Discovery and preliminary SAR of bisbenzylisoquinoline alkaloids as inducers of C/EBPα.

A high throughput in vitro screen has been developed to identify substances that induce expression of C/EBPα in tumor cells. An extract of the fruit of Gyrocarpus jacquinii showed induction of C/EBPα activity that was attributed to the bisbenzylisoquinoline (BBIQ) alkaloid pheanthine (13) by dereplication analysis. The research project was broadened to assess the effect of other natural BBIQ structural types occurring outside the genus Gyrocarpus. Several of the 28 compounds assayed showed enhancement of C/EBPα induction in U937 cells. The results of this study should encourage future efforts toward obtaining and screening a larger set of both natural and synthetic analogs of this interesting group of alkaloids.

Cysteine 81 is critical for the interaction of S100A4 and myosin-IIA.

Overexpression of S100A4, a member of the S100 family of Ca(2+)-binding proteins, is associated with a number of human pathologies, including fibrosis, inflammatory disorders, and metastatic disease. The identification of small molecules that disrupt S100A4/target interactions provides a mechanism for inhibiting S100A4-mediated cellular activities and their associated pathologies. Using an anisotropy assay that monitors the Ca(2+)-dependent binding of myosin-IIA to S100A4, NSC 95397 was identified as an inhibitor that disrupts the S100A4/myosin-IIA interaction and inhibits S100A4-mediated depolymerization of myosin-IIA filaments. Mass spectrometry demonstrated that NSC 95397 forms covalent adducts with Cys81 and Cys86, which are located in the canonical target binding cleft. Mutagenesis studies showed that covalent modification of just Cys81 is sufficient to inhibit S100A4 function with respect to myosin-IIA binding and depolymerization. Remarkably, substitution of Cys81 with serine or alanine significantly impaired the ability of S100A4 to promote myosin-IIA filament disassembly. As reversible covalent cysteine modifications have been observed for several S100 proteins, we propose that modification of Cys81 may provide an additional regulatory mechanism for mediating the binding of S100A4 to myosin-IIA.

Complex display of putative tumor stem cell markers in the NCI60 tumor cell line panel.

Tumor stem cells or cancer initiating cells (CICs) are single tumor cells that can regenerate a tumor or a metastasis. The identification and isolation of CICs remain challenging, and a variety of putative CIC markers have been described. We hypothesized that cell lines of the NCI60 panel contain CICs and express putative CIC markers. We investigated expression of putative CIC surface markers (CD15, CD24, CD44, CD133, CD166, CD326, PgP) and the activity of aldehyde dehydrogenase in the NCI60 panel singly and in combination by six-color fluorescence-activated cell sorting analysis. All investigated markers were expressed in cell lines of the NCI60 panel. Expression levels of individual markers varied widely across the 60 cell lines, and neither single marker expression nor simple combinations nor co-expression patterns correlated with the colony-formation capacity of cell lines. Rather, marker expression patterns correlated with tumor types in multidimensional analysis. Whereas some expression patterns correlated with tumor entities such as basal breast cancer, other expression patterns occurred across different tumor types and largely related to expression of a more mesenchymal phenotype in individual breast, lung, renal, and melanoma cell lines. Our data for the first time demonstrate that tumor cell lines display CIC markers in a complex pattern that relates to the tumor type. The complexity and tumor type specificity of marker display creates challenges for the application of cell sorting and other approaches to isolation of putative tumor stem cell populations and suggests that therapeutic targeting strategies will need to take this into account.

Identification of PDGFR as a receptor for AAV-5 transduction.

Understanding the process of vector transduction has important implications for the application and optimal use of a vector system for human gene therapy. Recent studies with vectors based on adeno-associated virus type 5 (AAV-5) have shown utility of this vector system in the lung, central nervous system, muscle and eye. To understand the natural tropism of this virus and to identify proteins necessary for AAV-5 transduction, we characterized 43 cell lines as permissive or nonpermissive for AAV-5 transduction and compared the gene expression profiles derived from cDNA microarray analyses of those cell lines. A statistically significant correlation was observed between expression of the platelet-derived growth factor receptor (PDGFR-alpha-polypeptide) and AAV-5 transduction. Subsequent experiments confirmed the role of PDGFR-alpha and PDGFR-beta as receptors for AAV-5. The tropism of AAV-5 in vivo also correlated with the expression pattern of PDGFR-alpha.

12 Arylstibonic acids that inhibit the DNA binding of five B-ZIP dimers.

Previously, we identified an arylstibonic acid, NSC13778 that specifically binds to the basic region of the C/EBPalpha B-ZIP domain and disrupts DNA binding. We now examine a panel of 14 additional arylstibonic acid derivatives of NSC13778 for their ability to inhibit the DNA binding of five B-ZIP dimers (c-Fos|JunD, VBP, C/EBPalpha, C/EBPbeta, and CREB). They show various specificities at inhibiting the DNA binding of five B-ZIP domains. NSC13746 inhibits the DNA binding of C/EBPbeta and CREB at 100nM and promiscuously inhibiting the DNA binding of all five proteins in the 1muM range. Dialysis experiments indicate that NSC 13746 binding to the B-ZIP domain is reversible. Thermal denaturation studies indicate that NSC13746 binds the B-ZIP domain. Some compounds specifically inhibit DNA binding, with VBP and c-Fos|JunD being most easily disrupted. These compounds inhibit, with similar specificities to the pure B-ZIP domains, the DNA binding of nuclear extract to the AP1 DNA sequence but no inhibition is observed to SP1 containing oligonucleotide. Transient transfection assays indicate that NSC13746 can inhibit the TPA induced activation of two B-ZIP dependent reporters. These experiments suggest that arylstibonic acids are promising leads for inhibiting the DNA binding of a group of B-ZIP proteins in cells.

Inhibitors of the NF-kappaB activation pathway from Cryptocarya rugulosa.

The nuclear factor-kappaB (NF-kappaB) signaling pathway is constitutively active in many types of cancers and is a potential therapeutic target. Using a cell-based assay for stability of inhibitor of kappa B (IkappaB), a critical regulator of NF-kappaB activity, we found that an organic solvent extract of the plant Cryptocarya rugulosa inhibited constitutive NF-kappaB activity in human lymphoma cell lines. The active components were identified as rugulactone, a new alpha-pyrone (1), and the known cryptocaryone (2). Rugulactone was the more active compound, exhibiting up to 5-fold induction of IkappaB at 25 microg/mL; maximal activity was observed with 10 h exposure of test cells to 1 or 2.

Cytotoxic and HIF-1alpha inhibitory compounds from Crossosoma bigelovii.

Cytotoxicity-guided fractionation of an organic solvent extract of the plant Crossosoma bigelovii led to the discovery of a new strophanthidin glycoside (1) and two new 2-methylchromone glycosides (2 and 3). Also isolated were the known chromones eugenin and noreugenin, the indole alkaloid ajmalicine, the dibenzylbutane lignan secoisolariciresinol, the dibenzylbutyrolactone lignan matairesinol, and the furanone 5-tetradec-5-enyldihydrofuran-2-one. Further investigation into the biological properties of strophanthidin glycosides revealed a connection between inhibition of HIF-1 activation and the glycosylation of the genin. This work is the first published study of the bioactive phytochemicals of the family Crossosomataceae.

Ligand-directed surface profiling of human cancer cells with combinatorial peptide libraries.

A collection of 60 cell lines derived from human tumors (NCI-60) has been widely explored as a tool for anticancer drug discovery. Here, we profiled the cell surface of the NCI-60 by high-throughput screening of a phage-displayed random peptide library and classified the cell lines according to the binding selectivity of 26,031 recovered tripeptide motifs. By analyzing selected cell-homing peptide motifs and their NCI-60 recognition patterns, we established that some of these motifs (a) are similar to domains of human proteins known as ligands for tumor cell receptors and (b) segregate among the NCI-60 in a pattern correlating with expression profiles of the corresponding receptors. We biochemically validated some of the motifs as mimic peptides of native ligands for the epidermal growth factor receptor. Our results indicate that ligand-directed profiling of tumor cell lines can select functional peptides from combinatorial libraries based on the expression of tumor cell surface molecules, which in turn could be exploited as "druggable" receptors in specific types of cancer.

Detailed DNA methylation profiles of the E-cadherin promoter in the NCI-60 cancer cells.

E-cadherin (E-cad) is a transmembrane adhesion glycoprotein, the expression of which is often reduced in invasive or metastatic tumors. To assess E-cad's distribution among different types of cancer cells, we used bisulfite-sequencing for detailed, base-by-base measurement of CpG methylation in E-cad's promoter region in the NCI-60 cell lines. The mean methylation levels of the cell lines were distributed bimodally, with values pushed toward either the high or low end of the methylation scale. The 38 epithelial cell lines showed substantially lower (28%) mean methylation levels compared with the nonepithelial cell lines (58%). The CpG site at -143 with respect to the transcriptional start was commonly methylated at intermediate levels, even in cell lines with low overall DNA methylation. We also profiled the NCI-60 cell lines using Affymetrix U133 microarrays and found E-cad expression to be correlated with E-cad methylation at highly statistically significant levels. Above a threshold of approximately 20% to 30% mean methylation, the expression of E-cad was effectively silenced. Overall, this study provides a type of detailed analysis of methylation that can also be applied to other cancer-related genes. As has been shown in recent years, DNA methylation status can serve as a biomarker for use in choosing therapy.

Transcript and protein expression profiles of the NCI-60 cancer cell panel: an integromic microarray study.

To evaluate the utility of transcript profiling for prediction of protein expression levels, we compared profiles across the NCI-60 cancer cell panel, which represents nine tissues of origin. For that analysis, we present here two new NCI-60 transcript profile data sets (A based on Affymetrix HG-U95 and HG-U133A chips; Affymetrix, Santa Clara, CA) and one new protein profile data set (based on reverse-phase protein lysate arrays). The data sets are available online at http://discover.nci.nih.gov in the CellMiner program package. Using the new transcript data in combination with our previously published cDNA array and Affymetrix HU6800 data sets, we first developed a "consensus set" of transcript profiles based on the four different microarray platforms. Using that set, we found that 65% of the genes showed statistically significant transcript-protein correlation, and the correlations were generally higher than those reported previously for panels of mammalian cells. Using the predictive analysis of microarray nearest shrunken centroid algorithm for functional prediction of tissue of origin, we then found that (a) the consensus mRNA set did better than did data from any of the individual mRNA platforms and (b) the protein data seemed to do somewhat better (P = 0.027) on a gene-for-gene basis in this particular study than did the consensus mRNA data, but both did well. Analysis based on the Gene Ontology showed protein levels of structure-related genes to be well predicted by mRNA levels (mean r = 0.71). Because the transcript-based technologies are more mature and are currently able to assess larger numbers of genes at one time, they continue to be useful, even when the ultimate aim is information about proteins.

Substituted purine analogues define a novel structural class of catalytic topoisomerase II inhibitors.

By screening 1,990 compounds from the National Cancer Institute diversity set library against human topoisomerase IIalpha, we identified a novel catalytic topoisomerase II inhibitor NSC35866, a S6-substituted analogue of thioguanine. In addition to inhibiting the DNA strand passage reaction of human topoisomerase IIalpha, NSC35866 also inhibited its ATPase reaction. NSC35866 primarily inhibited DNA-stimulated ATPase activity, whereas DNA-independent ATPase activity was less sensitive to inhibition. We compared the mode of topoisomerase II ATPase inhibition induced by NSC35866 with that of 12 other substituted purine analogues of different chemical classes. The ability of thiopurines with free SH functionalities to inhibit topoisomerase II ATPase activity was completely abolished by DTT, suggesting that these thiopurines inhibit topoisomerase II ATPase activity by covalently modifying free cysteine residues. In contrast, NSC35866 as well as two O6-substituted guanine analogues, O6-benzylguanine and NU2058, could inhibit topoisomerase II ATPase activity in the presence of DTT, indicating that they have a different mechanism of inhibition. NSC35866 did not increase the level of topoisomerase II covalent cleavable complexes with DNA, indicating that it is a catalytic inhibitor and not a poison. NSC35866 was also capable of inducing a salt-stable complex of topoisomerase II on closed circular DNA. In accordance with these biochemical data, NSC35866 could antagonize etoposide-induced cytotoxicity and DNA breaks in human and murine cancer cells, confirming that NSC35866 also functions as a catalytic topoisomerase II inhibitor in cells.

Integrating data on DNA copy number with gene expression levels and drug sensitivities in the NCI-60 cell line panel.

Chromosome rearrangement, a hallmark of cancer, has profound effects on carcinogenesis and tumor phenotype. We used a panel of 60 human cancer cell lines (the NCI-60) as a model system to identify relationships among DNA copy number, mRNA expression level, and drug sensitivity. For each of 64 cancer-relevant genes, we calculated all 4,096 possible Pearson's correlation coefficients relating DNA copy number (assessed by comparative genomic hybridization using bacterial artificial chromosome microarrays) and mRNA expression level (determined using both cDNA and Affymetrix oligonucleotide microarrays). The analysis identified an association of ERBB2 overexpression with 3p copy number, a finding supported by data from human tumors and a mouse model of ERBB2-induced carcinogenesis. When we examined the correlation between DNA copy number for all 353 unique loci on the bacterial artificial chromosome microarray and drug sensitivity for 118 drugs with putatively known mechanisms of action, we found a striking negative correlation (-0.983; 95% bootstrap confidence interval, -0.999 to -0.899) between activity of the enzyme drug L-asparaginase and DNA copy number of genes near asparagine synthetase in the ovarian cancer cells. Previous analysis of drug sensitivity and mRNA expression had suggested an inverse relationship between mRNA levels of asparagine synthetase and L-asparaginase sensitivity in the NCI-60. The concordance of pharmacogenomic findings at the DNA and mRNA levels strongly suggests further study of L-asparaginase for possible treatment of a low-synthetase subset of clinical ovarian cancers. The DNA copy number database presented here will enable other investigators to explore DNA transcript-drug relationships in their own domains of research focus.

Identification of small molecule inhibitors of hypoxia-inducible factor 1 transcriptional activation pathway.

Hypoxia-inducible factor 1 (HIF-1) is a master regulator of the transcriptional response to oxygen deprivation. HIF-1 has been implicated in the regulation of genes involved in angiogenesis [e.g., vascular endothelial growth factor (VEGF) and inducible nitric oxide synthase] and anaerobic metabolism (e.g., glycolytic enzymes). HIF-1 is essential for angiogenesis and is associated with tumor progression. In addition, overexpression of HIF-1 alpha has been demonstrated in many common human cancers. Therefore, HIF-1 is an attractive molecular target for development of novel cancer therapeutics. We have developed a cell-based high-throughput screen for the identification of small molecule inhibitors of the HIF-1 pathway. We have genetically engineered U251 human glioma cells to stably express a recombinant vector in which the luciferase reporter gene is under control of three copies of a canonical hypoxia-responsive element (U251-HRE). U251-HRE cells consistently expressed luciferase in a hypoxia- and HIF-1-dependent fashion. We now report the results of a pilot screen of the National Cancer Institute "Diversity Set," a collection of approximately 2000 compounds selected to represent the greater chemical diversity of the National Cancer Institute chemical repository. We found four compounds that specifically inhibited HIF-1-dependent induction of luciferase but not luciferase expression driven by a constitutive promoter. In addition, these compounds inhibited hypoxic induction of VEGF mRNA and protein expression in U251 cells. Interestingly, three compounds are closely related camptothecin analogues and topoisomerase (Topo)-I inhibitors. We show that concomitant with HIF-1 and VEGF inhibition, the activity of the Topo-I inhibitors tested is associated with induction of cyclooxygenase 2 mRNA expression. The luciferase-based high-throughput screen is a feasible tool for the identification of small molecule inhibitors of HIF-1 transcriptional activation. In addition, our results suggest that altered Topo-I function may be associated with repression of HIF-1-dependent induction of gene expression.

Karyotypic complexity of the NCI-60 drug-screening panel.

We used spectral karyotyping to provide a detailed analysis of karyotypic aberrations in the diverse group of cancer cell lines established by the National Cancer Institute for the purpose of anticancer drug discovery. Along with the karyotypic description of these cell lines we defined and studied karyotypic complexity and heterogeneity (metaphase-to-metaphase variations) based on three separate components of genomic anatomy: (a) ploidy; (b) numerical changes; and (c) structural rearrangements. A wide variation in these parameters was evident in these cell lines, and different association patterns between them were revealed. Analysis of the breakpoints and other specific features of chromosomal changes across the entire set of cell lines or within particular lineages pointed to a striking lability of centromeric regions that distinguishes the epithelial tumor cell lines. We have also found that balanced translocations are as frequent in absolute number within the cell lines derived from solid as from hematopoietic tumors. Important similarities were noticed between karyotypic changes in cancer cell lines and that seen in primary tumors. This dataset offers insights into the causes and consequences of the destabilizing events and chromosomal instability that may occur during tumor development and progression. It also provides a foundation for investigating associations between structural genome anatomy and cancer molecular markers and targets, gene expression, gene dosage, and resistance or sensitivity to tens of thousands of molecular compounds.

Diagnostic markers that distinguish colon and ovarian adenocarcinomas: identification by genomic, proteomic, and tissue array profiling.

Colon and ovarian cancers can be difficult to distinguish in the abdomen, and the distinction is important because it determines which drugs will be used for therapy. To identify molecular markers for that differential diagnosis, we developed a multistep protocol starting with the 60 human cancer cell lines used by the National Cancer Institute to screen for new anticancer agents. The steps included: (a) identification of candidate markers using cDNA microarrays; (b) verification of clone identities by resequencing; (c) corroboration of transcript levels using Affymetrix oligonucleotide chips; (d) quantitation of protein expression by "reverse-phase" protein microarray; and (e) prospective validation of candidate markers on clinical tumor sections in tissue microarrays. The two best candidates identified were villin for colon cancer cells and moesin for ovarian cancer cells. Because moesin stained stromal elements in both types of cancer, it would probably not have been identified as a marker if we had started with mRNA or protein profiling of bulk tumors. Villin appears at least as useful as the currently used colon cancer marker cytokeratin 20, and moesin also appears to have utility. The multistep process introduced here has the potential to produce additional markers for cancer diagnosis, prognosis, and therapy.

Components of the cell death machine and drug sensitivity of the National Cancer Institute Cell Line Panel.

PURPOSE: According to some studies, susceptibility of cells to anticancer drug-induced apoptosis is markedly inhibited by targeted deletion of genes encoding apoptotic protease activating factor 1 (Apaf-1) or certain caspases. Information about levels of these polypeptides in common cancer cell types and any possible correlation with drug sensitivity in the absence of gene deletion is currently fragmentary. EXPERIMENTAL DESIGN: Immunoblotting was used to estimate levels of Apaf-1 as well as procaspase-2, -3, -6, -7, -8, and -9 in the 60-cell-line panel used for drug screening by the National Cancer Institute. Sensitivity of the same lines to >80,000 compounds was determined with 48-hour sulforhodamine B binding assays. Additional 6-day assays were performed for selected agents. RESULTS: Levels of Apaf-1 and procaspases varied widely. Apaf-1 and procaspase-9, which are implicated in caspase activation after treatment of cells with various anticancer drugs, were detectable in all of the cell lines, with levels of Apaf-1 ranging from approximately 1 x 10(5) to 2 x 10(6) molecules per cell and procaspase-9 from approximately 5 x 10(3) to approximately 1.6 x 10(5) molecules per cell. Procaspase-8 levels ranged from 1.7 x 10(5) to 8 x 10(6) molecules per cell. Procaspase-3, a major effector caspase, varied from undetectable to approximately 1.6 x 10(6) molecules per cell. Correlations between levels of these polypeptides and sensitivity to any of a variety of experimental or conventional antineoplastic agents in either 2-day or 6-day cytotoxicity assays were weak at best. CONCLUSIONS: With the exception of caspase-3, all of the components of the core cell-death machinery are expressed in all of the cell lines examined. Despite variations in expression, levels of any one component are not a major determinant of drug sensitivity in these cells in vitro.

Application of high-throughput, molecular-targeted screening to anticancer drug discovery.

Increasing insight into the genetics and molecular biology of cancer has resulted in the identification of an increasing number of potential molecular targets for anti-cancer drug discovery and development. These targets can be approached through exploitation of emerging structural biology, "rational" drug design, screening of chemical libraries, or a combination of these methods. In this article we discuss the application of high-throughput screening to anti-cancer drug discovery, with special reference to approaches used at the U.S. National Cancer Institute.

A small molecule (pluripotin) as a tool for studying cancer stem cell biology: proof of concept.

BACKGROUND: Cancer stem cells (CSC) are thought to be responsible for tumor maintenance and heterogeneity. Bona fide CSC purified from tumor biopsies are limited in supply and this hampers study of CSC biology. Furthermore, purified stem-like CSC subpopulations from existing tumor lines are unstable in culture. Finding a means to overcome these technical challenges would be a useful goal. In a first effort towards this, we examined whether a chemical probe that promotes survival of murine embryonic stem cells without added exogenous factors can alter functional characteristics in extant tumor lines in a fashion consistent with a CSC phenotype. METHODOLOGY/PRINCIPAL FINDINGS: The seven tumor lines of the NCI60 colon subpanel were exposed to SC-1 (pluripotin), a dual kinase and GTPase inhibitor that promotes self-renewal, and then examined for tumorigenicity under limiting dilution conditions and clonogenic activity in soft agar. A statistically significant increase in tumor formation following SC-1 treatment was observed (p<0.04). Cloning efficiencies and expression of putative CSC surface antigens (CD133 and CD44) were also increased. SC-1 treatment led to sphere formation in some colon tumor lines. Finally, SC-1 inhibited in vitro kinase activity of RSK2, and another RSK2 inhibitor increased colony formation implicating a role for this kinase in eliciting a CSC phenotype. CONCLUSIONS/SIGNIFICANCE: These findings validate a proof of concept study exposure of extant tumor lines to a small molecule may provide a tractable in vitro model for understanding CSC biology.

Identification of CBX3 and ABCA5 as putative biomarkers for tumor stem cells in osteosarcoma.

Recently, there has been renewed interest in the role of tumor stem cells (TSCs) in tumorigenesis, chemoresistance, and relapse of malignant tumors including osteosarcoma. The potential exists to improve osteosarcoma treatment through characterization of TSCs and identification of therapeutic targets. Using transcriptome, proteome, immunophenotyping for cell-surface markers, and bioinformatic analyses, heterogeneous expression of previously reported TSC or osteosarcoma markers, such as CD133, nestin, POU5F1 (OCT3/4), NANOG, SOX2, and aldehyde dehydrogenase, among others, was observed in vitro. However, consistently significantly lower CD326, CD24, CD44, and higher ABCG2 expression in TSC-enriched as compared with un-enriched osteosarcoma cultures was observed. In addition, consistently higher CBX3 expression in TSC-enriched osteosarcoma cultures was identified. ABCA5 was identified as a putative biomarker of TSCs and/or osteosarcoma. Lastly, in a high-throughput screen we identified epigenetic (5-azacytidine), anti-microtubule (vincristine), and anti-telomerase (3,11-difluoro-6,8,13-trimethyl- 8H-quino [4,3,2-kl] acridinium methosulfate; RHPS4)-targeted therapeutic agents as candidates for TSC ablation in osteosarcoma.

Novel effect of antihelminthic Niclosamide on S100A4-mediated metastatic progression in colon cancer.

BACKGROUND: Metastasis formation in colon cancer severely reduces the survival rate in patients. S100A4, a calcium-binding protein, is implicated in promoting metastasis formation in colon cancer. METHODS: To identify a transcription inhibitor of S100A4, high-throughput screening of 1280 pharmacologically active compounds was performed using a human colon cancer cell line expressing a S100A4 promoter-driven luciferase (LUC) reporter gene construct (HCT116-S1004p-LUC). Niclosamide, an antihelminthic agent, was identified as a potential candidate. Colon cancer cell lines (HCT116, SW620, LS174T, SW480, and DLD-1) were treated with 1 µM niclosamide to analyze the effect on S100A4 mRNA and protein expression by quantitative reverse transcription-polymerase chain reaction and immunoblot assays, and effects on cell migration, invasion, proliferation, and colony formation were also assessed in vitro. The effect of niclosamide on liver metastasis was assessed in a xenograft mouse model of human colon cancer (n = 8 mice) by in vivo imaging. The long-term effect of niclosamide on metastasis formation after discontinued treatment was quantified by scoring, and overall survival (n = 12 mice) was analyzed by Kaplan-Meier method after discontinuation of treatment. All statistical tests were two-sided. RESULTS: Reduced S100A4 mRNA and protein expression, and inhibited cell migration, invasion, proliferation, and colony formation were observed in niclosamide-treated colon cancer cells in vitro. In vivo imaging of niclosamide-treated mice showed reduced liver metastasis compared with solvent-treated control mice (n = 4 mice per group). Compared with the control group, discontinuation of treatment for 26 days showed reduced liver metastasis formation in mice (n = 6 mice per group) (control vs discontinuous treatment, mean metastasis score = 100% vs 34.9%, mean difference = 65.1%; 95% confidence interval [CI] = 18.4% to 111.9%, P < .01) and increased overall survival (n = 6 mice per group; control vs discontinuous treatment, median survival = 24 vs 46.5 days, ratio = 0.52, 95% CI = 0.19 to 0.84, P = .001). CONCLUSION: Niclosamide inhibits S100A4-induced metastasis formation in a mouse model of colon cancer and has therapeutic potential.