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

Abstract

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.

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.

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.

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.

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.

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.

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.