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CBX8 Exhibits Oncogenic Activity Via AKT/ B-Catenin Activation in Hepatocellular Carcinoma

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CBX8 Exhibits Oncogenic Activity Via AKT/ B-Catenin Activation in Hepatocellular Carcinoma 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 proteins 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. Ó2017 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 locus (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 genes 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 Ó2017 American Association for Cancer Research. at Ser552, which enhances its transcriptional activity (18), or www.aacrjournals.org 51 Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2018 American Association for Cancer Research. Published OnlineFirst October 24, 2017; DOI: 10.1158/0008-5472.CAN-17-0700 Zhang et al. 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, gene 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 ÀDCt 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. Protein
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