Fu et al. Molecular Cancer (2018) 17:73 https://doi.org/10.1186/s12943-018-0783-3 RESEARCH Open Access Linc00210 drives Wnt/β-catenin signaling activation and liver tumor progression through CTNNBIP1-dependent manner Xiaomin Fu1,2†, Xiaoyan Zhu2†, Fujun Qin3, Yong Zhang1, Jizhen Lin4, Yuechao Ding5, Zihe Yang6, Yiman Shang1, Li Wang1, Qinxian Zhang2* and Quanli Gao1* Abstract Background: Liver tumor initiating cells (TICs) have self-renewal and differentiation properties, accounting for tumor initiation, metastasis and drug resistance. Long noncoding RNAs are involved in many physiological and pathological processes, including tumorigenesis. DNA copy number alterations (CNA) participate in tumor formation and progression, while the CNA of lncRNAs and their roles are largely unknown. Methods: LncRNA CNA was determined by microarray analyses, realtime PCR and DNA FISH. Liver TICs were enriched by surface marker CD133 and oncosphere formation. TIC self-renewal was analyzed by oncosphere formation, tumor initiation and propagation. CRISPRi and ASO were used for lncRNA loss of function. RNA pulldown, western blot and double FISH were used to identify the interaction between lncRNA and CTNNBIP1. Results: Using transcriptome microarray analysis, we identified a frequently amplified long noncoding RNA in liver cancer termed linc00210, which was highly expressed in liver cancer and liver TICs. Linc00210 copy number gain is associated with its high expression in liver cancer and liver TICs. Linc00210 promoted self-renewal and tumor initiating capacity of liver TICs through Wnt/β-catenin signaling. Linc00210 interacted with CTNNBIP1 and blocked its inhibitory role in Wnt/β-catenin activation. Linc00210 silencing cells showed enhanced interaction of β-catenin and CTNNBIP1, and impaired interaction of β-catenin and TCF/LEF components. We also confirmed linc00210 copy number gain using primary hepatocellular carcinoma (HCC) samples, and found the correlation between linc00210 CNA and Wnt/β-catenin activation. Of interest, linc00210, CTNNBIP1 and Wnt/β-catenin signaling targeting can efficiently inhibit tumor growth and progression, and liver TIC propagation. Conclusion: With copy-number gain in liver TICs, linc00210 is highly expressed along with liver tumorigenesis. Linc00210 drives the self-renewal and propagation of liver TICs through activating Wnt/β-catenin signaling. Linc00210 interacts with CTNNBIP1 and blocks the combination between CTNNBIP1 and β-catenin, driving the activation of Wnt/β-catenin signaling. Linc00210-CTNNBIP1-Wnt/β-catenin axis can be targeted for liver TIC elimination. Keywords: Linc00210, CTNNBIP1, Wnt/β-catenin, Liver TICs, Copy number alterations * Correspondence: [email protected]; [email protected] †Equal contributors 2Department of Histology and Embryology, College of Basic Medicine, Zhengzhou University, 100 Kexue Road, Zhengzhou, Henan 450052, China 1Department of Cancer Biology Immunotherapy, The Affiliated Cancer Hospital of Zhengzhou University and Henan Cancer Hospital, 127 Dongming Road, Zhengzhou, Henan 450003, China Full list of author information is available at the end of the article © The Author(s). 2018 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. Fu et al. Molecular Cancer (2018) 17:73 Page 2 of 14 Background Gene CNA plays critical roles in tumor formation and Liver cancer is the third leading cause of cancer related progression [21–23]. Gene copy number alterations are death all over the world, and 90% liver cancers are hepa- related to gene expression. In liver cancer, the oncogenic tocellular carcinoma (HCC) [1]. Liver tumorigenesis is a signaling pathways (including Wnt/β-catenin) with high complicated process, and the reason of tumorigenesis is expression along with tumorigenesis are frequently still elusive. The tumor initiating cell model proposed copy-number gained, while, lowly expressed genes (in- that only a small subset cancer cells termed tumor initi- cluding ARID1A and RPS6KA3) are copy-number de- ating cells (TICs) account for tumor initiation, metasta- leted [21]. The expression levels of another well-known sis and recurrence [2]. TICs can self-renew and copy number gained gene, c-Myc, were also positively differentiate into various cells within tumor bulk [3]. correlated to its copy number gain [24]. While, lncRNA Various surface markers have been found to identify and copy number alteration and its role in liver cancer and enrich liver TICs recently, including CD13, CD133, liver TICs haven’t been reported. Here, we focused on CD24, EPCAM and calcium channel α2δ1[4–6]. While, lncRNAs located on Chromatin 1q, a frequent copy the liver TIC biology remains largely unknown. number gained region in liver cancer [25]. We analyzed Several signaling pathways participate in liver cancer transcriptome data of liver TICs and non-TICs, and and liver TICs, including Wnt/β-catenin, Notch, Hedge- found a copy number gained lncRNAs, termed hog and NF-κB signaling pathways [7]. Among these linc00210, is highly expressed in liver cancer and liver pathways, Wnt/β-catenin signaling is most widely inves- TICs. Linc00210 interacts with CTNNBIP1, blocks its tigated in tumor initiating cells and many adult progeni- inhibitory role for Wnt/β-catenin signaling, and thus tor cells, including intestinal stem cells, liver progenitor drives the self-renewal of liver TICs. cells and so on [8, 9]. Wnt/β-catenin is also important for development, differentiation and many diseases, in- cluding various tumors [10]. As the core factor in Wnt/ Results β-catenin signaling, β-catenin can form APC-β-catenin Copy number gain of linc00210 in liver cancer complex and β-catenin-TCF complex, accounting for its DNA copy number alteration is a driver of tumorigen- stability and activity, respectively [11]. CTNNBIP1, a β- esis, and many oncogenes have increased copy numbers catenin interacting protein, can block the binding of β- in tumor cells, including c-Myc, FGFR, BCL2L1, DLC1, catenin and TCF/LEF, and thus functions as a negative PRKC1, Sox2 and so on [26]. Copy number gain often regulator of Wnt/β-catenin activation [12]. As the im- accompanies with high expression of transcripts, and portance of Wnt/β-catenin signaling in liver TICs, the copy number deletion results decreased expression. Al- regulatory mechanism of Wnt/β-catenin activation and though gene CNA is deeply explored, whether lncRNA the targeted therapy need further investigation. CNA occurs in tumorigenesis and its role remain un- As we know, many protein-coding genes participate in clear. For liver cancer, CNA of Chromatin 1q plays a tumor formation and tumor initiation, including onco- critical role in tumorigenesis. To investigate the role of genes and tumor suppresser genes [13]. Recently, long lncRNA CNA in liver tumorigenesis and liver TICs, we noncoding RNAs (LncRNAs) emerge as critical media- utilized online-available transcriptome dataset tors in many biological processes. LncRNAs are defined (GSE66529 [27]) and analyzed the expression levels of as transcripts that are longer than 200 nucleotides (nt) lncRNAs located on Chromatin 1q. From the 295 without protein coding ability [14]. LncRNAs exert their lncRNAs detected, many lncRNAs showed dysregulated roles through multi-layered regulation, including gene expression levels in liver TICs (Fig. 1a). To explore these transcription, translocation, mRNA stability, protein sta- lncRNAs in liver TIC self-renewal, we selected 10 bility, activity, subcellular location and so on. LncRNAs lncRNAs and silenced their expression in Hep3B and participate in gene expression by recruiting chromosome PLC with antisense oligos, and detected liver TIC self- remodeling complex into gene promoter through trans- renewal using sphere formation assay, a standard assay or cis- manners [14]. They also interact with some im- for TIC self-renewal. We found linc00210 knockdown portant proteins and regulate their stabilities or activities impaired the self-renewal of liver TICs (Fig. 1b). We [15, 16]. LncRNAs play critical roles in many physio- then confirmed the CNA of linc00210 using TCGA data- logical and pathological processes, including self-renewal set, and found about 13% liver cancer samples have regulation and tumorigenesis [17, 18], however, the role linc00210 copy number gain (Fig. 1c). To further con- of lncRNAs in liver TICs is largely unknown. firm the CNA of linc00210 in liver cancer, we collected Compared with normal cells, cancerous cells have 72 HCC samples, extracted tumor DNA, and detected more frequent mutations and instable chromosomes the copy number of linc00210 using realtime PCR, and [19]. Gene copy number alteration (CNA) and mutation found 16 samples had copy number gain, including are two common chromosome aberrances in tumor [20]. eleven 3-copy and five 4-copy samples (Fig. 1d). We also Fu et al. Molecular Cancer (2018) 17:73 Page 3 of 14 Fig. 1 Copy number gain of linc00210 in liver cancer. a Heat map of lncRNA expression levels in liver TICs and non-TICs.