Journal of Cancer 2019, Vol. 10 5862 Ivyspring International Publisher Journal of Cancer 2019; 10(23): 5862-5873. doi: 10.7150/jca.31599 Research Paper Oncogenic Wnt3a: A Candidate Specific Marker and Novel Molecular Target for Hepatocellular Carcinoma Wenjie Zheng1*, Min Yao2*, Miao Fang1, Liuhong Pan1, Li Wang3, Junling Yang1, Zhizhen Dong4, Dengfu Yao1 1. Research Center of Clinical Medicine, Affiliated Hospital of Nantong University, Nantong 226001, Jiangsu Province, China; 2. Department of Medical Immunology, Medical School of Nantong University, Nantong 226001, Jiangsu Province, China; 3. Department of Medical Informatics, Medical School of Nantong University, Nantong 226001, Jiangsu Province, China; 4. Department of Diagnostics, Affiliated Hospital of Nantong University, Nantong 226001, Jiangsu Province, China. * Equal contributors Corresponding author: Prof. Dengfu Yao, M.D. & Ph.D., Research Center of Clinical Medicine, Affiliated Hospital of Nantong University, 20 West Temple Rd, Jiangsu 226001, China. Tel: +0086-513-85052523; Fax: +0086-513-85052254; E-mail: [email protected] © The author(s). This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/). See http://ivyspring.com/terms for full terms and conditions. Received: 2019.06.12; Accepted: 2019.08.20; Published: 2019.10.08 Abstract Background and aim: It is of the utmost importance for the specific diagnosis and effective therapy of hepatocellular carcinoma (HCC). Abnormality of oncogenic Wingless-type MMTV integration site family member 3a (Wnt3a) has been associated with progression of HCC. In this study, we aimed to evaluate Wnt3a as a novel biomarker and target for HCC. Methods: Circulating Wnt3a levels were quantitatively detected in a cohort of chronic liver diseases by an enzyme-linked immune-absorbent assay (ELISA). Hepatic Wnt3a expression in HCC and para-cancerous tissues was analyzed by immunohistochemistry. Prognostic value of Wnt3a for HCC was discovered in the cohort from the Cancer Genome Atlas (TCGA). Dynamic alterations of Wnt3a levels were detected in the hepatocarcinogenesis model induced by 2-acetylaminofluorene. Effects of Wnt3a on biological behaviors were evaluated in vitro and in vivo based on Crispr/Cas9. Results: Up-regulated Wnt3a levels were observed in serum of HCC patients with high specificity and sensitivity for HCC diagnosis. Combination of Wnt3a and AFP could improve sensitivity to 93.9% in serological detection. In addition, Wnt3a expression in HCC tissues was significantly higher than that in para-cancerous tissues. The cohort of TCGA demonstrated that high Wnt3a expression led to a poor survival of HCC patients, especially in cases at advanced stages. Furthermore, the hepatocarcinogenesis model showed that Wnt3a dynamically increased in the development of HCC. Functionally, silencing Wnt3a by Crispr/Cas9 suppressed the proliferation, colony formation, induced cell cycle arrest of HCC cells by de-activating Wnt/β-catenin pathway in vitro, and inhibited xenograft tumor growth in vivo. Conclusions: Oncogenic Wnt3a could be considered as a candidate biomarker and novel target for HCC. Key words: Hepatocellular carcinoma; Wnt3a; Specific diagnosis; Target therapy; Biomarker Introduction Hepatocellular carcinoma (HCC) is still one of hepatitis C virus (HCV) infection, aflatoxin B1, and the most common cancers with leading cause of non-alcohol fat liver diseases (NAFLD)[3]. Chronic cancer in the world[1], particularly in the inshore area HBV carriers have a 5 ~ 15-fold increased risk of HCC of the Yangtze River[2]. The main etiological factors of compared with the general population, partially due HCC include chronic hepatitis B virus (HBV) or to the activation of cellular Wnt/β-catenin and http://www.jcancer.org Journal of Cancer 2019, Vol. 10 5863 transforming growth factor-β (TGF-β) pathways[4, 5]. University, China between Jan. 2012 and Jan. 2017 Early discovery and effective treatments are of the were enrolled in this study. They were divided into utmost importance for HCC therapy and prognosis. four groups: HCC (n = 180), liver cirrhosis (LC, n = Serological biomarkers are widely used in the early 80), chronic hepatitis (CH, n = 80), and NAFLD (n = diagnosis of HCC with the features of non-invasive, 60). HCC was diagnosed according to the diagnostic accuracy, and flexibility[6]. Despite of the wide criteria set by the 2012 Chinese National Collaborative application of alpha-fetoprotein (AFP), its Cancer Research Group. Among the 180 HCC (135 false-negative rate may be as high as 40 % for HCC males, 45 females; ages ranged 35-86 years old) patients at early stage[7]. To date, some novel cohort, no patients received any pretreatment and all biomarkers including circulating glypican-3 cases were confirmed by pathological examination. 59 (GPC-3)[8] and microRNAs[9] have been proposed. cases with AFP level more than 20 ng/ml; 84 cases However, the overall efficiency is still unsatisfactory, with lymph node metastasis; 47 cases with especially in HCC patients with low AFP level or postoperative recurrence, 121 cases with positive small-size tumor. hepatitis B surface antigen (HBsAg), and 81 cases with Accumulating evidence demonstrate that tumor size less than 3.0 cm. Serum AFP exceeding 20 Wnt/β-catenin signaling molecules play significant μg/L was considered positive. Written informed roles in physiology, consisting by total 19 highly consents were provided from all patients. Healthy conserved secreted cysteine-rich glycoproteins with persons with hepatitis B markers (HBsAg, HBcAb, distinct functions. For instance, Wingless-type MMTV and HBV-DNA) negative and normal serum alanine integration site family member 1 (Wnt1), Wnt2, Wnt3, aminotransferase (ALT) levels as normal controls Wnt3a, Wnt8a, Wnt8b, Wnt10a, and Wnt10b activate (NC, n = 80) were obtained from the Nantong Central the canonical Wnt signaling and promote tumor Blood Bank in China. The study was in accordance progression, while Wnt4, Wnt5a, Wnt5b, Wnt6, with the Declaration of Helsinki and approved by the Wnt7a, Wnt7b, and Wnt11 stimulate the Ethics Committee of Affiliated Hospital of Nantong non-canonical Wnt pathway as tumor University, China. suppressors[10]. The alterations of the Wnt/β-catenin signaling in pathology were related to numerous Liver tissue samples genetic/epigenetic abnormalities affecting cellular Human liver cancerous and their surrounding persistence, multiplication, migration, alteration and tissues in this study were obtained from 60 HCC genomic instability[11]. Recently, abnormal patients who underwent operations between Jan 2012 expression of the Wnt signaling was reported closely and Aug 2017 at the Affiliated Hospital of Nantong associated with the occurrence, progression, and University, China. All sections were reviewed for prognosis of HCC[12]. confirmation of the original pathological and clinical Wnt3a is one of the most well-known Wnt diagnosis. Related clinical information including ligands due to its critical roles in embryonic gender, age, tumor number, cirrhosis, HBV, AFP, and development as a pivotal component of the periportal cancer embolus were collected from their mesoderm gene. However, Wnt3a has recently been medical records. Tumor staging was based on the 6th considered as an oncogenic factor in cancers of colon, edition of tumor-node-metastasis (TNM) classification breast, lung, and esophageal squamous cell[13]. of the International Union Anti-Cancer. This study Notably, silencing Wnt3a could inhibit proliferation, was approved by the Ethics Committee of Affiliated invasion, and chemo-resistance in glioma derived Hospital of Nantong University, China stem-like cells[14]. Previously, we first discovered the (TDFY2013008), and in accordance with World abnormal expression of Wnt3a in sera and cancerous Medical Association Declaration of Helsinki. Written tissues of HCC patients[15]. Nevertheless, the exact informed consent was obtained from patients who underlying mechanisms of Wnt3a and its alteration participated in this study. The survival analysis was during malignant transformation of hepatocytes still conducted by the Kaplan-Meier plotter, which was remain to be explored. Therefore, this study aimed to built based on the data of Cancer Genome Atlas further investigate the role of Wnt3a in clinical value (TCGA). and molecular-target for HCC. Hepatocarcinogenesis model Methods Rat hepatocarcinogenesis model was made according to the previous method[16]. Male Serum samples Sprague-Dawley rats (4-wk-old, body weight 140-150 Total 400 patients with benign or malignant liver g) were supplied by the Animal Center of Nantong diseases from the Affiliated Hospital of Nantong University. Rats were maintained under http://www.jcancer.org Journal of Cancer 2019, Vol. 10 5864 pathogen-free conditions with a 12-h light-dark cycle. by trypsin and washed with pre-cold PBS. Following Malignant transformation of rat hepatocytes was fixed in 70 % ethanol for 12 h, cells were resuspended induced by 2-acetylaminofluorene (2-AAF; Sigma). in 50 μg/mL RNase A solution and incubated for 30 Sera and livers of rats were collected from rats min at 37 . Then samples were incubated with 10 sacrificed biweekly for immunochemistry and specific μg/mL propidium iodide (Solarbio, China) in the concentration of Wnt3a. All animal procedures were dark for 15℃ min. After that, cells were detected by a approved by the Animal Care and Use Committee of Flow Cytometer