Lysine-specific demethylase 5C promotes hepatocellular carcinoma cell invasion through inhibition BMP7 expression

Hepatocellular carcinoma (HCC) is the most common type of tumor and is associated with high morbidity and mortality rates. Patients with HCC routinely undergo surgery followed by adjuvant radiation therapy and chemotherapy.

Ji et al BMC Cancer (2015) 15:801 DOI 10.1186/s12885-015-1798-4 RESEARCH ARTICLE Open Access Lysine-specific demethylase 5C promotes hepatocellular carcinoma cell invasion through inhibition BMP7 expression Xuening Ji1†, Shi Jin2†, Xiaotong Qu3, Kejun Li2, Hongjiang Wang4, Hui He2, Fuchao Guo5* and Lei Dong2* Abstract Background: Hepatocellular carcinoma (HCC) is the most common type of tumor and is associated with high morbidity and mortality rates Patients with HCC routinely undergo surgery followed by adjuvant radiation therapy and chemotherapy Despite such aggressive treatment approaches, median survival times remain under year in most cases KDM5C is a member of the family of JmjC domain-containing proteins that removes methyl residues from methylated lysine on histone H3 lysine (H3K4) KDM5C has been proposed as an oncogene in many types of tumors; however, its role and underlying mechanisms in HCC remain unclear Methods: Expression level of KDM5C was examined by RT-PCR, and IHC Forced expression of KDM5C was mediated by retroviruses, and KDM5C was downregulated by shRNAs expressing lentiviruses Migration and invasion of HCC cells was measured by wound healing, Transwell and Matrigel assays respectively Results: In this study, we report that KDM5C is abundantly expressed in invasive human HCC cells Cellular depletion of KDM5C by shRNA inhibited HCC cell migration, invasion and epithelial-mesenchymal transition in vitro, and markedly decreased the metastasis capacity of invasive HCC cells in the liver and lung Furthermore, ectopic expression of KDM5C in HCC cells promoted cell migration, invasion and epithelial-mesenchymal transition via the inactivation of BMP7 Knockdown of BMP7 significantly promotes shKDM5C-induced cell migration inhibition Conclusions: Taken together, these data suggest that KDM5C-mediated BMP7 inactivation is essential for HCC cell invasion Keywords: KDM5C, BMP7, HCC, Metastasis Background Hepatocellular carcinoma (HCC) is a highly aggressive tumor characterized by its lack of response to conventional chemo-, radio-, and immunotherapies [1] HCC cells extensively invade normal tissues, which contribute to the continued poor prognosis for these tumors by preventing complete surgical resection [2, 3] Invading tumor cells are resistant to conventional therapies and invasiveness is enhanced by antiangiogenic approaches * Correspondence: wangyunshan135@163.com; dongleidoc@163.com † Equal contributors Department of general surgery, The first people’s Hospital of jinzhou District in Dalian City, No.683, Stalin Road, Jinzhou District, Dalian 116100, China Department of Laparoscopic Surgery, First Affiliated Hospital of Dalian Medical University, No.193, Lianhe Street, Shahekou District, Dalian 116001, China Full list of author information is available at the end of the article [4, 5] Thus, an effective strategy to prevent invasion of HCC cells into surrounding normal tissues is needed Lysine (K)-specific demethylase 5C (KDM5C) is a member of the family of JmjC domain-containing proteins that specifically removes methyl residues from tri-, di-, and monomethylated lysine on histone H3 lysine (H3K4) that are associated with active genes [6] KDM5C is a transcriptional repressor that harbors intrinsic histone demethylase activity [7] Trimethylation at H3K4 is an important histone mark associated with actively transcribed genes, and KDM5C specifically demethylates H3K4me3 to a transcriptionally inactive state [8] KDM5C is upregulated in multiple tumor cell lines [9–11] Furthermore, KDM5C promotes proliferation of cancer cells, and knockdown of KDM5C causes a significant delay in the G1/S transition [12, 13] KDM5C appears to © 2015 Ji et al Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated Ji et al BMC Cancer (2015) 15:801 promote tumorigenesis through the specific repression of antiproliferative genes [14, 15] Thus, KDM5C may act as an oncogene, but whether KDM5C plays a role in HCC metastasis remains unknown Bone morphogenetic proteins (BMPs) signal via specific serine/threonine kinase receptors on the cell surface known as bone morphogenetic protein receptors (BMPRs) [16] Both type-I and -II BMPRs are required for signal transduction The type-I receptor BMPRIA preferentially binds ligands of the Dpp class, such as BMP2 and BMP4, whereas the type-II receptor BMPRIB binds ligands of the 60A class, such as BMP7 [16–18] After ligand binding, the type-I/type-II receptor complex phosphorylates Smad1, and proteins that translocate to the nucleus to regulate target gene expression [19] Previous studies have shown that BMP-7 exert antiinvasive actions by inhibiting TGF-beta-induced expression of integrin beta [20, 21] In the current study, we show that KDM5C overexpression predicts poor prognosis in HCC patients undergoing curative resection Additionally, we present the evidence that KDM5C expression promotes HCC cells invasion, metastasis and EMT These functional effects of KDM5C were exerted through control of BMP7 transcriptional expression via H3K4me3 The down-regulation of BMP7 triggered by KDM5C therefore enforces HCC cells oncogenesis and metastasis Our findings provide a novel mechanistic role of KDM5C in HCC metastasis, suggesting that KDM5C may serve as a potential therapeutic target for advanced HCCs Methods Chemicals and antibodies Lipofectamine 2000 transfection and TRIZOL LS Reagents were purchased from Invitrogen (Grand Island, NY, USA) Antibodies against KDM5C, Histone H3, H3K4me1 (monomethylated K4), H3K4me2 (dimethylated K4), H3K4me3 (trimethylated K4), H3K27me3 (trimethylated K27) were purchased from Abcam (Cambridge, MA, USA) E-cadherin, N-cadherin, vimentin, BMP7, and β-actin antibodies were from Cell Signaling technology (Danvers, MA, USA) Anti-α-catenin antibody was from BD (Franklin Lakes, NJ, USA) Patients and specimens Eighteen tumor and para-cancerous tissues which, were used for qRT-PCR analysis, were randomly collected from HCC patients who underwent curative resection with informed consent between 2013 and 2014 at the department of laparoscopic surgery, First Affiliated Hospital of Dalian Medical University All tissues were collected immediately upon resection of the tumors in the operation theater, transported in liquid nitrogen, and then stored at −80 °C Another 221 hepatocellular carcinoma Page of 15 tissues which, were used for immunohistochemical analysis, were randomly collected from HCC patients who underwent curative resection with informed consent between 2008 and 2014 at the Department of Hepatobiliary Surgery, First Affiliated Hospital of Dalian Medical University Tumor staging was based on the 6th edition of the tumor-node-metastasis (TNM) classification of the International Union Against Cancer The clinicopathologic characteristics of the 221 hepatocellular carcinoma tissues are summarized in Table Follow-up data were summarized at the end of March 2015, with a median observation time of 62.4 months Study protocols were approved by the Hospital Ethics Committee of Dalian Medical University, and written informed consent was obtained from patients based on the Declaration of Helsinki Histological and immunohistochemical analysis The normal human liver tissues, human tumor tissues, and lungs dissected from mice were fixed in % paraformaldehyde in phosphate-buffered saline (PBS) overnight and subsequently embedded in paraffin wax Sections cut at a thickness of μm were stained with hematoxylin and eosin for histological analysis Immunohistochemical analysis was performed for different markers in these arrays as described previously The proportion of stained cells (lower, 50 99 (72 %) 39 (28 %) 138 Male 133 (73 %) 49 (27 %) 182 Female 31 (79 %) (21 %) 39 Sex 0.405 HBsAg 0.072 Negative 46 (80 %) 11 (20 %) 57 Positive 115 (60 %) 49 (40 %) 164 Negative 139 (66 %) 72 (34 %) 211 Positive (80 %) (20 %) 10 HCV 0.169 AFP 0.534 ≤ 20 73 (75 %) 25 (25 %) 98 > 20 91 (74 %) 32 (26 %) 123 γ-GT(U/L) ≤ 54 72 (67 %) 36 (33 %) 108 > 54 81 (72 %) 32 (28 %) 113 Liver cirrhosis No 29 (69 %) 13 (31 %) 42 Yes 122 (68 %) 57 (32 %) 179 ≤5 133 (68 %) 62 (32 %) 195 >5 12 (46 %) 14 (54 %) 26 Tumor diameter (cm) 0.187 Western blotting 0.566 Cells were lysed in lysis buffer and total protein contents were determined by the Bradford method 30 μg of lysis were separated by reducing SDS-PAGE and probed with specific antibodies Blots were washed and probed with respective secondary peroxidase-conjugated antibodies, and the bands visualized by chemoluminescence (Amersham Biosciences) 0.028 Microvascular invasion qRT-PCR