The Cullin-RING Ligase Inhibitor MLN4924 Induces Autophagy And Apoptosis To Suppress Liver Cancer Cell Growth In Vivo And In Vitro

[Background]Liver cancer is the sixth most frequently diagnosed cancer worldwide but the third most frequent cause of cancer death. An estimated 748,300 new liver cancer cases and 695,900 cancer deaths occurred worldwide in 2008. In China, chronic hepatitis B is the leading cause of liver cancer. There are 120 million carriers of hepatitis B virus, accounting for about 9% of total population. Patients with chronic hepatitis B and cirrhosis are more than 30 million. Approximately,10 to 20% of chronic hepatitis B patients may develop cirrhosis,1% to 5% may develop liver cancer. For early-stage liver cancer, surgical resection, transplantation and ablation are treatments that offer a high rate of complete responses and, thus, potential for cure. For advanced liver cancer,transarterialchemoembolisation and sorafenib are the only non-curative treatments that improve survival. Other treatments including radiotherapy,arterialembolisation without chemotherapy have some anti-tumor effects, but survival benefit remains to be proved. Sorafenib is the first FDA-approved drug for liver cancer; however, patients with hepatitis B appeared to have a poorer response. New target drug is in urgent needed for liver cancer targeted therapy, especially in advanced liver cancer.Ubiquitin-proteasomesystem (UPS), one of the two protein degradation pathways, regulates many cellular biological processes including proliferation, differentiation, apoptosis and stress response. The dysfunction of UPS may be involved in tumorigenesis, tumor progression and metastasis, and UPS has become one of the hot targets for anticancer therapy. Bortezomib is the first general proteasome inhibitor, approved by FDA as first line treatment for multiple myeloma. Although it has demonstrated certain anticancer activities, the drug is cytotoxic due to overall inhibition of proteolysis of a wide array of cellular proteins. In UPS, the substrate specificity is depended on the E3 ligase. Therefore, compared to Bortezomib, a specific E3 inhibitor would selectively stabilize a special number of proteins, and achieve a high level of specificity with less cytotoxicity.CRL (Cullin-Ring Ligase) is the largest E3 ubiquitin ligases family responsible for Ubiquitination of about 20% of cellular proteins doomed for UPS degradation. The core structure of CRL is Cullin-RBX.CRL is consisted of four subunits, namely Cullin, RING box protein (RBX), adaptor protein and receptor protein. In human genome, there are 8 Cullin proteins,2 RBX proteins,4 adaptor proteins and 69 F-box proteins. CRL as a large family for different combination of subunits regulates a special set of substrate proteins, including cell cycle regulators, transcription factors, signal transduction molecules, oncogene, tumor suppressor, DNA damage repair protein.CRL activity is possessed by Cullin-RBX complex; furthermore, Cullin neddylation is also indispensable for CRL activity. The Neddylation is a process of adding a NEED8 protein to the C terminal of a Cullin protein. The reaction involves the successive action of NEDD8-activating enzyme (NAE), NEDD8-conjugating enzyme (Ubcl2), and NEED8 E3 ligases. Recently, MLN4924, a specific inhibitor of NAE, was discovered via a high-throughput screening. Because of its significant anticancer efficacy in preclinical studies, MLN4924 has been advanced into several phase Ⅰ and phase Ⅱ clinical trials. In mechanism, MLN4924 inhibits NAE activities by binding to NAE at the active domain to form a covalent NEDD8-MLN4924 adduct. By doing so, MLN4924 inhibits Cullin neddylation, inactivates CRL, causes accumulation of a mass of CRL substrates, and triggers DNA rereplication stress and DNA damage response, as well as induces abnormal cell-cycle progression, apoptosis, and/or senescence to suppress the growth of cancer cells in vitro and in vivo.[Objectives]The study is devoted to elucidate the role and mechanism of MLN4924 suppressing liver cancer cells in vitro and in vivo. With better understanding of the mechanism of action, the study will provide scientific basis for clinical trials of this agent, and promote CRL as a potential target for anti-tumor drug development[Methods]1. The inhibitory of MLN4924 on Huh-7andHep G2 cells proliferation was measured by real-time cell monitoring system usinga Cell-IQ cell-culturing platform and by cell counting; 2. The inhibitory of MLN4924 on Huh-7andHep G2 cell colony formation was measured by a colony formation assay; 3. Cell cycle was measured by propidium iodide (PI) staining and fluorescence activated cellsorting analysis; 4. Cell lysates were prepared for Western blot, using antibodiesagainst Weel, Cullin1, p21, total CHK2 (t-CHK2), total H2A (t-H2A), mTOR, p-4E-BP1 (T37/46), NonP-4E-BP1, P-CHK2 (T68), p-H2A(Ser139), p27, Atg5, Atg7, Beclin 1, cleaved caspase-3, cleaved PARP, p-Histone H3 (Ser10), LC3, and Deptor and glyceraldehyde-3-phosphate dehydrogenase (GAPDH); 5. MLN4924 induced cellular senescence and the expression of senescence-associated β-galactosidase (SA-β-gal) in cells was determined by SA-β-gal staining; 6. Autophagy induced by MLN4924 was measured by Western blot,LC3-EGFP distribution, acridine orange staining and transmission electron microscopy; 7. The mechanism of MLN4924-induced autophagy is determined by Western blot; 8. The kinetics of autophagy and apoptosis responses upon treatment was evaluated by calculating the percentage of cells displaying punctuative distribution of EGFP-LC3 for autophagy and sub-G1 phase distribution for apoptosis, respectively; 9.To determine the effect of autophagy response on apoptosis induction, autophagy was blocked via siRNA silencing of autophagy genes ATG5 and Beclin 1 and apoptosis was evaluated; 10. Two pairsof mouse embryonic fibroblast (MEF) cells, MEF-Atg5-WT (autophagy competent) versus MEF-Atg5-KO (autophagy deficient), MEF-Atg7WT (autophagy competent) versus MEF-Atg7-KO (autophagy deficient), was introduced to evaluate the effect of autophagy response on apoptosis induction; 11. To evaluate the in vivo antitumor activity, MLN4924 was administered to Hep G2-GFP orthotopic xenografts, and the kinetic growth of tumors was monitored via a fluorescence-based imaging system; 12. To explore the in vivo anticancer mechanism of MLN4924, tumor proteins from treated group and control group were extracted and determined by western blot.[Results]MLN4924 suppressed liver cancer cell growth by inactivating CRL and causing cell cycle arrest, apoptosis and cellular senescence in Huh-7andHep G2 cells. MLN4924 completely inhibited Cullin neddylationand significantly suppressed the proliferation of Huh-7 and Hep G2 cells, regardless of p53 status. MLN4924 also notably suppressed cell clonogenic survival in these cells.MLN4924 triggered G2-M cell cycle arrest by PI staining. Furthermore, Weel significantly accumulated, whereas p-Histone H3 sharply decreased upon MLN4924 treatment, indicating that MLN4924-treated cells were arrested at the G2 and failed to enter M-phase. MLN4924 induced DDR in Huh-7 and Hep G2 cells, as shown by the appearance of phosphorylated H2A and CHK2,2 classical markers ofDDR, and induced apoptosis shown by sub-G1 of cell cycle assay and by cleaved PRAP and cleaved Caspase-3. MLN4924 induced cellular senescence in Huh-7 and Hep G2 cells. Mechanism study showed p21 was accumulated upon MLN4924 treatment, indicating MLN4924 induced p21-dependent cellular senescence.MLN4924 induced protective autophagy in liver cancer cells by Deptor accumulation and mTOR inactivation.MLN4924-induced autophagy was measured by following methods:1) MLN4924 induced the conversion of LC3-I to LC3-II, showed by the increasing accumulation of LC3II over time in Huh-7 and Hep G2 cells; 2) MLN4924 induced punctuative distribution of membrane-associated lipidated LC3-II in Huh-7-EGFP-LC3 and Hep G2-EGFP-LC3 cells; 3)MLN4924 induced AVO accumulation Significantly; 4) MLN4924 induced the appearance of double-membraned autophagosome, which contains engulfed bulk cytoplasm and cytoplasmic organelles, as golden hallmark of autophagy by transmission electronic "microscopy. Mechanistically, we found that MLN4924 blocked the turnover of Deptor and led to its accumulation in both Huh-7 and Hep G2 cells. The accumulation of Deptor by MLN4924 resulted in mTOR inactivation, indicated by a remarkable decrease in phosphorylated 4E-BP1 [p-4E-BP1 (T37/46)], which was coupled with remarkable increase in nonphosphorylated 4E-BP1 (NonP-4E-BP1).SiRNA-mediated Deptor knockdown significantly restored the phosphorylation of 4E-BP1 after MLN4924 treatment and attenuated the conversion of LC3-I to LC3-II in liver cancer cells, especially in Hep G2 cells, suggesting that Deptor accumulation is necessary to MLN4924-triggered autophagy.Autophagy occurred prior to apoptosis and played an antiapoptotic role during MLN4924 treatment. Autophagy started to occur as early as 24 hours after MLN4924 treatment, whereas apoptosis started to appear at 48 hours upon treatment in both Huh-7 and Hep G2 cells.Partial blockage of autophagy by siRNA knockdown of ATG5 and Beclin 1 enhanced apoptosis response upon MLN4924 treatment, as measured by caspase-3 activation in Hep G2 cells.MLN4924 induced autophagy response only in MEF-Atg5-WT or MEF-Atg7-WT but not in MEF-Atg5-KO MEF-Atg7-KO cells. Moreover, apoptosis could be obviously induced by MLN4924 even at a lower concentration (1 μmol/L) in autophagy-deficient Atg5-KO but not autophagy-competent Atg5-WT cells. Moreover, the intensity of apoptosis activationin autophagy-deficient cells was much stronger than that in autophagy-competent cells upon MLN4924 treatment at any dose. Similar result was obtained from autophagy-deficient Atg7-KO MEF cells.MLN4924 suppressed tumor growth in vivo by inducing autophagy and apoptosis with good tolerance. MLN4924-treated tumors progressed slowly, whereas control tumors grew rapidly over time, as shown by representative kinetic images of tumors and tumor growth curve.The size and weight of control tumors was significantly higher than that of treated tumors.During the whole treatment, no obvious treatment-related toxicity against body weight, liver function, and kidney function of animals was observed.Mechanistically MLN4924 significantly inhibited Cullin neddylation and led to accumulation of CRL/SCF substrates p21 and p27, indicating the efficient inactivation of CRL/SCF in vivo after treatment. Consistently, MLN4924 induced autophagy as shown by the accumulation of Deptor and conversion of LC3-I to LC3-Ⅱ. Meanwhile, it triggered apoptosis as shown by enhanced activation of PARP and caspase-3 in treated tumors.[Conclusions]1. MLN4924 specifically inhibited NAE, caused CRL inactivation and CRL substrate accumulation, inducing cell cycle arrest, apoptosis and cellular senescence, suppressing liver cancer cell growth.2. MLN4924 induced protective autophagy in liver cancer cells by Deptor accumulation and mTOR inactivation.3. MLN4924 suppressed the growth of liver cancer orthotopic xenografts model by inducing autophagy and apoptosis with good tolerance.