, respectively. These values are smaller than those obtained in lysates (see above). However, cells are known to concentrate isothiocyanates by factors of hundreds (4), which can account for this higher potency. Importantly, the inhibition of USP9x in cells is similar to the inhibition of cell proliferation and viability.
As in other cell types, Mcl-1 exists as two major bands in BaF3 cells (37). The fast-mobility (FM) isoform is an N-terminally truncated product formed from the full-length or slow-mobility (SM) isoform. The fast-mobility isoform has attenuated antiapoptotic activity as well as a significantly longer half-life (37). A corresponding decrease in total Mcl-1 was also observed when BaF3/p210 cells were treated with BITC and PEITC (Fig. 5A–C). In contrast to this isothiocyanate-induced decrease in Mcl-1, treatment with the proteasome inhibitors MG-132 and bortezomib increased Mcl-1 levels, as observed by others (Fig. 5B–D; refs. 38–40). Bortezomib also induced the appearance of a lower-molecular-weight caspase cleavage Mcl-1 fragment that was not observed with either isothiocyanate (Fig. 5D; ref. 41). MG-132 rescued Mcl-1 levels in isothiocyanate-treated cells, as expected if inhibition of USP9x increased ubiquitination and degradation (Fig. 5B and C). Importantly, BITC also decreased the levels of Mcl-1 in the presence of the translation inhibitor cycloheximide, further indicating that BITC promotes the degradation of Mcl-1 (Supplementary Fig. S7A–S7C). Similar results were obtained when BaF3 and K562 cells were treated with isothiocyanates (Supplementary Fig. S7D–S7F).
The inhibition of USP9x should increase the ubiquitination of Mcl-1 (33). COS1 cells expressing Flag-Mcl-1 were treated with isothiocyanates for 2 hours and Flag-Mcl-1 was isolated by immunoprecipitation. Both BITC and PEITC, as well as WP1130, increased the levels of ubiquitinated Flag-Mcl-1 in a concentration-dependent manner (Fig. 5E and Supplementary Fig. S8). Bortezomib alone also increased Flag-Mcl-1 ubiquitination, and the addition of bortezomib further increased ubiquitinated Flag-Mcl-1 in isothiocyanate-treated cells. These data indicate that the isothiocyanate-induced decrease in Mcl-1 results from increased ubiquitination, as expected when USP9x is inhibited. These observations provide additional evidence that the mechanism of isothiocyanates is distinct from proteasome inhibition or translation inhibition and suggest that USP9x inhibition can account for the anticancer activity of isothiocyanates in leukemia cells.
Isothiocyanates increase ubiquitination and degradation of Bcr-Abl
PEITC has been reported to cause the knockdown of Bcr-Abl kinase, the oncogenic fusion protein that causes chronic myelogenous leukemia (10). Both BaF3/p210 and K562 cells depend on Bcr-Abl, so the isothiocyanate sensitivity of these cells could derive from the depletion of Bcr-Abl as well as Mcl-1. We hypothesized that DUB inhibition could also be responsible for this isothiocyanate effect. Intriguingly, USP9x has been implicated in the regulation of Bcr-Abl (34, 42). Therefore we examined the correlation of Bcr-Abl ubiquitination, PARP cleavage, and USP9x inhibition.
Both BITC and PEITC decreased the levels of total Bcr-Abl in BaF3/p210 cells (Fig. 6A–C and Supplementary Fig. S9A). Bcr-Abl knockdown was essentially complete after only 2-hour treatment with BITC (5 μmol/L), whereas PARP cleavage was not observed until 6 hours (Fig. 6D). Likewise, Bcr-Abl was reduced to 20% of its initial level within 2 hours after treatment with PEITC (5 μmol/L), and PARP cleavage was observed at 4 hours (Fig. 6D). No aggregation of Bcr-Abl kinase was observed at low isothiocyanate concentrations (5 μmol/L), although aggregation was evident at high isothiocyanate concentrations (30 μmol/L; Supplementary Fig. S9B–S9D).
Two complimentary approaches were used to determine whether isothiocyanate treatment increases ubiquitination of Bcr-Abl. First, Bcr-Abl was immunoprecipitated from lysates of BaF3/p210 cells. Significantly, more ubiquitination was observed in Bcr-Abl isolated from isothiocyanate-treated cells than untreated cells (Fig. 6E and Supplementary Fig. S9E). Bcr-Abl ubiquitination predominantly involves K63 linkages, so we used RAP80-UIM–conjugated agarose resin to isolate K63-linked ubiquitinated proteins (34, 43). More polyubiquitinated Bcr-Abl was recovered from isothiocyanate-treated BaF3/p210 cells than from untreated cells (Fig. 6F and Supplementary Fig. S9F and S9G). A similar increase in Bcr-Abl ubiquitination was observed when K562 cells were treated with BITC and PEITC (Supplementary Fig. S10). Collectively, these observations demonstrate that isothiocyanate treatment increases the ubiquitination and degradation/aggregation of Bcr-Abl, strongly suggesting that the antileukemia program of isothiocyanates results from DUB inhibition. Importantly, USP9x inhibition occurred on the same time scale as Bcr-Abl knockdown at low isothiocyanate concentrations (Fig. 6C and Supplementary Fig. S7E). Thus, USP9x inhibition could be responsible for the increased ubiquitination of Bcr-Abl.
USP9x knockdown mimics the action of isothiocyanates
To further elucidate the role of USP9x inhibition in the knockdown of Mcl-1 and Bcr-Abl kinase, we used siRNA to decrease the level of USP9x in K562 cells. Optimal silencing occurred 24 hours posttransfection in these rapidly proliferating cells, resulting in a 70% decrease of USP9x (Fig. 7A and B and Supplementary Fig. S11A and S11B). The level of Mcl-1 decreased by approximately 30% in the USP9x silenced cells, as reported by others (Fig. 7B; refs. 33, 34). Bcr-Abl was also reduced by 30% in USP9x silenced cells. No insoluble Bcr-Abl aggregates were observed (Supplementary Fig. S11C). Mcl-1 and Bcr-Abl were also depleted when USP9x was silenced in BaF3/p210 cells (Fig. 7C and D and Supplementary Fig. 11D). Moreover, USP9x silencing resulted in significantly increased cell death in BaF3/p210 cells (Supplementary Fig. S11E), suggesting that the isothiocyanate-induced inhibition of USP9x can, at least partially, account for the reduced viability and increased cell death observed upon isothiocyanate treatment. 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 isothiocyanates (Fig. 7E vs. Supplementary Fig. S11F). We suggest two explanations for this observation: first, electroporation causes some cell death, which increased background and decreased assay sensitivity; second, viability was measured after a short isothiocyanate treatment (6 vs. 24 or 48 hours) to keep in the range of optimal siRNA-induced Bcr-Abl knockdown. Knockdown of Bcr-Abl was also observed when USP9x expression was silenced in NIH/3T3 cells stably transfected with the Bcr-Abl gene (3T3/p210 cells; Supplementary Fig. S11G). Thus, experiments in three cell lines, with RNAi targeting USP9x from both human and mouse cells lines, confirm the role of USP9x in maintaining the levels of Bcr-Abl.
The anticancer effects of BITC and PEITC are well established. Although isothiocyanates were generally believed to deplete glutathione, inducing ROS, this mechanism has recently been discredited (11, 12). We recognized that many of the effects attributed to isothiocyanates are also properties of DUB inhibitors. Our data demonstrate that BITC and PEITC inhibit at least two DUBs that are potential anticancer targets, UCH37 and USP9x (30–32). Moreover, a strong correlation exists between elevated USP9x levels and poor prognosis (31). While the role of UCH37 in cancer is not well understood, USP9x protects the antiapoptotic/prosurvival protein Mcl-1 from ubiquitination and depletion (32, 33). Thus, the inhibition of USP9x can account for the isothiocyanate-induced decrease in Mcl-1. Leukemia cells are dependent on Mcl-1 and especially sensitive to isothiocyanates. However, Mcl-1 levels are elevated in many cancers, so the inhibition of USP9x provides a molecular mechanism for the broader anticarcinogenic activity of BITC and PEITC.
Isothiocyanates also increase the ubiquitination of Bcr-Abl, causing depletion via degradation at low concentrations and aggregation at high concentrations. A partial knockdown of USP9x also depletes Bcr-Abl. The simplest explanation for this observation is that Bcr-Abl is a substrate for USP9x, although we cannot rule out the involvement of other DUBs. The DUB inhibitor WP1130 also increases the ubiquitination and aggregation of Bcr-Abl, although this work was unable to demonstrate that USP9x inhibition accounted for these effects (34).
In addition to inducing growth arrest and apoptosis in cancer cells, isothiocyanates perturb the inflammatory response (1) and DNA repair (3, 6). DUBs also regulate these processes. At least eight DUBs are involved in inflammation (CYLD, A20, Cezanne, 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 regulated by ubiquitination (6), DUB inhibition is likely to be the molecular mechanism underlying the pleiotropic effects of dietary isothiocyanates. It is important to note that our study focused on just two structurally similar isothiocyanates and two DUB targets. Other DUB targets remain to be identified. More structurally diverse isothiocyanates such as sulphoraphane could inhibit different cysteine proteases or even other enzymes with cysteine nucleophiles.
DUBS, like other cysteine proteases, are challenging targets for drug discovery because potent inhibition usually requires the presence of electrophillic “warheads” that can react nonspecifically with other proteins (46). Many DUB inhibitors fall into this category. For example, G5 (47) and b-AP15 (48) are highly reactive dienones, which can cause cross-linking. WP1130 (29) is an activated enone that has off-target effects (49). Only a handful of selective DUB inhibitors have emerged, including the USP14-specific DUB inhibitor IU1, the USP7-specific inhibitors P5091 and HBX 19,818, and the USP1 inhibitor ML323 (50). The reversible nature of isothiocyanate adducts and the relatively low intrinsic toxicity of this function offer a promising new avenue for the design of inhibitors for DUBs and other cysteine proteases. Since more than 120 isothiocyanate are available from both dietary and other natural sources, these compounds form a rich resource for lead compounds, drug discovery, and functional food design.
Disclosure of Potential Conflicts of Interest
F. El Oualid is a guest scientist (starting August 12, 2015) at the Netherlands Cancer Institute; has ownership interest in Shares in UbiQ (Bio) company; and gave expert testimony for Netherlands Cancer Institute (until August 11, 2015). No potential conflicts of interest were disclosed by the other authors.
Conception and design: A.P. Lawson, M.J.C. Long, R.T. Coffey, L. Hedstrom
Development of methodology: A.P. Lawson, M.J.C. Long, R.T. Coffey
Acquisition 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. Hedstrom
Writing, review, and/or revision of the manuscript: A.P. Lawson, M.J.C. Long, R.T. Coffey, F. El Oualid, L. Hedstrom
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): A.P. Lawson, M.J.C. Long, Y. Qian, F. El Oualid
Study supervision: L. Hedstrom
The authors thank Arifa Ahsan for the TOC graphic, the Ploegh laboratory for the ubiquitin intein plasmid, and Nelson Lau and Yuliya Sytnikova for assistance with the knockdown experiments.
This 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 was supported by a Howard Hughes Medical Institute International Student Research Fellowship.
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