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TP53INP1 Downregulation Activates a P73- Dependent DUSP10/ERK

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TP53INP1 Downregulation Activates a P73- Dependent DUSP10/ERK Published OnlineFirst July 3, 2017; DOI: 10.1158/0008-5472.CAN-16-3456 Cancer Tumor and Stem Cell Biology Research TP53INP1 Downregulation 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 Guan2,3, Nikki P Lee4, Yunfei Yuan5, Dan Xie5, Terence K Lee6, Nelson J Dusetti7, Alice Carrier7, and Stephanie Ma1,3 Abstract Identifying critical factors involved in the metastatic progres- phosphatase-mediated activation of the ERK pathway. The sion of hepatocellular carcinoma (HCC) may offer important DUSP10 promoter included putative binding sites for p73 therapeutic opportunities. Here, we report that the proapoptotic directly implicated in modulation by TP53INP1. Overall, our stress response factor TP53INP1 is often selectively downregu- findings show how TP53INP1 plays a critical role in limiting lated in advanced stage IV and metastatic human HCC tumors. the progression of early-stage HCC, with implications for Mechanistic investigations revealed that TP53INP1 downregula- developing new therapeutic strategies to attack metastatic HCC. tion in early-stage HCC cells promoted metastasis via DUSP10 Cancer Res; 77(17); 4602–12. Ó2017 AACR. Introduction Extracellular signal-regulated kinases (ERK) have been shown to play critical roles in malignant transformation and cancer Liver cancer remains one of the most prevalent and deadliest metastasis (3). Oncogenic activation of ERKs can be induced by cancer types worldwide. Hepatocellular carcinoma (HCC) various mechanisms including transcriptional overexpression, accounts for over 75% of all liver cancer cases. Metastasis and mutations in upstream components of the MAP kinase pathway, postsurgical recurrence are common and represent major obsta- such as RAS and BRAF, and downregulation of negative regulator cles to the improvement of patient survival. HCC patients are dual-specificity MAP kinase phosphatases (DUSP; ref. 4). ERK often diagnosed at an advanced stage when curative therapy is no plays a major role in invasion by inducing proteases that degrade longer available and even after surgery, the prognosis of HCC the basement membrane, enhances cell migration, and increases remains unsatisfactory, with a 5-year postrecurrence rate at >70%. cell survival. Activated ERK pathway has been shown to correlate Metastasis is a complex multistep process involving alterations in with the expression of epithelial–mesenchymal transition (EMT) the dissemination, invasion, survival, and growth of new cancer markers, a hallmark of metastasis. These findings collectively cell colonies, which are regulated by a complex network of intra- suggest that ERK plays a major role in tumor progression and and intercellular signal transduction cascades (1). However, metastasis. However, our knowledge of endogenous regulators of metastasis remains the most poorly understood component of DUSP/ERK remains limited and how they work to promote HCC cancer pathogenesis (2). Elucidation of the mechanisms under- metastasis is also not known. lying metastasis is fundamental for the development of new TP53INP1 is a stress-induced tumor suppressor gene with therapeutic treatments for advanced metastatic HCC. antiproliferative and proapoptotic activities (5, 6). It is an alter- natively spliced gene encoding two protein isoforms (a and b), and when overexpressed, both isoforms exert a tumor suppressor 1 School of Biomedical Sciences, The University of Hong Kong, Hong Kong. function, mainly by inducing the transcription of target genes 2Department of Clinical Oncology, The University of Hong Kong, Hong Kong. 3State Key Laboratory for Liver Research, Li Ka Shing Faculty of Medicine, The involved in cell-cycle arrest and p53-mediated apoptosis as part of fi University of Hong Kong, Hong Kong. 4Department of Surgery, The University of the cell responses to genotoxic stress. Signi cant reduction or loss Hong Kong, Hong Kong. 5State Key Laboratory of Oncology in Southern China, of TP53INP1 expression has been shown in a number of cancer Sun Yat-Sen University Cancer Center, Guangzhou, China. 6Department of types, including those of the stomach (7), breast (8), pancreas (9), Applied Biology and Chemical Technology, The Hong Kong Polytechnic Uni- esophagus (10), lung (11), melanocyte (12), colon (13), and 7 versity, Hong Kong. Aix Marseille University, CNRS, INSERM, Institut Paoli- blood (14). In relation to metastasis, TP53INP1 has only been Calmettes, CRCM, Marseille, France. implicated in a handful of studies including one report where they Note: Supplementary data for this article are available at Cancer Research found transcriptional levels of TP53INP1 to be downregulated in Online (http://cancerres.aacrjournals.org/). metastatic lung of brain cancers (15). A more recent study led by Corresponding Author: Stephanie Ma, The University of Hong Kong, Room 47, 1/F, our collaborator Dusetti and colleagues found TP53INP1 to Laboratory Block, Faculty of Medicine Building, 21 Sassoon Road, Pok Fu Lam, reduce pancreatic cancer cell migration by regulating SPARC Hong Kong. Phone: 852-3917-9238; Fax: 852-2817-0857; E-mail: [email protected] expression (16). TP53INP1 is a target gene of the transcription doi: 10.1158/0008-5472.CAN-16-3456 factor p53. Conversely, TP53INP1 has also been shown to play Ó2017 American Association for Cancer Research. a role in cellular homeostasis through p53-dependent and 4602 Cancer Res; 77(17) September 1, 2017 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2017 American Association for Cancer Research. Published OnlineFirst July 3, 2017; DOI: 10.1158/0008-5472.CAN-16-3456 TP53INP1 in HCC Metastasis p53-independent manners (5, 6). In addition to p53, TP53INP1, Phospho-kinase array profiling which is also a p73 target gene, can enhance transcriptional Proteome Profiler Human Phospho-Kinase Array Kit was pur- activity of p73 to induce cell-cycle arrest and cell death (17). chased from R&D Systems (ARY003B). Thus, TP53INP1 can exert its tumor suppressor function by inducing the transcription of both p53 and p73 target genes. Quantitative real-time PCR In our previous studies, we found that the initiation, growth Total RNA was extracted using RNAisoPlus (Takara). For quan- þ fi and self-renewal of CD133 liver tumors to be ne-tuned by a titative (q)RT-PCR of mRNA targets, cDNA was synthesized by balance of miR-130b overexpression and TP53INP1 downre- PrimeScript RT Master Mix (Takara) and amplified with EvaGreen gulation (18). This result suggests that TP53INP1 is a critical qPCR MasterMix-R (Applied Biological Materials) and primers effector driving hepatocarcinogenesis. Nevertheless, to date, no listed in Supplementary Table S1. b-Actin was amplified as an studies have determined the function of TP53INP1 in HCC internal control. Reactions were performed on an ABI Prism 7900 metastasis or the molecular mechanism by which TP53INP1 System (Applied Biosystems) with data analyzed using the ABI regulates invasion and metastasis in HCC. Here, we demon- SDS v2.3 software (Applied Biosystems). Relative expression ÀDD strate that TP53INP1 is frequently downregulated in advanced- differences were calculated using the 2 Ct method. stage and metastatic human HCC tumors and that downregu- lation of TP53INP1 in HCC functionally promotes metastasis through ERK activation via a p73-dependent DUSP10 regula- Western blot analysis fi tion. Findings from our study not only provide new insights Protein lysates were quanti ed and resolved on a SDS-PAGE into how HCC metastasis is regulated but also provide a new gel, transferred onto PVDF membrane (Millipore), and immuno- layer of mechanism by which DUSP10/ERK signaling is regu- blotted with a primary antibody, followed by incubation with a lated by p73/TP53INP1 and also identify DUSP10 as a new secondary antibody. Antibody signal was detected using an transcriptional effector of p73. enhanced chemiluminescence system (GE Healthcare). The fol- lowing antibodies were used: TP53INP1 (1:250, Genway Biotech, GWB-61D856), p-ERK1/2 (1:1,000, Cell Signaling Technology, Materials and Methods 9101), total ERK (1:1,000, Cell Signaling Technology, 9102), Gene expression profiling and patient samples DUSP10 (1:500, Cell Signaling Technology, 3483), p73 Gene expression profiling studies involving multiple clinical (1:1,000, Novus Biologicals, NB100-56674), BAX (1:1,000, Cell samples were performed analyzing the expression of specific Signaling Technology, 2772), MDM2 (1:500, Santa Cruz Biotech- transcripts in two datasets available through Gene Expression nology, sc-965), and b-actin (1:5,000, Sigma-Aldrich, A5316). Omnibus (GSE25097 and GSE40367; refs. 19, 20). In addition, human primary and matched metastatic HCC tissue samples were Expression plasmids and lentiviral transduction obtained from 37 patients undergoing hepatectomy at the Sun Expression plasmids for shRNAs were made in a pLKO.1-puro Yat-Sen University Cancer Centre in Guangzhou, China. Tissue vector (Sigma-Aldrich). The targeted sequences were: human samples were collected from patients who had not received any TP53INP1 (464, 50-CCGGCATAGATACTTGCACTGGTTTCTC- previous local or systemic treatment prior to operation. Use of GAGAAACCAGTGCAAGTATCTATGTTTTTTG-30) and (3834, 50- human samples was approved by the committee for ethical review CCGGGCGCCATGTTTCTCAAAGTTTCTCGAGAAACTTTGAGAA- of research involving human subjects at the Sun Yat-Sen Univer- ACATGGCGCTTTTTTG-30); human p73 (753, 50- CCGGATCC- sity Cancer Centre.
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