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CBX8 Exhibits Oncogenic Activity Via AKT/Β-Catenin Activation In

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CBX8 Exhibits Oncogenic Activity Via AKT/Β-Catenin Activation In Author Manuscript Published OnlineFirst on October 24, 2017; DOI: 10.1158/0008-5472.CAN-17-0700 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2017 American Association for Cancer Research. Author Manuscript Published OnlineFirst on October 24, 2017; DOI: 10.1158/0008-5472.CAN-17-0700 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2017 American Association for Cancer Research. Author Manuscript Published OnlineFirst on October 24, 2017; DOI: 10.1158/0008-5472.CAN-17-0700 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2017 American Association for Cancer Research. Author Manuscript Published OnlineFirst on October 24, 2017; DOI: 10.1158/0008-5472.CAN-17-0700 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2017 American Association for Cancer Research. Author Manuscript Published OnlineFirst on October 24, 2017; DOI: 10.1158/0008-5472.CAN-17-0700 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2017 American Association for Cancer Research. Author Manuscript Published OnlineFirst on October 24, 2017; DOI: 10.1158/0008-5472.CAN-17-0700 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2017 American Association for Cancer Research. Author Manuscript Published OnlineFirst on October 24, 2017; DOI: 10.1158/0008-5472.CAN-17-0700 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2017 American Association for Cancer Research. Author Manuscript Published OnlineFirst on October 24, 2017; DOI: 10.1158/0008-5472.CAN-17-0700 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. CBX8 exhibits oncogenic activity via AKT/β-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, Jing-Ping Yun1,2,* 1Sun Yat-sen University Cancer Center; State Key Laboratory of Oncology in South China; Collaborative Innovation Center for Cancer Medicine, Guangzhou 510060, China 2Department of Pathology, Sun Yat-sen University Cancer Center, Guangzhou 510060, China #These authors contributed equally to this work. Short title: CBX8 activates AKT/β-catenin pathway *To whom correspondence should be addressed: Jing-Ping Yun, M.D. Ph.D, Department of Pathology, Sun Yat-sen University Cancer Center, Guangzhou, 510060, Guangdong, China. Tel./Fax: +8620-8734-3693; Email: [email protected]. Conflict of interest: All authors declare no conflict of interest. Financial support: The study was supported by grants from the National Key R&D Program of China (2017YFC1309003 (D Xie and JP Yun)), the National Natural Science Foundation of China (No. 81572406 (JP Yun), 81572405 (CZ Zhang)). 1 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2017 American Association for Cancer Research. Author Manuscript Published OnlineFirst on October 24, 2017; DOI: 10.1158/0008-5472.CAN-17-0700 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Abstract Deregulation of Polycomb proteins influences the development and progression of hepatocellular carcinoma (HCC). Here we show that chromobox 8 (CBX8) expression is increased in HCC and correlates with poor outcome in two independent cohorts containing a total of 879 cases. Ectopic expression of CBX8 facilitated tumor growth and metastasis, whereas CBX8 silencing suppressed these effects. CBX8 efficiently activated AKT/β-catenin signaling via upregulation of the transcription factor EGR1 and miR-365-3p in a non-canonical manner: CBX8 directly bound the EGR1 promoter to enhance its activity. In the nucleus, CBX8 also interacted with EGR1 to prevent its degradation. Furthermore, CBX8 increased the transcription of miR-365a-3p, which promoted the nuclear localization of β-catenin by targeting the 3'-UTR ZNRF1. Inhibiting either EGR1 or miR-365a-3p partially rescued CBX8-mediated malignant phenotypes. In clinical samples, CBX8 expression closely correlated with EGR1, miR-365a-3p and nuclear β-catenin. Collectively, our results show that CBX8 functions as an oncogene to upregulate EGR1 and miR-365-3p to stimulate the AKT/β-catenin pathway. This newly identified signaling axis may suggest new therapeutic strategies against HCC. Keywords: CBX8; EGR1, miR-365a-3p; AKT/β-catenin; hepatocellular carcinoma 2 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2017 American Association for Cancer Research. Author Manuscript Published OnlineFirst on October 24, 2017; DOI: 10.1158/0008-5472.CAN-17-0700 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Introduction Hepatocellular carcinoma (HCC), accounting for most (70%–90%) of the primary liver cancers occurring worldwide, remains one of the most prevalent and deadliest human cancers (1). The five-year survival for patients with HCC generally did not improve during the last decades (2). Uncontrolled cell proliferation and metastasis are responsible for the high mortality and represent major obstacles to the clinical management of HCC (3-5). As a result, discovery of the mechanisms underlying tumor progression is important to develop new strategies for therapeutic treatment of HCC. Deregulation of genes involved in chromatin modification has been increasingly implicated in tumor development and progression. One of the chromatin modifiers is the Polycomb repressive complex (PRC1 and PRC2) (6). Under normal circumstances, PRC1 maintains the histone methylation induced by PRC2 to pass on the inactivation signals (7). Canonical PRC1 includes four subunits: Ring E3 ubiquitin ligase, Polyhomeotic, Posterior sex combs, and Polycomb (8). The cores of PRC1 complexes are Polycomb group (PcG) proteins, which were first identified as developmental regulators in Drosophila (9). Chromobox homolog 8 (CBX8), also known as human Polycomb 3 (HPC3), functions as a transcriptional repressor in PRC1. For example, CBX8 inhibited the expression of INK4a/ARF to bypass cell senescence in fibroblasts (10). However, a later study showed that PRC1 without CBX8 was capable of suppressing the INK4a/ARF locus (11), suggesting an unclear role of CBX8 in transcriptional regulation. Recently, CBX8 has been demonstrated to exert oncogenic functions in a non-canonical manner in human malignancies. Lee et al. reported that CBX8 cooperated with SIRT1 to suppress premature senescence and growth arrest in breast carcinoma (12). The PRC1-BCOR- CBX8 complex is required for BCL6-mediated lymphomagenesis (13). Chung and colleagues proposed that CBX8 transcriptionally activated genes involved in the Notch pathway promote breast cancer (14). However, the role of CBX8 in HCC remains unclear. The β-catenin signaling pathway is well known for its role in driving carcinogenesis (15). β-catenin can be frequently found at the cell surface. Following stimulation, β- catenin is activated, partly by phosphorylation at Ser552, to dissociate in the cytoplasm. The cytosolic accumulation of β-catenin leads to its localization to the nucleus where it triggers the transcription of various oncogenes, including LEF1/TCF, to promote cancer initiation and progression (16). The activation of β-catenin, one of the factors contributing to hepatocarcinogenesis (17), is typically mediated by Wnt. Recent studies showed that the β-catenin signaling axis can be triggered by AKT via either phosphorylation of β-catenin at Ser552, which enhances its transcriptional activity (18), or suppression of GSK-3β (19). However, the regulation of the AKT/β-catenin 3 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2017 American Association for Cancer Research. Author Manuscript Published OnlineFirst on October 24, 2017; DOI: 10.1158/0008-5472.CAN-17-0700 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. pathway in HCC is not fully understood. Using tissue microarray (TMA)-based immnunohistochemistry, gene microarrays, and biological function assays, we identified CBX8 as an oncogene in HCC. CBX8 expression is increased and associated with poor outcomes of patients with HCC. We further demonstrate that CBX8 promotes HCC progression in vitro and in vivo by up-regulating EGR1 and miR-365a-3p to subsequently activate the AKT/β-catenin pathway. Collectively, our functional and biochemical studies suggest CBX8 exhibits oncogenic activities towards HCC in a non-canonical fashion. Materials and Methods Cell culture Human HCC cell lines Huh7, MHCC-97H, and HepG2 were purchased from the American Type Culture Collection. The HCC cell lines SK-Hep1, QGY-7701, QGY-7703, Bel-7402, Bel-7404, and SMMC-7721 were obtained from the Cell Resource Center, Chinese Academy of Science Committee. All cell lines were authenticated by
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