title: Computational Discovery of Niclosamide Ethanolamine, a Repurposed Drug Candidate That Reduces Growth of Hepatocellular Carcinoma Cells In Vitro and in Mice by Inhibiting Cell Division Cycle 37 Signaling.
abstract: Drug repositioning offers a shorter approval process than new drug development. We therefore searched large public datasets of drug-induced gene expression signatures to identify agents that might be effective against hepatocellular carcinoma (HCC).
  
  
  
  We searched public databases of messenger RNA expression patterns reported from HCC specimens from patients, HCC cell lines, and cells exposed to various drugs. We identified drugs that might specifically increase expression of genes that are down-regulated in HCCs and reduce expression of genes up-regulated in HCCs using a nonparametric, rank-based pattern-matching strategy based on the Kolmogorov-Smirnov statistic. We evaluated the anti-tumor activity of niclosamide and its ethanolamine salt (NEN) in HCC cell lines (HepG2, Huh7, Hep3B, Hep40, and PLC/PRF/5), primary human hepatocytes, and 2 mouse models of HCC. In one model of HCC, liver tumor development was induced by hydrodynamic delivery of a sleeping beauty transposon expressing an activated form of Ras (v12) and truncated β-catenin (N90). In another mouse model, patient-derived xenografts were established by implanting HCC cells from patients into livers of immunocompromised mice. Tumor growth was monitored by bioluminescence imaging. Tumor-bearing mice were fed a regular chow diet or a chow diet containing niclosamide or NEN. In a separate experiment using patient-derived xenografts, tumor-bearing mice were given sorafenib (the standard of care for patients with advanced HCC), NEN, or niclosamide alone; a combination of sorafenib and NEN; or a combination sorafenib and niclosamide in their drinking water, or regular water (control), and tumor growth was monitored.
  
  
  
  Based on gene expression signatures, we identified 3 anthelmintics that significantly altered the expression of genes that are up- or down-regulated in HCCs. Niclosamide and NEN specifically reduced the viability of HCC cells: the agents were at least 7-fold more cytotoxic to HCCs than primary hepatocytes. Oral administration of NEN to mice significantly slowed growth of genetically induced liver tumors and patient-derived xenografts, whereas niclosamide did not, coinciding with the observed greater bioavailability of NEN compared with niclosamide. The combination of NEN and sorafenib was more effective at slowing growth of patient-derived xenografts than either agent alone. In HepG2 cells and in patient-derived xenografts, administration of niclosamide or NEN increased expression of 20 genes down-regulated in HCC and reduced expression of 29 genes up-regulated in the 274-gene HCC signature. Administration of NEN to mice with patient-derived xenografts reduced expression of proteins in the Wnt-β-catenin, signal transducer and activator of transcription 3, AKT-mechanistic target of rapamycin, epidermal growth factor receptor-Ras-Raf signaling pathways. Using immunoprecipitation assays, we found NEN to bind cell division cycle 37 protein and disrupt its interaction with heat shock protein 90.
  
  
  
  In a bioinformatics search for agents that alter the HCC-specific gene expression pattern, we identified the anthelmintic niclosamide as a potential anti-tumor agent. Its ethanolamine salt, with greater bioavailability, was more effective than niclosamide at slowing the growth of genetically induced liver tumors and patient-derived xenografts in mice. Both agents disrupted interaction between cell division cycle 37 and heat shock protein 90 in HCC cells, with concomitant inhibition of their downstream signaling pathways. NEN might be effective for treatment of patients with HCC.
authors.affiliation: Institute for Computational Health Sciences and Department of Pediatrics, University of California, San Francisco, California.
authors.forename: Bin
authors.initials: B
authors.lastname: Chen
authors.affiliation: Asian Liver Center and Department of Surgery, Stanford University School of Medicine, Stanford University, Stanford, California.
authors.forename: Wei
authors.initials: W
authors.lastname: Wei
authors.affiliation: Asian Liver Center and Department of Surgery, Stanford University School of Medicine, Stanford University, Stanford, California.
authors.forename: Li
authors.initials: L
authors.lastname: Ma
authors.affiliation: Department of Interventional Radiology, Beijing 302 Hospital, Beijing, China.
authors.forename: Bin
authors.initials: B
authors.lastname: Yang
authors.affiliation: Department of Pathology, University of California, San Francisco, California.
authors.forename: Ryan M
authors.initials: RM
authors.lastname: Gill
authors.affiliation: Asian Liver Center and Department of Surgery, Stanford University School of Medicine, Stanford University, Stanford, California. Electronic address: mchua@stanford.edu.
authors.forename: Mei-Sze
authors.initials: MS
authors.lastname: Chua
authors.affiliation: Institute for Computational Health Sciences and Department of Pediatrics, University of California, San Francisco, California. Electronic address: atul.butte@ucsf.edu.
authors.forename: Atul J
authors.initials: AJ
authors.lastname: Butte
authors.affiliation: Asian Liver Center and Department of Surgery, Stanford University School of Medicine, Stanford University, Stanford, California.
authors.forename: Samuel
authors.initials: S
authors.lastname: So
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000494:pharmacology; D006528:Carcinoma, Hepatocellular / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects*; D018833:Chaperonins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D019295:Computational Biology; D003198:Computer Simulation; D030541:Databases, Genetic; D004305:Dose-Response Relationship, Drug; D055808:Drug Discovery / Q000379:methods*; D058492:Drug Repositioning; D019856:Ethanolamine / Q000494:pharmacology*; D005260:Female; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism; D056945:Hep G2 Cells; D006801:Humans; D008113:Liver Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D008297:Male; D018832:Molecular Chaperones / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D009536:Niacinamide / Q000031:analogs & derivatives / Q000494:pharmacology; D009534:Niclosamide / Q000031:analogs & derivatives / Q000494:pharmacology*; D010671:Phenylurea Compounds / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects; D000077157:Sorafenib; D013997:Time Factors; D059467:Transcriptome; D047368:Tumor Burden / Q000187:drug effects; D023041:Xenograft Model Antitumor Assays
chemical_list: D000970:Antineoplastic Agents; C498362:CDC37 protein, human; C513271:Cdc37 protein, mouse; D018797:Cell Cycle Proteins; D018841:HSP90 Heat-Shock Proteins; D018832:Molecular Chaperones; D010671:Phenylurea Compounds; D009536:Niacinamide; D019856:Ethanolamine; D009534:Niclosamide; D000077157:Sorafenib; D018833:Chaperonins
keywords: Big Data; Liver Cancer; Mitochondrial Uncoupler; Treatment