DDR2 facilitates hepatocellular carcinoma invasion and metastasis via activating ERK signaling and stabilizing SNAIL1

Xie et al. Journal of Experimental & Clinical Cancer Research (2015) 34:101 DOI 10.1186/s13046-015-0218-6 RESEARCH Open Access DDR2 facilitates hepatocellular carcinoma invasion and metastasis via activating ERK signaling and stabilizing SNAIL1 Binhui Xie1†, Weihao Lin2†, Junming Ye3, Xiaonong Wang1, Bing Zhang4, Shiqiu Xiong5, Heping Li4* and Guosheng Tan4* Abstract Background: Several studies have found that DDR2 is up-regulated in many tumor types and facilitates tumor progression. However, the role of DDR2 in hepatocellular carcinoma (HCC) progression and its downstream signaling pathways remain unclear. Methods: DDR2 expression was assessed in several cell lines and 112 pairs of HCC and matched adjacent noncancerous liver tissues. Clinical significance of DDR2 in HCC was analyzed. Phosphorylated DDR2 (p-DDR2) expression was detected by immunoblotting to evaluate its correlation with DDR2. The effect of DDR2 on HCC cell migration and invasion were examined. Cycloheximide chase experiments were performed to detect the half-life of SNAIL1. Moreover, DDR2 expression was detected by immunohistochemistry to evaluate its correlation with SNAIL1. The regulatory effect of DDR2 on ERK signaling, SNAIL1, EMT, MT1-MMP and MMP2 was confirmed by immunoblotting. The effect of type I collagen on DDR2/ERK2/SNAIL1 signaling was assessed. Results: DDR2 was more highly expressed in HCC than in non-HCC tissues. DDR2 overexpression was correlated with clinicopathological features of poor prognosis. Clinical analysis revealed that DDR2 is an independent prognostic marker for predicting overall survival and disease free survival of HCC patients. Overexpression of DDR2 is associated with p-DDR2 amplification. In vitro studies showed that DDR2 facilitates HCC cell invasion, migration and EMT via activating ERK2 and stabilizing SNAIL1. DDR2 can up-regulate MT1-MMP and MMP2 expression through ERK2/SNAIL1 signaling in HCC. Additionally, collagen I can induce DDR2/ERK2/SNAIL1 signaling activation in HCC cells. Conclusions: Our findings suggest that DDR2 plays an important role in promoting HCC cell invasion and migration, and may serve as a novel therapeutic target in HCC. Keywords: DDR2, HCC, ERK, SNAIL1, EMT Background Hepatocellular carcinoma (HCC) is one of the most prevalent malignant tumors worldwide and is the second leading cause of cancer-related death in China [1–3]. In recent years, the incidence of HCC is increasing in many parts of the world including the United States, partly due to the rise in hepatitis C virus infection [4–6]. Hepatic * Correspondence: ; † Equal contributors 4 Department of Medical Imaging, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou 510080, China Full list of author information is available at the end of the article resection offers the chance of a cure for patients with HCC, but the prognosis remains very poor [7, 8]. Hence, understanding the molecular mechanisms involved in hepatocarcinogenesis and exploring prognostic markers and therapeutic targets of HCC is urgently needed. The discoidin domain receptors (DDRs), consisting of DDR1 and DDR2, are distinctive receptor tyrosine kinases (RTKs) and signal in response to collagens instead of soluble peptide growth factors [9–11]. Multiple studies have suggested that DDR2 can regulate cell adhesion, migration and matrix remodeling [12, 13]. DDR2 have also been shown to exhibit aberrant expression patterns © 2015 Xie 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. Xie et al. Journal of Experimental & Clinical Cancer Research (2015) 34:101 in several tumor types, including nasopharyngeal and prostate cancer [14–16]. Furthermore, it was found that the abnormal expression of DDR2 implicated in cancer progression and a poor prognosis [17, 18]. However, the role of DDR2 in HCC and the molecular mechanisms by which DDR2 exerts its biological function are still unclear. SNAIL1, a member of transcription factors, is critical for inducing and sustaining cancer epithelial–mesenchymal transition (EMT) [19, 20]. SNAIL1-induced EMT has been demonstrated essential for promoting tumor cell migration and invasion in multiple tumor types [21, 22]. Recently, Zhang et al. have found that SNAIL1 is regulated by the DDR2/ERK2 signaling pathway in breast cancer [23]. In the previous studies, ERK signaling pathway has been considered to promote the progression of HCC [24, 25]. It is also known that SNAIL1 plays an essential role in regulating HCC EMT [26, 27]. However, whether DDR2 is implicated in regulating the ERK signaling and SNAIL1 in HCC has not been clarified. As representative epithelial-specific and mesenchymalspecific biomarkers, E-cadherin and Vimentin are crucial factors in the invasion, migration and EMT of HCC [21]. Down-regulation of E-cadherin and up-regulation of Vimentin are used as biomarkers indicating an epithelial cell has undergone EMT in HCC [21]. Multiple studies have revealed that EMT associated with patients poor prognosis is an important step in the invasion and migration of HCC [21, 26]. Importantly, SNAIL1-induced E-cadherin down-regulation and Vimentin up-regulation have been demonstrated essential for triggering the EMT of HCC [27]. However, whether DDR2 is implicated in regulating the expression of E-cadherin and Vimentin in HCC are still unknown. In this study, we found that DDR2 was more highly expressed in HCC tissues than that in non-tumor tissues, and DDR2 overexpression was correlated with poor clinicopathological features and outcome of HCC patients. Our study demonstrated that DDR2 has an oncogenic role in HCC tumorigenesis by facilitating cancer cell invasion, migration and epithelial–mesenchymal transition via activating ERK signaling and stabilizing SNAIL1. Additionally, DDR2 can up-regulate membrane type-1 matrix metalloproteinase (MT1-MMP) and MMP2 expression through ERK2/SNAIL1 signaling in HCC. Our study identified that DDR2 is a novel regulator of EMT through stabilizing SNAIL1, indicating its potential therapeutic value for reducing HCC invasion and metastasis. Materials and methods Tissue samples A total of 112 pairs of HCC and corresponding adjacent non-tumorous liver tissues (>2.0 cm from the resection margin) were obtained from patients undergoing Page 2 of 13 hepatectomy between February 2007 and December 2009 at the Department of Hepatobiliary Surgery, the First Affiliated Hospital of Gannan Medical Univers (...truncated)