The Functional Mechanism Study Of Neddylation Pathway Overactivation And Targeted Therapy In Cholangiocarcinoma

BackgroundCholangiocarcinoma is a lethal hepatobiliary malignancy, which is secondary to hepatocellular carcinoma in liver cancers. It originates mainly from differentiated biliary epithelial cells and undifferentiated hepatic stem cells. Epidemiological investigation data indicates that the incidence and mortality incidence of cholangiocarcinoma are increasing, but the carcinogenesis of cholangiocarcinoma is still unknown. Because of the specific anatomic location and difficulties in early detection, patients with cholangiocarcinoma are always diagnosed at late stage. Traditional chemotherapy and radiation therapy have limited effect on advanced cholangiocarcinoma, and cannot prolong patients’survival effectively. Thus, it is urgent to identify novel molecules and pathways for cholangiocarcinoma targeted therapy.Neddylation is one new pattern ubiquitin-like posttranslational modification through an enzymatic cascade, and regulates biological functions of the targeted proteins by ligation with a ubiquitin-like protein NEDD8. CRL (Cullin RING Ligase) is one of the most well-characterized substrates, whose activation requires neddylation modification of its core component subunit Cullin. As one of the largest intracellular multi-unit ubiquitin ligases, CRL could regulate the degradation of around 20% proteins through ubiquitin proteasome systerm. MLN4924, a specific neddylation pathway inhibitor, efficiently blocks Cullin neddylation and inhibits CRL function. Due to its significant anti-tumor effects, MLN4924 is currently under phase I clinical trials for the treatment of several types of tumors.To investigate the role of neddylation pathway during the initiation and development of cholangiocarcinoma, we assessed the status of neddylation pathway through various methods and techniques, offering rational reference for neddylation pathway targeted therapy. Moreover, neddylation inhibitor MLN4924 for the treatment of cholangiocarcinoma was evaluated, which paves the way to the usage of MLN4924 in clinic.MethodsFirstly, the status of neddylation in cholangiocarcinoma was systematically evaluated through tumor tissue, including 1) global neddylation protein NEDD8, neddylation catalytic E1 enzyme, NAE and E2 enzyme UBC12; 2) patients follow-up tracking, case reports reviewing about pathological diagnosis and prognosis, providing proof-of-concept for targeting neddylation pathway for cholangiocarcinoma therapy.Secondly, the anticancer effect of MLN4924 on cholangiocarcinoma was evaluated through following experiments:the effect of MLN4924 on cholangiocarcinoma cell proliferation, the mechanism of MLN4924-induced tumor cell death and in vivo cholangiocarcinoma animal model in nude mouse.Resultes1. Neddylation is over-activated in cholangiocarcinoma tissue samples and correlated to cholangiocarcinoma recurrenceThe expression of key components, including NEDD8, NAE and UBC12 in cholangiocarcinoma tissue samples were evaluated through immunohistochemistry. Compared with normal bile duct tissues, global neddylation protein NEDD8 was overexpressed in 68.9%(222/322) of cholangiocarcinoma samples. The NEDD8 activation enzyme NAE subunits, NAE1 and UBA3, were overexpressed in 70.5% (227/322) and 67.1%(216/322) of tumor samples, respectively. And NEDD8 binding enzyme UBC12 was overexpressed in 72.0%(232/322) of cholangiocarcinoma samples. To sum up, these key proteins involved in neddylation pathway were highly expressed in more than 90% of cholangiocarcinoma samples indicateing that Neddylation pathway was over-activated in cholangiocarcinoma. Simutaneously, statistical methods were applied to analyze the correlation between staining results and clinical pathological data, and we found that overexpression of NEDD8, NAE, UBC12 were positively correlated with cholangiocarcinoma post-surgical recurrence.2. Neddylation inhibitor, MLN4924 inhibits cholangiocarcinoma in vitro(1) Neddylation activation in cholangiocarcinoma cell lines and primary cell linesNeddylation activation in cholangiocarcinoma cell lines and four primary cell lines was detected by western blot. NEDD8, NAE1, UBA3 and UBC12 were found activated in both cholangiocarcinoma cell lines and primary cell lines, indicating that neddylation pathway as attractive target for the treatment of cholangiocarcinoma.(2) MLN4924 inhibited cholangiocarcinoma cell growth in vitroAs a potent inhibitor of neddylation pathway, MLN4924 significantly inhibited the growth of human cholangiocarcinoma cell lines QBC939 and RBE detected by ATPlite and CCK8. Colony formation analysis revealed that the clone numbers of the two cholangiocarcinoma cell lines notably reduced with the treatment of MLN4924. These data indicated that MLN4924 can significantly inhibit the proliferation of QBC939 and RBE cells.(3) MLN4924 induced G2 cell cycle arrest and DNA damage response in cholangiocarcinoma cellsThrough flow cytometry, MLN4924 induced G2-M cell cycle arrest in QBC939 and RBE cells. By western blot, we found that Cullin-neddylation was inhibited in QBC939 and RBE cell lines. The inactivation of Cullin-neddylation induced 1) the accumulation of CRL substrates such as cell cycle inhibitory proteins p21, p27 and Weel, and the downregulation of M phase symbol protein p-H3; 2) the accumulation of DNA replications license proteins CDT1 and ORC1,the marker of DNA damage p-H2AX. Our data indicated that, in two cholangiocarcioma cell clines, MLN4924 inhibited the activity of CRL, leading to G2 cell cycle arrest and DNA damage response.(4) MLN4924 induced apoptosis or senescence in cholangiocarcioma cell clinesThrough morphological observation, Sub-G1 phase analysis and c-Caspase-3 and c-PARP detection, we found that MLN4924 induced apoptosis of QBC939 cells, while cellular senescence in RBE cells through SA-β-Gal staining, indicating MLN4924 can induce different cell death in a cell line dependent manner.3. MLN4924 inhibits cholangiocarcinoma in vivoCholangiocarcioma tumor model was established by subcutaneous injection of RBE cells in nude mice, which were then treated with MLN4924. We found that MLN4924 significantly inhibited the proliferation of tumor. Moreover, there are no obvious side effects, indicating high safety of MLN4924.ConclusionIn conclusion, we found that 1) Neddylation pathway was over-activated in cholangiocarcinoma, and was correlated with post-surgical recurrence; 2) MLN4924, the neddylation pathway inhibitor, could effectively inhibit the growth of cholangionoma with no obvious side effects.