Journal of Pathology J Pathol 2015 Published online in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/path.4492
ORIGINAL PAPER
CUL4B activates Wnt/𝛃-catenin signalling in hepatocellular carcinoma by repressing Wnt antagonists Jupeng Yuan,1 Bo Han,2 Huili Hu,1 Yanyan Qian,1 Zhaojian Liu,1 Zhao Wei,1 Xiaohong Liang,3 Baichun Jiang,1 Changshun Shao1 and Yaoqin Gong1* 1
Key Laboratory of Experimental Teratology, Ministry of Education, Institute of Molecular Medicine and Genetics, Shandong University School of Medicine, Jinan, 250012, China 2 Department of Pathology, Shandong University School of Medicine, Jinan, 250012, China 3 Department of Immunity, Shandong University School of Medicine, Jinan, 250012, China *Correspondence to: Yaoqin Gong, Institute of Molecular Medicine and Genetics, Shandong University School of Medicine, 44 Wenhuaxi Road, Jinan, Shandong 250012, PR China. E-mail: [email protected]
Abstract Activation of Wnt/𝛃-catenin signalling is frequently observed in many types of cancer including hepatocellular carcinoma (HCC). We recently reported that cullin 4B (CUL4B), a scaffold protein that assembles CRL4B ubiquitin ligase complexes, is overexpressed in many types of solid tumours and contributes to epigenetic silencing of tumour suppressors. In this study, we characterized the function of CUL4B in HCC and investigated whether CUL4B is involved in the regulation of Wnt/𝛃-catenin signalling. CUL4B and 𝛃-catenin were frequently up-regulated and positively correlated in HCC tissues. CUL4B activated Wnt/𝛃-catenin signalling by protecting 𝛃-catenin from GSK3-mediated degradation, achieved through CUL4B-mediated epigenetic silencing of Wnt pathway antagonists. Knockdown of CUL4B resulted in the up-regulation of Wnt signal antagonists such as DKK1 and PPP2R2B. Simultaneous knockdown of PPP2R2B partially reversed the down-regulation of 𝛃-catenin signalling caused by CUL4B depletion. Furthermore, CRL4B promoted the recruitment and/or retention of PRC2 at the promoters of Wnt antagonists and CUL4B knockdown decreased the retention of PRC2 components as well as H3K27me3. Knockdown of CUL4B reduced the proliferation, colony formation, and invasiveness of HCC cells in vitro and inhibited tumour growth in vivo, and these effects were attenuated by introduction of exogenous 𝛃-catenin or simultaneous knockdown of PPP2R2B. Conversely, ectopic expression of CUL4B enhanced the proliferation and invasiveness of HCC cells. We conclude that CUL4B can up-regulate Wnt/𝛃-catenin signalling in human HCC through transcriptionally repressing Wnt antagonists and thus contributes to the malignancy of HCC. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Keywords: CUL4B; PRC2; HCC; β-catenin; epigenetic regulation; Wnt antagonist
Received 27 May 2014; Revised 11 November 2014; Accepted 26 November 2014
No conflicts of interest were declared.
Introduction Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and is particularly prevalent in Eastern, Southeast Asia, and Africa [1,2]. Although the risk factors for HCC are well known [1–3], 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 malignancies [4,5], it is possible that aberrations in those core pathways may also be involved in the development of HCC. The Wnt/β-catenin signalling pathway belongs to this category for its critical role in embryonic development, cell proliferation, survival, self-renewal, and regeneration [6,7]. In the absence of Wnt ligands, cytoplasmic β-catenin is constitutively phosphorylated by a multi-protein destruction complex consisting of Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
APC, axin, and two kinases, GSK3 and CK1, resulting in its ubiquitination and subsequent proteosomal degradation [6,7]. When Wnt ligands are present, CK1 and GSK3 become inactivated and β-catenin is stabilized and translocates into the nucleus [6–8]. Nuclear β-catenin binds TCF transcription factors to initiate the expression of Wnt target genes, such as c-MYC, CCND1, and c-JUN, to promote tissue growth and development [6,7]. Aberrant activation of this pathway has been documented in several types of cancer [6,9–11] including HCC [12–17]. Abnormally activated Wnt/β-catenin signalling occurs in 40–70% of human HCC cases [12,17]. However, somatic mutations in the genes encoding the components of this signalling pathway, such as CTNNB1 (encoding β-catenin), APC, and AXIN, have been detected in only 12–32.8% of human HCCs [12,18,19]. These results suggest that abnormal activation of the Wnt/β-catenin pathway in HCC may also occur by other mechanisms. J Pathol 2015 www.thejournalofpathology.com
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Cullin–RING E3 ligase (CRL) complexes represent the largest known class of ubiquitin ligases and participate in a broad variety of physiologically and developmentally controlled processes such as cell cycle progression, transcription, and signal transduction [20–22]. In mammals, there are two cullin 4 proteins, CUL4A and CUL4B, which share 82% identity in protein sequences and are believed to be derived from one common ancestor, CUL4 [23]. Mutations in human CUL4B cause mental retardation, short stature, absence of speech, and other phenotypes, underscoring the functional importance of CUL4B [24,25]. Furthermore, in peripheral blood of human CUL4B heterozygotes, cells in which the mutant CUL4B resides on the active X chromosome were strongly selected against [24,26]. Recently, CUL4B has been shown to be up-regulated in various types of solid tumours [27–29]. By catalysing H2AK119 monoubiquitination and coordinating with Polycomb repressive complex 2 (PRC2), CUL4B–RING E3 ligase complex (CRL4B) can promote the transcriptional silencing of an array of tumour suppressor genes including p16 and PTEN [27,29]. In this study, we examined the expression of CUL4B and β-catenin in HCC and investigated the regulation of β-catenin by CUL4B. Our findings indicate that CUL4B positively regulates Wnt/β-catenin signalling through transcriptional repression of Wnt antagonists.
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(TMAs) were used in this study, including one commercial TMA from Superchip (Shanghai, China). The commercial TMA contained 55 hepatocellular carcinoma samples, 51 of them with paired adjacent tissues. These samples were collected from 2006 to 2008, and the information of these patients was well documented. A follow-up survey was made in 2012 to determine the survival of these patients. Using this commercial TMA, we started to examine the effect of CUL4B expression and evaluated the relationship between CUL4B expression levels and survival rates. To confirm these results, 110 pairs of tissue specimens were collected at Qilu Hospital from 2009 to 2011 and used to construct TMAs. The tissue slides were reviewed by a senior pathologist to identify and mark representative tumour areas. Duplicate cylinders (1.0 mm diameter) were taken from the corresponding tumour areas and adjacent non-malignant liver tissue of the individual donor’s tissue blocks. These cylinders were then re-embedded into a recipient paraffin block. Immunohistochemical staining was performed according to standard protocols using specific antibodies against CUL4B and β-catenin. The TMAs were scored for staining intensity using the following criteria: 0 (no signal); 1 (weak); 2 (moderate); and 3 (strong). Detailed clinical and pathological profiles were obtained from medical records.
Statistical analysis Materials and methods More details may be found in the Supplementary materials and methods.
Cell lines and cell culture HL-7702, LO2, HepG2, SMMC-7721, MHCC97H, and MHCC97L were purchased from the Shanghai Cell Collection (Chinese Academy of Sciences) and maintained in Dulbecco’s modified Eagle’s medium (Gibco, Grand Island, NY, USA). BEL-7402 was cultured in RPMI 1640 (Gibco). All media were supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, USA). Cells were maintained at 37 ∘ C in a humidified atmosphere with 5% CO2 .
Tissue specimens and immunohistochemistry The study was approved by the Institutional Review Board at Shandong University School of Medicine. Informed consent was obtained from each patient by the research team prior to surgery, with ethical approval received from Qilu Hospital. A total of 24 pairs of tumour and adjacent tissues were obtained from patients with primary HCC resected at Qilu Hospital of Shandong University (Jinan, China) from 2008 to 2010. Tissue samples were snap-frozen in liquid nitrogen immediately after resection and stored at −80 ∘ C until protein extraction and western blotting analysis, as described previously [27]. Four tissue microarrays Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
Data from the two groups were evaluated statistically by a two-tailed unpaired t-test (GraphPad Software, San Diego, CA, USA). The correlation between the clinical parameters of HCC and the CUL4B or β-catenin staining levels in tissue sections was analysed by the chi-square test. Survival differences were analysed using the log-rank test. In these analyses, p values less than 0.05 were considered significant (*p < 0.05, **p < 0.01, ***p < 0.001).
Results Up-regulation of CUL4B and β-catenin in human HCC cell lines and tissues We first examined CUL4B levels in four human HCC cell lines (HuH-7, HepG2, BEL-7402, and SMMC-7721) and two immortalized liver cell lines. Although CUL4B was detected in all the cell lines examined, its level was higher in the four HCC cell lines than in the immortalized cell lines (Figure 1A). We then determined the protein level of CUL4B in 24 matched pairs of human HCC tumours and their adjacent non-tumour tissues by western blotting analysis. Consistent with our previous observation of other solid tumours [27,29], CUL4B levels were higher in tumour than in adjacent non-tumour in 62.5% (15/24) of HCC tissue pairs examined (Figure 1B). To confirm these results, we performed CUL4B immunohistochemistry on 161 matched pairs of human HCC and their J Pathol 2015 www.thejournalofpathology.com
CUL4B promotes HCC through the Wnt/β-catenin pathway
adjacent non-tumour tissues. Significant up-regulation of CUL4B in HCC tumour tissues was observed in 51.5% (83/161) of the samples (Figure 1C). We did not detect any association of CUL4B expression levels with survival rates for HCC (Supplementary Figure 1A) or with clinicopathological features (Supplementary Table 1). Since aberrant activation of Wnt/β-catenin signalling has been reported in human HCCs [13–16], we then examined the expression pattern of β-catenin and the relationship between CUL4B and β-catenin expression in HCCs. The level of β-catenin was higher in tumour tissues than in the adjacent non-tumour tissues in 45.8% (11/24) of HCC tissue pairs (Figure 1B and Supplementary Figure 1B). Importantly, 10 out of 15 cases (66.7%) with overexpressed CUL4B also showed increased accumulation of β-catenin (Figure 1B and Supplementary Figure 1B, p = 0.008, chi-square test), indicating that CUL4B and β-catenin levels are positively correlated. To confirm this notion, we examined the level of β-catenin on HCC TMAs containing 110 samples. Similar to the observation for CUL4B, the level of β-catenin was found to be higher in tumour tissues than in the adjacent non-tumour tissues in 46.4% (51 out of 110) and higher levels of CUL4B were associated with elevated accumulation of β-catenin (Figures 1D and 1E) (p = 0.0048, chi-square test). Taken together, these results indicate that both CUL4B and β-catenin are up-regulated in HCC tissues and that the two are positively correlated. To determine the contribution of CTNNB1 gene mutation to the up-regulation of β-catenin, we sequenced a region in exon 3 encoding the destruction box in 60 HCC samples with aberrant expression of β-catenin and detected three different mutations in six samples (Figures 1F and 1G). CUL4B was up-regulated in five samples with CTNNB1 mutations (Figure 1G), suggesting that β-catenin-stabilizing mutation and CUL4B up-regulation are not necessarily mutually exclusive. That 39 out of 60 (65%) samples with aberrant expression of β-catenin exhibited CUL4B up-regulation and that the two are positively correlated in expression (Figure 1E) suggest that CUL4B up-regulation may be responsible for a proportion of HCC cases with aberrant expression of β-catenin.
CUL4B functions as a positive regulator of Wnt/β-catenin signalling in HCC cells We next determined the level of β-catenin in CUL4B knockdown HCC cells to test the effect of CUL4B on Wnt/β-catenin signalling in HCC cells. As shown in Figure 2A, the level of total β-catenin protein as well as that of the nuclear fraction was significantly reduced in CUL4B-depleted SMMC-7721 and HepG2 cells. Consistent with the reduction of β-catenin caused by depletion of CUL4B, overexpression of CUL4B resulted in up-regulation of β-catenin in SMMC-7721 and HepG2 cells (Figure 2B). These results suggest that CUL4B acts as a positive regulator of Wnt/β-catenin signalling. To strengthen the argument, we next examined Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
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the level of the active form of β-catenin, which mediates target gene activation. Our results in SMMC-7721 and HepG2 cells showed that activated β-catenin was significantly reduced when CUL4B was knocked down and increased when CUL4B was ectopically expressed (Figure 2C). The influence of CUL4B on β-catenin signalling activity was also determined through a well-established dual-luciferase TCF/β-catenin reporter assay, the TOP/FOP Flash assay. The TOP Flash reporter contains TCF-responsive sites, whereas the FOP Flash reporter, as a negative control, contains mutant TCF binding sites. Depletion of CUL4B significantly reduced TOP Flash luciferase activity, while exogenous CUL4B increased the transactivation of TCF reporter (Figures 2D and 2E). Next, we determined the expression level of the target genes of β-catenin, CCND1 and c-JUN. As shown in Figure 2D, knockdown of CUL4B resulted in a significant reduction in the expression of those target genes at both mRNA and protein level. On the other hand, ectopic expression of CUL4B led to significantly increased expression of both (Figure 2E). Altogether, these results indicate that CUL4B functions as a positive regulator of Wnt/β-catenin oncogenic activity.
CUL4B protects β-catenin from GSK3-mediated degradation in HCC cells To determine the mechanism by which CUL4B positively regulates β-catenin, we first examined the mRNA level of CTNNB1. No significant change in the mRNA level of CTNNB1 was detected in CUL4B-knockdown and CUL4B-overexpressing cells when compared with the corresponding control cells (Supplementary Figures 2A and 2B), suggesting that CUL4B status does not affect CTNNB1 transcription. Because β-catenin is known to be targeted for ubiquitin-mediated proteolysis, we next tested whether β-catenin proteolysis is affected by CUL4B. As shown in Figure 3A, the effect of CUL4B depletion on β-catenin and its target cyclin D1 was blocked in the presence of proteasome inhibitor MG132, implying that CUL4B protects β-catenin from proteasome-mediated degradation. Consistent with this notion, CUL4B depletion resulted in a significant decrease in the half-life of β-catenin (Figure 3B). Because phosphorylation of β-catenin by GSK3 is crucial for its degradation, we next examined the activity of GSK3. Although no significant change was detected in the total amount and the inactive form of GSK3β in CUL4B-depleted SMMC-7721 and HepG2 cells, the active forms of GSK3β and GSK3α appeared to be increased when compared with those in control cells (Figure 3C). These results suggest that the reduction of β-catenin caused by depletion of CUL4B was mediated by increased GSK3 activities. To confirm this notion, we tested whether the down-regulation of β-catenin by CUL4B depletion could be rescued by inhibition of GSK3 activity. As shown in Figure 3D, all three inhibitors of GSK3 activity, lithium chloride, J Pathol 2015 www.thejournalofpathology.com
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Figure 1. CUL4B and β-catenin are overexpressed in HCC cell lines and tissues. (A) Western blot of CUL4B protein in HCC cell lines (HuH-7, HepG2, BEL-7402, and SMMC-7721) and immortalized hepatocytes (HL-7702 and LO2). Numbers underneath the blot represent the fold change in CUL4B band intensity compared with HL-7702 cells, using GAPDH as a loading control. (B) CUL4B and β-catenin proteins in HCC tissues (T) compared with adjacent non-tumour tissues (N) determined by western blotting. (C) CUL4B immunohistochemical staining of tumour array. Staining intensity was classified using a 0–3 scale, corresponding to a, b, c, d (a: 0, negative; b: 1, weak; c: 2, moderate; d: 3, strong). Heat map of HCC CUL4B expression in tumour tissues (T) and paired non-tumour tissues (N). (D) Two representative pairs of tumour and non-tumour tissues showing concordant positive staining of CUL4B and β-catenin in the tumour. (E) Quantitative summary of CUL4B and β-catenin levels in 110 HCC samples (p = 0.0048, chi-square test). (F, G) Sixty HCC samples showing up-regulated expression of β-catenin were sequenced for mutations in exon 3 of CTNNB1. Mutations were detected in six patients (F). Of these, five patients showed a higher CUL4B level in the tumour than in the adjacent non-tumour tissues (G).
CHIR99021, and SB216763, efficiently attenuated the down-regulation of β-catenin and cyclin D1 caused by CUL4B depletion. Taken together, these results indicate that CUL4B protects β-catenin from GSK3-mediated degradation. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
CUL4B prevents β-catenin degradation by repressing Wnt antagonists As shown above, CUL4B can positively regulate Wnt/β-catenin signalling by protecting β-catenin from J Pathol 2015 www.thejournalofpathology.com
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Figure 2. CUL4B functions as a positive regulator of Wnt/β-catenin signalling in HCC cells. (A) Total protein or nuclear fractions from CUL4B-depleted (shCUL4B) and control (shControl) SMMC-7721 and HepG2 cells analysed by western blotting. (B) β-catenin levels in HCC cells overexpressing CUL4B. (C) Levels of the active form of β-catenin (*β-catenin) in HCC cells with knockdown or overexpression of CUL4B. (D, E) The activity of TCF/β-catenin reporter (TOP/FOP Flash) and the mRNA and protein levels of β-catenin target genes CCND1 and c-JUN in CUL4B-knockdown (D) and CUL4B-overexpressing cells (E). Results are represented as the fold change over control. Error bars represent the standard deviation (SD) of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001 (two-tailed unpaired t-test).
GSK3-mediated degradation. Wnt pathway antagonists are known either to interfere with the binding of Wnt proteins to their receptors or to organize the destruction complex for β-catenin degradation. Because epigenetic silencing of Wnt antagonists mediated by EZH2 has been shown to contribute to activation of Wnt signalling in HCC cells [14] and CRL4B is also capable of repressing transcription in cooperation with PRC2 [27], we next tested whether CUL4B can up-regulate the Wnt/β-catenin pathway through inhibiting the expression of these natural inhibitors of Wnt. To this end, we determined the mRNA levels Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
of ten well-characterized Wnt/β-catenin signalling antagonists that function in various physiological and pathological conditions such as cancers by a quantitative RT-PCR with the gene-specific primers (Supplementary Table 2) [6,30–33]. Of the antagonists tested, AXIN2, PPP2CB, DDK1, and PPP2R2B were elevated significantly in CUL4B-depleted cells. The transcript levels of PPP2R2B and DKK1 were increased 40and 20-fold, respectively, in CUL4B-depleted cells compared with controls (Figure 4A). To corroborate the negative regulation of these antagonists by CUL4B, we also analysed the effect of CUL4B overexpression, J Pathol 2015 www.thejournalofpathology.com
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Figure 3. CUL4B protects β-catenin from GSK-mediated degradation in HCC cells. (A) Western blotting of whole cell lysates of the indicated cells treated with or without 30 μM MG132 for 24 h. (B) CUL4B-knockdown and control SMMC-7721 cells were treated with 50 μg/ml cycloheximide, harvested at the indicated time points, and subjected to western blotting. Below the blots: β-catenin protein levels were quantified by densitometric analysis using Quantity One. Expression is represented as the percentage relative to 0 h. (C) Active GSK3, inactive GSK3β, and total GSK3β were analysed by western blotting from CUL4B-knockdown and control SMMC-7721 or HepG2 cells. (D) CUL4B-knockdown SMMC-7721 and control cells were treated with and without indicated GSK3 inhibitors, LiCl, CHIR99021 or SB216763. Cell lysates were collected after 24 h and analysed by western blotting. *β-catenin: active form of β-catenin.
which reduced the mRNA levels of AXIN2, PPP2CB, DKK1, and PPP2R2B (Figure 4B). Western blotting results confirmed the effect of CUL4B on the expression of PPP2R2B and DKK1 (Figure 4C). These results indicate that the expression of these antagonists is negatively regulated by CUL4B. To confirm that activation Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
of Wnt signalling by CUL4B is mediated by repression of antagonists, we performed rescue experiments by knocking down PPP2R2B in CUL4B-depleted cells. As shown in Figure 4D, down-regulation of β-catenin and cyclin D1 and activation of GSK3 in CUL4B-depleted cells were efficiently attenuated by J Pathol 2015 www.thejournalofpathology.com
CUL4B promotes HCC through the Wnt/β-catenin pathway
knockdown of PPP2R2B. Furthermore, double knockdown of PPP2R2B and CUL4B blocked the reduction in the half-life of β-catenin caused by CUL4B depletion (Supplementary Figure 3). These results suggest that the activation of Wnt signalling by CUL4B is through repression of Wnt antagonists. To characterize the molecular mechanisms responsible for repression of PPP2R2B and DKK1 expression by CUL4B further, we used chromatin immunoprecipitation (ChIP) to examine whether these genes are bound by the CUL4B complex using the primers specific for the promoters of PPP2R2B and DKK1 (Supplementary Table 3). ChIP showed that CUL4B and EZH2 directly bound to the promoters of PPP2R2B and DKK1. Notably, H3K27me3 and H2AK119ub1, two histone marks of transcriptional repression, were also enriched in the same region (Figure 4E). Furthermore, quantitative ChIP showed that depletion of CUL4B decreased CUL4B and EZH2 recruitment to the promoters of PPP2R2B and DKK1 (Figure 4F). Consistently, the levels of H2AK119ub1 and H3K27me3 were also markedly decreased at PPP2R2B and DKK1 promoters, providing further support that CUL4B is required for PRC2-mediated transcriptional repression of Wnt antagonists (Figure 4F). On the other hand, knockdown of EZH2 did not affect the recruitment of CUL4B to PPP2R2B and DKK1 promoters and H2AK119 monoubiquitination (Figure 4G), despite the fact that it derepressed PPP2R2B and DKK1 expression and down-regulated β-catenin, cyclin D1, and c-Jun (Supplementary Figure 4), supporting the notion that CRL4B promotes the recruitment and/or retention of PRC2 at the promoters of targeted genes. These results are consistent with our previous observation in oesophageal cancer cells [27]. Taken together, our results demonstrated that CRL4B/PRC2 complexes can repress Wnt antagonists such as PPP2R2B and DKK1 by promoting H2AK119 monoubiquitination and H3K27 trimethylation, and consequently activate Wnt/β-catenin signalling.
CUL4B promotes HCC cell proliferation and migration by repressing Wnt/β-catenin signalling antagonists Frequent overexpression of CUL4B in HCC cell lines and primary tumours indicated that CUL4B might function as an oncogene in HCC. We thus examined the effect of CUL4B on HCC cell proliferation and migration by manipulating its expression in HCC cell lines. As shown in Figure 5A, knockdown of CUL4B reduced cell proliferation in SMMC-7721 cells, whereas overexpression led to enhanced proliferation. Colony formation assays further showed that the CUL4B expression level was positively correlated with the number of clones formed (Figure 5B). Importantly, while knockdown of CUL4B decreased cell proliferation and colony numbers, this was efficiently blocked by introduction of exogenous β-catenin or partially blocked by simultaneous knockdown of PPP2R2B (Figures 5C, Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
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5D, and Supplementary Figure 5A and 5B), suggesting that the growth-promoting effect of CUL4B in HCC cells is achieved through suppressing Wnt/β-catenin antagonists. Next, we investigated whether CUL4B could increase the motility of HCC cells. Overexpression of CUL4B caused a more than 2-fold increase in cell migration, while depletion of CUL4B resulted in an approximately 5-fold decrease (Figure 5E). The decreased motility upon CUL4B depletion was attenuated when exogenous β-catenin was expressed or PPP2R2B was concomitantly knocked down (Figure 5F and Supplementary Figure 5C), indicating that CUL4B can promote the migration of HCC cells by repressing Wnt/β-catenin signalling antagonists.
Knockdown of CUL4B suppresses tumour growth in vivo To evaluate the tumour-promoting ability of CUL4B in vivo, tumour formation in nude mice was assessed by s.c. injection of CUL4B-depleted HCC cells and their control cells followed by measuring the size every 2 days. The results showed that tumour growth was suppressed by CUL4B knockdown (Figure 6A). Furthermore, introduction of exogenous β-catenin could efficiently rescue the reduced tumour growth caused by CUL4B knockdown (Figure 6B). Immunohistochemical staining for CUL4B, β-catenin, c-Jun, and PPP2R2B on serial xenograft tumour sections showed that, compared with the control, tumours derived from CUL4B-depleted HCC cells exhibited significantly lower levels of β-catenin and c-Jun, while PPP2R2B was elevated (Figure 6C). These results further support the notion that CUL4B positively regulates Wnt/β-catenin signalling in conferring its oncogenic activity in HCC.
Discussion By assembling the CRL4B ubiquitin ligase complex with DDB1 and ROC1, CUL4B participates in the regulation of a broad spectrum of biological processes, such as cell cycle progression, DNA replication, and DNA damage response [34]. Previous studies showed that CRL4B can target different substrates for proteosomal degradation or protein modification [27,35–38] and that CUL4B is overexpressed in many types of solid tumours [27–29]. However, the role of CUL4B in hepatocarcinogenesis is unknown. In this study, we demonstrated that CUL4B is up-regulated in HCC cell lines as well as in clinical specimens and that CUL4B exerts its oncogenic effect by positively regulating the Wnt/β-catenin signalling pathway. Moreover, we showed that CUL4B functions as a positive regulator of Wnt/β-catenin by transcriptionally repressing several Wnt signalling antagonists such as DKK1 and PPP2R2B, thus protecting β-catenin from GSK3-mediated degradation. J Pathol 2015 www.thejournalofpathology.com
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Figure 4. CUL4B transcriptionally represses Wnt antagonists. Expression of Wnt pathway antagonists measured by real-time RT-PCR in (A) CUL4B-knockdown and (B) CUL4B-overexpressing SMMC-7721 cells and controls. The mRNA levels of Wnt pathway antagonists were normalized to that of GADPH. *p < 0.05; **p < 0.01; ***p < 0.001 (two-tailed unpaired t-test). (C) Western blotting of PPP2R2B and DKK1 in CUL4B-knockdown, CUL4B-overexpressing, and control SMMC-7721 cells. (D) Western blotting assays of SMMC-7721 cells with PPP2R2B and CUL4B knockdown. (E) ChIP experiments of the PPP2R2B and DKK1 promoters in SMMC-7721 cells using the indicated antibodies. p16 was used as a positive control. (F, G) qChIP analysis of the PPP2R2B and DKK1 promoters in CUL4B-knockdown, EZH2-knockdown, and control SMMC-7721 cells using the indicated antibodies. Results are represented as the fold change over control. Error bars represent the SD of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001 (two-tailed unpaired t-test).
Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
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Figure 5. CUL4B promotes HCC cell proliferation and migration in vitro. (A) MTT assays of CUL4B-knockdown, CUL4B-overexpressing cells, and control SMMC-7721 cells. Error bars represent the SD of three independent experiments. (B) Colony formation efficiency of HCC cells with CUL4B depletion or overexpression. Representative photographs are shown on the left and statistical analysis is on the right. Error bars represent the SD of three independent experiments. The effect of ectopic expression of β-catenin on cell proliferation (C) and colony formation (D) of CUL4B-knockdown SMMC-7721 cells. (E) Cell migration assays of CUL4B-depleted or -overexpressing cells. Representative photographs are shown on the left and statistical analysis is on the right. Error bars represent the SD of three independent experiments. (F) Motility of CUL4B depletion and exogenous β-catenin expression or PPP2R2B knockdown. *p < 0.05; **p < 0.01; ***p < 0.001 (two-tailed unpaired t-test).
We propose that under physiological conditions, the Wnt signalling antagonists are expressed to restrain the activity of Wnt/β-catenin signalling. When CUL4B is up-regulated, the CRL4B–PRC2 complex represses Wnt antagonist expression, thus unleashing Wnt/β-catenin signalling and driving tumourigenesis. Evidence supporting the oncogenic role of CUL4B is accumulating. Our recent studies showed CUL4B overexpression in several types of solid tumours including oesophageal, lung, gastric, colon, pancreas, and cervical carcinomas [27]. More recently, using a large cohort of colon cancers, Jiang et al found that high CUL4B expression was associated with depth of tumour invasion, lymph node metastasis, distant metastasis, histological differentiation, vascular invasion, and advanced tumour stage [28]. Positive correlations between CUL4B and histological grade Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
were also detected in oesophageal and cervical carcinomas [27,29]. However, while we demonstrated a tumour-promoting role of CUL4B in HCC, CUL4B expression in clinical samples was not associated with any of the clinicopathological features examined. Lack of an association between the CUL4B level and the degree of malignancy is in agreement with studies on β-catenin accumulation in HCC [12,15,16,39]. Conceivably, other factors may confound the tumour-promoting roles of CUL4B in clinical tissues, such as oncogenic alterations of Wnt-related genes. These questions need further investigation. Aberrant activation of Wnt/β-catenin signalling is well documented to be closely associated with carcinogenesis in different cancers [9]. In this study, we showed that CUL4B and β-catenin were often concordantly overexpressed in human HCC tissues. In contrast J Pathol 2015 www.thejournalofpathology.com
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Figure 6. Down-regulation of CUL4B suppresses HCC tumour growth in vivo. (A, B) Xenograft tumour growth in vivo of CUL4B-depleted SMMC-7721 and HepG2 cells (A) and of these cells with ectopic expression of β-catenin (B). Each group contained nine mice. Error bars represent the SEM. *p < 0.05; **p < 0.01; ***p < 0.001 (two-tailed unpaired t-test). (C) Immunohistochemical staining of CUL4B, β-catenin, c-Jun, and PPP2R2B in serial xenograft tumour sections.
to findings that CRL4B may target β-catenin for degradation in an AhR ligand-dependent manner [40], our results demonstrated CUL4B as a positive regulator of β-catenin. Knockdown of CUL4B significantly reduced the level of β-catenin as well as the expression of its target genes CCND1 and c-JUN, while overexpression of CUL4B increased β-catenin accumulation and target gene expression. These results reveal a novel mechanism responsible for activation of the Wnt/β-catenin pathway in human HCC. While mutations in the Wnt pathway-related genes such as CNNTB1, APC, and AXIN have been found to contribute to Wnt/β-catenin signalling activation, these mutations only account for a small proportion of human HCCs with aberrantly activated Wnt/β-catenin [12,17]. Previous observations made with a large cohort of HCC patients (n = 421) Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
from Taiwan showed that the CTNNB1 gene was less frequently mutated in HBV-related HCC than in non-HBV-related HCC (9.3% versus 25%) [41]. Consistent with that observation, the vast majority (90 out of 105, 85.7%) of patients in our cohort were HBV carriers and only 10% of tumour samples with up-regulated β-catenin carried mutations within the degradation box in exon 3 of the CTNNB1 gene, indicating that CTNNB1 mutations are not the main cause of dysregulation of Wnt/β-catenin signalling in our cohort. In addition to genetic alterations, epigenetic aberrations including DNA methylation, histone modifications, and microRNA dysregulation can also lead to dysregulation of Wnt/β-catenin signalling in tumours [6,10,14,42]. Our previous studies have shown that CRL4B can catalyse H2AK119 monoubiquitination J Pathol 2015 www.thejournalofpathology.com
CUL4B promotes HCC through the Wnt/β-catenin pathway
and coordinate with PRC2 to repress the expression of a set of tumour suppressors. In addition, EZH2, the core component of the PRC2 complex, is frequently overexpressed in HCC [43,44] and has been shown to up-regulate Wnt/β-catenin signalling by repressing the expression of Wnt antagonists [14]. Consistent with these observations, we observed that Wnt antagonists such as PPP2R2B and DKK1 were up-regulated when CUL4B was depleted and down-regulated when it was overexpressed. Simultaneous knockdown of the Wnt antagonist PPP2R2B attenuated the down-regulation of β-catenin caused by CUL4B depletion. Furthermore, the ChIP assay confirmed that CUL4B and EZH2 co-occupied the promoters of PPP2R2B and DKK1. Knockdown of CUL4B led to a significant reduction in the promoter occupancy by CUL4B, EZH2, H2AK119ub1, and H3K27me3, leading to the transcriptional up-regulation of the PPP2R2B and DKK1 genes. Altogether, our results establish a novel mechanistic link between CUL4B and Wnt/β-catenin signalling in human HCC. Our results indicate that CUL4B acts as an epigenetic modifier in HCC. Up-regulated Wnt signalling antagonists due to CUL4B depletion may inhibit Wnt/β-catenin signalling in different subcellular compartments and through different processes. For example, DKK1 inhibits the interaction between receptor and ligand, whereas AXIN2, PPP2CB, and PPP2R2B are components of the destruction complex that targets β-catenin for proteosomal degradation [6,14]. Our results showed that CUL4B depletion increased the levels of active GSK3α and GSK3β, which in turn decreased β-catenin levels, suggesting that active GSK3 is important for β-catenin reduction in CUL4B-depleted cells. This is supported by the rescue experiments with GSK3 inhibitors. Therefore, CUL4B activates Wnt/β-catenin by protecting β-catenin from GSK3-mediated degradation. In conclusion, CUL4B contributes to hepatocarcinogenesis by activating Wnt/β-catenin signalling through repressing Wnt pathway antagonists and preventing GSK3-mediated β-catenin degradation. Our findings may be helpful for the development of new diagnostic and therapeutic strategies for HCC.
Acknowledgments We thank Dr Chunhong Ma for her critical suggestion and technical assistance. This work was supported by National Basic Research Program of China (973 Program) grants (2011CB966200, 2013CB910900) and grants (81330050, 81321061, 81101522) from the National Natural Science Foundation of China.
Author contribution statement JY performed experiments, including cell culture, western blotting, real-time RT-PCR, immunohistochemistry, Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
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MTT assays, colony formation assays as well as transwell migration assays; analysed data; and wrote the first draft of the manuscript. BH provided tissue microarrays, performed clinical studies, and participated in data interpretation. HH performed ChIP. YQ performed in vivo studies and statistical analysis. ZL and ZW assisted with viral production. XL provided HCC tissue and performed clinical studies. BJ participated in in vivo studies and data interpretation. CS interpreted data and critically revised the manuscript. YG conceived the concept, designed experiments, interpreted data, and critically revised the manuscript.
Abbreviations ChIP, chromatin immunoprecipitation; CRL4B, cullin 4B–RING E3 ligase complex; EZH2, enhancer of zeste homologue 2; H3K27me3, H3K27 trimethylation; HCC, hepatocellular carcinoma; PRC2, Polycomb repressive complex 2
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31. Kimelman D, Xu W. β-catenin destruction complex: insights and questions from a structural perspective. Oncogene 2006; 25: 7482-7491. 32. Kikuchi A. Roles of Axin in the Wnt signalling pathway. Cell Signal 1999; 11: 777-788. 33. Kawano Y, Kypta R. Secreted antagonists of the Wnt signalling pathway. J Cell Sci 2003; 116: 2627-2634. 34. Jackson S, Xiong Y. CRL4s: the CUL4–RING E3 ubiquitin ligases. Trends Biochem Sci 2009; 34: 562-570. 35. He F, Lu D, Jiang B, et al. X-linked intellectual disability gene CUL4B targets Jab1/CSN5 for degradation and regulates bone morphogenetic protein signaling. Biochim Biophys Acta 2013; 1832: 595-605. 36. Li X, Lu D, He F, et al. Cullin 4B protein ubiquitin ligase targets peroxiredoxin III for degradation. J Biol Chem 2011; 286: 32344-32354. 37. Lan L, Nakajima S, Kapetanaki MG, et al. Monoubiquitinated histone H2A destabilizes photolesion-containing nucleosomes with concomitant release of UV-damaged DNA-binding protein E3 ligase. J Biol Chem 2012; 287: 12036-12049. 38. Nakagawa T, Xiong Y. X-linked mental retardation gene CUL4B targets ubiquitylation of H3K4 methyltransferase component WDR5 and regulates neuronal gene expression. Mol Cell 2011; 43: 381-391. 39. Ban KC, Singh H, Krishnan R, et al. GSK-3β phosphorylation and alteration of β-catenin in hepatocellular carcinoma. Cancer Lett 2003; 199: 201-208. 40. Kawajiri K, Kobayashi Y, Ohtake F, et al. Aryl hydrocarbon receptor suppresses intestinal carcinogenesis in ApcMin/+ mice with natural ligands. Proc Natl Acad Sci U S A 2009; 106: 13481-13486. 41. Hsu HC, Jeng YM, Mao TL, et al. β-catenin mutations are associated with a subset of low-stage hepatocellular carcinoma negative for hepatitis B virus and with favorable prognosis. Am J Pathol 2000; 157: 763-770. 42. Liu Y, Huang T, Zhao X, et al. MicroRNAs modulate the Wnt signaling pathway through targeting its inhibitors. Biochem Biophys Res Commun 2011; 408: 259-264. 43. Sudo T, Utsunomiya T, Mimori K, et al. Clinicopathological significance of EZH2 mRNA expression in patients with hepatocellular carcinoma. Br J Cancer 2005; 92: 1754-1758. 44. Chen Y, Lin MC, Yao H, et al. Lentivirus-mediated RNA interference targeting enhancer of zeste homolog 2 inhibits hepatocellular carcinoma growth through down-regulation of stathmin. Hepatology 2007; 46: 200-208. 45. Peters SR (ed.). A Practical Guide to Frozen Section Technique. Springer: New York, 2010.
SUPPORTING INFORMATION ON THE INTERNET The following supporting information may be found in the online version of this article: Supplementary materials and methods. Figure S1. CUL4B expression level is not associated with survival rates of HCC, but is associated with β-catenin level in HCCs. Figure S2. The mRNA level of CTNNB1 is not regulated by CUL4B. Figure S3. CUL4B prevents β-catenin degradation through repressing Wnt pathway antagonists. Figure S4. EZH2 transcriptionally repressed Wnt antagonists. Figure S5. CUL4B enhances the colony formation efficiency and motility of HCC cells through the Wnt pathway. Table S1. Statistical analysis of CUL4B expression in HCC samples. Table S2. Primers used for real-time PCR. Table S3. Primers used for ChIP PCR.
Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
J Pathol 2015 www.thejournalofpathology.com
ORIGINAL PAPER
CUL4B activates Wnt/𝛃-catenin signalling in hepatocellular carcinoma by repressing Wnt antagonists Jupeng Yuan,1 Bo Han,2 Huili Hu,1 Yanyan Qian,1 Zhaojian Liu,1 Zhao Wei,1 Xiaohong Liang,3 Baichun Jiang,1 Changshun Shao1 and Yaoqin Gong1* 1
Key Laboratory of Experimental Teratology, Ministry of Education, Institute of Molecular Medicine and Genetics, Shandong University School of Medicine, Jinan, 250012, China 2 Department of Pathology, Shandong University School of Medicine, Jinan, 250012, China 3 Department of Immunity, Shandong University School of Medicine, Jinan, 250012, China *Correspondence to: Yaoqin Gong, Institute of Molecular Medicine and Genetics, Shandong University School of Medicine, 44 Wenhuaxi Road, Jinan, Shandong 250012, PR China. E-mail: [email protected]
Abstract Activation of Wnt/𝛃-catenin signalling is frequently observed in many types of cancer including hepatocellular carcinoma (HCC). We recently reported that cullin 4B (CUL4B), a scaffold protein that assembles CRL4B ubiquitin ligase complexes, is overexpressed in many types of solid tumours and contributes to epigenetic silencing of tumour suppressors. In this study, we characterized the function of CUL4B in HCC and investigated whether CUL4B is involved in the regulation of Wnt/𝛃-catenin signalling. CUL4B and 𝛃-catenin were frequently up-regulated and positively correlated in HCC tissues. CUL4B activated Wnt/𝛃-catenin signalling by protecting 𝛃-catenin from GSK3-mediated degradation, achieved through CUL4B-mediated epigenetic silencing of Wnt pathway antagonists. Knockdown of CUL4B resulted in the up-regulation of Wnt signal antagonists such as DKK1 and PPP2R2B. Simultaneous knockdown of PPP2R2B partially reversed the down-regulation of 𝛃-catenin signalling caused by CUL4B depletion. Furthermore, CRL4B promoted the recruitment and/or retention of PRC2 at the promoters of Wnt antagonists and CUL4B knockdown decreased the retention of PRC2 components as well as H3K27me3. Knockdown of CUL4B reduced the proliferation, colony formation, and invasiveness of HCC cells in vitro and inhibited tumour growth in vivo, and these effects were attenuated by introduction of exogenous 𝛃-catenin or simultaneous knockdown of PPP2R2B. Conversely, ectopic expression of CUL4B enhanced the proliferation and invasiveness of HCC cells. We conclude that CUL4B can up-regulate Wnt/𝛃-catenin signalling in human HCC through transcriptionally repressing Wnt antagonists and thus contributes to the malignancy of HCC. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Keywords: CUL4B; PRC2; HCC; β-catenin; epigenetic regulation; Wnt antagonist
Received 27 May 2014; Revised 11 November 2014; Accepted 26 November 2014
No conflicts of interest were declared.
Introduction Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and is particularly prevalent in Eastern, Southeast Asia, and Africa [1,2]. Although the risk factors for HCC are well known [1–3], 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 malignancies [4,5], it is possible that aberrations in those core pathways may also be involved in the development of HCC. The Wnt/β-catenin signalling pathway belongs to this category for its critical role in embryonic development, cell proliferation, survival, self-renewal, and regeneration [6,7]. In the absence of Wnt ligands, cytoplasmic β-catenin is constitutively phosphorylated by a multi-protein destruction complex consisting of Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
APC, axin, and two kinases, GSK3 and CK1, resulting in its ubiquitination and subsequent proteosomal degradation [6,7]. When Wnt ligands are present, CK1 and GSK3 become inactivated and β-catenin is stabilized and translocates into the nucleus [6–8]. Nuclear β-catenin binds TCF transcription factors to initiate the expression of Wnt target genes, such as c-MYC, CCND1, and c-JUN, to promote tissue growth and development [6,7]. Aberrant activation of this pathway has been documented in several types of cancer [6,9–11] including HCC [12–17]. Abnormally activated Wnt/β-catenin signalling occurs in 40–70% of human HCC cases [12,17]. However, somatic mutations in the genes encoding the components of this signalling pathway, such as CTNNB1 (encoding β-catenin), APC, and AXIN, have been detected in only 12–32.8% of human HCCs [12,18,19]. These results suggest that abnormal activation of the Wnt/β-catenin pathway in HCC may also occur by other mechanisms. J Pathol 2015 www.thejournalofpathology.com
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Cullin–RING E3 ligase (CRL) complexes represent the largest known class of ubiquitin ligases and participate in a broad variety of physiologically and developmentally controlled processes such as cell cycle progression, transcription, and signal transduction [20–22]. In mammals, there are two cullin 4 proteins, CUL4A and CUL4B, which share 82% identity in protein sequences and are believed to be derived from one common ancestor, CUL4 [23]. Mutations in human CUL4B cause mental retardation, short stature, absence of speech, and other phenotypes, underscoring the functional importance of CUL4B [24,25]. Furthermore, in peripheral blood of human CUL4B heterozygotes, cells in which the mutant CUL4B resides on the active X chromosome were strongly selected against [24,26]. Recently, CUL4B has been shown to be up-regulated in various types of solid tumours [27–29]. By catalysing H2AK119 monoubiquitination and coordinating with Polycomb repressive complex 2 (PRC2), CUL4B–RING E3 ligase complex (CRL4B) can promote the transcriptional silencing of an array of tumour suppressor genes including p16 and PTEN [27,29]. In this study, we examined the expression of CUL4B and β-catenin in HCC and investigated the regulation of β-catenin by CUL4B. Our findings indicate that CUL4B positively regulates Wnt/β-catenin signalling through transcriptional repression of Wnt antagonists.
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(TMAs) were used in this study, including one commercial TMA from Superchip (Shanghai, China). The commercial TMA contained 55 hepatocellular carcinoma samples, 51 of them with paired adjacent tissues. These samples were collected from 2006 to 2008, and the information of these patients was well documented. A follow-up survey was made in 2012 to determine the survival of these patients. Using this commercial TMA, we started to examine the effect of CUL4B expression and evaluated the relationship between CUL4B expression levels and survival rates. To confirm these results, 110 pairs of tissue specimens were collected at Qilu Hospital from 2009 to 2011 and used to construct TMAs. The tissue slides were reviewed by a senior pathologist to identify and mark representative tumour areas. Duplicate cylinders (1.0 mm diameter) were taken from the corresponding tumour areas and adjacent non-malignant liver tissue of the individual donor’s tissue blocks. These cylinders were then re-embedded into a recipient paraffin block. Immunohistochemical staining was performed according to standard protocols using specific antibodies against CUL4B and β-catenin. The TMAs were scored for staining intensity using the following criteria: 0 (no signal); 1 (weak); 2 (moderate); and 3 (strong). Detailed clinical and pathological profiles were obtained from medical records.
Statistical analysis Materials and methods More details may be found in the Supplementary materials and methods.
Cell lines and cell culture HL-7702, LO2, HepG2, SMMC-7721, MHCC97H, and MHCC97L were purchased from the Shanghai Cell Collection (Chinese Academy of Sciences) and maintained in Dulbecco’s modified Eagle’s medium (Gibco, Grand Island, NY, USA). BEL-7402 was cultured in RPMI 1640 (Gibco). All media were supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, USA). Cells were maintained at 37 ∘ C in a humidified atmosphere with 5% CO2 .
Tissue specimens and immunohistochemistry The study was approved by the Institutional Review Board at Shandong University School of Medicine. Informed consent was obtained from each patient by the research team prior to surgery, with ethical approval received from Qilu Hospital. A total of 24 pairs of tumour and adjacent tissues were obtained from patients with primary HCC resected at Qilu Hospital of Shandong University (Jinan, China) from 2008 to 2010. Tissue samples were snap-frozen in liquid nitrogen immediately after resection and stored at −80 ∘ C until protein extraction and western blotting analysis, as described previously [27]. Four tissue microarrays Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
Data from the two groups were evaluated statistically by a two-tailed unpaired t-test (GraphPad Software, San Diego, CA, USA). The correlation between the clinical parameters of HCC and the CUL4B or β-catenin staining levels in tissue sections was analysed by the chi-square test. Survival differences were analysed using the log-rank test. In these analyses, p values less than 0.05 were considered significant (*p < 0.05, **p < 0.01, ***p < 0.001).
Results Up-regulation of CUL4B and β-catenin in human HCC cell lines and tissues We first examined CUL4B levels in four human HCC cell lines (HuH-7, HepG2, BEL-7402, and SMMC-7721) and two immortalized liver cell lines. Although CUL4B was detected in all the cell lines examined, its level was higher in the four HCC cell lines than in the immortalized cell lines (Figure 1A). We then determined the protein level of CUL4B in 24 matched pairs of human HCC tumours and their adjacent non-tumour tissues by western blotting analysis. Consistent with our previous observation of other solid tumours [27,29], CUL4B levels were higher in tumour than in adjacent non-tumour in 62.5% (15/24) of HCC tissue pairs examined (Figure 1B). To confirm these results, we performed CUL4B immunohistochemistry on 161 matched pairs of human HCC and their J Pathol 2015 www.thejournalofpathology.com
CUL4B promotes HCC through the Wnt/β-catenin pathway
adjacent non-tumour tissues. Significant up-regulation of CUL4B in HCC tumour tissues was observed in 51.5% (83/161) of the samples (Figure 1C). We did not detect any association of CUL4B expression levels with survival rates for HCC (Supplementary Figure 1A) or with clinicopathological features (Supplementary Table 1). Since aberrant activation of Wnt/β-catenin signalling has been reported in human HCCs [13–16], we then examined the expression pattern of β-catenin and the relationship between CUL4B and β-catenin expression in HCCs. The level of β-catenin was higher in tumour tissues than in the adjacent non-tumour tissues in 45.8% (11/24) of HCC tissue pairs (Figure 1B and Supplementary Figure 1B). Importantly, 10 out of 15 cases (66.7%) with overexpressed CUL4B also showed increased accumulation of β-catenin (Figure 1B and Supplementary Figure 1B, p = 0.008, chi-square test), indicating that CUL4B and β-catenin levels are positively correlated. To confirm this notion, we examined the level of β-catenin on HCC TMAs containing 110 samples. Similar to the observation for CUL4B, the level of β-catenin was found to be higher in tumour tissues than in the adjacent non-tumour tissues in 46.4% (51 out of 110) and higher levels of CUL4B were associated with elevated accumulation of β-catenin (Figures 1D and 1E) (p = 0.0048, chi-square test). Taken together, these results indicate that both CUL4B and β-catenin are up-regulated in HCC tissues and that the two are positively correlated. To determine the contribution of CTNNB1 gene mutation to the up-regulation of β-catenin, we sequenced a region in exon 3 encoding the destruction box in 60 HCC samples with aberrant expression of β-catenin and detected three different mutations in six samples (Figures 1F and 1G). CUL4B was up-regulated in five samples with CTNNB1 mutations (Figure 1G), suggesting that β-catenin-stabilizing mutation and CUL4B up-regulation are not necessarily mutually exclusive. That 39 out of 60 (65%) samples with aberrant expression of β-catenin exhibited CUL4B up-regulation and that the two are positively correlated in expression (Figure 1E) suggest that CUL4B up-regulation may be responsible for a proportion of HCC cases with aberrant expression of β-catenin.
CUL4B functions as a positive regulator of Wnt/β-catenin signalling in HCC cells We next determined the level of β-catenin in CUL4B knockdown HCC cells to test the effect of CUL4B on Wnt/β-catenin signalling in HCC cells. As shown in Figure 2A, the level of total β-catenin protein as well as that of the nuclear fraction was significantly reduced in CUL4B-depleted SMMC-7721 and HepG2 cells. Consistent with the reduction of β-catenin caused by depletion of CUL4B, overexpression of CUL4B resulted in up-regulation of β-catenin in SMMC-7721 and HepG2 cells (Figure 2B). These results suggest that CUL4B acts as a positive regulator of Wnt/β-catenin signalling. To strengthen the argument, we next examined Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
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the level of the active form of β-catenin, which mediates target gene activation. Our results in SMMC-7721 and HepG2 cells showed that activated β-catenin was significantly reduced when CUL4B was knocked down and increased when CUL4B was ectopically expressed (Figure 2C). The influence of CUL4B on β-catenin signalling activity was also determined through a well-established dual-luciferase TCF/β-catenin reporter assay, the TOP/FOP Flash assay. The TOP Flash reporter contains TCF-responsive sites, whereas the FOP Flash reporter, as a negative control, contains mutant TCF binding sites. Depletion of CUL4B significantly reduced TOP Flash luciferase activity, while exogenous CUL4B increased the transactivation of TCF reporter (Figures 2D and 2E). Next, we determined the expression level of the target genes of β-catenin, CCND1 and c-JUN. As shown in Figure 2D, knockdown of CUL4B resulted in a significant reduction in the expression of those target genes at both mRNA and protein level. On the other hand, ectopic expression of CUL4B led to significantly increased expression of both (Figure 2E). Altogether, these results indicate that CUL4B functions as a positive regulator of Wnt/β-catenin oncogenic activity.
CUL4B protects β-catenin from GSK3-mediated degradation in HCC cells To determine the mechanism by which CUL4B positively regulates β-catenin, we first examined the mRNA level of CTNNB1. No significant change in the mRNA level of CTNNB1 was detected in CUL4B-knockdown and CUL4B-overexpressing cells when compared with the corresponding control cells (Supplementary Figures 2A and 2B), suggesting that CUL4B status does not affect CTNNB1 transcription. Because β-catenin is known to be targeted for ubiquitin-mediated proteolysis, we next tested whether β-catenin proteolysis is affected by CUL4B. As shown in Figure 3A, the effect of CUL4B depletion on β-catenin and its target cyclin D1 was blocked in the presence of proteasome inhibitor MG132, implying that CUL4B protects β-catenin from proteasome-mediated degradation. Consistent with this notion, CUL4B depletion resulted in a significant decrease in the half-life of β-catenin (Figure 3B). Because phosphorylation of β-catenin by GSK3 is crucial for its degradation, we next examined the activity of GSK3. Although no significant change was detected in the total amount and the inactive form of GSK3β in CUL4B-depleted SMMC-7721 and HepG2 cells, the active forms of GSK3β and GSK3α appeared to be increased when compared with those in control cells (Figure 3C). These results suggest that the reduction of β-catenin caused by depletion of CUL4B was mediated by increased GSK3 activities. To confirm this notion, we tested whether the down-regulation of β-catenin by CUL4B depletion could be rescued by inhibition of GSK3 activity. As shown in Figure 3D, all three inhibitors of GSK3 activity, lithium chloride, J Pathol 2015 www.thejournalofpathology.com
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Figure 1. CUL4B and β-catenin are overexpressed in HCC cell lines and tissues. (A) Western blot of CUL4B protein in HCC cell lines (HuH-7, HepG2, BEL-7402, and SMMC-7721) and immortalized hepatocytes (HL-7702 and LO2). Numbers underneath the blot represent the fold change in CUL4B band intensity compared with HL-7702 cells, using GAPDH as a loading control. (B) CUL4B and β-catenin proteins in HCC tissues (T) compared with adjacent non-tumour tissues (N) determined by western blotting. (C) CUL4B immunohistochemical staining of tumour array. Staining intensity was classified using a 0–3 scale, corresponding to a, b, c, d (a: 0, negative; b: 1, weak; c: 2, moderate; d: 3, strong). Heat map of HCC CUL4B expression in tumour tissues (T) and paired non-tumour tissues (N). (D) Two representative pairs of tumour and non-tumour tissues showing concordant positive staining of CUL4B and β-catenin in the tumour. (E) Quantitative summary of CUL4B and β-catenin levels in 110 HCC samples (p = 0.0048, chi-square test). (F, G) Sixty HCC samples showing up-regulated expression of β-catenin were sequenced for mutations in exon 3 of CTNNB1. Mutations were detected in six patients (F). Of these, five patients showed a higher CUL4B level in the tumour than in the adjacent non-tumour tissues (G).
CHIR99021, and SB216763, efficiently attenuated the down-regulation of β-catenin and cyclin D1 caused by CUL4B depletion. Taken together, these results indicate that CUL4B protects β-catenin from GSK3-mediated degradation. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
CUL4B prevents β-catenin degradation by repressing Wnt antagonists As shown above, CUL4B can positively regulate Wnt/β-catenin signalling by protecting β-catenin from J Pathol 2015 www.thejournalofpathology.com
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Figure 2. CUL4B functions as a positive regulator of Wnt/β-catenin signalling in HCC cells. (A) Total protein or nuclear fractions from CUL4B-depleted (shCUL4B) and control (shControl) SMMC-7721 and HepG2 cells analysed by western blotting. (B) β-catenin levels in HCC cells overexpressing CUL4B. (C) Levels of the active form of β-catenin (*β-catenin) in HCC cells with knockdown or overexpression of CUL4B. (D, E) The activity of TCF/β-catenin reporter (TOP/FOP Flash) and the mRNA and protein levels of β-catenin target genes CCND1 and c-JUN in CUL4B-knockdown (D) and CUL4B-overexpressing cells (E). Results are represented as the fold change over control. Error bars represent the standard deviation (SD) of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001 (two-tailed unpaired t-test).
GSK3-mediated degradation. Wnt pathway antagonists are known either to interfere with the binding of Wnt proteins to their receptors or to organize the destruction complex for β-catenin degradation. Because epigenetic silencing of Wnt antagonists mediated by EZH2 has been shown to contribute to activation of Wnt signalling in HCC cells [14] and CRL4B is also capable of repressing transcription in cooperation with PRC2 [27], we next tested whether CUL4B can up-regulate the Wnt/β-catenin pathway through inhibiting the expression of these natural inhibitors of Wnt. To this end, we determined the mRNA levels Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
of ten well-characterized Wnt/β-catenin signalling antagonists that function in various physiological and pathological conditions such as cancers by a quantitative RT-PCR with the gene-specific primers (Supplementary Table 2) [6,30–33]. Of the antagonists tested, AXIN2, PPP2CB, DDK1, and PPP2R2B were elevated significantly in CUL4B-depleted cells. The transcript levels of PPP2R2B and DKK1 were increased 40and 20-fold, respectively, in CUL4B-depleted cells compared with controls (Figure 4A). To corroborate the negative regulation of these antagonists by CUL4B, we also analysed the effect of CUL4B overexpression, J Pathol 2015 www.thejournalofpathology.com
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Figure 3. CUL4B protects β-catenin from GSK-mediated degradation in HCC cells. (A) Western blotting of whole cell lysates of the indicated cells treated with or without 30 μM MG132 for 24 h. (B) CUL4B-knockdown and control SMMC-7721 cells were treated with 50 μg/ml cycloheximide, harvested at the indicated time points, and subjected to western blotting. Below the blots: β-catenin protein levels were quantified by densitometric analysis using Quantity One. Expression is represented as the percentage relative to 0 h. (C) Active GSK3, inactive GSK3β, and total GSK3β were analysed by western blotting from CUL4B-knockdown and control SMMC-7721 or HepG2 cells. (D) CUL4B-knockdown SMMC-7721 and control cells were treated with and without indicated GSK3 inhibitors, LiCl, CHIR99021 or SB216763. Cell lysates were collected after 24 h and analysed by western blotting. *β-catenin: active form of β-catenin.
which reduced the mRNA levels of AXIN2, PPP2CB, DKK1, and PPP2R2B (Figure 4B). Western blotting results confirmed the effect of CUL4B on the expression of PPP2R2B and DKK1 (Figure 4C). These results indicate that the expression of these antagonists is negatively regulated by CUL4B. To confirm that activation Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
of Wnt signalling by CUL4B is mediated by repression of antagonists, we performed rescue experiments by knocking down PPP2R2B in CUL4B-depleted cells. As shown in Figure 4D, down-regulation of β-catenin and cyclin D1 and activation of GSK3 in CUL4B-depleted cells were efficiently attenuated by J Pathol 2015 www.thejournalofpathology.com
CUL4B promotes HCC through the Wnt/β-catenin pathway
knockdown of PPP2R2B. Furthermore, double knockdown of PPP2R2B and CUL4B blocked the reduction in the half-life of β-catenin caused by CUL4B depletion (Supplementary Figure 3). These results suggest that the activation of Wnt signalling by CUL4B is through repression of Wnt antagonists. To characterize the molecular mechanisms responsible for repression of PPP2R2B and DKK1 expression by CUL4B further, we used chromatin immunoprecipitation (ChIP) to examine whether these genes are bound by the CUL4B complex using the primers specific for the promoters of PPP2R2B and DKK1 (Supplementary Table 3). ChIP showed that CUL4B and EZH2 directly bound to the promoters of PPP2R2B and DKK1. Notably, H3K27me3 and H2AK119ub1, two histone marks of transcriptional repression, were also enriched in the same region (Figure 4E). Furthermore, quantitative ChIP showed that depletion of CUL4B decreased CUL4B and EZH2 recruitment to the promoters of PPP2R2B and DKK1 (Figure 4F). Consistently, the levels of H2AK119ub1 and H3K27me3 were also markedly decreased at PPP2R2B and DKK1 promoters, providing further support that CUL4B is required for PRC2-mediated transcriptional repression of Wnt antagonists (Figure 4F). On the other hand, knockdown of EZH2 did not affect the recruitment of CUL4B to PPP2R2B and DKK1 promoters and H2AK119 monoubiquitination (Figure 4G), despite the fact that it derepressed PPP2R2B and DKK1 expression and down-regulated β-catenin, cyclin D1, and c-Jun (Supplementary Figure 4), supporting the notion that CRL4B promotes the recruitment and/or retention of PRC2 at the promoters of targeted genes. These results are consistent with our previous observation in oesophageal cancer cells [27]. Taken together, our results demonstrated that CRL4B/PRC2 complexes can repress Wnt antagonists such as PPP2R2B and DKK1 by promoting H2AK119 monoubiquitination and H3K27 trimethylation, and consequently activate Wnt/β-catenin signalling.
CUL4B promotes HCC cell proliferation and migration by repressing Wnt/β-catenin signalling antagonists Frequent overexpression of CUL4B in HCC cell lines and primary tumours indicated that CUL4B might function as an oncogene in HCC. We thus examined the effect of CUL4B on HCC cell proliferation and migration by manipulating its expression in HCC cell lines. As shown in Figure 5A, knockdown of CUL4B reduced cell proliferation in SMMC-7721 cells, whereas overexpression led to enhanced proliferation. Colony formation assays further showed that the CUL4B expression level was positively correlated with the number of clones formed (Figure 5B). Importantly, while knockdown of CUL4B decreased cell proliferation and colony numbers, this was efficiently blocked by introduction of exogenous β-catenin or partially blocked by simultaneous knockdown of PPP2R2B (Figures 5C, Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
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5D, and Supplementary Figure 5A and 5B), suggesting that the growth-promoting effect of CUL4B in HCC cells is achieved through suppressing Wnt/β-catenin antagonists. Next, we investigated whether CUL4B could increase the motility of HCC cells. Overexpression of CUL4B caused a more than 2-fold increase in cell migration, while depletion of CUL4B resulted in an approximately 5-fold decrease (Figure 5E). The decreased motility upon CUL4B depletion was attenuated when exogenous β-catenin was expressed or PPP2R2B was concomitantly knocked down (Figure 5F and Supplementary Figure 5C), indicating that CUL4B can promote the migration of HCC cells by repressing Wnt/β-catenin signalling antagonists.
Knockdown of CUL4B suppresses tumour growth in vivo To evaluate the tumour-promoting ability of CUL4B in vivo, tumour formation in nude mice was assessed by s.c. injection of CUL4B-depleted HCC cells and their control cells followed by measuring the size every 2 days. The results showed that tumour growth was suppressed by CUL4B knockdown (Figure 6A). Furthermore, introduction of exogenous β-catenin could efficiently rescue the reduced tumour growth caused by CUL4B knockdown (Figure 6B). Immunohistochemical staining for CUL4B, β-catenin, c-Jun, and PPP2R2B on serial xenograft tumour sections showed that, compared with the control, tumours derived from CUL4B-depleted HCC cells exhibited significantly lower levels of β-catenin and c-Jun, while PPP2R2B was elevated (Figure 6C). These results further support the notion that CUL4B positively regulates Wnt/β-catenin signalling in conferring its oncogenic activity in HCC.
Discussion By assembling the CRL4B ubiquitin ligase complex with DDB1 and ROC1, CUL4B participates in the regulation of a broad spectrum of biological processes, such as cell cycle progression, DNA replication, and DNA damage response [34]. Previous studies showed that CRL4B can target different substrates for proteosomal degradation or protein modification [27,35–38] and that CUL4B is overexpressed in many types of solid tumours [27–29]. However, the role of CUL4B in hepatocarcinogenesis is unknown. In this study, we demonstrated that CUL4B is up-regulated in HCC cell lines as well as in clinical specimens and that CUL4B exerts its oncogenic effect by positively regulating the Wnt/β-catenin signalling pathway. Moreover, we showed that CUL4B functions as a positive regulator of Wnt/β-catenin by transcriptionally repressing several Wnt signalling antagonists such as DKK1 and PPP2R2B, thus protecting β-catenin from GSK3-mediated degradation. J Pathol 2015 www.thejournalofpathology.com
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Figure 4. CUL4B transcriptionally represses Wnt antagonists. Expression of Wnt pathway antagonists measured by real-time RT-PCR in (A) CUL4B-knockdown and (B) CUL4B-overexpressing SMMC-7721 cells and controls. The mRNA levels of Wnt pathway antagonists were normalized to that of GADPH. *p < 0.05; **p < 0.01; ***p < 0.001 (two-tailed unpaired t-test). (C) Western blotting of PPP2R2B and DKK1 in CUL4B-knockdown, CUL4B-overexpressing, and control SMMC-7721 cells. (D) Western blotting assays of SMMC-7721 cells with PPP2R2B and CUL4B knockdown. (E) ChIP experiments of the PPP2R2B and DKK1 promoters in SMMC-7721 cells using the indicated antibodies. p16 was used as a positive control. (F, G) qChIP analysis of the PPP2R2B and DKK1 promoters in CUL4B-knockdown, EZH2-knockdown, and control SMMC-7721 cells using the indicated antibodies. Results are represented as the fold change over control. Error bars represent the SD of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001 (two-tailed unpaired t-test).
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Figure 5. CUL4B promotes HCC cell proliferation and migration in vitro. (A) MTT assays of CUL4B-knockdown, CUL4B-overexpressing cells, and control SMMC-7721 cells. Error bars represent the SD of three independent experiments. (B) Colony formation efficiency of HCC cells with CUL4B depletion or overexpression. Representative photographs are shown on the left and statistical analysis is on the right. Error bars represent the SD of three independent experiments. The effect of ectopic expression of β-catenin on cell proliferation (C) and colony formation (D) of CUL4B-knockdown SMMC-7721 cells. (E) Cell migration assays of CUL4B-depleted or -overexpressing cells. Representative photographs are shown on the left and statistical analysis is on the right. Error bars represent the SD of three independent experiments. (F) Motility of CUL4B depletion and exogenous β-catenin expression or PPP2R2B knockdown. *p < 0.05; **p < 0.01; ***p < 0.001 (two-tailed unpaired t-test).
We propose that under physiological conditions, the Wnt signalling antagonists are expressed to restrain the activity of Wnt/β-catenin signalling. When CUL4B is up-regulated, the CRL4B–PRC2 complex represses Wnt antagonist expression, thus unleashing Wnt/β-catenin signalling and driving tumourigenesis. Evidence supporting the oncogenic role of CUL4B is accumulating. Our recent studies showed CUL4B overexpression in several types of solid tumours including oesophageal, lung, gastric, colon, pancreas, and cervical carcinomas [27]. More recently, using a large cohort of colon cancers, Jiang et al found that high CUL4B expression was associated with depth of tumour invasion, lymph node metastasis, distant metastasis, histological differentiation, vascular invasion, and advanced tumour stage [28]. Positive correlations between CUL4B and histological grade Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
were also detected in oesophageal and cervical carcinomas [27,29]. However, while we demonstrated a tumour-promoting role of CUL4B in HCC, CUL4B expression in clinical samples was not associated with any of the clinicopathological features examined. Lack of an association between the CUL4B level and the degree of malignancy is in agreement with studies on β-catenin accumulation in HCC [12,15,16,39]. Conceivably, other factors may confound the tumour-promoting roles of CUL4B in clinical tissues, such as oncogenic alterations of Wnt-related genes. These questions need further investigation. Aberrant activation of Wnt/β-catenin signalling is well documented to be closely associated with carcinogenesis in different cancers [9]. In this study, we showed that CUL4B and β-catenin were often concordantly overexpressed in human HCC tissues. In contrast J Pathol 2015 www.thejournalofpathology.com
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Figure 6. Down-regulation of CUL4B suppresses HCC tumour growth in vivo. (A, B) Xenograft tumour growth in vivo of CUL4B-depleted SMMC-7721 and HepG2 cells (A) and of these cells with ectopic expression of β-catenin (B). Each group contained nine mice. Error bars represent the SEM. *p < 0.05; **p < 0.01; ***p < 0.001 (two-tailed unpaired t-test). (C) Immunohistochemical staining of CUL4B, β-catenin, c-Jun, and PPP2R2B in serial xenograft tumour sections.
to findings that CRL4B may target β-catenin for degradation in an AhR ligand-dependent manner [40], our results demonstrated CUL4B as a positive regulator of β-catenin. Knockdown of CUL4B significantly reduced the level of β-catenin as well as the expression of its target genes CCND1 and c-JUN, while overexpression of CUL4B increased β-catenin accumulation and target gene expression. These results reveal a novel mechanism responsible for activation of the Wnt/β-catenin pathway in human HCC. While mutations in the Wnt pathway-related genes such as CNNTB1, APC, and AXIN have been found to contribute to Wnt/β-catenin signalling activation, these mutations only account for a small proportion of human HCCs with aberrantly activated Wnt/β-catenin [12,17]. Previous observations made with a large cohort of HCC patients (n = 421) Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
from Taiwan showed that the CTNNB1 gene was less frequently mutated in HBV-related HCC than in non-HBV-related HCC (9.3% versus 25%) [41]. Consistent with that observation, the vast majority (90 out of 105, 85.7%) of patients in our cohort were HBV carriers and only 10% of tumour samples with up-regulated β-catenin carried mutations within the degradation box in exon 3 of the CTNNB1 gene, indicating that CTNNB1 mutations are not the main cause of dysregulation of Wnt/β-catenin signalling in our cohort. In addition to genetic alterations, epigenetic aberrations including DNA methylation, histone modifications, and microRNA dysregulation can also lead to dysregulation of Wnt/β-catenin signalling in tumours [6,10,14,42]. Our previous studies have shown that CRL4B can catalyse H2AK119 monoubiquitination J Pathol 2015 www.thejournalofpathology.com
CUL4B promotes HCC through the Wnt/β-catenin pathway
and coordinate with PRC2 to repress the expression of a set of tumour suppressors. In addition, EZH2, the core component of the PRC2 complex, is frequently overexpressed in HCC [43,44] and has been shown to up-regulate Wnt/β-catenin signalling by repressing the expression of Wnt antagonists [14]. Consistent with these observations, we observed that Wnt antagonists such as PPP2R2B and DKK1 were up-regulated when CUL4B was depleted and down-regulated when it was overexpressed. Simultaneous knockdown of the Wnt antagonist PPP2R2B attenuated the down-regulation of β-catenin caused by CUL4B depletion. Furthermore, the ChIP assay confirmed that CUL4B and EZH2 co-occupied the promoters of PPP2R2B and DKK1. Knockdown of CUL4B led to a significant reduction in the promoter occupancy by CUL4B, EZH2, H2AK119ub1, and H3K27me3, leading to the transcriptional up-regulation of the PPP2R2B and DKK1 genes. Altogether, our results establish a novel mechanistic link between CUL4B and Wnt/β-catenin signalling in human HCC. Our results indicate that CUL4B acts as an epigenetic modifier in HCC. Up-regulated Wnt signalling antagonists due to CUL4B depletion may inhibit Wnt/β-catenin signalling in different subcellular compartments and through different processes. For example, DKK1 inhibits the interaction between receptor and ligand, whereas AXIN2, PPP2CB, and PPP2R2B are components of the destruction complex that targets β-catenin for proteosomal degradation [6,14]. Our results showed that CUL4B depletion increased the levels of active GSK3α and GSK3β, which in turn decreased β-catenin levels, suggesting that active GSK3 is important for β-catenin reduction in CUL4B-depleted cells. This is supported by the rescue experiments with GSK3 inhibitors. Therefore, CUL4B activates Wnt/β-catenin by protecting β-catenin from GSK3-mediated degradation. In conclusion, CUL4B contributes to hepatocarcinogenesis by activating Wnt/β-catenin signalling through repressing Wnt pathway antagonists and preventing GSK3-mediated β-catenin degradation. Our findings may be helpful for the development of new diagnostic and therapeutic strategies for HCC.
Acknowledgments We thank Dr Chunhong Ma for her critical suggestion and technical assistance. This work was supported by National Basic Research Program of China (973 Program) grants (2011CB966200, 2013CB910900) and grants (81330050, 81321061, 81101522) from the National Natural Science Foundation of China.
Author contribution statement JY performed experiments, including cell culture, western blotting, real-time RT-PCR, immunohistochemistry, Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
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MTT assays, colony formation assays as well as transwell migration assays; analysed data; and wrote the first draft of the manuscript. BH provided tissue microarrays, performed clinical studies, and participated in data interpretation. HH performed ChIP. YQ performed in vivo studies and statistical analysis. ZL and ZW assisted with viral production. XL provided HCC tissue and performed clinical studies. BJ participated in in vivo studies and data interpretation. CS interpreted data and critically revised the manuscript. YG conceived the concept, designed experiments, interpreted data, and critically revised the manuscript.
Abbreviations ChIP, chromatin immunoprecipitation; CRL4B, cullin 4B–RING E3 ligase complex; EZH2, enhancer of zeste homologue 2; H3K27me3, H3K27 trimethylation; HCC, hepatocellular carcinoma; PRC2, Polycomb repressive complex 2
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SUPPORTING INFORMATION ON THE INTERNET The following supporting information may be found in the online version of this article: Supplementary materials and methods. Figure S1. CUL4B expression level is not associated with survival rates of HCC, but is associated with β-catenin level in HCCs. Figure S2. The mRNA level of CTNNB1 is not regulated by CUL4B. Figure S3. CUL4B prevents β-catenin degradation through repressing Wnt pathway antagonists. Figure S4. EZH2 transcriptionally repressed Wnt antagonists. Figure S5. CUL4B enhances the colony formation efficiency and motility of HCC cells through the Wnt pathway. Table S1. Statistical analysis of CUL4B expression in HCC samples. Table S2. Primers used for real-time PCR. Table S3. Primers used for ChIP PCR.
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