Inhibition of neddylation modification by MLN4924 sensitizes hepatocellular carcinoma cells to sorafenib.

Sorafenib remains the standard care for patients with hepatocellular carcinoma (HCC) even though it has low antitumor efficacy. Protein neddylation is abnormally activated in many types of human cancer. However, whether dysregulation of neddylation is involved in HCC progression and whether targeting neddylation sensitizes HCC cells to sorafenib need to be ascertained. In the present study, it was demonstrated that high expression of neddylation components, neural precursor cell expressed, developmentally downregulated 8 (NEDD8) and NEDD8‑activating enzyme 1 (NAE1), were associated with poor survival of patients with HCC. Inhibition of neddylation by MLN4924, a small‑molecule inhibitor of NAE1, significantly inhibited HCC growth, reduced clonogenic survival, increased apoptosis, and decreased migration capacity. Sorafenib alone exhibited minimal anticancer efficacy. However, a combination of sorafenib with MLN4924 at a low concentration significantly enhanced the inhibition of cell proliferation and migration as well as the induction of apoptosis induced by sorafenib. In vivo HCC xenograft mouse models also showed that MLN4924 increased the antitumor efficacy of sorafenib. Mechanistically, MLN4924 enhanced the antitumor activity of sorafenib in HCC cells via upregulation of cullin‑RING E3 ubiquitin ligase (CRL)/Skp1‑Cullin1‑F box (SCF) E3 ubiquitin ligase substrates p21, p27, Deptor and IκBɑ. Taken together, these findings suggest that combination therapy of MLN4924 with sorafenib appears to present an additive effect with a maximal in the treatment of HCC.

Introduction

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related morbidity and mortality worldwide. Sorafenib has long been the standard chemotherapy for HCC (1), and recent clinical trials have given rise to more effective regimens such as modified FOLFOX combined with sorafenib for the treatment of advanced HCC (2). The past few decades have witnessed a surge in the development and application of targeted therapies for a wide range of malignancies (3). With regard to HCC, only transarterial chemoembolization (TACE) has shown a modest increase in efficacy when combined with sorafenib (4). Hence there is a critical need to develop novel targeted or combination therapies for HCC.

The ubiquitin-proteasome system (UPS) is a selective proteolytic system that conjugates ubiquitin to substrates to induce degradation by the 26S proteasome. UPS regulates almost all cellular processes including apoptosis, cell division, differentiation, response to stress, DNA repair and signal transduction (5). Bortezomib, a proteasome inhibitor, has been approved for the treatment of patients with multiple myeloma (6) and mantle cell lymphoma (7), suggesting UPS inhibition is an attractive antitumor approach.

Proteins are targeted for degradation within the UPS via a three-step cascade mechanism. The ubiquitin-activating enzyme (E1) activates ubiquitin via ATP to form ubiquitin adenylate. The activated ubiquitin is transferred to the ubiquitin-transferring enzyme (E2) through a thioester bond. The ubiquitin ligase (E3) subsequently promotes the transfer of ubiquitin from E2 to the Lys of substrates (5). The Cullin-Ring ligases (CRLs) are the largest family of E3 ligases (8). Activation of CRLs requires the covalent binding of neural precursor cell expressed, developmentally downregulated 8 (NEDD8) to the core scaffolds named as cullin proteins by NEDD8-activating enzyme (NAE) (9). Therefore, inhibition of NAE would inhibit CRL-mediated UPS.

MLN4924 (TAK-924/Pevonedistat) is a first-in-class highly selective NAE inhibitor that has been evaluated in several phase I/II clinical trials (10–13). MLN4924 prevents NAE from processing NEDD8 for CRL conjugation, resulting in CRL inhibition and substrate accumulation, such as p21/p27 (14), IκBα (15) and Deptor (16). As demonstrated in several studies, MLN4924 was found to present with antitumor activities towards a variety of solid and hematologic malignancies, possibly by inducing p21 and p27 accumulation (17–19). In addition, MLN4924 was found to activate NF-κB and mammalian target of rapamycin (mTOR) activation which resulted from the accumulation of CRL substrates IκBα and Deptor (15,16,20,21). NF-κB and mTOR are two critical oncogenes required for proliferation and migration that are commonly activated in a wide variety of cancers including HCC (22–24). A previous study demonstrated that MLN4924 could inhibit HCC cell growth (25). In the present study, MLN4924 and sorafenib alone at low concentrations weakly inhibited cell proliferation, induced apoptosis and suppressed migration. Given that sorafenib has weak antitumor activity, we hypothesized that a combination of MLN4924 with sorafenib would have superior antitumor efficacy, especially towards HCC. We tested our hypothesis in vitro and in vivo. We found that MLN4924 enhanced the antitumor activity of sorafenib, possibly by inhibiting cell proliferation and migration via the upregulation of p21, p27, IκBα and Deptor.

Materials and methods

Reagents and antibodies

MLN4924 (cat. no. S7109) and sorafenib (cat. no. S7397) were purchased from Selleck Industries LLC (Shanghai, China). Fetal bovine serum (FBS) medium (cat. no. 11995500) was obtained from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Cell Counting Kit-8 (CCK-8) (cat. no. CK04) was purchased from Dojindo Laboratories (Kumamoto, Japan). Annexin V/PI kit (cat. no. 556547) was from BD Biosciences (Franklin Lakes, NJ, USA). Anti-mouse lgG-HRP (cat. no. sc-2005), anti-rabbit lgG-HRP (cat. no. sc-2004) and IκBα (cat. no. sc-371) antibodies were obtained from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). Chemiluminescent HRP substrate (cat. no. WBKLS0500) was obtained from EMD Millipore (Billerica, MA, USA). Cleaved caspase-3 (cat. no. 9661S), cleaved PARP (cat. no. 6987S), p-ERK (cat. no. 4370S), ERK (cat. no. 4695S), p21 (cat. no. 2947S), p27 (cat. no. 3686S), E-cadherin (cat. no. 14472S), N-cadherin (cat. no. 14215S), vimentin (cat. no. 3932S), Noxa (cat. no. 14766S), Deptor (cat. no. 11816S), p-4EBP1 (cat. no. 2855S), p-S6K (cat. no. 9202S), NF-κB p65 (cat. no. 8242S), NEDD8 (cat. no. 2745S) and p-mTOR (cat. no. 5536S) antibodies were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). MMP9 antibody (cat. no. 10375-2-AP) was obtained form Proteintech (Wuhan, China). NAE1 (NHA2310) was provided by Novogene Co., Ltd. (Beijing, China).

Cell culture

The human HCC LM3 and 97H cell lines were purchased from the Cell Bank of the Type Culture Collection of Fudan University (Shanghai, China) and they are neither misidentified nor contaminated. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM). All experiments were carried out in DMEM containing 10% FBS, 100 U/ml penicillin and 100 mg/ml streptomycin (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C in 5% CO2.

Cytotoxicity and clonogenic survival assays

For the cell viability assay, the cells were seeded into 96-well microtiter plates at a density of 3,000 cells/well and allowed to adhere for 12 h. Cells were then treated with different chemicals for the indicated durations and then exposed to CCK-8 (10 µl/well) for 2 h at 37°C. Absorbance was measured at 450 nm on a Tecan Sunrise microplate reader (Tecan Group AG, Männedorf, Switzerland).

For the clonogenic assay, 800 cells were plated in triplicate in 6-well plates. After overnight incubation at 37°C, different chemicals were added into the medium to reach various concentrations (0, 25, 50, 100, 300 and 1,000 nM). Medium was changed every 3–4 days while maintaining the previous concentrations. After 9–10 days of culture, cell colonies were fixed with ice-cold methanol, followed by 0.05% crystal violet staining for 15 min. Colonies containing >50 cells in each well were counted manually under an Olympus BX41 light microscope (Olympus Corp., Tokyo, Japan). A gridded plastic sheet was attached to the bottom of each well to keep track of colonies counted (26). All fields were counted.

Western blot analysis

Cells were harvested and lysed with lysis buffer [20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM DTT, 1 mM sodium orthovanadate, 1 µg/ml leupeptin and 1 mM phenylmethylsulfonyl fluoride] on ice for 1 h. Afterwards, cell lysates were centrifuged for 15 min at 11,000 × g at 4°C. Protein concentrations of the supernatants were determined using the bicinchoninic acid assay (BCA) assay. A total of 50 mg of protein loaded per lane and separated using 8–12% SDS-PAGE and then transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were then blocked with 5% non-fat milk and incubated 4°C for 12 h with the specified primary and secondary antibodies. All antibodies were diluted at 1:1,000 for western blot analysis. Protein bands were visualized using an ECL detection kit (EMD Millipore).

Annexin V and propidium iodide (PI) staining

Cells were treated with indicated chemicals and then washed twice with phosphate-buffered saline (PBS). After incubation with Annexin V-FITC in binding buffer [10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2, 0.1% bovine serum albumin (BSA) and pH 7.4] for 15 min, the cells were immediately exposed to 2 µg/ml PI for 5 min before analysis by flow cytometry. Annexin V-positive cells represented apoptotic cells and were quantified as previously described (27).

Small interfering RNA (siRNA) transfection

The siRNA oligos were purchased from Shanghai GenePharma Co., Ltd., (Shanghai, China) as follows: siNFκB p65, 5′-GAUUGAGGAGAAACGUAAAdTdT-3′; a non-target siRNA (siControl), 5′-UCUACGAGGCACGAGACUU-3′. Briefly, cells were transfected with various siRNAs in MEM medium with 90 nM of each siRNA duplex, using Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific, Inc.) following the manufacturer's protocol. After transfection for 48 h, cells were harvested for western blot analysis.

Xenograft tumor assay in vivo

Twenty-four 5 week-old male BALB/c nude mice with body weight of ~18.5 g were purchased from Beijing Bioscience Co., Ltd. (Beijing, China) and maintained with 12-h light/12-h dark cycles at a temperature of 25°C with a humidity level of 40–60% with food and water provided ad libitum in the Laboratory Animal Center of Army Medical University (Chongqing, China). Animal studies were approved by the Institutional Animal Care and Use Committee of Southwest Hospital (Chongqing, China). 97H cells (1×107 cells in 100 µl serum-free DMEM medium) were inoculated subcutaneously into the right flank of the nude mice (n=6/group). After the third day, the mice were randomized and treated with 60 mg/kg MLN4924 in 10% cyclodextrin (13), 30 mg/kg sorafenib in ethanol/castor oil (v/v 1:1) or the combination of the two chemicals via daily oral gavage.

Tumor growth (6 for each group) was measured in all three dimensions once a week for three weeks. Tumor volume was calculated using the formula V = 4/3(π)XYZ, where X, Y and Z represent the radius of the tumor in each dimension. After three weeks, the mice were placed in the euthanasia chamber before turning on gas from a CO2 tank. The flow rate of CO2 (~30% of the euthanasia chamber volume per min) was added to the existing air in the chamber. Mice were usually expected to reach unconsciousness within 2–3 min, followed by waiting at least 2 min without seeing a breath and a heartbeat. To ensure death, cervical dislocation was used following CO2 death. Tumors were harvested, photographed, weighed and the results were plotted.

Immunohistochemistry (IHC) staining

The NEDD8 and NAE expression at mRNA levels for 337 patients with HCC were obtained from the TCGA database and analyzed (Tables I and II) [The results shown here are part based upon data generated by TCGA Research Network (http://cancergenome.nih.gor)]. In order to validate the TCGA data, the HCC specimens used in this study for IHC staining were obtained from 26 patients with HCC who underwent curative resection at the Department of Hepatobiliary Surgery, Southwest Hospital. This study was approved by the Ethics Committee of Southwest Hospital. Patient information as shown in Table SI. For each patient, the diagnosis of HCC was confirmed by pathologic examination. The HCC specimens were harvested and then fixed in 10% formalin for 48 h. Then the tumor tissues were embedded and sliced at 5-µm thickness. Immunohistochemistry was performed using the ABC Vectastain kit (Vector Laboratories Inc., Burlingame, CA, USA) with rabbit anti-NEDD8 and anti-NAE1 antibodies All antibodies were diluted at 1:200 for immunohistochemistry staining. Sections were developed with DAB and counterstained with hematoxylin. Finally, the sections were photographed under a light microscope (Olympus BX41; Olympus Corp.) with magnification at ×200. The staining was evaluated by different specialized pathologists and was performed without any knowledge of the patient characteristics (28,29). For the staining intensity if no significant difference was noted for paired HCC tissues and adjacent normal tissues, the staining intensity was determined by selecting same object area using Spot Denso function of an AlphaEaseFC software (Protein Simple, San Jose, CA, USA), and the integrated density value (IDV) was compared between cancer tissues and adjacent normal tissues.

Table I.

Association of NEDD8 expression and clinicopathologic characteristics of the HCC patients.

Variables	No. of cases	High NAE1 level	Low NAE1 level	P-value
Age (years)				0.313
<55	110	45	65
≥55	227	80	147
Sex				0.575
Female	107	42	65
Male	230	83	147
Recurrence				0.183
Present	154	63	91
Absent	183	62	121
Histologic grade				0.011[a]
G1-G2	210	67	143
G3-G4	127	58	69
Tumor stage				0.763
T1-T2	253	95	158
T3-T4	84	30	54
Clinical stage				0.743
I–II	250	94	156
III–IV	87	31	56

P<0.05. HCC, hepatocellular carcinoma.

Table II.

Association of NAE1 expression and clinicopathologic characteristics of the HCC cases.

Variables	No. of cases	High NAE1 level	Low NAE1 level	P-value
Age (years)				0.261
<55	110	50	60
≥55	227	118	109
Sex
Female	107	56	51	0.534
Male	230	112	118
Recurrence				0.114
Present	154	84	70
Absent	183	84	99
Histologic grade				0.016[a]
G1-G2	210	94	116
G3-G4	127	74	53
Tumor				0.041[a]
T1-T2	253	118	135
T3-T4	84	50	34
Clinical stage				0.017[a]
I–II	250	115	135
III–IV	87	53	34

P<0.05. HCC, hepatocellular carcinoma.

Statistical analysis

The Pearson's Chi-squared test (χ2) test was used to analyze the relationship between NEDD8/NAE1 expression and the clinicopathological features of HCC cases using the SPSS 15.0 software (SPSS, Inc., Chicago, IL, USA). For cell proliferation, migration, apoptotic cells, patient survival, tumor volume as well as tumor weight, statistical analysis was performed using the Student's t-test for comparison of two groups or one-way analysis of variance (ANOVA) for comparison of more than two groups followed by Tukey's multiple comparison test. For multiple testing, a Bonferroni post hoc test of P-values was made using GraphPad Prism 6 (GraphPad, Inc., San Diego, CA, USA). Data are expressed as mean ± SEM of at least three independent experiments. A P-value <0.05 was considered to be statistically significant.

Results

High expression of NEDD8 and NAE1 is associated with poor survival of HCC patients

As activation of CRLs requires the covalent binding of NEDD8, we aimed to ascertain whether NEDD8 is related to HCC patients. We analyzed the relationship between NEDD8 expression and clinicopathologic characteristics of the HCC patients with complete information using TCGA data. The result indicated that NEDD8 expression is associated with histologic grade (Table I). Similar to this finding, NEDD8-activating enzyme (NAE) expression was found to be related to histologic grade, tumor size as well as clinical stage of HCC patients (Table II). Kaplan-Meier analysis showed that the overall survival rate was significantly low in HCC patients with high expression of NEDD8 (Fig. 1A, P=0.0472, n=337) or NAE1 (Fig. 2A, P=0.0146, n=337) compared to patients with low expression of these proteins, respectively. This suggests that high expression levels of NEDD8 and NAE1 in HCC are significantly associated with worse patient prognosis. In order to further confirm this finding, we used our department patient samples to detect NEDD8 and NAE expression levels by IHC assay. The results demonstrated that protein levels of NEDD8 and NAE1 were highly expressed in tumor tissues compared to matched adjacent non-tumor tissues (Figs. 1B and 2B).

Figure 1.

High expression level of NEDD8 is associated with poor survival of HCC patients. (A) Overall survival rate of HCC patients (n=337) based on NEDD8 as analyzed using log-rank (Mantel-Cox) test. (B) Representative images of NEDD8 expression levels in HCC tissue vs. normal adjacent tissue (n=26). Magnification, ×100. HCC, hepatocellular carcinoma. NEDD8, neural precursor cell expressed, developmentally downregulated 8.

Figure 2.

MLN4924 inhibits cell proliferation in HCC cells. (A) Overall survival rate of HCC patients based on NAE1 as analyzed using log-rank (Mantel-Cox) test. (B) Representative images of NAE1 expression levels in HCC tissue vs. normal adjacent tissue. Magnification at ×100. (C) LM3 and 97H cells were treated with various concentrations of NAE1 inhibitor MLN4924 for 48 h and then harvested for western blot analysis. (D) HCC cells were treated with various concentrations of MLN4924 for 72 h, exposed to CCK-8 for 2 h, followed by absorbance measurement at 450 nm. (E) LM3 and 97H cells were plated into 6-well plate at a density of 800 cells/well, treated with various concentrations of MLN4924 for 9 days, followed by 0.05% crystal violet staining. HCC, hepatocellular carcinoma; NAE1, NEDD8-activating enzyme 1; CCK-8, Cell Counting Kit-8.

MLN4924 inhibits cell proliferation in HCC cells

As shown in Fig. 2C, MLN4924, a small molecular inhibitor of NAE, inhibited cullin-1 neddylation. We next determined whether inactivation of neddylation modification by MLN4924 would inhibit cell growth and clonogenic survival in HCC LM3 and 97H cell lines. The results showed that MLN4924 could significantly inhibit cancer cell growth and clonogenic survival in a dose-dependent manner (Fig. 2D and E).

MLN4924 enhances the inhibition of cell proliferation by sorafenib in HCC cells

In order to determine whether MLN4924 further inhibits cell proliferation by sorafenib, LM3 and 97H cells were treated with MLN4924 and sorafenib alone or in combination. CCK-8 and clonogenic assays were then performed to evaluate cell proliferation and survival. MLN4924 significantly inhibited HCC cell proliferation with IC10 and IC15 concentrations of 50 and 100 nM, respectively (Fig. 3A). Sorafenib weakly inhibited cell proliferation even at 2 µM, but when combined with low concentrations of MLN4924 the cytotoxicity of sorafenib was enhanced in a dose-dependent manner (P<0.05) (Fig. 3B). In addition, MLN4924 at 50 nM similar to sorafenib at 2 µM weakly inhibited the colony formation in both LM3 and 97H cells. However, following the combination treatment, enhanced inhibition of colony formation of sorafenib was noted (Fig. 3C and D). Taken together, these findings suggest that the combination of sorafenib and MLN4924 appears to cause an additive effect with a maximal antitumor activity in the treatment of HCC.

Figure 3.

MLN4924 enhances cell proliferation inhibition induced by sorafenib. (A) LM3 and 97H cells were treated with various concentrations of MLN4924 for 48 h. The cell proliferation was analyzed using CCK-8 kit. (B) HCC cells were treated with various concentrations of sorafenib in the presence or absence of MLN4924 for 48 h, followed by CCK-8 kit analysis. (C) Cells were plated into 6-well plate at a density of 800 cells/well, and then treated with MLN4924 (MLN, 50 nM), sorafenib (Soraf, 2 µM), or a combination of these two agents for 9 days, followed by 0.05% crystal violet staining. Scale bar, 500 mm. (D) Colonies containing >50 cells were counted. Data are shown as mean ± SEM. **P<0.01; ***P<0.001. CCK-8, Cell Counting Kit-8.

MLN4924 promotes sorafenib-mediated caspase-3-dependent apoptosis in HCC cells

Annexin V/PI staining assay was used to determine apoptosis levels in the LM3 and 97H cell lines. Cells were grown as monolayers and exposed to sorafenib alone or in combination with MLN4924 for up to 48 h. Apoptosis levels were determined by assessing the cells that were positive for Annexin V staining. As shown in Fig. 4A and B, MLN4924 and sorafenib alone at low concentrations did not significantly induce apoptosis, whereas the combination of both drugs significantly induced apoptotic cells up to 25%. In order to further validate this finding, we analyzed apoptosis-related proteins cleaved caspase-3 and its downstream target PARP using western blot analysis. As shown in Fig. 4C, sorafenib alone induced cleaved caspase-3 and cleaved PARP accumulation in a dose-dependent manner, and this was enhanced by MLN4924 in a dose-dependent manner. Cell growth inhibition induced by these two drugs alone or in combination appears to be mediated by the induction of apoptosis.

Figure 4.

MLN4924 promotes sorafenib-mediated caspase-3-dependent apoptosis in HCC cells. (A and B) LM3 and 97H cells were treated with MLN4924 (MLN, 50 nM), sorafenib (Soraf, 2 µM), or combination of these two drugs for 48 h, followed by Annexin V-FITC/PI staining. (A) Annexin V-positive cells represent apoptotic cells as shown by flow cytometry. (B) Data are shown as mean ± SEM. ***P<0.001. (C) LM3 and 97H cells were treated with various concentrations of sorafenib (Soraf) in the presence or absence of MLN4924 for 48 h, followed by western blot analysis. HCC, hepatocellular carcinoma.

MLN4924 further suppresses cell migration induced by sorafenib via upregulation of E-cadherin and downregulation of N-cadherin and vimentin in HCC cells

We determined the effect of sorafenib alone or in combination with MLN4924 on HCC cell migration capacity. In LM3 cells, sorafenib at 2 µM induced an ~15% inhibition, whereas MLN4924 at 50 nM induced a 10% inhibition of cell migration. The combination of both drugs induced a 60% inhibition in cell migration, which was statistically significant (Fig. 5A). Likewise, in 97H cells, single drug treatment induced a 15–30% inhibition of cell migration for sorafenib at 5 µM and MLN4924 at 100 nM. Whereas the combination of the two drugs induced up to a 65% inhibition of cell migration, which was again statistically significant (Fig. 5A). Subsequently, migration markers were analyzed by western blot analysis. Sorafenib upregulated E-cadherin, and downregulated N-cadherin and vimentin in a dose-dependent manner. Compared to sorafenib alone treatment, the combination treatment further enhanced this action (Fig. 5B). Taken together, MLN4924 further suppresses cell migration induced by sorafenib by upregulating E-cadherin and downregulating N-cadherin and vimentin.

Figure 5.

MLN4924 further suppresses cell migration induced by sorafenib in HCC cell lines. (A) Cells were seeded into Transwell plates, treated with MLN4924 (MLN; 0, 50 and 100 nM), sorafenib (Soraf; 0, 2 and 5 µM), or combination of these two drugs, followed by 0.05% crystal violet staining to determine the proportion of migrated cells. ***P<0.001. Magnification, ×200. (B) Cells were treated with various concentrations of sorafenib (Soraf) in the presence or absence of MLN4924 for 48 h, followed by western blot analysis for E-cadherin, N-cadherin and vimentin. HCC, hepatocellular carcinoma.

MLN4924 increases the antitumor efficacy of sorafenib in an in vivo xenograft tumor model

We assessed the efficacy of sorafenib, MLN4924, and the combination of both in an in vivo 97H xenograft mouse model. As shown in Fig. 6A and B, sorafenib and MLN4924 alone moderately inhibited tumor growth in nude mice, while the combination of the two drugs had a nearly complete inhibition of tumor growth. At the end of the experiment, the average tumor weight in the Control group, the sorafenib group, the MLN4924 group and the combination therapy group was 1.52, 0.80, 0.89 and 0.22 g, respectively, with a statistical difference between the sorafenib group and the combination therapy group (P<0.05) (Fig. 6B). The tumor growth index in the sorafenib group, the MLN4924 group and the combination therapy group was 47.0, 65.4 and 21.2%, respectively, with statistical difference between each group (P<0.05) (Fig. 6C). Finally, the drug dosages used were not toxic to the animals as indicated by the minimal loss of body weight (Fig. 6D). Taken together, the results demonstrate that MLN4924 indeed sensitizes HCC tumors to sorafenib in an in vivo xenograft mouse tumor model.

Figure 6.

MLN4924 enhances antitumor activity of sorafenib in an in vivo HCC xenograft mouse tumor model. Cells (1×107) were inoculated subcutaneously into the right flank of nude mice. The mice were then randomized and treatment was initiated two days post inoculation for 3 weeks. Tumors were then harvested, photographed (A) and weighed (B) and the results plotted. (C) The growth of tumors (6 for each group) was measured once a week for 4 weeks and results plotted. (D) The weight of animals was monitored once a week and results plotted. Data are shown as the mean ± SEM. *P<0.05; **P<0.01; ***P<0.001. HCC, hepatocellular carcinoma; MLN, MLN4924; Soraf, sorafenib.

MLN4924 increases the accumulation of SCF E3 ligase substrates to sensitize HCC to sorafenib

To determine the potential mechanism of MLN4924 as a sorafenib sensitizer, cell proliferation- and migration-associated substrates of CRL/SCF E3 ligase were analyzed by western blot analysis. We found that MLN4924 inhibited cullin neddylation, and induced the accumulation of Noxa (a pro-apoptotic protein, shown to be a CRL5 substrate). However, we did not observe a further increase in Noxa levels after combination treatment, suggesting it was not critical for sorafenib sensitization. However, the levels of proliferation- and migration-associated proteins Deptor (an mTOR inhibitor), IκBα (an inhibitor of NF-κB), p21 and p27 (two inhibitors of cyclin-dependent kinases) were higher in the combination treatment group compared to the MLN4924 or sorafenib treatment only group (Fig. 7A). These findings were further validated by decreases in p-mTOR, p-4EBP1 and p-S6K (two downstream targets of mTOR) and NF-κB p65 (a downstream target of IκBα). Given that p-ERK is the main target of sorafenib and that MLN4924 did not have any effect on p-ERK levels, this suggests that ERK was not involved in MLN4924-mediated sorafinib sensitization. A previous study reported that MMP9, a downstream target of NF-κB, was important for regulating cell migration (30). However, we found that MMP9 was not associated with sorafineb sensitization triggered by MLN4924 in HCC cells, as indicated by no significant changes to MMP9 levels in the presence or absence of MLN4924. Furthermore, we found that silencing of NF-κB p65 and mTOR inhibitor rapamycin could significantly inhibit cell proliferation and migration (Fig. 7B-D), which are consistent with the effect of the combination treatment group. Taken together, these results indicate that the accumulation of SCF E3 ligase substrates p21, p27, Deptor and IκBα induced by MLN4924 was associated with the chemo-sensitization effects of sorafenib on HCC cell lines (Fig. 7E).

Figure 7.

MLN4924 induces p21, p27, Deptor and IkBa accumulation that are important for sorafenib sensitization. (A) LM3 cells were treated with different concentrations of sorafenib (Soraf) in the presence or absence of MLN4924 (MLN) for 48 h, followed by western blot analysis. (B-D) LM3 cells were transfected with NF-κB p65 siRNA (siNFκB p65) or treated with mTOR inhibitor rapamycin (100 nM). Forty-eight hours later, a portion of cells were harvested for (B) western blot analysis or (C) cell proliferation using CCK-8. (D) Other cells were used for Transwell assay to determine the migratory ability. Magnification at ×100. (E) MLN4924 inhibits CRL/SCF E3 ubiquitin ligase activity by inhibiting NAE resulting in the accumulation of p21/p27 to inhibit proliferation and migration; IκBα to inhibit NF-κB, and block cell migration by decreasing N-cadherin and vimentin; Deptor to inhibit mTORC1 activation thus inhibiting cell proliferation and migration. Arrows represent promotion events, blunt arrows indicate suppression events. *P<0.05; **P<0.01; ***P<0.001. CCK-8, Cell Counting Kit-8; mTOR, mammalian target of rapamycin; NAE1, NEDD8-activating enzyme 1; CRL, Cullin-Ring ligase; SCF, Skp1-Cullin1-F box; MMP9, metalloproteinase 9.

Discussion

Hepatocellular carcinoma (HCC) is the third leading cause of death worldwide (31). This is due to the lack of efficacious therapeutic drugs for HCC treatment. Sorafenib is the only anticancer drug approved by the FDA for advanced HCC (32,33). However, its anticancer efficacy is poor (34). Ubiquitin proteasome system (UPS) participates in a wide variety of biological processes which include cell cycle, cell proliferation, signal transduction, DNA repair and apoptosis (35). UPS disorders may lead to the development of tumors or other diseases (5). Recent studies have demonstrated that targeting the ubiquitin and ubiquitin-like activating enzymes is a novel approach for cancer treatment. MLN4924, the most widely used NAE inhibitor, has entered phase I and II clinical trials for acute myeloid leukaemia and myelodysplastic syndromes (36). MLN4924 has been reported to have anticancer properties in a wide range of solid cancers including head and neck, squamous cell carcinoma, gastric cancer, and metastatic melanoma in vitro and in vivo (37). Previous studies have focused on the sensitizing effects of MLN4924 to existing chemotherapy agents (38–40) or radiation therapy (41,42), but the anticancer activities of MLN4924-sorafenib combination has not been reported to date. We hypothesized that MLN4924 could enhance the sensitivity of HCC to sorafenib. We evaluated the effects of a combination therapy of MLN4924 and sorafenib in vitro and in vivo.

Consistent with a previous study (25), we found that MLN4924 alone at high doses was a potential tumor growth inhibitor, while sorafenib alone weakly inhibited HCC cell proliferation even at high doses. However, MLN4924 significantly enhanced the antitumor efficacy of sorafenib both in vitro and in vivo. At the doses tested, there was no significant toxicity for all the treatment groups. Similar to the efficacy of the proteasome inhibitor bortezomib, the combination of MLN4924 with sorafenib has potential for the clinical application to treat HCC.

Deptor/mTOR and IκBɑ/NF-κB signaling pathways are two well-established signaling pathways that are significantly activated in HCC (43–45). Cell proliferation and migration of HCC cells are correlated with mTOR activation and NF-κB overexpression (22–24). MLN4924 was found to significantly inhibit cell proliferation and migration in lung cancer cells via inhibition of mTOR activation or via downregulation of NF-κB expression (19,46). Consistent with these findings, MLN4924 indeed further inactivated mTORC1 and downregulated NF-κB in HCC cells compared to sorafenib alone. This was mediated by the increase in Deptor and IκBɑ, known substrates of SCF-βTrCP, as demonstrated in melanoma and HCC cancer cell lines (47,48). However, we did not observe an increase in Noxa, a known substrate of CRL5, in the MLN4924 only treatment group in HCC cells.

Previous studies have shown that NF-κB is an upstream regulator of MMP9, a known matrix metalloproteinase (MMP) that is correlated with the aggressiveness of cancer. NF-κB positively regulates MMP9 expression levels in colon carcinoma and gastric cancer cells (49–51). However, in the present study, the decrease in NF-κB levels induced by MLN4924 did not decrease MMP9 expression levels in HCC cell lines, suggesting that MMP9 was not associated with MLN4924-mediated sorafenib sensitization. However, Xu et al reported that NF-κB could upregulate vimentin and N-cadherin expression levels that are the central mediators of cellular adhesion junctions (52). Based on these findings, it is possible that the combination treatment of MLN4924 and sorafenib inhibited the migration of HCC cells via the downregulation of vimentin and N-cadherin expression through the IκBɑ/NF-κB pathway (Fig. 7E).

The application of MLN4924 in combination with chemotherapy or radiotherapy for the treatment of multiple solid tumors has been reported. MLN4924 has been shown to enhance the antitumor effects of gemcitabine for pancreatic cancer and oxaliplatin for colorectal cancer (53,54). Oladghaffari et al reported that MLN4924 and 2-deoxy-D-glucose (2DG) combination increased the efficacy of radiotherapy for breast cancer (42). MLN4924 indeed was found to have an inhibitory effect on HCC cells (25). Whether the combination of MLN4924 and sorafenib is efficacious against HCC has not been reported. In the present study, we provide preclinical evidence that MLN4924 enhances the anticancer effects of sorafenib on HCC via upregulation of CRL/SCF E3 ubiqutin ligase substrates p21, p27, Deptor and IκBɑ, suggesting its use as a novel treatment strategy for HCC.