Published OnlineFirst October 24, 2017; DOI: 10.1158/0008-5472.CAN-17-0700

Cancer Molecular Cell Biology Research

CBX8 Exhibits Oncogenic Activity via AKT/ b-Catenin Activation in Hepatocellular Carcinoma Chris Zhiyi Zhang1,2, Shi-Lu Chen1,2, Chun-Hua Wang1,2, Yang-Fan He1,2, Xia Yang1,2, Dan Xie1,2, and Jing-Ping Yun1,2

Abstract

Deregulation of polycomb influences the develop- 3p, which promoted the nuclear localization of b-catenin by ment and progression of hepatocellular carcinoma. Here we show targeting the 30-UTR ZNRF1. Inhibiting either EGR1 or miR- that chromobox 8 (CBX8) expression is increased in hepatocel- 365a-3p partially rescued CBX8-mediated malignant phenotypes. lular carcinoma and correlates with poor outcome in two inde- In clinical samples, CBX8 expression closely correlated with pendent cohorts containing a total of 879 cases. Ectopic expres- EGR1, miR-365a-3p, and nuclear b-catenin. Collectively, our sion of CBX8 facilitated tumor growth and metastasis, whereas results show that CBX8 functions as an oncogene to upregulate CBX8 silencing suppressed these effects. CBX8 efficiently activated EGR1 and miR-365-3p to stimulate the AKT/b-catenin pathway. AKT/b-catenin signaling via upregulation of the transcription This newly identified signaling axis may suggest new therapeutic factor EGR1 and miR-365-3p in a noncanonical manner: CBX8 strategies against hepatocellular carcinoma. directly bound the EGR1 promoter to enhance its activity. In the Significance: Elucidation of a key new element of the b-catenin nucleus, CBX8 also interacted with EGR1 to prevent its degrada- signaling pathway in liver cancer may suggest new therapeutic tion. Furthermore, CBX8 increased the transcription of miR-365a- targets. Cancer Res; 78(1); 51–63. 2017 AACR.

Introduction Canonical PRC1 includes four subunits: ring E3 ubiquitin ligase, polyhomeotic, posterior sex combs, and polycomb Hepatocellular carcinoma, accounting for most (70–90%) of (8). The cores of PRC1 complexes are polycomb group (PcG) the primary liver cancers occurring worldwide, remains one of the proteins, which were first identified as developmental regula- most prevalent and deadliest human cancers (1). The 5-year tors in Drosophila (9). Chromobox homolog 8 (CBX8), also survival for patients with hepatocellular carcinoma generally did known as human polycomb 3 (HPC3), functions as a tran- not improve during the last decades (2). Uncontrolled cell pro- scriptional repressor in PRC1. For example, CBX8 inhibited the liferation and metastasis are responsible for the high mortality expression of INK4a/ARF to bypass cell senescence in fibro- and represent major obstacles to the clinical management of blasts (10). However, a later study showed that PRC1 without hepatocellular carcinoma (3–5). As a result, discovery of the CBX8 was capable of suppressing the INK4a/ARF (11), mechanisms underlying tumor progression is important to devel- suggesting an unclear role of CBX8 in transcriptional regula- op new strategies for therapeutic treatment of hepatocellular tion. Recently, CBX8 has been demonstrated to exert oncogenic carcinoma. functions in a noncanonical manner in human malignancies. Deregulation of involved in chromatin modification Lee and colleagues reported that CBX8 cooperated with has been increasingly implicated in tumor development and SIRT1 to suppress premature senescence and growth arrest in progression. One of the chromatin modifiers is the polycomb breast carcinoma (12). The PRC1–BCOR–CBX8 complex is repressive complex (PRC1 and PRC2; ref. 6). Under normal required for BCL6-mediated lymphomagenesis (13). Chung circumstances, PRC1 maintains the histone methylation and colleagues proposed that CBX8 transcriptionally activated inducedbyPRC2topassontheinactivationsignals(7). genes involved in the Notch pathway promote breast cancer (14). However, the role of CBX8 in hepatocellular carcinoma remains unclear. 1 Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in The b-catenin signaling pathway is well known for its role in South China, Collaborative Innovation Center for Cancer Medicine, Guangzhou, 2 driving carcinogenesis (15). b-Catenin can be frequently found at China. Department of Pathology, Sun Yat-sen University Cancer Center, b Guangzhou, China. the cell surface. Following stimulation, -catenin is activated, partly by phosphorylation at Ser552, to dissociate in the cyto- Note: Supplementary data for this article are available at Cancer Research b Online (http://cancerres.aacrjournals.org/). plasm. The cytosolic accumulation of -catenin leads to its local- ization to the nucleus where it triggers the transcription of various C.Z. Zhang, S.-L. Chen, and C.-H. Wang contributed equally to this article. oncogenes, including LEF1/TCF, to promote cancer initiation and Corresponding Author: Jing-Ping Yun, Sun Yat-sen University Cancer Center, progression (16). The activation of b-catenin, one of the factors No. 651 Dongfeng Road East, Guangzhou, 510060 Guangdong, China. Phone: contributing to hepatocarcinogenesis (17), is typically mediated 86-020-87342258; Fax: 86-020-87342258; E-mail: [email protected] by Wnt. Recent studies showed that the b-catenin signaling axis doi: 10.1158/0008-5472.CAN-17-0700 can be triggered by AKT via either phosphorylation of b-catenin 2017 American Association for Cancer Research. at Ser552, which enhances its transcriptional activity (18), or

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suppression of GSK-3b (19). However, the regulation of the AKT/ sized with the miRCURY LNATM Universal cDNA Synthesis Kit b-catenin pathway in hepatocellular carcinoma is not fully (Exiqon). For mRNA analyses, cDNA was synthesized using understood. Moloney murine leukemia virus reverse transcriptase (Pro- Using tissue microarray (TMA)-based IHC, microarrays, mega). qRT-PCR was performed with SYBR Premix ExTaq and biological function assays, we identified CBX8 as an onco- (TaKaRa) with the Stratagene Mx3000P real-time PCR system gene in hepatocellular carcinoma. CBX8 expression is increased (Agilent Technologies, Inc.). Expression levels of miR-365a-3p and associated with poor outcomes of patients with hepato- were normalized against the endogenous snRNA U6 control, cellular carcinoma. We further demonstrate that CBX8 pro- whereas 18s rRNA was used as internal control for mRNA motes hepatocellular carcinoma progression in vitro and in vivo quantification. The relative expression ratio of genes in each by upregulating EGR1 and miR-365a-3p to subsequently acti- paired tumor and non-tumorous tissue was calculated by the vate the AKT/b-catenin pathway. Collectively, our functional DCt method. The sequences of the PCR primers are shown in and biochemical studies suggest CBX8 exhibits oncogenic Supplementary Table S1. activities towards hepatocellular carcinoma in a noncanonical fashion. Immunohistochemistry Antibodies for IHC and Western blot analyses are listed in Materials and Methods Supplementary Table S2. Immunohistochemical staining for CBX8, EGR1, and p-b-catenin was performed on an hepa- Cell culture tocellular carcinoma tissue microarray. The expression levels Human hepatocellular carcinoma cell lines Huh7, MHCC- were scored as a proportion of the immunopositive staining 97H, and HepG2 were purchased from the ATCC. The hepato- area (0%, 0; 1%–25%, 1; 26%–50%, 2; 51%–75%, 3; and 76%– cellular carcinoma cell lines SK-Hep1, QGY-7701, QGY-7703, 100%, 4) multiplied by intensity of the staining (0, negative; 1, Bel-7402, Bel-7404, and SMMC-7721 were obtained from the weak; 2, moderate; and 3, intense). The scores were indepen- Cell Resource Center, Chinese Academy of Science Committee. All dently rendered by two pathologists. The median IHC score cell lines were authenticated by Genecreate Company 5 months waschosenasthecut-offvaluefordefining high and low before this study. Cells were maintained in DMEM (Gibco) expression. supplemented with 10% heat-inactivated FBS (Hyclone) at 37C fi in a humidi ed incubator containing 5% CO2. Cells were treated Immunofluorescence with RING inhibitor PRT4165 (MedChemExpress Company) or Cells were fixed for 20 min in PBS containing 4% paraformal- transfected with overexpression vectors, siRNA, shRNA, miR- dehyde, permeabilized in 0.1% Triton X-100 two times, 5 min 365a-3p mimics, or corresponding empty vectors as described in each, incubated in blocking buffer (3% donkey serum in TBS) Supplementary Table S1. for 1 h, and then incubated with antibody for 2 h at room temperature. After washing in PBS three times, 8 min each, cells Patients and tissue specimens were incubated with the appropriate fluorochrome-conjugated A cohort of 514 patients with hepatocellular carcinoma who secondary antibody for 1 h, and observed under a fluorescence received surgery between January 2005 and January 2009 was microscope. recruited at the Sun Yat-sen University Cancer Center, Guangz- fi hou, China. Paraf n-embedded tissues and clinical information Coimmunoprecipitation were collected. None of the patients had received radiotherapy or Proteins were extracted by radioimmunoprecipitation assay chemotherapy before surgery. Written informed consents from buffer supplemented with proteinase inhibitor cocktail. Pri- the patients were obtained. Another 56 pairs of fresh hepatocel- mary antibodies were added for 2.5 h. A/G beads lular carcinoma and adjacent nontumorous liver specimens were were added for an additional 2 h. Precipitated proteins were collected from the patients at the time of surgical resection. All dissolved in SDS loading buffer and fractionated by SDS- fresh samples were anonymous. This study was approved by the PAGE. Sun Yat-sen University Cancer Center Institute Research Ethics Committee and conducted in accordance with International Tetrazolium dye (MTT) and colony formation assays Ethical Guidelines for Biomedical Research Involving Human For MTT assays, 3.0 103 cells were seeded in 96-well plates Subjects (CIOMS). The prognostic value of CBX8 was further after transfection. MTT assays were performed by adding 100 mL validated in a cohort of The Cancer Genome Atlas (TCGA) of MTT (5 mg/mL, AMRESCO) for 4 h at 37C. The formazan dataset (http://www.cbioportal.org). crystals were dissolved in DMSO (150 mL/well) and the absor- bance at 490 nm was measured. Results were recorded and Microarray and RNA sequencing normalized against the control. For colony formation assays, MicroRNA microarrays (SYSUCC) was used to detect the stable cells were seeded in six-well plates at a density of 1 103 changes of microRNAs in MHCC-97H cells transfected with CBX8 per well, then incubated at 37C for 10 days. Colonies were siRNAs. The RNA-sequencing was performed in Bel-7402 cells fixed with methanol, stained with 0.1% crystal violet, and with or without CBX8 overexpression. The data are deposited at counted. the Sequence Read Archive (SRA) database under accession no. SRP116159. Cell migration assays About 2 103 cells were resuspended in 200 mLofserum- qRT-PCR free medium and then placed in the upper compartment Total RNA was extracted by Trizol Reagent (Invitrogen). For of a Transwell chamber (Corning; 24-well insert, pore size: miR-365a-3p detection, reverse-transcribed cDNA was synthe- 8 mm). The lower chamber was filled with 15% FBS as a

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chemoattractant and incubated for 48 h. At the end of the Luciferase reporter assay experiments, the cells on the upper surface of the membrane For the binding of CBX8 to the EGR1 or miR-365a-3p promot- were removed, and the cells on the lower surface were fixed er, and miR-365a-3p to the ZNRF1 30-UTR, Bel-7402 cells were with methanol and stained with 0.1% crystal violet and cotransfected with the overexpression or empty vector. Cells were counted. Five visual fields were randomly chosen and the collected 48 h after transfection and luciferase activity was ana- number of cells was counted under a microscope. lyzed with the Dual-Luciferase Reporter Assay System (Promega).

Figure 1. CBX8 expression is increased in hepatocellular carcinoma and associated with poor outcome. A, The expression of CBX8 in hepatocellular carcinoma cell lines was examined. The immortalized liver cell lines (L-02 and MiHA) were used as controls. B, The mRNA level of CBX8 in 56 paired fresh hepatocellular carcinoma specimens was evaluated by qRT-PCR. U6 was used as the loading control. C, The protein expression of CBX8 in another 27 hepatocellular carcinoma samples was determined by Western blot analysis. The relative protein densities were measured and are shown. D, CBX8 expression in 514 hepatocellular carcinoma paraffin-embedded tissues was examined by IHC. Images representing the expression of CBX8 in hepatocellular carcinoma (T) and nontumorous (N) tissue are shown. The IHC score is indicated and compared. E, Kaplan–Meier survival analyses were conducted to evaluate the significance of CBX8 in overall survival, based on the protein expression in our cohort (top) and the mRNA level in TCGA's cohort (bottom). F, Representative images of hematoxylin and eosin (H&E) and IHC staining of CBX8 in nontumor (N), tumor (T), and metastatic (M) tissues are shown. G, The implication of CBX8 in disease-free survival of patients with hepatocellular carcinoma was determined in our cohort (top) and TCGA's cohort (bottom).

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Chromosome immunoprecipitation (Supplementary Figs. S1B and S12). The mRNA and protein levels The immunoprecipitation (ChIP) assay was per- were obviously higher in hepatocellular carcinoma cells than formed using the SimpleChIP Enzymatic Chromatin IP Kit those in the nontumorous tissues (Fig. 1A). In 56 pairs of fresh (#9002; Cell Signaling Technology). Hepatocellular carcinoma specimens, CBX8 mRNA was significantly upregulated in tumor cells with CBX8 modulation were lysed using SDS lysis buffer and tissues, compared to that in the nontumorous ones (Fig. 1B). DNA was sheared by sonication. Protein–DNA complexes were Similarly, CBX8 protein expression was increased by 4.35-fold in precipitated by control immunoglobulin G or CBX8 antibody, hepatocellular carcinoma cells (Fig. 1C). Clear upregulation, followed by eluting the complex from the antibody. qRT-PCR was comparable expression and clear downregulation of CBX8 pro- carried out with primers specific for EGR1 promoter region. The tein expression in hepatocellular carcinoma samples were primers used in ChIP assay were described in Supplementary detected in 55.6% (15/27), 25.9% (7/27), and 18.5% (5/27), Table S1. respectively. TMA-based IHC analysis showed positive staining of CBX8 Animal experiments in 27.2% (140/514) of the hepatocellular carcinoma tissues Stable hepatocellular carcinoma cells with CBX8 overexpres- but in only 2.5% (13/514) of the nontumorous tissues sion or depletion were implanted subcutaneously under the right (Fig.1D).Significantly association of CBX8 and clinical stage armpits and into the flanks of male BALB/c- nude mice aged 3 to 4 was found (Supplementary Table S3). Among the benign weeks. Tumor size and body weight were measured every 4 days. tumors in the liver, positive expression of CBX8 was found Four weeks later, the mice were sacrificed and tumor weight and in 16.7% of PEComa, 6.7% of hepatic adenoma and 12.5% of size were measured. Volumes were calculated using the following focal nodular hyperplasia (Supplementary Fig. S3A). Further formula: volume ¼ length width2 0.5. For metastasis obser- study indicated that CBX8 expression was significantly vation, 4-week-old male nude BALB/c athymic mice were injected increased in cholangiocarcinoma, lung cancer, colorectal can- with 5 105 cells via the tail vein. Six weeks later, mice were killed. cer, and breast cancer, but was decreased in renal cell carci- Lungs of the mice were fixed and stained with hematoxylin and noma (Supplementary Fig. S3B). Kaplan–Meier analysis eosin. Lung metastasis was quantified by counting the number of revealed that CBX8 expression was correlated with poor post- tumor foci in 10 randomly selected high-power fields. All animal surgical survival in patients in both our cohort and TCGA's studies were conducted with the approval of the Medical Exper- cohort (Fig. 1E), which was validated by stratified survival imental Animal Care Commission of Sun Yat-sen University analyses (Supplementary Fig. S4). Cancer Center. We noticed that CBX8 expression in the portal vein embolus was higher than that in the primary tumor (Fig. 1F). TMA-based Statistical analysis IHC staining of 95 patients with tumor metastasis showed that Data from at least three experiments were detected as mean cells in the embolus expressed more CBX8 than in primary values SEM. Differences among various groups were compared tumors in 85.3% (81/95) of the cases (Supplementary Fig. S5). by Student t test. Spearman's correlation test was used to evaluate In the cohort of 514 patients with hepatocellular carcinoma the correlations between parameters. Survival was analyzed using and TCGA's cohort, patients with high expression of CBX8 the Kaplan–Meier method, whereas factors associated with sur- experienced a shorter period until tumor recurrence or metas- vival were identified by the Cox proportional hazard regression tasis (Fig. 1G). Multivariate analyses indicated CBX8 as an model. Differences were considered significant when the P value independent prognostic factor of both overall and disease-free was less than 0.05. All analysis was performed in statistical survival in hepatocellular carcinoma (Supplementary Table software SPSS Statistics 19.0 (IBM). S4). Taken together, our data suggest that CBX8 overexpression may serve as a promising biomarker for prognosis in hepato- Results cellular carcinoma. CBX8 expression is increased and associated with poor outcomes in hepatocellular carcinoma CBX8 exerts oncogenic activity towards hepatocellular The expressions of Chromobox family genes (CBX1-8) were carcinoma cells determined in hepatocellular carcinoma fresh tissues. Results Next, we performed in vitro functional assays to assess the role of showed that the mRNA expression of CBX8, as well as CBX1, CBX8 in hepatocellular carcinoma progression. CBX8 was either CBX4, and CBX6, was significantly upregulated in hepatocellular overexpressed in Bel-7402 and SMMC-7721 cells or knocked carcinoma (Supplementary Fig. S1A). Data from TCGA datasets down in MHCC-97H cells (Supplementary Fig. S6A). Cell viabil- revealed that genomic amplification of the CBX8 gene being more ity was increased in CBX8-transfected cells but decreased in CBX8- frequently found in hepatocellular carcinoma and other cancers silenced cells (Supplementary Fig. S6B). Ectopic CBX8 expression

Figure 2. CBX8 promotes tumor growth and metastasis in hepatocellular carcinoma. A, Cells with CBX8 overexpression or depletion were cultured in 6-well plates for 10 days. The number of colonies formed by hepatocellular carcinoma cells was calculated and is indicated by the histogram. Data are the mean SEM of three independent experiments. B, Cell proliferation was measured by EdU assays. C, Stable cells were injected into the right flank of the null mice. Tumor volumes were measured every 4 days. The tumors were dissected at day 27 and weighted. Growth curves are summarized at the bottom. , P < 0.05; , P < 0.01. C, Transwell assays were used to determine the effect of CBX8 on cell migration. The migrated cells were stained with 0.1% crystal violet and counted undera microscope. The fold changes of cell migration were calculated and are shown in the histogram. D, Cells with CBX8 overexpression or silencing were injected into the mice through the tail vein. The metastatic nodules in the lungs were sectioned and counted. Representative micrographs of hematoxylin and eosin staining and the number of lung metastasis are shown. , P < 0.05; , P < 0.01.

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significantly enhanced the ability to form foci. In contrast, to investigate the effect of CBX8 on tumorigenesis in vivo. Tumors the CBX8-depleted cells failed to form colonies (Fig. 2A). The were found in 5/7 and 3/7 of mice injected with CBX8-expressing 5-ethynyl-20-deoxyuridine (EdU)-positive cells were noticeably and empty vector-transfected Bel-7402 cells, respectively. The induced by CBX8 overexpression, but were reduced by CBX8 tumor volumes and sizes were significantly larger in the CBX8- siRNAs (Fig. 2B). Subcutaneous xenograft models were applied overexpression groups (Fig. 2C). Conversely, tumors formed by

Figure 3. CBX8 triggers the AKT/b-catenin pathway in hepatocellular carcinoma cells. A, Immunofluorescence staining was performed to indicate the cellular localization of b-catenin. DAPI was used to stain the nucleus. B, Proteins obtained from cells with or without CBX8 were subjected to Western blot analysis to examine the activation of the AKT/b-catenin pathway. C, Representative IHC images for CBX8 and phosphorylated b-catenin are shown. Their correlation in 514 patients with hepatocellular carcinoma is presented at the bottom. D, Cells with CBX8 overexpression were treated with either b-catenin siRNA or its inhibitor XAV-939. Colony formation was performed to evaluate the effect of inhibition of b-catenin on CBX8-promoted cell growth. E, The impact of b-catenin suppression on cell migration was determined in cells treated as described in E. The fold change of migrated cells is indicated by histograms. , P < 0.05.

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CBX8-silenced MHCC-97H cells were dramatically smaller than showed that cell migration was drastically increased by CBX8 those in the control group (Fig. 2C). Furthermore, the tumors introduction, but decreased by CBX8 knockdown (Fig. 2D). were much heavier in the CBX8-overexpression groups but much Wound-healing assays demonstrated that CBX8-expressing lighter in the CBX8-depletion groups, compared to the corre- cells filled the gap faster than control cells. By contrast, CBX8 sponding control groups (Supplementary Fig. S7). depletion hindered cell movement (Supplementary Fig. S6D). We next questioned whether CBX8 could influence the A caudal vein injection model was used to evaluate the effect of metastatic ability of hepatocellular carcinoma cells. Cell shapes CBX8 on tumor metastasis in vivo. Lung metastasis was more were identified by phallotoxin staining to visualize cytoskel- often detected in the CBX8-overexpression groups, but hardly eton F-actin. The formation of cell pseudopodium was found in the CBX8-depletion groups, compared to the control enhanced by CBX8 overexpression but impaired by CBX8 groups (Fig. 2E). The expression of vimentin, a metastatic silencing (Supplementary Fig. S6C). The transwell assays marker, was induced by CBX8 overexpression, but reduced by

Figure 4. CBX8 exhibits oncogenic effects via EGR1. A, Cells were transfected with CBX8 overexpression vector and ERG1 siRNA for 48 hours. A coimmunoprecipitation assay, using EGR1 antibody, was used to detect the binding of EGR1 and b-catenin. B, The relationship between CBX8 mRNA and EGR1 mRNA was determined in 56 hepatocellular carcinoma samples. r, Pearson correlation coefficient. C, Plots showed the connection of CBX8 protein and EGR1 mRNA in 27 hepatocellular carcinoma cases. D, The mRNA level of EGR1 was determined in cells transfected with CBX8 overexpression vector and siRNAs. E, The protein expression of CBX8 and EGR1 was examined by Western blot analysis. F, Cells with CBX8 overexpression were treated with Ring1b siRNA or RING inhibitor PRT4165 for 24 hours. The mRNA expression of EGR1 was determined by qRT-PCR. G, Luciferase activity of EGR1 promoter was determined in Bel-7402 cells with CBX8 overexpression or knockdown. H, ChIP assay was performed in Bel-7402 cells. PCR was performed using primers specific for the EGR1 promoter. Data are shown by mean SEM of three independent experiments. , P < 0.05; , P < 0.01; , P < 0.001.

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CBX8 depletion (Supplementary Fig. S8). Collectively, these overexpression. CBX8-promoted hepatosphere formation was findings indicate that CBX8 greatly contributes to hepatocel- largely abolished by treatment with XAV-939 or b-catenin siRNA lular carcinoma progression. (Fig. 3D). Similarly, cell motility was weakened in cells cultured with the inhibitor and siRNA (Fig. 3E). Combinative suppressions CBX8 augments hepatocellular carcinoma proliferative and of b-catenin and MAPK pathways results in complete inhibition of metastatic potential via AKT/b-catenin pathway CBX8-mediated phenotypes (Supplementary Fig. S11). These To unveil the underlying mechanisms of CBX8-mediated onco- data indicate that CBX8 exerts its protumor functions via activa- genic features, we conducted transcriptional profiling by RNA- tion of the AKT/b-catenin pathway. seq. Thirty-six genes were jointly altered by forced CBX8 expres- sion in Bel-7402 and SMMC-7721 cells (Supplementary Fig. S9). CBX8 triggers AKT/b-catenin pathway via EGR1 Pathway enrichment analyses showed that both b-catenin and Because EGR1 is interacted with b-catenin and was upregu- MAPK pathways were significantly activated by CBX8 overexpres- lated by CBX8 overexpression, we next tested whether EGR1 sion (Supplementary Fig. S10). Interestingly, CBX8 overexpres- was involved in the CBX8-mediated activation of b-catenin. sion caused the nuclear localization of b-catenin, supported by The IP experiments showed that CBX8 overexpression in hepa- immunofluorescence staining (Fig. 3A). The phosphorylations of tocellular carcinoma cells strengthened the interaction between b- catenin at Ser552 and AKT were increased in cells transfected EGR1 and b-catenin (Fig. 4A). Interestingly, EGR1 mRNA with CBX8, but blocked in cells with CBX8 depletion (Fig. 3B), was positively correlated with CBX8 mRNA and protein in suggesting a potential role of AKT in b-catenin activation. In 56 and 27, respectively, pairs of hepatocellular carcinoma clinical samples, nuclear b-catenin was significantly correlated tissues (Fig. 4B and C). As determined by IHC staining, high with CBX8 expression (Fig. 3C). EGR1 expression was frequently found in the hepatocellular The profound impact of CBX8 on activation of b-catenin carcinoma cases with positive CBX8 expression (Supplemen- signaling prompted us to examine the effect of b-catenin on tary Fig. S12). Upon the CBX8 transfection, EGR1 mRNA CBX8-mediated malignant activities. The b-catenin inhibitor expression was induced in a dose-dependent manner. In con- (XAV-939) and siRNA were introduced into cells with CBX8 trast, EGR1 mRNA was downregulated by CBX8 siRNAs

Figure 5. CBX8 interacts with EGR1 to maintain its protein stability. A, Proteins extracted from hepatocellular carcinoma cells were incubated with antibody for EGR1 or CBX8. After pelleting by Protein A/G-agarose, samples were subjected to Western blot analysis to investigate the interaction of CBX8 and EGR1. B, HEK293T cells were transfected with Flag-EGR1 and HA-CBX8 overexpression vectors for 24 hours. Coimmunoprecipitation assays were performed using an antibody for HA or Flag. C, Bel-7402 and SMMC-7721 cells were fixed by 4% PFA and incubated with antibodies overnight at 4C. After staining with fluorescent secondary antibodies and DAPI, cells were observed under a confocal fluorescence microscope. D, MHCC-97H cells with CBX8 siRNAs were treated with cycloheximide (CHX) for the indicated periods. The expression of EGR1 was examined by Western blot analysis. The relative EGR1 protein intensities were calculated and are shown by the curve. E, The half-life of EGR1 protein was measured in Bel7402 and SMMC-7721 cells with CBX8 overexpression.

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(Fig. 4D). Results of Western blot analysis confirmed that carcinoma samples (Fig. 6D). These data raised the possibility that EGR1 protein level was altered by CBX8 (Fig. 4E). To test CBX8 may directly regulate the expression of miR-365a-3p. To test whether CBX8 modulates EGR1 in the context of PRC1, Ring1b this, we performed a luciferase reporter assay. The relative lucif- was suppressed by siRNA or RING inhibitor PRT4165. The erase activity of miR-365a-3p promoter was enhanced by CBX8 upregulation of EGR1 mRNA by CBX8 was not affected by the overexpression but reduced by CBX8 silencing (Fig. 6E). Loss of treatment of Ring1b siRNA or PRT4165 (Fig. 4F). Suppression Ring1b did not affect the upregulation of miR-365a-3p by CBX8 of Ring1b did not compromise CBX8-mediated clonogenicity overexpression (Fig. 6F), indicating a PRC1-independent manner and cell migration (Supplementary Fig. S13). This suggests that for CBX8-modulated miR-365a-3p. the role of CBX8 in promoting hepatocellular carcinoma may The effect of miR-365a-3p on CBX8-mediated phenotypes be independent of canonical PRC1. A dual-luciferase reporter was next examined. Treatment of miR-365a-3p inhibitor in assay demonstrated that increased CBX8 significantly stimu- CBX8-expressing Bel-7402 cells partly attenuated cell growth lated, whereas decreased CBX8 significantly inhibited, the and migration. In contrast, miR-365a-3p introduction into cells transcriptional activity of the EGR1 promoter (Fig. 4G). ChIP with CBX8 depletion partly regained the clonogenicity and cell assays showed the direct binding of CBX8 to the EGR1 pro- movement (Fig. 6G and H). It was noticed that combined moter (Fig. 4H). suppressions or combined overexpressions of EGR1 and In addition, endogenous EGR1 and CBX8 were detectable in miR-365a-3p slightly rescued the CBX8-mediated phenotypes the precipitate mediated by the antibody of EGR1 or CBX8 in both (Fig. 6G and H). Following the combined treatments, the Bel-7402 and SMMC-7721 cells (Fig. 5A). Using anti-Flag or anti- phosphorylation and the nuclear translocation of b-catenin HA antibody, we found that HA-CBX8 coprecipitated with Flag- were decreased in CBX8-expressing cells, but increased in EGR1 in HEK-293T cells (Fig. 5B). The colocalization of CBX8 and CBX8-silencing cells, compared with the single modulation of EGR1 in the nucleus of hepatocellular carcinoma cells was EGR1 or miR-365a-3p expression (Fig. 6I and J). These data observed by confocal immunofluorescence (Fig. 5C). We next may suggest an additive effect of EGR1 and miR-365a-3p on intended to explore the consequences of this interaction. Using CBX8-mediated phenotypes. cycloheximide (20 mg/mL), the half-life of the EGR1 protein was To dissect the molecular mechanism of miR-365a-3p, we determined. In MHCC-97H cells, EGR1 was degraded within 30 performed in silico prediction using bioinformatics tools (Targets- min. Following CBX8 knockdown, the degradation of EGR1 can, miRanda, PITA, and picTar). The Venn diagram revealed protein was significantly enhanced (Fig. 5D). In Bel-7402 and that 18 genes, including ZNRF1 that has been demonstrated in SMMC-7721 cells that express less CBX8, the half-life of EGR1 was AKT/b-catenin pathway were predicted by all four bioinformat- significantly shorter than that in MHCC-97H cells with high CBX8 ic algorithms (Supplementary Fig. S16A and Supplementary expression. The introduction of CBX8 resulted in a prolonged Table S6). One putative binding site at the 30-UTR of ZNRF1 half-life of EGR1 protein (Fig. 5E). Collectively, these data suggest was found (Supplementary Fig. S16B). The luciferase activity of that CBX8 promotes cell proliferation and migration via modu- the wild-type reporter containing seed sequence was signifi- lation of EGR1 expression. cantly reduced by miR-365a-3p transfection in Bel-7402 cells, We reasoned that if EGR1 is the downstream effector of CBX8, whereas it remained unchanged in mutant reporters (Fig. 7A). then the inhibition of EGR1 should rescue CBX8-promoted Ectopic miR-365a-3p expression resulted in a significant malignant phenotypes. EGR1 was silenced by its siRNAs in Bel- decrease of ZNRF1 at both the mRNA and protein levels (Fig. 7402 and SMMC-7721 cells (Supplementary Fig. S14). As 7B and C). In 27 pairs of clinical samples, miR-365a-3p expected, CBX8-induced enhancement of cell viabilities was expression was inversely associated with ZNRF1 protein expres- abolished by ERG1 siRNAs (Supplementary Fig. S15A). The sion (Fig. 7D). These data suggest ZNRF1 is a direct target of percentage of EdU-positive cells with CBX8 overexpression and miR-365a-3p. EGR1 knockdown decreased to the control levels (Supplementary We next determined whether miR-365a-3p was involved Fig. S15B). Furthermore, cells transfected with CBX8 and EGR1 in CBX8-mediated AKT/b- catenin activation. Similar to the less efficiently formed colonies, compared to those with CBX8 effect of CBX8 overexpression, enforced miR-365a-3p led to (Supplementary Fig. S15C). The CBX8-enhanced migration abil- the nuclear localization of b-catenin (Fig. 7E). miR-365a-3p ity was partly attenuated by EGR1 siRNAs (Supplementary Fig. upregulated phospho-AKT and phospho-b-catenin, which was S15D). These data suggest a requirement of EGR1 in CBX8- mimicked by ZNRF1 siRNA (Fig. 7F). In hepatocellular car- mediated malignancy. cinoma patients with high expression of miR-365a-3p, low ZNRF1 expression and nuclear b-catenin were likely to be CBX8 triggers the AKT/b-catenin pathway via detected. Statistically, ZNRF1 expression was inversely corre- miR-365a-3p-targeting ZNRF1 lated with nuclear b-catenin expression in 514 patients with microRNA microarray was used to test whether CBX8 hepatocellular carcinoma (Fig. 7G). Collectively, these find- could affect the expression of microRNAs in hepatocellular car- ings implicate that CBX8-elevated miR-365a-3p suppresses the cinoma cells. A total of 34 microRNAs were deregulated in expression of ZNRF1 to consequently trigger the AKT/b-cate- response to CBX8 knockdown in MHCC-97H cells (Fig. 6A and nin signaling in hepatocellular carcinoma cells. Supplementary Table S5). miR-365a-3p was chosen for further studies, because it was the most downregulated microRNA by CBX8 siRNAs. CBX8 silence resulted in significant decrease of Discussion miR-365a-3p in MHCC-97H cells (Fig. 6B). In contrast, enforced Efforts have been increasingly made to discover the mechan- CBX8 expression in Bel-7402 and SMMC-7721 cells led to an isms of the unlimited growth of hepatocellular carcinoma increase of miR-365a-3p (Fig. 6C). A positive correlation between cells over the last decades. Several biomarkers have been CBX8 and miR-365a-3p was found in 36 pairs of hepatocellular found and applied to the daily managements of hepatocellular

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CBX8 Activates AKT/b-Catenin Pathway

carcinoma (20, 21). Here, we identified CBX8 as a promising we find that CBX8 physically binds to EGR1 and subsequently prognostic and therapeutic factor in hepatocellular carcinoma. enhances the interaction of EGR1 and b-catenin. This may be a We also elucidated that CBX8 manifested its protumor activities novel way in which CBX8 modulates the proliferation and through activating the AKT/b-catenin pathway by simultaneously migration of cancer cells. enhancing the expression of EGR1 and miR-365a-3p (Fig. 7H). Aberrant activation of b-catenin signaling contributes to the Misregulation of CBX8 has been reported in human cancers, initiation and progression of hepatocellular carcinoma. Over- but its clinical significance is not well understood. Our data expression of b-catenin has been noted in our and other studies, provide compelling biological and clinical evidence that CBX8 showing that b-catenin is upregulated in more than half of the is overexpressed in hepatocellular carcinoma. Clear upregula- hepatocellular carcinoma cases (25). Accumulating cytosolic tion of CBX8 was identified in 27.4% of paraffin-embedded b-catenin results in its nuclear localization and transcriptional samples by IHC and 55.6% of fresh tissues by Western blot activity. Another mechanism of activation of b-catenin medi- analysis. Further studies showed that CBX8 expression was ated by AKT has been proposed: phospho-AKT phosphorylates significantly increased in cholangiocarcinoma, lung cancer, b-catenin at the Ser552 residue to increase nuclear b-catenin colorectal cancer, and breast cancer, but was decreased in renal (18). Our data showed that b-catenin was transported to the cell carcinoma. In line with our study, the increase in CBX8 was nucleus in hepatocellular carcinoma cells with overexpression also found in glioblastoma and esophageal squamous cell of CBX8. Ectopic CBX8 induced the phosphorylation of AKT carcinomas in other studies. In our and TCGA's cohorts, and b-catenin. Furthermore, CBX8 protected the protein sta- patients with CBX8 expression likely experienced shorter over- bility of EGR1 to enhance the interaction of EGR1 and b-cate- all and disease-free survival, compared with those without nin, leading to the nuclear accumulation of b-catenin. It should CBX8 expression. A recent report showed that high expression be noticed that PRC1 has been was essential to maintain the of CBX8 was correlated with poor overall survival in breast leukemia cell identity and intestinal stem cell identity via cancer (14). However, high CBX8 expression was connected to activation of b-catenin signaling (26, 27). Our data suggest favorable prognoses in colorectal cancer, although CBX8 was the independency of PRC1 in CBX8-mediated phenotypes in frequently upregulated (22). Taken together, these findings hepatocellular carcinoma. These data may suggest that CBX8 is suggest misregulation of CBX8 is sensitive in human cancers not involved in the regulation of PRC1 activity in hepatocel- and CBX8 is a potential biomarker for clinical surveillance of lular carcinoma cells. tumor progression. The relationship between polycomb proteins and microRNA The typical manner for CBX8 to exert its functions in PRC1 is has been rarely determined. O'Loghlen and colleagues showed as a transcriptional repressor of other genes, such as INK4a/ARF that CBX7 was downregulated by miR-125 and miR-181 in (10). Our data demonstrate that CBX8 functions as a transcrip- embryonic stem cells (28). Zheng and colleagues identified tional activator in a PRC1-independent manner to upregulate miR-195 as the upstream regulator of CBX4 to suppress hepato- the expression of EGR1 and miR-365a-3p in hepatocellular cellular carcinoma (29). However, whether microRNAs can be carcinoma, which was also confirmed in colorectal and breast regulated by Polycomb proteins remains unknown. Here, we cancers (Supplementary Fig. S17). Chromobox proteins were suggest that aberrant CBX8 results in altered expression of micro- reported to function synergistically or antagonistically. In our RNAs in hepatocellular carcinoma cells. Especially, CBX8 knock- study, downregulation of EGR1 or miR-365a-3p was not pre- down reduced the expression of miR-365a-3p, which targets sented in hepatocellular carcinoma cells with silence of CBX1, ZNRF1 to degrade AKT. CBX4, or CBX7. Furthermore, the cell growth and migration Luciferase reporter assays implicated CBX8 as a modulator of were attenuated by the knockdown of CBX4 and CBX8, but not the promoter activity of miR-365a-3p. However, CBX8 was further CBX1 and CBX7 (Supplementary Fig. S18). The noncanonical identified as a downstream effector of miR-410-3p (Supplemen- manner of CBX8 is supported by other studies. CBX8 interacted tary Fig. S19 and Supplementary Table S7). Inverse correlations of with either MLL-AF9 to activate HOX gene transcription in CBX8 and miR-410-3p were found in hepatocellular carcinoma, leukemogenesis (23) or Wdr5 to induce expression of genes colorectal and breast cancers. Reintroduction of miR-410-3p involved in the Notch pathway in breast carcinoma (14). significantly decreased the mRNA expression of CBX8. Luciferase Furthermore, CBX8 was able to facilitate the activation of genes assays demonstrated that the activity of CBX8 30-UTR was mod- involved in ES cell differentiation (24). These findings suggest a ulated by CBX8. Together, our data show that CBX8 is capable of dual role of CBX8 in transcriptional regulation. However, the regulating the profile of microRNAs in hepatocellular carcinoma previous studies showed that the interaction of CBX8 with cells, suggesting a new approach for CBX8 to affect the progression other proteins enhances transcriptional activity. In this study, of human cancers.

Figure 6. CBX8 transcriptionally upregulates miR-365a-3p in hepatocellular carcinoma cells. A, MicroRNA microarrays were used to examine the altered expression of microRNAs by CBX8 siRNAs in MHCC-97H cells. A total of 34 microRNAs was found to be deregulated. B, The decrease of miR-365a-3p induced by CBX8 siRNAs was confirmed in MHCC-97H cells. C, Bel-7402 and SMMC-7721 cells were transfected with CBX8 overexpression vector for 48 hours. The expression of miR-365a-3p was determined by qRT-PCR. D, The correlation between CBX8 mRNA and miR-365a-3p was determined in 36 fresh hepatocellular carcinoma samples. r, Pearson correlation coefficient. E, Luciferase assays were performed to measure the effect of CBX8 overexpression or knockdown on the activity of the miR-365a-3p promoter in Bel-7402 cells. All data are means SEM of three independent experiments. , P < 0.05; , P < 0.01. F, Cells with CBX8 overexpression were treated with Ring1b siRNA or RING inhibitor PRT4165 for 24 hours. The mRNA expression of miR-365a-3p was determined by qRT-PCR. G, Colony formation assays were performed. Cells with CBX8 overexpression were treated with EGR1 siRNA, miR-365a-3p inhibitor (miR In), or both. Cells with CBX8 depletion were treated with EGR1, miR-365a-3p mimic (miR), or both. H, Transwell assays were performed in cells described in G. I, Proteins obtained from cells described in G were subjected to Western blot for the examination of phosphor-b-catenin. J, Confocal assays were performed to indicate the nuclear localization of b-catenin.

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Figure 7. miR-365a-3p targets ZNRF1 to activate the AKT/b-catenin pathway in hepatocellular carcinoma cells. A, The effect of miR-365a-3p on the promoter activity of ZNRF1 was determined in Bel-7402 cells. B, The overexpression of miR-365a-3p in hepatocellular carcinoma cell lines was confirmed by qRT-PCR. C, Cells were transfected with miR-365a-3p mimics for 48 hours. The mRNA and protein expression levels of ZNRF1 were examined. D, The correlation of miR-365a-3p and ZNRF1 mRNA was tested in 27 hepatocellular carcinoma samples. r, Pearson correlation coefficient. E, The cellular localization of b-catenin was indicated by immunofluorescence staining in cells incubated with miR-365a-3p mimics for 48 hours. F, The activation of the AKT/b-catenin pathway was confirmed by Western blot analysis in cells with miR-365a-3p overexpression or ZNRF1 depletion. G, Representative IHC images for ZNRF1 and phosphorylated b-catenin in hepatocellular carcinoma tissues with low or high expression of miR-365a-3p are shown. The correlation of ZNRF1 and phosphorylated b-catenin in 514 patients with hepatocellular carcinoma is presented at bottom. H, Schematic diagram of the CBX8/EGR1 or the CBX8/miR-365a-3p/ZNRF1 signaling axis in driving AKT/b-catenin activation in hepatocellular carcinoma cells.

In summary, we identified CBX8 as an oncogene with prog- and miR-365a-3p. In light of the development and progress nostic significance in hepatocellular carcinoma. CBX8 pro- of compounds with the ability to inhibit the function of CBX motes hepatocellular carcinoma progression via a non-canon- family proteins (30, 31), our study provides, for further eval- ical mechanism, which triggers the AKT/b-catenin pathway uation, a novel therapeutic target for hepatocellular carcinoma through transcriptionally activating the expression of EGR1 treatment.

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CBX8 Activates AKT/b-Catenin Pathway

Disclosure of Potential Conflicts of Interest Acknowledgments No potential conflicts of interest were disclosed. The study was supported by grants from the National Key R&D Program of China (No. 2017YFC1309003 to D. Xie and J.P. Yun), the National Natural Science Foundation of China (No. 81572406 to J.P. Yun; 81572405 Authors' Contributions to C.Z. Zhang). The authenticity of this article has been validated by uploading the key raw Conception and design: C.Z. Zhang, J.-P. Yun data onto the Research Data Deposit public platform (www.researchdata.org. Developmentofmethodology:C.Z. Zhang, S.-L. Chen, C.-H. Wang, cn), with the approval RDD number as RDDB201700. Y.-F. He Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): C.Z. Zhang, S.-L. Chen, C.-H. Wang, Y.-F. He, X. Yang, The costs of publication of this article were defrayed in part by the payment of advertisement D. Xie, J.-P. Yun page charges. This article must therefore be hereby marked in Analysis and interpretation of data (e.g., statistical analysis, biostatistics, accordance with 18 U.S.C. Section 1734 solely to indicate this fact. computational analysis): C.Z. Zhang, S.-L. Chen, C.-H. Wang, Y.-F. He, J.-P. Yun Writing, review, and/or revision of the manuscript: C.Z. Zhang, D. Xie, J.-P. Yun Received March 22, 2017; revised September 5, 2017; accepted October 18, Study supervision: J.-P. Yun 2017; published OnlineFirst October 24, 2017.

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CBX8 Exhibits Oncogenic Activity via AKT/β-Catenin Activation in Hepatocellular Carcinoma

Chris Zhiyi Zhang, Shi-Lu Chen, Chun-Hua Wang, et al.

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