Pathogenic Roles Of CUL4B In Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and is particularly prevalent in China. Although the risk factors for HCC are well known, the cellular and molecular mechanisms contributing to hepatocarcinogenesis are poorly understood. Since many core pathways that regulate proliferation and survival are commonly dysregulated in a broad variety of malignancy, characterization of possible dysregulation in such pathways may also shed new insights into the development of HCC.Cullin-Ring E3ligase complexes (CRLs), which contain Cullin protein, RING protein, and substrate-recognition subunit as the core components, represent the largest known class of unbiquitin ligases and participate in a broad variety of biological processes, including development, tumorigenesis, cell cycle, signal transduction, transcriptional regulation and viral infection. In mammalians, there are two cullin4proteins, CUL4A and CUL4B, which share more than80%identity in protein. Mutations in human CUL4B cause mental and growth retardation, underscoring the functional importance of CUL4B. Recently, CUL4B has been shown to be substantially upregulated in various types of human cancers including esophageal cancer, lung cancer, stomach cancer, colon cancer, pancreatic cancer and cervical cancer. We have also demonstrate that CUL4B exerts its oncogenic effect by repressing tumor suppressors. By catalyzing H2AK119monoubiquitination, CUL4B-Ring E3ligase complex (CRL4B) can coordinate with Polycomb-Repressive Complex2(PRC2) or SUV39H1/HP1/DNMT3A to promote histone and DNA methylation, leading to transcriptional silencing of tumor suppressor genes including p16, PTEN, and IGFBP3. However, the role of CUL4B in hepatocarcinogenesis remains to be determined. In this study, we aim to characterize the role of CUL4B in occurrence and development of hepatocellular carcinoma using HCC cells, clinical specimens and CUL4B transgenic mice.Part ⅠCUL4B positively regulates Wnt/β-Catenin signaling pathway by repressing Wnt antagonists in hepatocellular carcinomaAberrant activation of Wnt/β-Catenin is frequently observed in many types of cancers including HCC. However, somatic mutations in the genes encoding the components of this signaling pathway have been detected in only30%of human HCC, suggesting other alterations in the pathway may also contribute to the abnormal activation of Wnt/β-Catenin pathway in HCC. To investigate the function of CUL4B and whether CUL4B is involved in regulation of Wnt/β-Catenin signaling, we did the following analysis:1) CUL4B is upregulated in human HCC cell lines and tissues:We first examined the expression level of CUL4B in four human HCC cell lines (HuH-7, HepG2, BEL-7402, SMMC-7721) and two immortalized liver cell lines(HL-7702, LO2). Although CUL4B was detected in all cell lines examined, its level was higher in the four HCC cell lines than in the other two cell lines. We then determined the protein level of CUL4B in24matched pairs of human HCC tumors and their adjacent non-tumor tissues by Western blotting analysis. Consistent with our previous observation made with other solid tumors, CUL4B level was higher in tumor tissues than in the adjacent non-tumor tissues in62.5%(15/24) of HCC tissue pairs examined. To further confirm these results, we next performed immunohistochemistry to evaluate the protein level of CUL4B on161matched pairs of human HCC tumors and their adjacent non-tumor tissues.Significantly increased CUL4B in HCC tumor tissues was observed in51.5%(83/161) of the samples.2) P-Catenin was also upregulated in human HCC tissues and positively correlated with CUL4B:We then examined the expression pattern of β-Catenin and the relationship between CUL4B and β-Catenin expression in HCCs.10out of15cases (66.7%) with overexpressed CUL4B also showed increased accumulation of p-Catenin, suggesting that the expression level of CUL4B and β-Catenin are positively correlated. To further confirm this notion, we examined the level of β-Catenin on HCC tissue arrays containing110samples. Similar to the observation for CUL4B, the level of P-Catenin was found to be higher in tumor tissues than in the adjacent non-tumor tissues in46.4%(51out of110) of the samples. Furthermore, HCC tissues with higher level of CUL4B tend to have elevated accumulation of β-Catenin. Taken together, these results indicate that both CUL4B and P-Catenin are upregulated in HCC tissues and that the two are positively correlated.3) CUL4B functions as a positive regulator of Wnt/β-Catenin signaling pathway in HCC cells:To test the effect of CUL4B on Wnt/β-Catenin signaling in HCC cells, we next determined the level of β-Catenin in CUL4B-knockdown HCC cells. The level of total β-Catenin protein as well as that of the nuclear fraction was significantly reduced in CUL4B-knockdown SMMC-7721and HepG2cells. Consistent with the reduction of P-Catenin caused by depletion of CUL4B, overexpression of CUL4B resulted in up-regulation of P-Catenin in SMMC-7721and HepG2cells. To determine the mechanism by which CUL4B positively regulates β-Catenin, we first examined the mRNA level of CTNNB1(the gene encoding β-Catenin). However, no significant change on the mRNA level of CTNNB1was detected in CUL4B-knockdown cells and CUL4B-overexpressing cells when compared to the corresponding control cells, suggesting that regulation of P-Catenin by CUL4B does not occur at transcriptional level. The effect of CUL4B depletion on β-Catenin and its target gene Cyclin D1was blocked in the presence of proteasome inhibitor MG132, implying that CUL4B may protect β-Catenin from proteasome-mediated degradation. Consistent with this notion, CUL4B depletion resulted in significant increase in β-Catenin degradation.4) CUL4B protects P-Catenin from GSK3-mediated degradation:Because phosphorylation of β-Catenin by GSK3β is crucial for its degradation, we next examined the activity of GSK3β by checking the level of its active and inactive form as well as the total amount. Although no significant change was detected in the total amount and the inactive form of GSK3β in CUL4B-depleted SMMC-7721cells and HepG2cells, the active forms of GSK3β and GSK3a appeared to be increased when compared to those in control cells. We further used three different inhibitors of GSK3activity (lithium chloride, CHIR99021and SB216763). All three inhibitors blocked suppression of β-Catenin signaling caused by CUL4B depletion. Taken together, these results indicate CUL4B protects β-Catenin from GSK3-mediated degradation.5) CUL4B prevents β-Catenin degradation by repressing Wnt antagonists:To further explore the mechanism of regulation of GSK3β by CUL4B, we tested whether CUL4B can activate Wnt/β-Catenin pathway through repressing expression of these natural inhibitors of Wnt pathway. To this end, we determined the mRNA level of10well-characterized Wnt/β-Catenin signaling antagonists that function in various physiologic and pathological conditions such as cancers. Among the antagonists tested, the expression level of AXIN2, PPP2CB, DDK1and PPP2R2B were elevated significantly in CUL4B-depleted cells. To corroborate the negative regulation of these antagonists by CUL4B, we also analyzed the effect of CUL4B overexpression on the expression of Wnt antagonists. As expected, CUL4B overexpression led to decrease in the level of AXIN2, PPP2CB, DKK1and PPP2R2B. To further confirm that activation of Wnt signaling by CUL4B is mediated by repression of antagonists, we performed the rescue experiments by knocking down PPP2R2B in CUL4B-depleted cells. As expected, down-regulation of β-Catenin and its target gene Cyclin D1, and increased active form of GSK3in CUL4B-depleted cells were efficiently attenuated by knockdown of PPP2R2B. Furthermore, double knockdown of PPP2R2B and CUL4B blocked the reduction in the half-life of P-Catenin caused by CUL4B depletion. These results suggest that activation of Wnt signaling and inhibition of β-Catenin degradation by CUL4B is through repression of Wnt antagonists.6) CRL4B coordinates with PRC2to repress transcription of Wnt antagonists:To further characterize the molecular mechanisms responsible for repression of PPP2R2B and DKK1expression by CUL4B, we used chromatin immunoprecipitation (ChIP) to examine whether these genes are bound by CRL4B complex. As expected, ChIP assay showed that CUL4B and EZH2directly bound to the promoters of PPP2R2B and DKK1. Notably, H3K27me3and H2AK119ub1, two histone markers of transcriptional repression, were also enriched in the same region. Furthermore, quantitative ChIP assay showed that depletion of CUL4B led to evident decrease in the recruitment of CUL4B and EZH2to the promoters of PPP2R2B and DKK1. Consistently, the level of H2AK119ubl and H3K27me3were also markedly decreased at PPP2R2B and DKK1promoters, providing further support that CUL4B is required for PRC2-mediated transcriptional repression of Wnt antagonists. Taken together, our results demonstrated that CRL4B/PRC2complexes can repress Wnt antagonists such as PPP2R2B and DKK1by promoting H2AK119monoubiquitination and H3K27trimethylation, and consequently activate Wnt/β-Catenin signaling pathway.7) CUL4B promotes proliferation, colony formation, and invasiveness of HCC cells in vitro as well as tumor growth in vivo. Knockdown of CUL4B resulted in inhibition of cell proliferation, colony formation, and invasion ability in HCC cell lines as well as reduction of tumor growth in xenograft mouse model. Conversely, exogenous overexpression of CUL4B promotes cell proliferation, colony formation and invasiveness in HCC cells. Co-knockdwon Wnt antagonist PPP2R2B could partially rescue the reduced growth inhibition caused by CUL4B down-regulation. These results further support the notion that CUL4B positively regulates Wnt/β-Catenin signaling in conferring its oncogenic activity in HCC.Taken together, we conclude that CUL4B can upregulate Wnt/β-Catenin signaling pathway in human HCCs through transcriptionally repressing Wnt signal antagonists, and thus contributes to the malignancy of HCC. Part IICUL4B promotes hepatocellular carcinoma in CUL4B transgenic miceIn the last three decades, technological advances have facilitated the generation of genetically engineered mouse models (GEMMs) to mimic the alterations frequently observed in human cancers or to conduct intervention studies and assess the relevance of candidate gene networks in tumorigenesis. To clarify the roles of CUL4B in liver tumorigenesis, we generated transgenic mice expressing human CUL4B under the control of the CMV promoter and evaluate its role in hepatocellular carcinoma.1) Generation of CUL4B transgenic mice:To generate CUL4B transgenic mice, an EGFP-CUL4B expression construct was linearized by ApaLI and MluI and purified for pronuclear microinjection. Three transgenic founders were generated based on PCR genotyping. Expression pattern of CUL4B (both human and mouse) in the transgenic mice was examined by real-time RT-PCR. The results showed that CUL4B was highly expressed in heart, liver, colon, lung, thymus, spleen and blood of CUL4B transgenic mice compared to those of littermate control mice. Western bloting showed both human and mouse CUL4B were highly expressed in mouse embryonic fibroblasts (MEF) and livers of CUL4B transgenic mice, but not in those of WT mice. These data indicated that human CUL4B was overexpressed in liver of CUL4B transgenic mice.2) Accelerated hepatocyte proliferation by CUL4B overexpression:We examined the cell proliferation of hepatocytes in CUL4B transgenic mice by BrdU incorporation assay. The BrdU-positive cells in liver of2-week-old CUL4B transgenic mice were significantly increased compared with that in littermate control mice. However, TdT-mediated dUTP nick end labeling (TUNEL) assay failed to detect any significant differences in hepatocyte apoptosis between CUL4B transgenic and littermate control mice. These data suggested that overexpression of CUL4B could greatly accelerate the cell proliferation of hepatocytes. To investigate the molecular mechanism of increased hepatocyte proliferation caused by CUL4B overexpression, the moleculars associated with cell proliferation were detected in livers of2-week-old CUL4B transgenic mice and littermate control mice. Western blotting showed that expressions of Cyclin D1, Cdkl and Cdk4were upregulated, while p16was downregulated, in CUL4B transgenic mice compared to that in littermate control mice.3) Aged CUL4B transgenic mice developed liver tumors:CUL4B overexpression accelerates hepatocyte proliferation, which prompts us to investigate whether overexpression of CUL4B could induce hepatocarcinogenesis in mice. CUL4B transgenic mice developed normally and exhibited no signs of obvious abnormalities until approximately2years, when more than80%(4/5) of mice developed liver tumors, while none of the littermate control mice (0/5) developed liver tumors at the same age. The liver/body weight ratio was not changed because of the small sizes of the tumors. These data suggested that CUL4B overexpression promotes the initiation of liver tumors, which may partially be caused by accelerated hepatocytes proliferation.4) Overexpression of CUL4B strongly promotes DEN-induced hepatocarcinogenesis:DEN is a commonly used chemical carcinogen for the liver, and was often used together with phenobarbital (PB) as a tumor promoter. Mice are sacrificed at26weeks and52weeks post-DEN treatment, CUL4B transgenic mice showed significant increase in the number of tumors, tumor maximum diameter, the liver weight/body weight ratio, levels of alanine aminotransferase (Alanine aminotransferase, ALT) and aspartate aminotransferase (Aspartate aminotransferase, AST).5) Overexpression of CUL4B exacerbates DEN-induced liver injury and compensatory proliferation:The results that overexpression of CUL4B strongly promotes DEN-induced hepatocarcinogenesis prompted us to examine the early effects of DEN on cell behavior and signal transduction. After DEN injection, serum ALT and AST levels as well as the compensatory growth increased significantly in CUL4B transgenic mice compared to those in littermate control mice. We also found that after DEN injection, the ROS levels in the liver of CUL4B transgenic mice was significantly increased compared to that in littermate control mice. Further experiments proved that the increased ROS level in CUL4B transgenic mice was mediated by Prdx3after DEN-induced liver injury.In summary, we generated CUL4B transgenic mice and investigated their susceptibility to hepatocarcinogenesis. We found that overexpression of CUL4B accelerated hepatocyte proliferation and strongly promoted DEN-induced hepatocarcinogenesis. Aged CUL4B transgenic mice developed liver tumors even without DEN induction. Mechanistically, overexpression of CUL4B increased compensatory proliferation and ROS level. These results demonstrated a critical role of CUL4B during hepatocarcinogenesis in mice as well.