The Neddylation-Cullin 2-RBX1 E3 Ligase Axis Targets Tumor Suppressor RhoB for Degradation in Liver Cancer

Abstract

The neddylation-cullin-RING E3 ligase (CRL) pathway has recently been identified as a potential oncogenic event and attractive anticancer target; however, its underlying mechanisms have not been well elucidated. In this study, RhoB, a well known tumor suppressor, was identified and validated with an iTRAQ-based quantitative proteomic approach as a new target of this pathway in liver cancer cells. Specifically, cullin 2-RBX1 E3 ligase, which requires NEDD8 conjugation for its activation, interacted with RhoB and promoted its ubiquitination and degradation. In human liver cancer tissues, the neddylation-CRL pathway was overactivated and reversely correlated with RhoB levels. Moreover, RhoB accumulation upon inhibition of the neddylation-CRL pathway for anticancer therapy contributed to the induction of tumor suppressors p21 and p27, apoptosis, and growth suppression. Our findings highlight the degradation of RhoB via the neddylation-CRL pathway as an important molecular event that drives liver carcinogenesis and RhoB itself as a pivotal effector for anticancer therapy targeting this oncogenic pathway.

Post-translational protein neddylation is a process of the covalent attachment of NEDD8,¹ a ubiquitin-like small molecule, to lysine residues of substrate proteins and thus regulates their function through modulating their conformation, stability or subcellular localization (1, 2). NEDD8 conjugation to substrates is catalyzed by a three-step enzymatic cascade mediated by NEDD8-activating enzyme (E1, NAE1, and UBA3 form a heterodimer), NEDD8-conjugating enzyme (E2, UBC12 or UBE2F), and NEDD8 E3 ligases sequentially (1–3). So far, the well recognized neddylation substrates are the cullin family, and they serve as the fundamental components of multiunit cullin-RING E3 ligase (CRL) as well as the potential anticancer target (4, 5). Functionally, NEDD8 conjugation to cullins changes the conformation of CRL and leads to its activation for protein ubiquitination and degradation (4, 5). In addition, NEDD8 conjugation has been reported to stabilize oncoproteins Mdm2 (6) and HuR (7) while to repress the transcriptional activity of tumor suppressor p53 (6). Most recently, we reported that the entire neddylation pathway, including NEDD8-activating enzyme E1, NEDD8-conjugating enzyme E2, and global neddylation of substrates, is overactivated in human lung cancer and associates with worse overall survival of patients (8). These findings highlight a pivotal role of neddylation in carcinogenesis and tumor progression; however, whether neddylation is overactivated in other types of tumors, such as liver cancer, and how it facilitates tumor development remain elusive.

Inhibition of the neddylation-CRL pathway has recently emerged as a promising anticancer strategy (9). MLN4924, a small molecule inhibitor of NAE, has been discovered as a first-in-class anticancer agent by high throughput screening and has been advanced into several Phase I clinical trials due to its significant anticancer efficacy and tolerated toxicity in preclinical studies (9–12). Mechanistically, MLN4924 blocks cullin neddylation, inactivates CRL, induces the accumulation of CRL substrates and eventually causes DNA damage, cell cycle defects, senescence, apoptosis, and autophagy (13–16). So far, several classes of CRL substrates have been reported to accumulate upon neddylation inhibition and mediate MLN4924-induced cellular responses in a cell context-dependent manner. They include but are not limited to 1) DNA replication licensing proteins CDT1 and ORC1, inducing DNA rereplication stress and DNA damage (13, 15); 2) cell cycle inhibitors such as p21, p27, and Wee1, inducing cell cycle arrest and senescence (13, 15, 17); 3) NF-κB inhibitor IκB-α, resulting in inhibition of NF-κB activity (16); and 4) mTOR inhibitory protein Deptor, leading to inactivation of the mammalian target of rapamycin and induction of autophagic responses (18, 19). Given that CRL, the largest multiunit ubiquitin ligase family in cells, is in charge of degradation of ∼20% of ubiquitinated cellular proteins (13), the identification of novel CRL substrates will not only broaden our understanding of how the neddylation-CRL pathway regulates tumor development, but also provide a new insight into how cancer cells respond to anticancer therapy targeting this oncogenic pathway.

RhoB, a member of the Rho family of small GTPases, participates in the modulation of numerous essential cellular processes, including actin organization, gene transcription, cell adhesion, proliferation, apoptosis and vesicle traffic (20–22). Moreover, RhoB controls a variety of important cancer-associated signaling pathways through modulation of many downstream effectors. For example, RhoB triggers cell cycle arrest or apoptosis through inducing the expression of p21 and p27 (23, 24). RhoB also regulates the traffic of several critical cancer-associated molecules, such as epidermal growth factor receptor, platelet-derived growth factor receptor, chemokine receptor, PDK1, Akt, and Src (25–29). Acting as an important tumor suppressor, RhoB is significantly down-regulated in many human cancers, including lung, stomach, breast, brain, ovary, and head and neck carcinomas (30–35). Moreover, RhoB expression is further decreased or lost in invasive tumors and poorly differentiated tumors (35, 36). In contrast, the revivification of RhoB expression inhibits the malignant phenotypes of cancer cells, such as cell proliferation, migration, and invasion (33, 37). These findings highlight that down-regulation of RhoB is an important molecular event that drives carcinogenesis; however, the molecular mechanisms by which RhoB expression is regulated during the process are largely unknown.

In this study, RhoB was identified and validated via iTRAQ-based quantitative proteomic analysis as a new substrate of the neddylation-CRL pathway. Moreover, the expression of this pathway, mediating the degradation of RhoB, was overactivated in human liver cancer. Inhibition of this pathway induced RhoB accumulation, which led to tumor-suppressive cellular responses. Our studies revealed a previously unknown regulatory mechanism of RhoB loss in liver cancer and validated RhoB as a novel target of the neddylation-CRL pathway in human liver carcinoma.

MATERIALS AND METHODS

Cell Culture and Reagents

Human hepatocellular carcinoma cell lines HepG2 and Huh7, lung cancer cell line A549, breast cancer cell line MCF-7, pancreatic cancer cell line MiaPaCa-2, colon cancer cell line HCT116, and human umbilical vein endothelial cells (HUVECs) were obtained from the American Type Culture Collection, and routinely cultured_. MLN4924 was synthesized and used for in vitro studies as described previously (38). For in vitro studies, MLN4924 was dissolved in dimethyl sulfoxide (DMSO) and kept at −20°C.

Protein Extraction, Digestion, and iTRAQ Labeling for HUVECs

HUVEC extracts were prepared following our published methods (39). Briefly, proteins of HUVECs were extracted with 8 m urea and 50 mm NH₄HCO₃, and 200 μg of protein was reduced by adding 2.96 μl of 0.1 m dithiotheitol for 4 h at 37 °C and then alkylated by adding 3.29 μl of 0.5 m iodoacetamide for 60 min at room temperature in the dark. The protein sample was diluted to 1 m urea with 50 mm NH₄HCO₃ in water and digested with trypsin at a mass ratio of 1:50 enzyme/protein overnight at 37 °C, followed by termination with 1% formic acid. Peptide mixtures (80 μg) from each condition were labeled with iTRAQ 4plex reagent (Applied Biosystems, Foster City, CA), with tag 114 for the DMSO condition and tag 116 for the MLN4924 condition (40). The reaction was terminated by adding an equal volume of distilled water (41). The labeled peptides were combined, desalted with a C₁₈ solid-phase extraction column (Waters Associates, Milford, MA), and lyophilized for further analysis.

Peptide Separation and LC-MS/MS Analysis in HUVECs

Peptide mixtures were separated by off-line high-pH reversed phase chromatography first following the parameters, and 19 fractions were generated for further LC-MS/MS analysis, for which a nanoflow HPLC instrument (EASY-nLC 1000 system, Thermo Fisher Scientific, Waltham, MA) coupled to an on-line Q Exactive mass spectrometer (Thermo Fisher Scientific) with a nanoelectrospray ion source (Thermo Fisher Scientific) was used. The chromatography columns were packed in-house with Ultimate XB-C18 3-μm resin (Welch Materials, Shanghai, China). The peptide mixtures were loaded onto the C₁₈ reversed phase column (10-cm length, 75-μm inner diameter) with buffer A (99.5% water and 0.5% formic acid) and separated with a 75-min linear gradient of 5–100% buffer B (99.5% acetonitrile and 0.5% formic acid) at a flow rate of 300 nl/min. Including the loading and washing steps, the total time for an LC-MS/MS run was ∼90 min. The electrospray voltage was 2.0 kV. Peptides were analyzed by data-dependent MS/MS acquisition with dynamic exclusion duration of 18 s. In MS1, the resolution was 70,000, the automatic gain control (AGC) target was 3e6, and the maximum injection time was 20 ms. In MS2, the resolution was 17,500, the automatic gain control target was 1e6, and the maximum injection time was 60 ms. The scan range was 300–1400 m/z, and the top 75 intensive precursor ions were selected for MS/MS analysis.

The raw data were processed using the proteomic workflow of Proteome Discoverer 1.3. The fragmentation spectra were searched against the UniProt reviewed human database (20130415, 20268 sequences) using the Mascot search engine (version 2.2.06) with the precursor and fragment mass tolerances set to 15 ppm and 20 milli-mass units (mmu), respectively. Two missed cleavage sites were allowed. The fixed modification was carbamidomethylation (Cysteine), and the variable modifications were oxidation (Methionine), acetylation (protein N terminus), and iTRAQ labeling (Tyrosine and Lysine, N-terminal residue). Peptide ions were filtered using the cutoff scores of Percolator based on p < .01. The false discovery rate was set to 1% for peptide identifications. An additional filter was applied with the removal of spectrum matches with scores lower than 10. The iTRAQ quantization values were automatically calculated on the basis of the intensity of the iTRAQ reporter ions in the dissociation scans with higher collision energy using Proteome Discoverer (41). All protein iTRAQ ratios were exported to an Excel file, the Gaussian distribution of ratios of 116:114 was recalculated manually, and all ratios were transformed to base 10 logarithm values. A confidence interval of 99% was used to determine the cutoff values for statistically significant changes (42).

RNA Interference

HepG2 or Huh7 cells were transfected with siRNA oligonucleotides using Lipofectamine 2000 reagent (Life Technologies, Invitrogen, CA) according to the manufacturer's instructions. The sequences of siRNAs are as follows: for RBX1 (43), 5′-GACUUUCCCUGCUGUUACCUAATT-3′ and 5′-GGACAACAGAGAGUGGGAATT-3′; for RBX2 (44), 5′-GAGGACUGUGUUGUGGUCUTT-3′; for NAE1 (45), 5′-GGGUUGUGCUUUAGUCUGUTT-3′; for UBA3 (46), 5′-UGUUCUGGUAGCCUGGGCAUAGAUGTT-3′; for UBC12 (46), 5′-GGGCUUCUACAAGAGUGGGAAGUTT-3′; and for control scrambled siRNA 5′-UUCUCCGAACGUGUCACGUTT-3′. All of the above siRNAs were purchased from GenePharma (Shanghai, China).

Immunoblot Analysis

Cell lysates were prepared for immunoblot analysis using antibodies against RhoB (ABclonal, Cambridge, MA); RBX1 and ubiquitin (Abcam, Cambridge, MA); RBX2 and cullin 4B (Proteintech, Chicago, IL); cullin 7 and NAE1 (Sigma, St. Louis, MO); UBA3, UBC12, and p21 (Epitomics, Burlingame, CA); NEDD8, Wee1, p27, p21, cleaved caspase-3, cleaved poly(ADP) ribose polymerase, cullin 3, and cullin 4A (Cell Signaling Technology, Beverly, MA); cullin 2 (BD Biosciences, San Jose, CA); cullin 1, cullin 5, and HA (Santa Cruz Biotechnology, Santa Cruz, CA); FLAG (Genomics, Shanghai, China); and GAPDH (Kangwei, Shanghai, China).

Collection of Liver Cancer Tissues

Human hepatocellular carcinoma tissues and paired normal tissues were obtained from Zhongshan Hospital (Fudan University, Shanghai, China) in 2014. Human hepatocellular carcinoma diagnosis was based on the World Health Organization criteria (47). Ethical approval was obtained from the research ethics committee of Zhongshan Hospital, and written informed consent was obtained from each patient.

In Vivo Ubiquitination Assay

To detect endogenous RhoB ubiquitination, HepG2 cells were transfected with siRNA oligonucleotide targeting RBX1 or cullin 2, along with scrambled control siRNA. At 96 h post-transfection, cells were harvested and subjected to immunoprecipitation with anti-RhoB Ab and immunoblotting with anti-ubiquitin Ab. To determine RhoB ubiquitination by MLN4924, cells were treated with MLN4924 along with a DMSO control, followed by immunoprecipitation with anti-RhoB Ab and immunoblotting with anti-ubiquitin Ab.

Statistical Analysis

All data are presented as mean ± S.E. Student's t test was used for comparison of parameters between two groups, and the statistical significance of differences between groups was assessed using GraphPad Prism 5 software. Three levels of significance (*, p < .05; **, p < .01; ***, p < .001) were used, and p < .05 was considered to be significant.

RESULTS

Identification and Validation of RhoB as a Downstream Target of the Neddylation Pathway

A quantitative proteomic strategy based on the iTRAQ stable isotope labeling technique was performed to determine the up- and down-regulated proteins upon neddylation inhibition with MLN4924 in HUVECs (Fig. 1A). The results showed that 6,886 human proteins were identified with high confidence, and 6,850 of them had at least one unique pair of quantifiable 116:114 ions. Information on the peptides and proteins quantified is provided in supplemental Tables S1 and S2.

Fig. 1.

Identification and validation of RhoB as a downstream target of the neddylation pathway. A, schematic view of the quantitative proteomics strategy based on iTRAQ labeling. Up- or down-regulation of a protein was reported as the ratio of the peak intensity from the reporter ions 114 and 116. B, representative tandem MS spectrum of peptide IVVVGDGACGK, in which the cysteine was carbamidomethylated and the amino groups in the N terminus and the lysine were modified by the iTRAQ reagents. The inset shows the region of iTRAQ reporter ions. C, Western blot analysis confirmed RhoB accumulation from iTRAQ proteomics data in HUVECs. Wee1 and p27 are known substrates and were used as positive controls. D, LC-MS/MS analysis of RhoB up-regulation upon treatment with 1 μm MLN4924 in HepG2 cells. The iBAQ algorithm was used to quantify RhoB protein abundance. p < .05. E, MLN4924 treatment induced RhoB accumulation in a dose- and time-dependent manner. HepG2, Huh7, MiaPaCa-2, or HCT116 cells were treated with a different dosage of MLN4924 for the indicated hours and then harvested and subjected to Western blot analysis using antibody against RhoB or GAPDH as a loading control. F, down-regulation of NAE1, UBA3, or UBC12 induced RhoB accumulation in HepG2 cells. Cells were transfected with control (siCtrl), NAE1, UBA3, or UBC12 siRNA for 120 h and harvested for Western blot analysis. Endogenous NAE1, UBA3, or UBC12 expression level is presented as the efficacy of siRNA.

By modeling the normal distribution of the protein ratios (supplemental Fig. S1A), a confidence interval of 99% (0.770–1.286) was applied to find the protein with significant up- or down-regulation after MLN4924 treatment (supplemental Tables S3 and S4). The results revealed 77 up-regulated proteins and 18 down-regulated proteins (supplemental Fig. S1B). Among these up-regulated proteins (supplemental Table S3), CDKN1A (p21) and MYC were well established targets upon MLN4924 treatment as a positive control (13, 48).

Rho-related GTP-binding protein RhoB was identified for three matching unique peptides and two shared peptides (supplemental Table S2). To avoid the disturbance from the iTRAQ ratio of the shared peptides, the quantitative value was averaged only from the ratios of the three unique peptides. There were six spectra from these three peptides, which gave an average 116:114 ratio of 1.387 with a coefficient of variation of 7.8%. Three representative spectra and their matching information are presented in Fig. 1B and supplemental Figs. S1C and S1D. The intensities of the 116 and 114 peaks in the figure insets indicated that the 116:114 ratios were very stable among different peptides, thus strongly suggesting that RhoB is a positive target upon MLN4924 treatment. To verify iTRAQ-based quantitative proteomic results, we determined the expression of RhoB by immunoblot analysis and found that RhoB was indeed accumulated upon MLN4924 treatment in HUVECs (Fig. 1C). In addition, LC-MS/MS analysis also revealed a significant accumulation of RhoB in MLN4924-treated HepG2 liver cancer cells (Fig. 1D). Together, these findings indicate that RhoB is a true downstream target of the neddylation pathway.

Given that RhoB serves as an important tumor suppressor, we further validated RhoB expression upon inhibition of the neddylation pathway in multiple human cancer cell lines, including the HepG2 and Huh7 liver cancer cell lines, MiaPaCa-2 pancreatic cancer cell line, HCT116 colon cancer cell line, A549 lung cancer cell line, and MCF-7 breast cancer cell line. We found that RhoB was significantly up-regulated in MLN4924-treated cells in a dose- and time-dependent manner (Fig. 1E and supplemental Figs. S2A and S2B). Finally, we determined whether genetic inactivation of the neddylation pathway also induced RhoB accumulation. As shown in Fig. 1F, down-regulation of NAE1, UBA3 or UBC12 by siRNA silencing induced the up-regulation of RhoB in HepG2 cells. Together, these findings indicate that RhoB is a novel target of the neddylation pathway.

The Neddylation Pathway Regulates the Degradation and Transcription of RhoB

To determine the underlying mechanism of RhoB regulation by the neddylation pathway, we first applied cycloheximide to block protein translation and determined RhoB turnover rate upon MLN4924 treatment. As shown in Fig. 2A, neddylation inactivation by MLN4924 significantly delayed RhoB turnover (left panel) and extended the half-life of RhoB (right panel) in both HepG2 and Huh7 cells. Similarly, treatment of cells with MG-132, a classical proteasome inhibitor, also dramatically extended the half-life of RhoB compared with control cells (Fig. 2B). These findings indicate that the neddylation pathway regulates RhoB degradation through the ubiquitin-proteasome system in liver cancer cells.

Fig. 2.

Neddylation pathway regulates the degradation and transcription of RhoB. A, MLN4924 treatment extended the half-life of RhoB. HepG2 or Huh7 cells were pretreated with 1 μm MLN4924 for 12 h and then switched to fresh medium comprising 25 μg/ml cycloheximide (CHX) and 1 μm MLN4924 for the indicated times and harvested for Western blot analysis. B, MG-132 blocked RhoB turnover in HepG2 and Huh7 cells. HepG2 or Huh7 cells were pretreated with 1 μm MLN4924 for 12 h and then switched to fresh medium comprising 25 μg/ml cycloheximide and 10 μm MG-132 for the indicated times and harvested for Western blot analysis. Note that the expression of RhoB was quantified by densitometric analysis using ImageJ software.

Previous studies indicated that genotoxic agents can induce RhoB expression transcriptionally (49). Because we found that MLN4924 induced DNA rereplication stress and triggered DNA damage in liver cancer cells (14), we hypothesized that MLN4924 may also regulate RhoB expression at the transcriptional level. To test this hypothesis, quantitative PCR was performed to determine RhoB mRNA expression upon MLN4924 treatment. Compared with MLN4924-induced accumulation of RhoB protein (supplemental Figs. S3A and S3B, left panel), MLN4924 also increased RhoB mRNA but to a lesser extent (supplemental Figs. S3A and S3B, right panel). We therefore conclude that MLN4924 regulates RhoB at both the transcriptional and post-translational levels.

Neddylation Substrate Cullin 2 Mediates RhoB Ubiquitination and Degradation

Because CRL, the well known substrate of neddylation, targets protein for ubiquitination and degradation, we hypothesized that the neddylation pathway regulates RhoB degradation through CRL. To test this hypothesis, we first determined which cullin family member interacts with RhoB by co-immunoprecipitation assay. As shown in Fig. 3A, endogenous RhoB exclusively interacted with cullin 2 but not with other cullin family proteins. Similarly, Overexpression of Myc-cullin 2 in transfected cells also interacted with RhoB (Fig. 3B). We next determined whether down-regulation of cullin 2 blocks RhoB turnover and found that siRNA silencing of cullin 2 induced the accumulation of RhoB (Fig. 3C). Finally, we determined whether inhibition of cullin neddylation and activation by MLN4924 affect RhoB ubiquitination. As shown in Fig. 3 (D and E), inactivation of cullin 2 with MLN4924 or down-regulation of cullin 2 via siRNA silencing strongly inhibited the polyubiquitination of RhoB in both HepG2 and Huh7 cells. These findings indicate that cullin 2 interacts with RhoB and targets RhoB for ubiquitination and degradation in liver cancer cells.

Fig. 3.

Neddylation substrate cullin 2 mediates RhoB ubiquitination and degradation. A, endogenous RhoB interacted with cullin 2 conjugated with NEDD8. HepG2 cells were serum-starved for 24 h, followed by serum addition to MG-132 for 2 h. Cells were harvested and subjected to immunoprecipitation (IP) with anti-RhoB Ab and immunoblotting with anti-cullin Abs. B, overexpressed Myc-cullin 2 interacts with RhoB. Interaction between Myc-cullin 2 and RhoB was detected using anti-Myc Ab for immunoprecipitation and anti-RhoB Ab for immunoblotting. C, down-regulation of cullin 2 induced the accumulation of RhoB expression. HepG2 cells were transfected with control (siCtrl) or cullin 2 siRNA for 120 h and harvested for Western blot analysis. Endogenous cullin 2 expression level is presented as the efficacy of siRNA. D, MLN4924 inhibited RhoB polyubiquitination in HepG2 or Huh7 cells. Cells were treated with DMSO or 1 μm MLN4924 for 24 h and followed with MG-132 for 2 h. Cells were extracted and subjected to immunoprecipitation with anti-RhoB Ab and immunoblotting (IB) with anti-ubiquitin Ab. E, down-regulation of cullin 2 inhibited RhoB polyubiquitination in HepG2 or Huh7 cells. Cells were transfected with control or cullin 2 siRNA for 120 h and followed with MG-132 for 2 h. Cells were extracted and subjected to immunoprecipitation with anti-RhoB Ab and immunoblotting with anti-ubiquitin Ab.

CRL Component RBX1 Regulates RhoB Ubiquitination and Degradation

RBX1 and RBX2 are two RING finger proteins of CRL, either of which can interact with cullins to constitute the core complex of CRL (50). To determine which RING finger protein modulates the turnover of RhoB in cancer cells, we first performed a co-immunoprecipitation assay and found that endogenous RhoB bound RBX1, but not RBX2 in HepG2 cells (Fig. 4A). Consistently, overexpression of HA-RBX1 also interacted with exogenous FLAG-RhoB in these cells (Fig. 4B). Moreover, down-regulation of RBX1, but not RBX2, significantly induced RhoB accumulation (Fig. 4C) and blocked RhoB degradation in the presence of cycloheximide (CHX) (Fig. 4D). Finally, we found that down-regulation of RBX1 significantly impaired RhoB polyubiquitylation in both HepG2 and Huh7 cells (Fig. 4, E and F). These findings indicate that CRL component RBX1 targets RhoB for ubiquitination and degradation in liver cancer cells.

Fig. 4.

CRL component RBX1 regulates RhoB ubiquitination and degradation. A, endogenous RhoB interacted with RBX1 in HepG2 cells. Cells were serum-starved for 24 h, followed by serum addition to MG-132 for 2 h. Cells were harvested and subjected to immunoprecipitation (IP) with anti-RhoB Ab and immunoblotting with anti-RBX Abs. B, interaction between HA-RBX1 and FLAG-RhoB was detected using anti-HA Ab for immunoprecipitation and anti-FLAG Ab for immunoblotting. Note that the IgG control had a nonspecific band. C, down-regulation of RBX1 induced RhoB up-regulation in HepG2 cells. Cells were transfected with control (siCtrl), RBX1, or RBX2 (siRNA) for 120 h and harvested for Western blot analysis. Endogenous RBX1 or RBX2 expression level is presented as the efficacy of siRNA. D, down-regulation of RBX1 inhibited RhoB degradation in HepG2 cells. Cells were transfected with control, RBX1, or RBX2 siRNA for 96 h and switched to fresh medium comprising DMSO or 25 μg/ml cycloheximide (CHX) for 12 h. E, down-regulation of RBX1 reduced RhoB polyubiquitination in HepG2 cells. Cells were transfected with control or RBX1 siRNA for 96 h and with MG-132 for 2 h. Cells were extracted and subjected to immunoprecipitation with anti-RhoB Ab and immunoblotting (IB) with anti-ubiquitin (Ub) Ab. F, down-regulation of RBX1 reduced RhoB polyubiquitination in Huh7 cells. Cells were transfected with control or RBX1 siRNA for 96 h and with MG-132 for 2 h. Cells were extracted and subjected to immunoprecipitation with anti-RhoB Ab and immunoblotting with anti-ubiquitin Ab. **, p < .01.

Inverse Correlation between the Neddylation Pathway and RhoB, the Accumulation of Which Contributes to the Anticancer Effect of Neddylation-CRL Pathway Inhibition

On the basis of our above findings that the neddylation-CRL pathway targets RhoB for degradation and that tumor suppressor RhoB is frequently down-regulated in human cancer, we further hypothesized that this pathway may be overactivated in liver cancer and therefore repress RhoB expression in vivo. We first determined the expression status of NEDD8-activating enzyme (E1, NAE1 and UBA3 heterodimer) and NEDD8-conjugating enzyme (E2, UBC12) in liver tumor versus adjacent normal tissues by immunoblotting. As shown in Fig. 5A, overexpression of these essential components of the neddylation pathway was seen in the majority of tumor tissues compared with adjacent tissues. As a result, global neddylation of substrates was enhanced in liver tumor tissues due to overactivation of neddylation enzymes (Fig. 5B). In contrast to overactivation of the neddylation pathway, the expression of RhoB was significantly down-regulated in liver tumor tissues compared with adjacent normal tissues (Fig. 5C). These findings indicate that the overactivated neddylation pathway targets RhoB for degradation in human liver cancer.

Fig. 5.

Inverse correlation between the neddylation pathway and RhoB, the accumulation of which contributes to the anticancer effect of neddylation-CRL pathway inhibition. A, the neddylation pathway (NAE1, UBA3, and UBC12) was up-regulated in hepatocellular carcinoma tissues. Representative results of 4 of 20 pairs of tissues are shown. T, tumor tissues; N, adjacent normal tissues. Shown are the results from the quantification of neddylation pathway protein expression in hepatocellular carcinoma tissues compared with adjacent normal tissues (n = 20; ***, p < .001). B, global NEDD8-conjugated proteins were dramatically up-regulated in hepatocellular carcinoma. Global NEDD8-conjugated proteins were determined by immunoblotting using NEDD8-specific antibodies. Representative results of three pairs of tissues are shown. C, RhoB was expressed at significantly low levels in hepatocellular carcinoma tissues. The expression of RhoB protein was determined by immunoblotting using RhoB-specific antibodies. Representative results of 4 of 18 pairs of tissues are shown. Shown are the results from the quantification of RhoB protein expression in hepatocellular carcinoma tissues compared with adjacent normal tissues (n = 18; ***, p < .001). D, down-regulation of RhoB decreased cellular responses to MLN4924 treatment. HepG2 cells were transfected with control (siCtrl) or RhoB siRNA for 96 h and then treated with 0.3 μm MLN4924 for the indicated times and harvested for Western blot analysis. E, down-regulation of RhoB rescued the anticancer efficacy of MLN4924 in cell proliferation. HepG2 cells were treated with 0.3 μm MLN4924 or DMSO for 48 h, followed by ATPlite cell viability assay. **, p < .01. F, working model. During liver cancer development, the neddylation-CRL pathway is overactivated (red), which promotes the degradation of RhoB as a tumor suppressor (green) and facilitates carcinogenesis and tumor progression. In contrast, inhibition of the neddylation-CRL pathway by MLN4924 blocks cullin neddylation and therefore inactivates CRL to induce the accumulation of RhoB as a novel CRL substrate to trigger apoptosis and inhibit the growth of liver cancer cells.

In our previous study, we demonstrated that the neddylation-CRL pathway is essential to maintain the malignant phenotypes of liver cancer, whereas disruption of this pathway suppresses the growth of liver cancer cells by inducing cell cycle arrest and apoptosis, highlighting this pathway as a therapeutic target in liver cancer (14). To investigate whether RhoB plays a role during anticancer therapy by targeting this pathway, the expression of RhoB was down-regulated via siRNA silencing, and its effect on cellular responses to MLN4924 treatment was determined. As shown in Fig. 5D, MLN4924 induced significant accumulation of CRL substrates (e.g. RhoB, p21, p27, and phosphorylated IκBα) and apoptosis at 48 h post-treatment in liver cancer cells. Intriguingly, we found that down-regulation of RhoB substantially reduced the accumulation of p21/p27 and therefore impaired apoptotic induction upon MLN4924 treatment (Fig. 5D). In contrast, a non-RhoB downstream target such as phosphorylated IκBα was not changed upon RhoB down-regulation in MLN4924-treated cells (Fig. 5D). Furthermore, we found that down-regulation of RhoB attenuated cell viability impairment induced by MLN4924 (Fig. 5E).Together, these findings indicate that RhoB plays an important role in cellular responses to neddylation-CRL-targeted anticancer therapy.

DISCUSSION

Liver cancer still ranks as one of the most common and deadly human malignancies and is the second leading cause of cancer-related death worldwide (51). In our previous study, we demonstrated that the neddylation pathway is required for the proliferation and survival of liver cancer cells, whereas inhibition of this pathway suppresses the growth of liver cancer cells (14). Here, we further report that this pathway, including neddylation enzymes and global protein neddylation, is overactivated in liver cancer, which provides a strong rationality for further pursuing this pathway as a new therapeutic target. Similarly, we found that in lung cancer, the overall neddylation pathway is also overexpressed and serves as an attractive antitumor target (8). These findings indicate that the neddylation pathway serves as a general oncogenic molecular event and a novel anticancer target.

RhoB, an important tumor suppressor, is frequently down-regulated or absent in human cancers, and the underlying mechanisms are elusive (30–35). A previous study indicated that the degradation of RhoB is regulated by TGF-β in the 26 S proteasome-dependent pathway (52). However, Pérez-Sala et al. (53) reported that RhoB degradation may be mediated through the endolysosomal, but not proteasomal pathway in bovine aortic endothelial cells. In this study, we demonstrated that in a broad panel of human cancer cells, the degradation of RhoB is mediated by the ubiquitin-proteasome pathway, because inhibition of this pathway with a classical proteasome inhibitor, MG-132, almost completely blocked RhoB turnover. Furthermore, we demonstrated that CRL E3 ligase, which requires NEDD8 modification for its activation, targets RhoB for degradation. These findings highlight a previously unrecognized role of the neddylation-CRL axis in the regulation of tumor development and progression by targeting RhoB for proteasome-dependent degradation.

CRL is required for the acquisition and maintenance of malignant phenotypes of cancer cells and serves as an attractive anticancer target (11, 54). The core structure of CRL is a complex of cullin-RING finger proteins (RBX1 or RBX2) in which one of seven cullin family members interacts with either RBX1 or RBX2 to activate CRL (50, 55). Unlike other cullin family members whose substrates have been frequently identified (56), only a few substrates of cullin 2, such as HIF-1α (57, 58), have been revealed. In this study, we defined RhoB as a new substrate of cullin 2. As far as we know, this is the first tumor suppressor substrate of cullin 2. Moreover, we further demonstrated that RING finger protein RBX1, which has been shown to be overexpressed in human liver cancer (59), accompanies cullin 2 to target RhoB for degradation. We identified cullin 2-RBX1 E3 ligase as the major modulator of the ubiquitination and degradation of RhoB in cancer cells, which addressed a fundamental question of how the down-regulation of RhoB, an important tumor suppressor, is achieved in human cancer.

Anticancer therapy targeting the neddylation-CRL axis with MLN4924 induces the accumulation of tumor-suppressive CRL substrates to suppress the growth of cancer cells (8, 13). Our previous study demonstrated that MLN4924 induces apoptosis in liver cancer cells (14). The findings obtained from this study further highlight a critical role of RhoB during this process, because 1) RhoB is significantly accumulated upon MLN4924 treatment in a broad panel of cancer cells, and 2) down-regulation of RhoB via siRNA silencing significantly attenuates apoptotic induction and partially rescues growth suppression induced by MLN4924. Mechanistically, we found that ablation of RhoB reduced the expression of tumor suppressors p21 and p27, two targets of RhoB (23, 24). These findings indicate modulation of RhoB expression as a new mechanism of action for neddylation-CRL-targeted anticancer therapy.

On the basis the findings obtained from this study, we propose a working model regarding the potential role of the neddylation-CRL-RhoB axis in liver cancer (Fig. 5F). During liver cancer development, the neddylation-CRL pathway is overactivated, promotes the degradation of tumor suppressor RhoB, and therefore facilitates the liver carcinogenesis and tumor progression. In contrast, inhibition of the neddylation-CRL pathway by MLN4924 blocks cullin neddylation, inactivates CRL, and thus induces the accumulation of RhoB to trigger apoptosis and inhibits the growth of liver cancer cells. Altogether, our study highlights a pivotal role of the neddylation-CRL-RhoB axis in liver carcinogenesis and anticancer therapy against this deadly malignancy.