1

2 Mini-review

4 Multi-targeted therapy of cancer by niclosamide: A new application5 for an old drug6

7

8 Yonghe Li a,!Q1 , Pui Kai Li b, Michael J. Roberts c, Rebecca Arend d, Rajeev S. Samant e, Donald J. Buchsbaum f

9 a Drug Discovery Division, Southern Research Institute, Birmingham, AL, United States10 b Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, OH, United States11 c Drug Development Division, Southern Research Institute, Birmingham, AL, United States12 d Department of Gynecologic Oncology, The University of Alabama at Birmingham, Birmingham, AL, United States13 e Department of Pathology, The University of Alabama at Birmingham, Birmingham, AL, United States14 f Department of Radiation Oncology, The University of Alabama at Birmingham, Birmingham, AL, United States

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1 7a r t i c l e i n f o

18 Article history:19 Received 2 February 201420 Received in revised form 24 March 201421 Accepted 5 April 201422 Available online xxxx

23 Keywords:24 Niclosamide25 FDA-approved drug26 Multi-targeted therapy27 Drug discovery28 Cancer stem cells29

3 0a b s t r a c t

31The rapid development of new anticancer drugs that are safe and effective is a common goal shared by32basic scientists, clinicians and patients. The current review discusses one such agent, namely niclosamide,33which has been used in the clinic for the treatment of intestinal parasite infections. Recent studies repeat-34edly identified niclosamide as a potential anticancer agent by various high-throughput screening cam-35paigns. Niclosamide not only inhibits the Wnt/b-catenin, mTORC1, STAT3, NF-jB and Notch signaling36pathways, but also targets mitochondria in cancer cells to induce cell cycle arrest, growth inhibition37and apoptosis. A number of studies have established the anticancer activities of niclosamide in both38in vitro and in vivo models. Moreover, the inhibitory effects of niclosamide on cancer stem cells provide39further evidence for its consideration as a promising drug for cancer therapy. This article reviews various40aspects of niclosamide as they relate to its efficacy against cancer and associated molecular mechanisms.41! 2014 Published by Elsevier Ireland Ltd.42

43

44

45 Introduction

46 Niclosamide (trade name Niclocide), a teniacide in the anthel-47 mintic family which is especially effective against cestodes, has48 been approved for use in humans for nearly 50 years (Fig. 1)49 [1,2]. Niclosamide inhibits oxidative phosphorylation and stimu-50 lates adenosine triphosphatase activity in the mitochondria of ces-51 todes (eg. tapeworm), killing the scolex and proximal segments of52 the tapeworm both in vitro and in vivo [2]. Niclosamide is well tol-53 erated in humans. TheQ2 treatment of Taenia saginata (beef tape-54 worm), Diphyllobothrium latum (fish tapeworm) and Dipylidium55 caninum (dog tapeworm) in adult is 2 g as a single oral dose. For56 the treatment of Hymenolepis nana (dwarf tapeworm), the same57 oral dose is used for 7 days [2].58 Drug development, from the initial lead discovery to the final59 medication, is an expensive, lengthy and incremental process [3].60 Finding new uses for old or failed drugs is much faster and more61 economical than inventing a new drug from scratch, as existing

62drugs have known pharmacokinetics and safety profiles and have63often been approved for human use, therefore any newly identified64use(s) can be rapidly evaluated in clinical trials [4]. In the last655 years niclosamide has been identified as a potential anticancer66agent by various high-throughput screening campaigns. This arti-67cle reviews the current studies regarding various aspects of niclo-68samide as they relate to its potential new use in cancer therapy.

69Niclosamide – a multiple pathway inhibitor for anti-cancer70efficacy

71Recently, several studies reported the inhibitory effects of niclo-72samide on multiple intracellular signaling pathways. The signaling73molecules in these pathways are either over-expressed, constitu-74tively active or mutated in many cancer cells, and thus render75niclosamide as a potential anticancer agent. The effects of niclosa-76mide on these pathways are described below.

77The Wnt/b-catenin pathway

78The Wnt/b-catenin signaling pathway regulates cancer progres-79sion, including tumor initiation, tumor growth, cell senescence, cell80death, differentiation and metastasis [5–7]. In the absence of Wnt,

http://dx.doi.org/10.1016/j.canlet.2014.04.0030304-3835/! 2014 Published by Elsevier Ireland Ltd.

! Corresponding author. Address: Drug Discovery Division, Southern ResearchInstitute, 2000 Ninth Avenue South, Birmingham, AL 35205, United States. Tel.: +1(205) 581 2750; fax: +1 (205) 581 2903.

E-mail address: y.li@southernresearch.org (Y. Li).

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81 b-catenin is sequestered in a complex that consists of the ade-82 nomatous polyposis coli (APC) tumor suppressor, axin, glycogen83 synthase kinase-3b (GSK3b), and casein kinase 1 (CK1). This com-84 plex formation induces the phosphorylation of b-catenin by CK185 and GSK3b, which results in the ubiquitination and subsequent86 degradation of b-catenin by the 26S proteasome. Conversely, when87 Wnt proteins form a ternary complex with the cell surface recep-88 tors, low-density lipoprotein receptor-related protein5/6 (LRP5/6)89 and Frizzled (Fzd), signaling from Wnt receptors proceeds through90 the proteins dishevelled (Dvl) and axin, leading to the inhibition of91 GSK3b and the stabilization of cytosolic b-catenin. The b-catenin92 then translocates into the nucleus where it interacts with T-cell93 factor/lymphoid enhancing factor (TCF/LEF) to induce the expres-94 sion of specific target genes [5–7] (Fig. 2A).95 Chen et al. performed a high-throughput screening of a library96 containing approximately 1200 FDA-approved drugs and drug-like97 molecules with a primary imaged-based green fluorescent protein98 (GFP) fluorescence assay that used Fzd1 endocytosis as the readout99 in human osteosarcoma U2OS cells, and identified niclosamide as a

100 small molecule inhibitor of Wnt/b-catenin signaling [8]. Niclosa-101 mide promoted Wnt receptor Fzd1 endocytosis, downregulated102 Dvl2 protein, and inhibited Wnt3A-stimulated b-catenin stabiliza-103 tion and TCF/LEF reporter activity in U2OS cells [8]. It also104 decreased the cytosolic expression of endogenous Dvl2 and b-cate-105 nin in human colorectal cancer cell lines and in human colorectal106 cancer cells isolated by surgical resection of metastatic disease,107 regardless of mutations in APC [9]. Moreover, we recently demon-108 strated that niclosamide was able to inhibit Wnt/b-catenin signal-109 ing by promoting Wnt co-receptor LRP6 degradation, but had no110 effect on Dvl2 expression in prostate and breast cancer cells111 [10,11], suggesting that the mechanism underlying niclosamide-112 mediated inhibition of Wnt/b-catenin signaling could be cell113 type-dependent (Fig. 2A). In addition, the inhibitory effects of114 niclosamide on Wnt/b-catenin signaling were also demonstrated115 in primary human glioblastoma cells [12].116 More than 90% of colorectal cancers bear mutations that result117 in the activation of the Wnt/b-catenin pathway [13]. S100 calcium118 binding protein A4 (S100A4) is a target gene of the Wnt/b-catenin119 pathway in colorectal cancers [14]. Sack et al. performed a high-120 throughput screening of the LOPAC chemical library containing121 1280 compounds with a cell-based luminescence assay that used122 S100A4 promoter-driven luciferase activity as the readout in123 human colorectal cancer HCT116 cells, and identified niclosamide124 as an inhibitor of S100A4 through reduction of S100A4 mRNA125 and protein expression in colorectal cancer cells [15]. It was pro-126 posed that niclosamide inhibits b-catenin/TCF complex formation127 and thereby interrupts target gene transcription, an alternative128 model of Wnt/b-catenin inhibition by niclosamide in colorectal129 cancer cells [15] (Fig. 2A).

130 The mTORC1 pathway

131 The mammalian target of rapamycin complex 1 (mTORC1) is a132 heterotrimeric protein kinase that consists of the mTOR catalytic133 subunit and two associated proteins, raptor (regulatory associated134 protein of mTOR) and mLST8 (mammalian lethal with sec-13). The135 mTORC1 activity is regulated by upstream signals from growth

136factors, amino acids, stresses and energy state, and its activation137induces the phosphorylation of p70S6 kinase (p70S6K) and eukary-138otic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1), leading139to the enhanced translation of a subset of mRNAs that are critical140for cell growth and metabolism [16,17] (Fig. 2B). Activation of141either the serine/threonine protein kinase Akt (also known as pro-142tein kinase B or PKB) or the extracellular signal-regulated kinase143(ERK) pathway, or inhibition of the adenosine monophosphate-144activated protein kinase (AMPK) pathway, leads to activated145mTORC1signaling. As a downstream effector of Akt, mTORC1 has146been described as the most essential effector in driving cell prolif-147eration and susceptibility to oncogenic transformation. This leads148to the targeting of mTORC1 as a therapeutic strategy in many types149of cancer [16,17].150Balgi et al. performed a high-throughput screening of a collec-151tion of >3500 chemicals with a primary imaged-based enhanced152GFP (EGFP) fluorescence assay that used EGFP-LC3 punctate stain-153ing as the readout in human breast cancer MCF-7 cells, and identi-154fied niclosamide as an inhibitor of mTORC1 signaling in cells155maintained in nutrient-rich conditions [18]. Niclosamide did not156inhibit mTORC2, which also contains mTOR as a catalytic subunit,157suggesting that niclosamide does not inhibit mTOR catalytic activ-158ity but rather inhibits signaling to mTORC1 [18]. Tuberous sclerosis159complex (TSC2), a negative regulator of mTORC1, was not required160for the inhibition of mTORC1 signaling by niclosamide, as niclosa-161mide was able to suppress mTORC1 signaling in TSC2-deficient162cells where mTORC1 activity was elevated [18]. Further studies163demonstrated that niclosamide does not impair PI3K/Akt signaling,164nor does it interfere with mTORC1 assembly and mTORC1 kinase165activity [19]. It had been proposed that increased cytosolic acidifi-166cation is responsible for the mTORC1 inhibition, and that the struc-167tural features of niclosamide required for protonophoric activity168are essential for the mTORC1 inhibition [19]. Lysosome is the169degrading machine for autophagy, but has also been linked to170mTORC1 activation through the Rag/RRAG GTPase pathway [20].171More recently, Li et al. proposed that mTORC1 inhibition by niclo-172samide is caused by lysosomal dysfunction [21]. Niclosamide173inhibits the lysosomal degradative function likely by altering lyso-174somal permeability and the pH gradient [21], which is consistent175with the finding that niclosamide can cause dispersion of protons176from the lysosomes to the cytosol, leading to cytosolic acidification177[19] (Fig. 2B).

178The STAT3 pathway

179Signal transducers and activators of transcription 3 (STAT3), a180member of a family of six different transcription factors, is one of181the down-stream signaling proteins for cytokine and growth factor182receptors [22]. Engagement of cell-surface cytokine or growth fac-183tor receptors activates the janus kinase (JAK) family of protein184kinases, which in turn phosphorylates STAT3 at tyrosine residue185705, leading to the dimerization of two STAT3 monomers through186the SH2 domains of the proteins [23,24]. The activated STAT3187dimers then translocate into the nucleus and activate the tran-188scription of a panel of genes that control cell proliferation, apopto-189sis, angiogenesis and other cell functions [25,26]. This JAK2/STAT3190signaling pathway is one of the three major modules that play an191essential role in transmitting external signals from the surface192membrane to target genes in the nucleus, controlling processes193such as growth, differentiation, senescence and apoptosis [27,28].194Importantly, STAT3 is the major intrinsic pathway for cancer195inflammation owing to its frequent activation in malignant cells196and key role in regulating many genes crucial for cancer inflamma-197tion in the tumor microenvironment [22].198Ren et al. demonstrated that niclosamide is a potent STAT3 inhib-199itor that suppresses STAT3 transcriptional activity by blocking its

Fig. 1. The chemical structure of niclosamide.

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200 phosphorylation and nuclear translocation in prostate cancer201 DU145 cells [29] (Fig. 2C). The inhibitory effect of niclosamide on202 STAT3 signaling was subsequently reported in multiple myeloma203 cells [30]. STAT3 can mediate cancer cell survival by activating anti-204 apoptotic genes such as Bcl-2 and Bcl-XL. Recently, it was reported205 that inhibition of STAT3 by niclosamide blocked the erlotinib acti-206 vated STAT3/Bcl-2/Bcl-XL pathway and reversed erlotinib resistance207 of human head and neck cancer cells and non-small cell lung cancer208 cells [31,32]. Furthermore, niclosamide not only blocked the ioniz-209 ing radiation-induced activation of the STAT3/Bcl-2/Bcl-XL pathway210 in both radiosensitive and radioresistant human lung cancer cells,211 but also reversed the radioresistance and restored the sensitivity212 of radioresistant cells to ionizing radiation [33].

213 The NF-jB pathway

214 Nuclear factor-kappaB (NF-jB) is an important transcription215 factor associated with cancer, and has been implicated in many216 hallmarks of cancer development, including growth factor inde-217 pendent proliferation, inhibition of apoptosis, tumor angiogenesis,218 limitless replicative potential and tissue invasion and metastasis219 [34,35]. NF-jB is activated by several agents, including cytokines,220 oxidant free radicals, inhaled particles, ultraviolet irradiation, and221 bacterial or viral products. In response to these stimuli, IjB kinase

222(IKK) is activated and can phosphorylate the inhibitory IjBa sub-223unit of the NF-jB!IjBa complex in the cytoplasm. This phosphory-224lation marks IjBa for degradation by the proteasome and releases225NF-jB from the inhibitory complex, and the freed NF-jB proteins226are then transported into the nucleus where they bind to their tar-227get sequences and activate gene transcription [36,37].228Jin et al. demonstrated that niclosamide blocked tumor necrosis229factor a (TNF-a)-induced IjBa phosphorylation, translocation of230NF-jB p65 subunit, and expression of NF-jB-regulated genes in231acute myelogenous leukemia cells [38]. The inhibitory effects of232niclosamide on NF-jB signaling were also reported in multiple233myeloma cells [30] and primary human glioblastoma cells [12].234Transforming growth factor-b (TGF-b) activated kinase1 (TAK1)235mediates IKK activation in the TNF-a signaling pathway [39,40].236It was demonstrated that niclosamide inhibited the steps of237TAK1 ? IKK and IKK ? IjBa of the NF-jB activation [38]238(Fig. 2D). In addition, niclosamide inhibited the DNA binding of239NF-jB to the promoter of its target genes [38].

240The Notch pathway

241The Notch signaling pathway represents a critical component in242the molecular circuits that control cell fate during development.243Under physiological conditions, the Notch ligand binds to its

Fig. 2. The intracellular signaling pathways inhibited by niclosamide in cancer cells. (A) Niclosamide blocks Wnt/b-catenin signaling by enhancing Wnt receptor Fzd1internalization, promoting Wnt co-receptor LRP6 degradation, suppressing Wnt signaling regulator Dvl2 expression, and inhibiting b-catenin/TCF complex formation.(B) mTORC1 inhibition by niclosamide is associated with cytosolic acidification and lysosomal dysfunction. (C) Niclosamide inhibits STAT3 transcriptional activity by blockingits phosphorylation and nuclear translocation. (D) Niclosamide ablates TNF-induced NF-jB activation at TAK1 and IKK steps. (E) Niclosamide inhibits Notch signaling bysuppressing the expression of cleaved activated Notch1 receptor.

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244 receptor to initiate Notch signaling by releasing the intracellular245 domain of the Notch receptor (Notch-IC) through a cascade of pro-246 teolytic cleavages by both a-secretase and c-secretase. The247 released intracellular Notch-IC then translocates into the nucleus248 where it modulates gene expression primarily by binding to a249 ubiquitous transcription factor, CBF1, and suppressor of hairless,250 Lag-1 (CSL). In cancers, molecular genetic alterations cause251 increased levels of intracellular Notch-IC and subsequent constitu-252 tive activation of the Notch pathway. Aberrant activation of this253 pathway contributes to tumorigenesis, and Notch inhibitory254 agents, such as

255 c-secretase inhibitors, are being investigated as candidate cancer256 therapeutic agents [41–43].257 Wang et al. developed a cell based luciferase reporter gene258 assay to measure the endogenous CBF1-dependent Notch signaling259 in human erythroleukemia K562 cells, and identified niclosamide260 as a potent inhibitor of Notch signaling [44]. Niclosamide inhibited261 the luciferase activity of CBF1-dependent reporter gene, and sup-262 pressed the expression of cleaved activated Notch1 receptor and263 Notch target Hes-1 in K562 cells [44] (Fig. 2E). In addition, the264 inhibitory effects of niclosamide on Notch signaling were also265 reported in primary human glioblastoma cells [12].

266 Niclosamide targeting of mitochondria

267 Mitochondria are vital for cellular bioenergetics and play a cen-268 tral role in determining the point-of-no-return of the apoptotic269 process. It has been proposed that targeting mitochondria is an270 efficient strategy for cancer chemotherapy [45]. Khanim et al.271 screened a panel of 100 off-patent licensed oral drugs for anti-272 myeloma activity, and identified niclosamide as a killer of multiple273 myeloma cell lines and primary multiple myeloma cells [30]. Inter-274 estingly, niclosamide anti-multiple myeloma activity could be275 mainly mediated through the mitochondria with rapid loss of276 mitochondrial membrane potential, uncoupling of oxidative phos-277 phorylation and production of mitochondrial superoxide [30].278 Moreover, Park et al. screened the LOPAC chemical library and279 identified niclosamide as a potent inducer of mitochondrial fission,280 which induced mitochondria fragmentation and promoted both281 apoptotic and autophagic cell death [46]. In addition, it was282 reported that inactivation of NF-jB by niclosamide caused mito-283 chondrial damage and the generation of reactive oxygen species284 (ROS), leading to apoptosis of acute myelogenous leukemia cells285 [38]. Finally, Yo et al. performed a genome-wide gene expression286 array and demonstrated that niclosamide was able to disrupt mul-287 tiple metabolic pathways affecting biogenetics, biogenesis, and288 redox regulation in ovarian-cancer-initiating cells. These disrup-289 tions presumably lead to the activation of the intrinsic mitochon-290 drial apoptosis pathway, loss of tumor stemness, and growth291 inhibition [47].

292 Anti-cancer activity of niclosamide: in vitro studies

293 Anti-cancer activity of niclosamide has been demonstrated in294 human breast cancer [10,11,18,48,49], prostate cancer [10,29],295 colon cancer [9,15], ovarian cancer [47], multiple myeloma [30],296 acute myelogenous leukemia [38], glioblastoma [12], head and297 neck cancer [21] and lung cancer cells [31]. As summarized in298 the earlier sections, niclosamide is able to block the multiple sig-299 naling pathways that govern cancer initiation and progression,300 thus it is not surprising that niclosamide has a potent activity to301 induce cancer cell cycle arrest, growth inhibition and apoptotic302 death across multiple cancer types [50].303 The NCI-60 human tumor cell line anticancer drug screen was304 developed in the late 1980s as an in vitro drug-discovery tool,

305and was rapidly recognized as a rich source of information about306the mechanisms of growth inhibition and tumor-cell kill [51]. To307further characterize the anticancer activities of niclosamide, we308performed a 60 human tumor cell line anticancer drug screen with309niclosamide. We found that niclosamide inhibited cell proliferation310of all tested cancer cell lines, and that the IC50 values for most cell311lines were less than 1 lM (Table 1). These results indicate that312niclosamide has a significant broad-spectrum and potent in vitro313anticancer activity.314Niclosamide is able to potentiate the cytotoxicity towards315tumor cells of different anticancer agents in several in vitro cancer316models. It enhanced the anti-proliferative activity of oxaliplatin, a317commonly used drug for colorectal cancer, in human colorectal318cancer Caco2 cells and colorectal cancer explant cells [9]. Further-319more, inhibition of STAT3 by niclosamide blocked erlotinib-320induced STAT3 phosphorylation and sensitized human head and321neck cancer cells and non-small cell lung cancer cells to erlotinib322[31,32]. In addition, combinatorial drug testing established that a323heterozygous deletion of the NFKBIA locus in glioblastoma samples324could serve as a genomic biomarker for predicting synergistic325activity of niclosamide with temozolomide, the current standard326in glioblastoma therapy [12]. Finally, synergistic effects of niclosa-327mide in combination with Ara-C, VP-16, and DNR, the frontline328chemotherapeutic agents for acute myelogenous leukemia, have329been reported in acute myelogenous leukemia stem cells [38].

330Anti-cancer activity of niclosamide: in vivo studies

331The efficacy of niclosamide has been shown against tumor332growth and metastases in several xenograft models. Jin et al. dem-333onstrated for the first time that niclosamide has in vivo anti-cancer334activities [38]. As niclosamide has limited solubility in water, Jin

Table 1Effects of niclosamide on growth of 60 human tumor cell lines.

IC50 (lM) IC50 (lM) IC50 (lM)

Leukemia Renal OvarianCCRF-CEM 0.51 786-0 1.01 IGR-OV1 1.01HL-60 (TB) 0.14 A498 0.52 OVCAR-3 0.35K562 0.18 ACHN 0.54 OVCAR-4 0.61MOLT-4 0.22 CAKI-1 1.11 OVCAR-5 0.76RPMI-8226 0.05 RXF 393 0.86 OVCAR-8 0.81SR 0.13 SN12C 0.58 NCI/ADR-RES 0.38

TK-10 0.51 SK-OV-3 0.72UO-31 0.55

Non-small cell lung Colon MelanomaA549/ATCC 0.96 COLO 205 0.98 LOX IMVI 0.60EKVX 0.50 HCC-2998 1.25 MALME-3M 0.74HOP-62 0.41 HCT-116 0.374 M14 0.74HOP-92 0.61 HCT-15 1.06 MDA-Mb-435 0.52NCL-H226 0.73 HT29 4.17 SK-MEL-2 1.32NCL-23 0.22 KM12 1.07 SK-MEL-28 0.95NCL-H322M 0.61 SW-620 0.54 SK-Mel-5 0.40NCL-H460 0.16 UACC-257 1.07NCL-H522 0.26 UACC-62 0.22

Breast CNS ProstateMCF-7 0.38 SF-268 0.53 PC-3 0.62MDA-MB-231 0.86 SF-295 1.05 DU-145 0.60MDA-Mb-468 0.24 SF-539 2.25HS 578T 0.43 SNB-19 0.29BT-549 0.86 SNB-75 1.14T-47D 0.43 U251 0.34

Cells were seeded into 96-well tissue culture treated microtiter plates at a den-sity of 5000–20,000 cells/well (depending on cell line) in a total volume of 50 ll.After overnight incubation, the cells were treated with niclosamide for 72 h intriplicate by adding 50 ll of 2X stock solutions to appropriate wells alreadycontaining 50 ll of cells and medium to expose cells to the final concentrations ofniclosamide required. Cell viability was measured by the Cell Titer Glo Assay(Promega).

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335 et al. synthesized a niclosamide analog – phosphate of niclosamide336 (p-niclosamide), and found p-niclosamide showed significant inhi-337 bition of xenograft tumor growth of acute myeloid leukemia HL-60338 cells by suppressing the NF-jB pathway [38]. Subsequently, Osada339 et al. reported that niclosamide inhibited the growth of colorectal340 cancer cells in NOD/SCID mice by down-regulating Dvl2 and341 b-catenin expression [9]. Moreover, niclosamide in combination342 with erlotinib potently repressed erlotinib-resistant head and neck343 cancer xenografts and lung cancer xenografts by inhibiting STAT3344 signaling [31,32]. In addition, niclosamide alone or in combination345 with radiation overcame radioresistance in lung cancer xenografts346 [33]. Niclosamide was also able to significantly diminish the malig-347 nant potential of primary human glioblastoma cells in vivo by348 suppressing intracellular Wnt/b-catenin-, NOTCH-, mTORC1-, and349 NF-kB signaling cascades [12]. TRA-8 is an agonistic monoclonal350 antibody (mAb) to TRAIL death receptor 5 (DR5) [52,53]. We351 recently found that niclosamide in combination with TRA-8 sup-352 pressed the growth of basal-like breast cancer 2LMP orthotopic353 tumor xenografts by inhibiting Wnt/b-catenin and STAT3 signaling354 [11]. Importantly, niclosamide was able to inhibit the tumor for-355 mation of ovarian-cancer-initiating cells derived from cancer cell356 lines in vivo [47] and tumor growth of breast cancer stem-like cell357 subpopulations in vivo [49]. Finally, it was found that niclosamide358 significantly reduced liver metastasis formation in mice bearing359 xenografted intrasplenic colon tumors by suppressing the expres-360 sion of the Wnt/b-catenin signaling target S100A4 [15], as361 S100A4 plays a critical role in metastasis formation in colon cancer362 [14,54–56]. Overall, these studies reveal the in vivo anticancer363 activities of niclosamide, and provide a rationale for its use in clin-364 ical trials.

365 Niclosamide as a drug for targeting CSCs

366 Current cancer treatments such as chemotherapy, targeted367 therapy and radiotherapy are successful at destroying bulk cancer368 cells, but fail to eliminate cancer stem cells (CSCs). CSCs are char-369 acterized by tumorigenic properties and the ability to self-renew,370 form differentiated progeny, and develop resistance to therapy.371 The inability to eradicate CSCs is thought to be the reason for can-372 cer relapse and chemo-resistance, the major obstacles in current373 cancer therapy [57–59]. The Wnt/b-catenin, Notch, and Hedgehog374 pathways are three major pathways which have been implicated in375 CSC tumorigenic properties and the ability to self-renew, form dif-376 ferentiated progeny, and develop resistance to therapy [43,60–62].377 However, many other pathways such as the mTORC1, STAT3 and378 NF-jB pathways are also involved in CSC self-renewal and tumor379 initiation [63].380 Many studies have been performed on surface markers for381 potential identification and isolation of CSCs [59]. In some cases,382 stem-like cancer cells were identified using the flow cytometry-383 based side population (SP) technique [64,65]. Wang et al. isolated384 the SP of breast cancer MCF-7 cells, generated SP spheres, and per-385 formed a high-throughput drug screening using these SP spheres386 and the LOPAC chemical library, and identified niclosamide as an387 inhibitor of breast cancer stem-like cells [49]. Niclosamide down-388 regulated several stem cell signaling pathways including the389 Wnt/b-catenin, Notch, and Hedgehog pathways, inhibited the for-390 mation of spheroids, and induced apoptosis in breast cancer SP391 spheres. Animal studies also confirmed this therapeutic effect392 [49]. Using a similar approach, the same group further demon-393 strated that niclosamide was an inhibitor of stem-like ovarian-can-394 cer-initiating cells, and suppressed the tumor formation of395 ovarian-cancer-initiating cells in vivo [47]. Moreover, it was396 reported that niclosamide was effective in killing acute myeloid397 leukemia (AML) stem cells (CD34+ CD38"), but had minimal

398cytotoxicity against progenitor cells in normal bone marrow [38].399In addition, STAT3-mediated stem cell marker OCT-4 gene expres-400sion was effectively suppressed by treatment with niclosamide401during mammosphere culture of breast cancer MDA-MB-231 cells402[48]. Aldehyde dehydrogenase (ALDH) is a known marker of CSCs403[66,67]. Recently, we isolated a non-adherent population of cells404that have high ALDH expression, and found that niclosamide sup-405pressed Wnt/b-catenin and STAT3 signaling, and showed cytotox-406icity against these non-adherent ALDH expressing cells in407addition to the adherent cells from four basal-like breast cancer408cell lines [11]. The inhibitory effects of niclosamide on CSCs pro-409vide compelling evidence for its consideration as a promising drug410for cancer therapy.411Salinomycin, a polyether ionophore antibiotic isolated from412Streptomyces albus, is used as an antibiotic in animal husbandry413[68]. Recently, Gupta et al. performed a high throughput screening414to discover agents with specific toxicity for epithelial CSCs, and415identified salinomycin as a selective inhibitor of breast CSCs [69].416Promising results from preclinical trials in human xenograft mice417and a few clinical pilot studies reveal that salinomycin could be a418promising novel anti-cancer agent [70–72]. Ketola et al. performed419connectivity map analysis to identify compounds with similar or420opposite effects to salinomycin [73]. The differentially expressed421genes in response to salinomycin exposure for 6 h in prostate carci-422noma VCaP cells were compared with the 47,000 expression profiles423representing drug responses to 41,309 compounds. Interestingly,424niclosamide was one of the two top enriched compounds altering425gene expression in the same direction as salinomycin [73], provid-426ing further evidence that niclosamide, like salinomycin, can work427as a CSC killer.

428Challenges to using niclosamide as an anti-cancer agent in429humans

430The oral dose of niclosamide for adult in cestocidal treatment is4312 g as a single dose, leading to maximal serum concentrations of4320.25–6.0 lg/ml (corresponding to 0.76–18.35 lM) [1], which is433well within the anti-cancer active concentration range. The reason434for this wide range of serum concentrations is considered to be due435to intra-individual difference in absorption rate [1]. Niclosamide436has poor water solubility, and the oral bioavailability of niclosa-437mide was only 10% in male Sprague-Dawley rats [74]. The plasma438half-life (t1/2) of niclosamide was 6.7 ± 2.0 h in rats [74]. However,439no specific data for pharmacokinetic parameters of niclosamide in440humans is available in the literature [2]. Overall, the low oral bio-441availability and wide range of serum concentrations of niclosamide442could result in variation of its anti-cancer efficacy when it is used443in clinical studies.444Studies in animals have suggested that niclosamide has no445mutagenic, oncogenic, or embryotoxic activity [1]. Long term446experiments in rats (1000 or 2500 mg/kg b.w. oral daily for 55 or44764 days, thereafter 10,000 and 25,000 mg/kg feed; total duration448365–381 days) and dogs (100 mg/kg b.w. orally in capsules for449366–393 days) found no indication of a higher incidence of tumor450occurrence [1]. Niclosamide has been proven to be without effect451on hematological and urinalysis parameters and not to influence452liver and kidney function tests [1]. Recent studies have also indi-453cated that no significant toxicity was shown against non-tumor454cells in vitro and no obvious side effects were observed in niclosa-455mide-treated mice [9,30,38]. The fate of niclosamide in humans,456which closely resembles that in animals, and the low acute toxicity457in humans and animals suggest that even a prolonged contact with458niclosamide will not result in cumulative toxic effects in humans459[1]. However, niclosamide is poorly absorbed from the intestinal460tract, which explains its good tolerability in humans [1,2].

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461 Therefore, additional safety studies on niclosamide with a well462 absorbed formulation are required before definitive conclusions463 can be drawn for cancer therapy.

464 Future directions

465 While considerable high-throughput screening campaigns have466 identified niclosamide is a potent inhibitor of a number of biological467 signaling pathways that mediate niclosamide’s anti-cancer effects468 in vitro and in vivo, direct targets of niclosamide still remain unclear.469 Niclosamide was tested in vitro against a panel of 95 protein kinases,470 and it did not significantly inhibit any of these kinases at concentra-471 tions (1 or 10 lM) efficiently inhibiting the Wnt/b-catenin,472 mTORC1, STAT3, NF-jB and Notch signaling pathways in cancer473 cells, indicating that the mechanism of action of niclosamide is474 probably not direct kinase inhibition [19]. Among the techniques475 currently available, protein affinity isolation using suitable small-476 molecule probes (pulldown) and subsequent mass spectrometric477 analysis of the isolated proteins appears to be the most powerful478 and most frequently applied [75]. Therefore, the targets of niclosa-479 mide could be identified using affinity reagents – biotinylated deriv-480 atives of niclosamide in the future.481 Although many studies have demonstrated that niclosamide482 has potent in vitro and in vivo anti-tumor growth activities, the483 cancer chemopreventive efficacy of niclosamide has not been stud-484 ied yet. Therefore, there is a great need for further research to485 examine cancer chemopreventive activity of niclosamide in vari-486 ous animal carcinogenesis models and transgenic models.487 Niclosamide is an FDA-approved drug that is given orally to488 helminthosis patients, however its applications in cancer therapy489 could be limited, as niclosamide is only partially absorbed from490 the gastrointestinal tract. Thus, there is great interest in conduct-491 ing studies to elucidate the structure-activity relationship of niclo-492 samide and to identify novel derivatives of niclosamide with493 improved bioavailability as additional clinical candidates for can-494 cer therapy [76,77].495 Finally, intravenous administration of niclosamide to rats gave496 rise to a peak plasma concentration of 25 lM [74]. Thus, an intrave-497 nous injectable niclosamide formulation would be desirable. How-498 ever, the safety and possibility of systemic intravenous application499 of niclosamide needs to be comprehensively investigated before500 niclosamide could be administered intravenously in the clinic.

501 Conflict of Interest

502 The authors declare that they have no conflict of interest.

503 Acknowledgments

504 We thank Meredith S. Plaxco and Tommie A. Gamble for run-505 ning the 60 human tumor cell line anticancer drug screen. This506 work was completed with the support of Grants from the National507 Institute of Health (R01CA124531 and R21CA182056 to Y. Li).

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