BRG1 promotes hepatocarcinogenesis by regulating proliferation and invasiveness

RESEARCH ARTICLE BRG1 promotes hepatocarcinogenesis by regulating proliferation and invasiveness Benedikt Kaufmann1, Baocai Wang1, Suyang Zhong2, Melanie Laschinger1, Pranali Patil1, Miao Lu3, Volker Assfalg1, Zhangjun Cheng3, Helmut Friess1, Norbert Hüser1, Guido von Figura2☯*, Daniel Hartmann1☯* a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 Department of Surgery, Klinikum rechts der Isar, Technische Universität München, Munich, Germany, 2 II. Medizinische Klinik und Poliklinik, Klinikum rechts der Isar, Technische Universität München, Munich, Germany, 3 Department of General Surgery, the Affiliated Zhongda Hospital, Southeast University, Nanjing, China ☯ These authors contributed equally to this work. * (DH); (GvF) Abstract OPEN ACCESS Citation: Kaufmann B, Wang B, Zhong S, Laschinger M, Patil P, Lu M, et al. (2017) BRG1 promotes hepatocarcinogenesis by regulating proliferation and invasiveness. PLoS ONE 12(7): e0180225. https://doi.org/10.1371/journal. pone.0180225 Editor: Aamir Ahmad, University of South Alabama Mitchell Cancer Institute, UNITED STATES Received: March 2, 2017 Accepted: June 12, 2017 Published: July 12, 2017 Copyright: © 2017 Kaufmann et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by the Wilhelm Sander-Stiftung (to D.H. and G.v.F.). Work in GVF’s laboratory was supported by funding from the Deutsche Forschungsgemeinschaft (Emmy Noether Program, FI1719/2-1). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The chromatin remodeler complex SWI/SNF plays an important role in physiological and pathological processes. Brahma related gene 1(BRG1), a catalytic subunit of the SWI/SNF complex, is known to be mutated in hepatocellular carcinoma (HCC). However, its role in HCC remains unclear. Here, we investigate the role of BRG1 on cell growth and invasiveness as well as its effect on the expression of putative target genes. Expression of BRG1 was examined in human liver tissue samples and in HCC cell lines. In addition, BRG1 was silenced in human HCC cell lines to analyse cell growth and invasiveness by growth curves, colony formation assay, invasion assay and the expression of putative target genes. BRG1 was found to be significantly increased in HCC samples compared to non-HCC samples. In addition, a declined proliferation rate of BRG1-silenced human HCC cell lines was associated with a decrease of expression of cyclin family members. In line with a decreased invasiveness of BRG1-siRNA-treated human HCC cell lines, down-regulation of MMP7 was detected. These results support the hypothesis that overexpression of BRG1 increases cell growth and invasiveness in HCC. Furthermore, the data highlight cyclin B, E and MMP7 to be associated with BRG1 during hepatocarcinogenesis. Introduction Liver cancer is the fifth most common cancer in men and seventh most common cancer in women worldwide [1]. Accounting for more than 85%, hepatocellular carcinoma (HCC) is the most common histopathological type of primary liver cancer [2]. A large number of mutations in different genes have been identified in HCC to date [3]. There is growing evidence for the importance of the SWI/SNF chromatin remodeling complex during hepatocarcinogenesis based on the detection of mutations and gene alterations in various subunits of the SWI/SNF chromatin remodeling complex in HCC [4]. Chromatin remodeling complexes modify chromatin structure and regulate transcription of genes to control different cellular processes. Mammalian SWI/SNF chromatin remodeling PLOS ONE | https://doi.org/10.1371/journal.pone.0180225 July 12, 2017 1 / 14 BRG1 in HCC Competing interests: The authors have declared that no competing interests exist. complexes are the most mutated chromatin regulators in human cancer [5]. Evolutionarily conserved, the mammalian SWI/SNF complexes are separated into two groups: the brahma related gene 1 (BRG1)-associated factor complex (BAF) and the polybromo BRG1-associated complex (PBAF). These two complexes differ in their respective catalytic ATPase subunits. The BAF complex utilises either BRG1 or BRM as the catalytic subunit, whereas the PBAF complex is composed of BRG1 exclusively. In association with these catalytic subunits, various other proteins contribute to the SWI/SNF complexes that are finally composed of 9 to 12 subunits [6,7,8]. The mutational landscape of human cancer reveals different subunits of the SWI/ SNF complexes including BRG1 to be frequently mutated and altered [3,4,9,10]. However, the role of mutated BRG1 in tumourigenesis remains largely unknown. Various human cancers reveal an overexpression of BRG1, whereas a similar number of malignant tumours show the suppression of BRG1 expression [11–24]. In addition, BRG1 is known to interact with both proliferation-promoting and -inhibiting genes, including cyclins and pRB [16,17,19,25]. This implies that BRG1 not only acts as a tumour suppressor gene, but also as an oncogene. However, at present it is not clear when BRG1 acts as a tumour suppressor gene and when it acts as an oncogene. In HCC, BRG1 reveals four different somatic heterozygous, missense mutations, causing overexpression [11]. One of these somatic mutations was found in the catalytic ATPase domain. This domain enables mechanical movement by converting ATP energy. Two somatic mutations were detected in the bromodomain, a domain that is involved in the recognition of acetylated lysines in histone tails [11]. While Endo et al. (2013) [11] observed no correlation in HCC for BRG1 expression and overall survival or any other clinicopathological parameters, Zhu et al. (2016) [12] detected a positive correlation between increased BRG1 expression and the severity of HCC as well as metastasis. Moreover, BRG1 plays an important role in the regulation of liver cancer stem cells [12]. However, the specific role of BRG1 in HCC remains largely unclear at present. In this study, the role of BRG1 on proliferation and invasion in human HCC cancer cell lines was investigated. In addition, target genes regulating the cell cycle and the ability of invasion were analysed. Our findings support the hypothesis that BRG1 promotes proliferation as well as invasion in HCC and highlight the correlation between the expression of BRG1 and members of the cyclin family as well as matrix metalloproteinases. Materials and methods Cell lines and cell cultures All cell experiments were performed by using the the human HCC cell line HuH7 and the human hepatoblastoma cell line HepG2 purchased from the Japanese Collection of Research Bioresources Cell B (...truncated)