Published OnlineFirst November 20, 2019; DOI: 10.1158/1541-7786.MCR-19-0759 MOLECULAR CANCER RESEARCH | NEW HORIZONS IN CANCER BIOLOGY A Novel Zebrafish Model of Metastasis Identifies the HSD11b1 Inhibitor Adrenosterone as a Suppressor of Epithelial–Mesenchymal Transition and Metastatic Dissemination Joji Nakayama1,2,3, Jeng-Wei Lu1, Hideki Makinoshima2,4, and Zhiyuan Gong1 ABSTRACT ◥ Metastasis of cancer cells is multi-step process and dissemination 1 (HSD11b1), has a suppressor effect on cell dissemination in this is an initial step. Here we report a tamoxifen-controllable Twist1a- model. Pharmacologic and genetic inhibition of HSD11b1 sup- ERT2 transgenic zebrafish line as a new animal model for metastasis pressed metastatic dissemination of highly metastatic human cell research, and demonstrate that this model can serve as a novel lines in a zebrafish xenotransplantation model. Through down- platform for discovery of antimetastasis drugs targeting metastatic regulation of Snail and Slug, adrenosterone-treated cells recovered dissemination of cancer cells. By crossing Twist1a-ERT2 with xmrk expression of E-cadherin and other epithelial markers and lost (a homolog of hyperactive form of EGFR) transgenic zebrafish, partial expression of mesenchymal markers compared with vehicle- which develops hepatocellular carcinoma, approximately 80% of treated cells. Taken together, our model offers a useful platform for the double transgenic zebrafish showed spontaneous cell dissem- the discovery of antimetastasis drugs targeting metastatic dissem- ination of mCherry-labeled hepatocytes from the liver to the entire ination of cancer cells. abdomen region and the tail region. The dissemination is accom- plished in 5 days through induction of an epithelial-to- Implications: This study describes a transgenic zebrafish model for mesenchymal transition. Using this model, we conducted in vivo liver tumor metastasis and it has been successfully used for iden- drug screening and identified three hit drugs. One of them, adre- tification of some drugs to inhibit metastatic dissemination of nosterone, an inhibitor for hydroxysteroid (11-beta) dehydrogenase human cancer cells. Introduction of cell polarity, loss of cell–cell adhesions, and induction of epithelial- to-mesenchymal transition (EMT; ref. 6). Overt metastases, the end result of malignant alteration of cancer EMT plays a central role in early embryonic morphogenesis, its cells, are responsible for approximately 90% of cancer-associated program enables various types of epithelial cells to convert into mortality. Metastasis consists of multiple processes: invasion, intra- mesenchymal cells through a downregulation of epithelial markers vasation, survival in the circulatory system, extravasation, coloniza- such as E-cadherin, and an upregulation of mesenchymal markers tion, andtumor formation insecondary organs withangiogenesis (1, 2). such as vimentin. Experimental studies demonstrated that EMT also Metastatic dissemination of cancer cells was traditionally viewed as a contributes to metastatic progression by conferring invasiveness, late stage event of cancer progression (3). However, this paradigm has motility, and an increased resistance to chemotherapy and apoptosis been challenged by recent studies, in which mammary epithelial cells on cancer cells. EMT is initiated and orchestrated by multiple signaling disseminated systemically from early neoplastic lesions of Her2 and pathways and transcriptional factor networks: TGFb, BMP, and Wnt- PyMT transgenic mice and from ductal carcinoma in situ in patients mediated signaling; and Snail, Slug, and Twist-mediated transcrip- with breast cancer (4, 5). Molecular mechanisms that promote dis- tional networks (7–9). semination involve the breakdown of local basement membrane, loss Twist, a basic helix-loop-helix transcription factor, plays a critical role in inducing the EMT program. It was first identified in Drosophila melanogaster as an organizer of EMT during fly gastrulation and a 1Department of Biological Sciences, National University of Singapore, Singapore. regulator of mesoderm differentiation (10). However, in the last 2Tsuruoka Metabolomics Laboratory, National Cancer Center, Tsuruoka, Japan. decade, experimental studies have demonstrated an additional role 3 4 Shonai Regional Industry Promotion Center, Tsuruoka, Japan. Division of of Twist—ectopic expression of Twist confers metastatic properties on Translational Research, Exploratory Oncology Research and Clinical Trial Center, cancer cells through induction of EMT. Moreover, clinical studies have National Cancer Center, Kashiwa, Japan. revealed elevated expression of Twist is associated with poor survival Note: Supplementary data for this article are available at Molecular Cancer rates in patients with cancer (8, 11, 12). Research Online (http://mcr.aacrjournals.org/). Cancer research using zebrafish as a model has attracted attention Corresponding Authors: Zhiyuan Gong, National University of Singapore, because this model offers many unique advantages that are not readily 14 Science Drive 4, Singapore 115473, Singapore. Phone: 656-516-2860; provided by other animal models (13). Moreover, the zebrafish has Fax: 656-779-2486; E-mail: [email protected]; and Joji Nakayama, Tsuruoka become a popular platform for drug screening to discover anticancer Metabolomics Laboratory, National Cancer Center, Tsuruoka, Japan. fi E-mail: [email protected] drugs (14); however, a zebra sh model that develops spontaneous metastases has not previously been reported. Mol Cancer Res 2020;18:477–87 Here we report a tamoxifen-controllable Twist1a-ERT2 transgenic doi: 10.1158/1541-7786.MCR-19-0759 zebrafish as a new animal model for metastasis research, and show that Ó2019 American Association for Cancer Research. this model serves as a novel platform for discovery of antimetastasis AACRJournals.org | 477 Downloaded from mcr.aacrjournals.org on September 27, 2021. © 2020 American Association for Cancer Research. Published OnlineFirst November 20, 2019; DOI: 10.1158/1541-7786.MCR-19-0759 Nakayama et al. drugs targeting metastatic dissemination of cancer cells. Through and Y27632 were purchased from Cayman Chemical and R&D in vivo drug screening using this model, we found that adrenosterone Systems, respectively. Doxycycline, 4-hydroxytamoxifen (4-OHT), has suppressor effects on cell dissemination in both this model and adrenosterone, and rabeprazole were purchased from Sigma-Aldrich. human cancer cells. Finally, we demonstrated that pharmacologic and Olmesartan was purchased from Cayman Chemical. genetic inhibition of hydroxysteroid (11-beta) dehydrogenase 1 (HSD11b1), a primary target of adrenosterone, suppressed metastatic In vivo drug screen dissemination of highly metastatic human cancer cells in a zebrafish Larvae of Twist1a-ERT2/xmrk double transgenic zebrafish at 8 days xenotransplantation model. postfertilization (dpf) were treated with 30 mg/mL of doxycycline in E3 medium for 3 days to induce xmrk expression and then aliquoted Materials and Methods approximately 20 larvae into each well of a 6-well plate with 8 mL of E3 medium containing doxycycline. Drugs from the Prestwick Chemical fi Zebra sh Library were added to each well of the plates at a final concentration of fi Transgenic zebra sh lines Tg(fabp10a:mCherry-T2A-Twist1a- 5 mmol/L. Twelve hours after drug addition, 4-OHT was added to each T2 T2 ER ) and Tg(fabp10a:mCherry-T2A-ER ) were generated through well of the plates at a final concentration of 0.1 mmol/L to induce Ac/Ds transposon system (15). Five to 10 pg of either fabp10a: Twist1a-ERT2 biological activity. Five days after drug addition, the T2 T2 mCherry-T2A-Twist1a-ER or fabp10a:mCherry-T2A-ER plas- larvae were investigated under a fluorescence microscope and the fi – mid was coinjected into wild-type zebra sh embryos with 25 50 pg pattern of cell dissemination was quantified. The number of the larvae – of in vitro synthesized AC transposase mRNA at the 1 2 cell stage. Tg showing each dissemination pattern of mCherry-labeled cells from the fi (fabp10a:TA; TRE:xmrk; krt4:GFP) transgenic zebra sh line known as liver were counted. xmrk was described previously (16). Tg(kdrl:eGFP) zebrafish was provided by Dr. Stainier (Max Planck Institute for Heart and Lung Immunoblotting Research). The study protocol was approved by the Institutional Western blotting was performed as described previously (20). Animal Care and Use Committee of the National University of Anti-E-cadherin and anti-b-actin antibodies were purchased Singapore (protocol number: 096/12). from BD Biosciences and Sigma, respectively. Anti-GAPDH, anti-Snail, anti-Slug, and anti-Histon H3 antibodies were purchased Plasmids þ þ from Cell Signaling Technology. Anti-HSD11b1, anti-H /K -ATPase DNA fragments coding for mCherry-T2A, ERT2, and zebrafish beta, anti-Vimentin, and Twist1 antibodies were purchased from Twist1a were amplified by PCR with primers containing restriction Abcam. Anti-PCNA, anti-a-tubulin, anti-Occludin, anti-Claudin6, enzyme recognition sequences. mCherry-T2A–coding fragment was anti-KRT14, anti-KRT19, anti-MMP1, anti-MMP2, and anti- amplified from hsp70l:mCherry-T2A-CreERT2 plasmid (17). ERT2 S100A4 antibodies were purchased from Santa Cruz Biotechnology. coding DNA fragment was amplified from pCAG-Cre-ERT2 plas- fi fi mid (18). The fragments of zebra sh Twist1a was ampli ed from Cell culture and cell viability assay cDNA that was prepared from wild-type zebrafish embryos at sphere MCF7, MDA-MB-231, MDA-MB-435, MIA-PaCa2, PC3, and stage.