Author Manuscript Published OnlineFirst on July 3, 2017; DOI: 10.1158/0008-5472.CAN-16-3456 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. TP53INP1 down-regulation activates a p73-dependent DUSP10/ERK signaling pathway to promote metastasis of hepatocellular carcinoma Kai-Yu Ng1, Lok-Hei Chan1, Stella Chai1, Man Tong1, Xin-Yuan Guan2,4, Nikki P Lee3, Yunfei Yuan5, Dan Xie5, Terence K Lee6, Nelson J Dusetti7, Alice Carrier7, Stephanie Ma1,4 1School of Biomedical Sciences, Departments of 2Clincial Oncology and 3Surgery, 4State Key Laboratory for Liver Research, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong; 5State Key Laboratory of Oncology in Southern China, Sun Yat-Sen University Cancer Center, Guangzhou, China; 6Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hong Kong; 7Aix Marseille University, CNRS, INSERM, Institut Paoli- Calmettes, CRCM, Marseille, France Corresponding author: Stephanie Ma, PhD, School of Biomedical Sciences, Li Ka Shing Faculty of Medicine, The University of Hong Kong, 1/F, Laboratory Block, 21 Sassoon Road, Pok Fu Lam, Hong Kong. E-mail: [email protected]; Tel: 852-3917-9238; Fax: 852-2817-0857 Short title: TP53INP1 in HCC metastasis Keywords: metastasis; TP53INP1; p73; DUSP10; ERK; liver cancer Abbreviations: DUSP, dual-specificity MAP kinase phosphatases; EV, empty vector; HCC, hepatocellular carcinoma; IHC, immunohistochemistry; KD, knockdown; MKP, MAP kinase phosphatases; NTC, non-target control; OE, overexpression; qRT-PCR, quantitative real time 1 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2017 American Association for Cancer Research. Author Manuscript Published OnlineFirst on July 3, 2017; DOI: 10.1158/0008-5472.CAN-16-3456 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. polymerase chain reaction; shRNA, short hairpin RNA; TP53INP1, tumor protein 53 inducible nuclear 1 Conflicts of interest: The authors disclose no conflicts. Funding: This work was supported in part by grants from Research Grants Council – General Research Fund (HKU_773412M), – Collaborative Research Fund (C7027-14G) and the Croucher Foundation Innovation Award to S. Ma. 2 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2017 American Association for Cancer Research. Author Manuscript Published OnlineFirst on July 3, 2017; DOI: 10.1158/0008-5472.CAN-16-3456 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Abstract Identifying critical factors involved in the metastatic progression of hepatocellular carcinoma (HCC) may offer important therapeutic opportunities. Here we report that the pro-apoptotic stress response factor TP53INP1 is often selectively down-regulated in advanced stage IV and metastatic human HCC tumors. Mechanistic investigations revealed that TP53INP1 down- regulation in early stage HCC cells promoted metastasis via DUSP10 phosphatase-mediated activation of the ERK pathway. The DUSP10 promoter included putative binding sites for p73 directly implicated in modulation by TP53INP1. Overall, our findings showed how TP53INP1 plays a critical in limiting progression of early stage HCC, with implications for developing new therapeutic strategies to attack metastatic HCC. 3 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2017 American Association for Cancer Research. Author Manuscript Published OnlineFirst on July 3, 2017; DOI: 10.1158/0008-5472.CAN-16-3456 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Introduction Liver cancer remains one of the most prevalent and deadliest cancer types worldwide. Hepatocellular carcinoma (HCC) accounts for over 75% of all liver cancer cases. Metastasis and post-surgical recurrence are common and represent major obstacles to the improvement of patient survival. HCC patients are often diagnosed at an advanced stage when curative therapy is no longer available and even after surgery, the prognosis of HCC remains unsatisfactory, with a five year post-recurrence rate at >70%. Metastasis is a complex multistep process involving alterations in the dissemination, invasion, survival and growth of new cancer cell colonies, which are regulated by a complex network of intra- and inter-cellular signal transduction cascades (1). However, metastasis remains the most poorly understood component of cancer pathogenesis (2). Elucidation of the mechanisms underlying metastasis is fundamental for the development of new therapeutic treatments for advanced metastatic HCC. Extracellular signal-regulated kinases (ERKs) have been shown to play critical roles in malignant transformation and cancer metastasis (3). Oncogenic activation of ERKs can be induced by various mechanisms including transcriptional overexpression, mutations in upstream components of the MAP kinase pathway, such as RAS and BRAF, and down-regulation of negative regulator dual- specificity MAP kinase phosphatases (DUSPs) (4). ERK plays a major role in invasion by inducing proteases that degrade the basement membrane, enhances cell migration, and increases cell survival. Activated ERK pathway has been shown to correlate with the expression of epithelial- mesenchymal transition (EMT) markers, a hallmark of metastasis. These findings collectively suggest that ERK plays a major role in tumor progression and metastasis. However, our knowledge of endogenous regulators of DUSP/ERK remains limited and how they work to promote HCC metastasis is also not known. 4 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2017 American Association for Cancer Research. Author Manuscript Published OnlineFirst on July 3, 2017; DOI: 10.1158/0008-5472.CAN-16-3456 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. TP53INP1 is a stress-induced tumor suppressor gene with anti-proliferative and pro-apoptotic activities (5-6). It is an alternatively spliced gene encoding two protein isoforms (α and β), and when overexpressed, both isoforms exert a tumor suppressor function, mainly by inducing the transcription of target genes involved in cell-cycle arrest and p53-mediated apoptosis as part of the cell responses to genotoxic stress. Significant reduction or loss of TP53INP1 expression has been shown in a number of cancer types including those of the stomach (7), breast (8), pancreas (9), esophagus (10), lung (11), melanocyte (12), colon (13) and blood (14). In relation to metastasis, TP53INP1 has only been implicated in a handful of studies including one report where they found transcriptional levels of TP53INP1 to be down-regulated in metastatic lung of brain cancers (15). A more recent study led by our collaborator Dusetti et al. found TP53INP1 to reduce pancreatic cancer cell migration by regulating SPARC expression (16). TP53INP1 is a target gene of the transcription factor p53. Conversely, TP53INP1 has also been shown to play a role in cellular homeostasis through p53-dependent and p53-independent manners (5-6). In addition to p53, TP53INP1, which is also a p73 target gene, can enhance transcriptional activity of p73 to induce cell cycle arrest and cell death (17). Thus, TP53INP1 can exert its tumor suppressor function by inducing the transcription of both p53 and p73 target genes. In our previous studies, we found that the initiation, growth and self-renewal of CD133+ liver tumors to be fine-tuned by a balance of miR-130b overexpression and TP53INP1 down-regulation (18). This result suggests that TP53INP1 is a critical effector driving hepatocarcinogenesis. Nevertheless, to date, no studies have determined the function of TP53INP1 in HCC metastasis or the molecular mechanism by which TP53INP1 regulates invasion and metastasis in HCC. Here, we demonstrate that TP53INP1 is frequently down-regulated in advanced stage and metastatic 5 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2017 American Association for Cancer Research. Author Manuscript Published OnlineFirst on July 3, 2017; DOI: 10.1158/0008-5472.CAN-16-3456 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. human HCC tumors and that down-regulation of TP53INP1 in HCC functionally promotes metastasis through ERK activation via a p73-dependent DUSP10 regulation. Findings from our study not only provides new insight into how HCC metastasis is regulated but also provides a new layer of mechanism by which DUSP10/ERK signaling is regulated by p73/TP53INP1 and also identifies DUSP10 as a new transcriptional effector of p73. 6 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2017 American Association for Cancer Research. Author Manuscript Published OnlineFirst on July 3, 2017; DOI: 10.1158/0008-5472.CAN-16-3456 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Materials and Methods Gene expression profiling and patient samples. Gene expression profiling studies involving multiple clinical samples were performed analyzing the expression of specific transcripts in two datasets available through Gene Expression Omnibus (GSE25097 and GSE40367) (19-20). In addition, human primary and matched metastatic HCC tissue samples were obtained from 37 patients undergoing hepatectomy at the Sun Yat‐Sen University Cancer Centre in Guangzhou, China. Tissue samples were collected from patients who