- Catenin Degradation β of Hepatocellular Carcinoma by Modifying Ubiquitin-like Protein FAT 10 Promotes the Invasion and Metastasis

The ubiquitin-like protein FAT10 and the homeobox protein HOXB9 each promote metastatic progression in hepatocellular carcinoma (HCC). In this study, we investigated the clinicopathologic significance of FAT10 and HOXB9 in HCC and investigated a mechanistic role for FAT10 in HOXB9-mediated invasiveness and metastasis. Relative to adjacent normal tissues, FAT10 and HOXB9 were markedly overexpressed in HCC, where a positive correlation in their expression and associated malignant characteristics were found. RNAi-mediated silencing of FAT10 decreased HOXB9 expression and inhibited HCC invasion andmetastasis in vitro and in vivo. The effects of FAT10 silencing were reversed by HOXB9 overexpression, whereas RNAi-mediated silencing of HOXB9 decreased HCC invasion and metastasis driven by FAT10 overexpression. Mechanistically, FAT10 regulated HOXB9 expression by modulating the b-catenin/TCF4 pathway, directly binding to b-catenin and preventing its ubiquitination and degradation. Together, our results identified a novel HCC regulatory circuit involving FAT10, b-catenin/TCF4, and HOXB9, the dysfunction of which drives invasive and metastatic character in HCC. Cancer Res; 74(18); 5287–300. 2014 AACR. Introduction Hepatocellular carcinoma (HCC) is one of the most prevalent malignancies and leading causes of death worldwide (1). Although some recent progress has been made with regard to the clinical detection of and treatment methods for HCC, the metastasis and recurrence rates after radical resection of HCC remain high (2, 3). The elucidation of themechanisms involved in HCC progression and metastasis is important for the development of HCC therapeutic agents. HLA-F locus adjacent transcript 10 (FAT10) is a member of the ubiquitin-like protein family (Ubl), which covalently modifies target substrates by binding via their C-termini containing conserved Gly–Gly motifs (4, 5). FAT10 has been implicated in cell growth and survival; the deregulation of FAT10 could induce abnormal alterations in the cell cycle, apoptosis, and immune response (5–7). Recently, there have been an increasing number of studies on FAT10 in malignant tumors. The FAT10 gene is overexpressed in various cancers, such as gastrointestinal cancer, HCC, pancreatic ductal adenocarcinoma, and human glioma (8–12). In the liver, FAT10 overexpression is an epigeneticmarker for liver neoplasia (13). In addition, FAT10 overexpression in HCC tissues is an independent poor prognostic factor in patients withHCC (14). These studies have suggested that FAT10 may play an important role in the tumorigenesis and progression of HCC. However, its specific mechanism of action in the process of HCC invasion and metastasis is still unclear. Homeobox B9 (HOXB9), a member of the class I homeobox (HOX) genes, is critical for embryonic segmentation and limb patterning (15). Recently, emerging evidence links the biologic function of HOXB9 to tumor metastasis. For example, the overexpression of HOXB9 in breast cancer could increase angiogenesis and distal metastasis, and the knockdown of HOXB9 significantly decreased the ability of lung cancer cells to form bone and brainmetastases (16, 17). Previously, we used cDNAmicroarray analysis to determine that the knockdown of FAT10 could inhibit HOXB9 expression in HCC cells (4.912fold; see Supplementary Table S1). Thus, we presumed that FAT10 could affect HCC invasion andmetastasis by regulating HOXB9. In addition, previous studies have shown that HOXB9 is regulated by the Wnt/b-catenin/TCF4 pathway (17, 18). The Wnt/b-catenin signaling plays a critical role in promoting HCC Department of General Surgery, Second Affiliated Hospital of Nanchang University, Nanchang, China. Jiangxi Province Engineering Research Center of Hepatobiliary Disease, Nanchang, China. Jiangxi Province Key Laboratory of Molecular Medicine, Nanchang, China. Department of Digestion, Second Affiliated Hospital of Nanchang University, Nanchang, China. Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). R. Yuan and K. Wang contributed equally to this work. Corresponding Authors: Jianghua Shao, Second Affiliated Hospital of NanchangUniversity, No.1,MindeRoad,Nanchang330006,China. Phone: 86-791-86265564; Fax: 86-791-86262262; E-mail: shao5022@163.com; and Kui Hong, Jiangxi Province Key Laboratory of Molecular Medicine, No. 1MindeRoad,Nanchang330006,China. E-mail: Hongkui88@163.com doi: 10.1158/0008-5472.CAN-14-0284 2014 American Association for Cancer Research. Cancer Research www.aacrjournals. org 5287 on November 2, 2014. © 2014 American Association for Cancer Research. cancerres.aacrjournals. org Downloaded from Published OnlineFirst July 23, 2014; DOI: 10.1158/0008-5472.CAN-14-0284