Inhibition of LEF1-Mediated DCLK1 by Niclosamide Attenuates Colorectal

Published OnlineFirst November 16, 2018; DOI: 10.1158/1078-0432.CCR-18-1232

Cancer Therapy: Preclinical Clinical Cancer Research Inhibition of LEF1-Mediated DCLK1 by Niclosamide Attenuates Colorectal Cancer Stemness So-Yeon Park1,2, Ji-Young Kim3, Jang-Hyun Choi1, Jee-Heun Kim1, Choong-Jae Lee1, Pomila Singh4, Shubhashish Sarkar4, Jeong-Heum Baek5, and Jeong-Seok Nam1,2,6

Abstract

Purpose: Niclosamide, an FDA-approved anthelmintic pathway, specifically lymphoid enhancer-binding factor 1 drug, has been characterized as a potent Wnt inhibitor that (LEF1) expression, which is critical for regulating stemness. can suppress tumor growth and cancer stem-like cell (CSC) Subsequently, we identified that the doublecortin-like kinase 1 populations. However, the underlying molecular mechanisms (DCLK1)-B is a target of LEF1 and upregulates cancer stemness remain poorly understood. This study aimed to examine how in colorectal cancer cells. We first documented that niclosa- Wnt inhibition by niclosamide preferentially targets CSCs. mide blocks the transcription of DCLK1-B by interrupting the Experimental Design: The mechanistic role of niclosamide binding of LEF1 to DCLK1-B promoter. DCLK1-B depletion in CSC inhibition was examined in public databases, human impairs cancer stemness resulting in reduced survival potential colorectal cancer cells, colorectal cancer xenografts, and azox- and increased apoptosis, thus sensitizing colorectal cancer to ymethane/dextran sulfate sodium (AOM/DSS)-induced colo- chemoradiation. rectal cancer model. Conclusions: Disruption of the LEF1/DCLK1-B axis by Results: Niclosamide suppresses CSC populations and their niclosamide eradicates cancer stemness and elicits therapeutic self-renewal activities in colorectal cancer cells, and this CSC- effects on colorectal cancer initiation, progression, and resis- targeting effect leads to irreversible disruption of tumor- tance. These findings provide a preclinical rationale to broaden initiating potential in vivo. Mechanistically, niclosamide the clinical evaluation of niclosamide for the treatment of downregulates multiple signaling components of the Wnt colorectal cancer.

Introduction a small subpopulation, collectively referred to as cancer stem-like cells (CSC), which re-initiate tumor growth and colonize distant Colorectal cancer is a major health problem worldwide owing organs resulting in relapse and metastasis (4, 5). CSCs engage in to its high prevalence and mortality rates (1, 2). Although earlier self-renewal, induce tumors at low-cell density, and produce diagnosis by advanced technology and new treatment regimens tumors with differentiated and heterogeneous cell profiles. An have considerably improved the survival of patients with colo- increasing number of studies suggest that CSCs promote cancer rectal cancer in the past decades, nearly 50% of patients with progression at various stages including tumor initiation, growth, colorectal cancer still face recurrence at local or distant sites after invasion, and metastasis, and notably, they are more resistant to conventional therapy (3). Initially, conventional therapy kills anticancer therapy than are differentiated non-CSCs (5). In this most cancer cells and immediately shrinks tumor while sparing regard, compounds or designed drugs that hinder CSC-specific pathways or interfere with CSC-specific targets have been recently

1 recognized as promising combinatorial therapies for permanent School of Life Sciences, Gwangju Institute of Science and Technology, Gwangju, cure. To actualize this possibility, we need to understand molec- Republic of Korea. 2Cell Logistics Research Center, Gwangju Institute of Science and Technology, Gwangju, Republic of Korea. 3Laboratory Animal Resource ular mechanism by which CSCs maintain stemness and promote Center, Gwangju Institute of Science and Technology, Gwnagju, Republic of resistance, leading to recurrence after chemoradiotherapy. Korea. 4Department of Neuroscience and Cell Biology, University of Texas CSCs display many features of embryonic or tissue stem cells Medical Branch, Galveston, Texas. 5Division of Colon and Rectal Surgery, and typically demonstrate persistent activation of one or more Department of Surgery, Gil Medical Center, Gachon University College of 6 highly conserved signal transduction pathways involved in devel- Medicine, Incheon, Republic of Korea. Silver Health Bio Research Center, opment and tissue homeostasis, including Notch, Hedgehog, and Gwangju Institute of Science and Technology, Gwangju, Republic of Korea. Wnt pathways. In particular, the aberrant activation of Wnt/ Note: Supplementary data for this article are available at Clinical Cancer b-catenin signaling pathway is involved in both development Research Online (http://clincancerres.aacrjournals.org/). and progression of colorectal cancer. During colon carcinogene- S.-Y. Park, J.-Y. Kim, and J.-H. Choi have contributed equally to this article. sis, hyperactivation of Wnt/b-catenin signaling drives adenoma- Corresponding Author: Jeong-Seok Nam, Gwangju Institute of Science and tous polyp formation (6, 7), and differentiated colon epithelial Technology, 123 Cheomdan-gwagiro (Oryong-dong), Buk-gu, Gwangju 500- cells with constitutive activation of Wnt/b-catenin signaling can 712, Republic of Korea. Phone: 82-62-715-2893; Fax: 82-62-715-2484; E-mail: re-acquire stem cell-like properties and give rise to poorly differ- [email protected] entiated colorectal cancer (8, 9). Colorectal cancer tissues display doi: 10.1158/1078-0432.CCR-18-1232 more intense nuclear accumulation of b-catenin than do normal 2018 American Association for Cancer Research. tissues. Moreover, CSC populations harbor more enhanced

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such as colorectal (20, 21), prostate (22), breast cancer (23), and Translational Relevance glioblastoma (24). More recently, accumulating evidence has Constitutive activation of Wnt signaling has been implicat- suggested that niclosamide can be a potential CSC-targeting drug. ed in cancer stem cells (CSC), leading to cancer initiation and A drug screening of a LOPAC chemical library first identified that progression. A number of Wnt inhibitors have been proposed niclosamide efficiently suppressed spheroid formation of side as CSC-targeting drugs; yet, a lack of understanding of molec- populations in breast CSCs (26). Likewise, niclosmiade has ular regulation on CSC survival and self-renewal remains an been reported to decrease CSC population, such as Aldefluorþ þ obstacle. Niclosamide inhibits multiple components of Wnt melanoma CSCs (27) and CD133 ovarian CSCs (28). Although signaling and CSC; thus we explored the downstream targets of these previous data suggest that niclosamide can reduce CSC niclosamide by using public genomic databases, human colo- populations by interrupting Wnt/b-catenin pathway, how Wnt rectal cancer cells, and colorectal cancer mouse models. We inhibition by niclosamide results in the reduction of CSC popula- found that doublecortin-like kinase 1 (DCLK1)-B is predom- tions remains elusive. Therefore, we need to identify the molec- inantly expressed in colorectal cancer cells, and it is more ular mechanism of CSC inhibition by niclosamide to enable the enriched in CSCs than in non-CSCs. DCLK1-B is transcrip- use of niclosamide in clinics. tionally activated by lymphoid enhancer-binding factor 1 Taking all of these findings from previous studies into consid- (LEF1) and this LEF1/DCLK1-B axis is critical for CSC survival eration, we hypothesized that Wnt-targeting niclosamide can and self-renewal activity. Disruption of the LEF1/DCLK1-B attenuate CSC properties in colorectal cancer and that its effects axis by niclosamide can impair the tumor-initiating and can possibly be extended as a potential adjuvant therapeutics with survival potential of CSCs. These findings between LEF1 and chemoradiotherapy to reduce therapy resistance in CSCs. Mech- DCLK1-B may improve understanding of the signaling net- anistically, multitargeting Wnt/b-catenin by niclosamide leads to work of CSCs. the reduction in lymphoid enhancer factor 1 (LEF1) expression, which is correlated with therapy resistance and poor prognosis in patients with colorectal cancer. By genomic analysis of metastatic colorectal cancer patient tumors, we identified a set of enriched transcriptional activity of Wnt/b-catenin signaling and express LEF1 target genes among the stemness-related genes, and discov- higher levels of Wnt target gene than non-CSCs (10, 11). This ered that doublecortin-like kinase1 (DCLK1) displayed the most activated Wnt/b-catenin signaling is critical for CSC to self-renew dramatic decrease following niclosamide treatment. Moreover, and repopulate tumor, resulting in chemoresistance and metas- we found that colorectal CSCs predominantly expressed DCLK1-B tasis (10, 12–15). Thus, Wnt signaling inhibitors are gaining rather than DCLK1-A. Mechanistically, DCLK1-B transcription is b interest as potential CSC-targeting agents and are under preclin- directly activated by Wnt/ -catenin signaling and LEF1 mediates ical or clinical investigation (16–18). Wnt-induced CSC properties by enhancing proliferation, Thus far, Wnt/b-catenin inhibitors have not been approved in survival, anti-apoptosis, and self-renewal potential of CSCs. clinics, and only few, such as the anti-frizzled7 (FZD7) antibody Our results thus provide a good understanding of how niclosa- vantictumab (NCT01345201) and the porcupine inhibitor mide selectively targets CSCs, suggesting DCLK1-B as a potential fi LGK974 (NCT01351103), have made it to early clinical trials. CSC-speci c target for preventing cancer progression and Clinical trials of new lead compounds are expensive and lengthy therapy resistance. and need incremental process. In this regard, repurposing the existing FDA-approved drugs is being considered a promising Materials and Methods strategy because finding new uses for old drugs is much faster and Ethics, cell culture, and reagents more economical than developing a new drug from scratch; All animal experiments were carried out according to the additionally, because existing drugs have known pharmacokinetic Institutional Animal Care and Use Committee (IACUC) of the and safety profiles and have often been approved for human use, Gwangju Institute of Science and Technology (GIST-2017-038). any newly identified uses can be rapidly evaluated in clinical trials. All work related to human tissues was preapproved by Institu- Niclosamide, a teniacide from anthelmintic family that is espe- tional Review Board (IRB) at Gwangju Institute of Science and cially effective against cestodes, has been approved for use in Technology (#20170410-BR-28-03-02) and the Lee Gil Ya Cancer humans for nearly 50 years. In the past decade, niclosamide has and Diabetes Institute of Gachon University (GCIRB-2013-66), been identified as a potential anticancer agent by multiple inde- and was conducted in accordance with the Helsinki Declaration. pendent high-throughput screening efforts. The Wnt-targeting Informed consent forms were signed and obtained from all effect of niclosamide was first identified through high-throughput subjects prior to participation. Detailed information of patient screening of a library of FDA-approved drugs that detect frizzled1 with colorectal cancer samples, cell lines, including from where (FZD1) endocytosis in human osteosarcoma (19). Accumulating and when they were obtained, and reagents were described in evidence suggests that niclosamide interrupts Wnt/b-catenin Supplementary Materials and Methods. pathway by targeting multiple signaling components such as Dvl2 protein (20, 21), b-catenin (20, 21), b-catenin/TCF complex Luciferase reporter assay formation (21), and LRP6 (22–24). Collectively, in contrast to The luciferase reporter experiments using TopFlash/FopFlash other Wnt inhibitors, niclosamide can target various components plasmids were performed as reported previously (29). Promoter- involved in Wnt/b-catenin pathway, thus it is a strong multi- reporter constructs for DCLK1-B and DCLK1-A are described in targeting Wnt inhibitor (25). Wnt/b-catenin inhibition by niclo- previous report (30). To measure luciferase activity, luminescence samide has shown anticancer effects including tumor cell growth was detected by a luminometer (Glomax, Promega) according to inhibition and tumor burden reduction in various types of cancer manufacturer's recommendations.

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Statistical analysis the therapy resistance of CSCs. Indeed, the surviving colorectal All statistical data are expressed as mean SD (n ¼ 3). cancer cells following 5-fluorouracil (5-FU) or radiation expo- Statistical comparisons were determined by Student t test between sure, the first-line therapies for colorectal cancer, displayed the þ two groups or by one-way ANOVA with Dunnett's multiple increased CD44v6 CSC populations (Fig. 1E; Supplementary comparison among multiple groups. In case of in vivo experi- Fig. S1F) with the enhanced self-renewal activities (Fig. 1F; ments, the number of mice was indicated in each legends. Supplementary Fig. S1G). Surprisingly, combinatorial treat- Log-rank test was used for Kaplan–Meier analyses. , , and ment of niclosamide prevented the increase of stemness in indicate P < 0.05, P < 0.01, and P < 0.001, respectively. surviving cells following conventional anticancer. Of note, More information about methods, including the cell prolifer- these CSC-targeting effects of niclosamide finally resulted in ation assay, the limiting dilution assay, the establishment of sensitization of colorectal cancer cells to chemotherapy (Fig. 1G knockdown/knockout cell lines, is provided in Supplementary and H) and radiotherapy (Fig. 1I). Therefore, niclosamide Materials and Methods. inhibits CSC-like properties by inhibiting Wnt/b-catenin signaling and thus can be a potential agent to overcome therapy resistance in colorectal cancer cells. Results Niclosamide inhibits cancer stemness and therapy resistance by LEF1, a regulator of cancer stemness, is a potent target of inhibiting Wnt/b-catenin signaling in colorectal cancer cells niclosamide To examine whether niclosamide inhibits the colorectal cancer Next, to investigate the molecular mechanism of niclosamide, proliferation, we evaluated the IC50 values in multiple cells we performed qPCR screening of Wnt/b-catenin pathway genes in including HCT116, HT29, SW480, and patient-derived colorectal niclosamide-treated HCT116 cells (Fig. 2A). Various Wnt com- cancer cells (P#21257113 and P#14005083). Patient-derived ponents were suppressed by niclosamide, and LEF1 showed the colorectal cancer cells were isolated from primary tumors of most potent reduction following niclosamide treatment. In fur- patients with colorectal cancer and pathologically validated based ther experiments, niclosamide dose-dependently reduced both on IHC markers. They were determined to express the colon- mRNA and protein levels of LEF1 in multiple colorectal cancer þ specific cytokeratin (CK) pattern, CK20 /CK7 (31), but not to cells (Fig. 2B and C). Consistently, immunofluorescence analysis express the fibroblast marker vimentin (Supplementary Fig. S1A; revealed that LEF1 were increased in nuclear regions by Wnt m ref. 32). The IC50 values of niclosamide were below 6 mol/L in all activation, and niclosamide could reduce both the basal and tested colorectal cancer cells, whereas niclosamide showed min- Wnt-induced nuclear LEF1 (Fig. 2D). In fact, histopathologic imal effect in a normal colon epithelial cell line (CCD-18Co) even analysis determined that colon epithelial cells expressed LEF1 at a maximum concentration 20 mmol/L (Fig. 1A). These data are more intensely in cancerous tissues than in normal tissues consistent with previous report that niclosamide does not exert a (Fig. 2E). And fluorescence-activated cell sorting (FACS) analysis þ significant effect against proliferation in normal cells (20), sug- indicated that LEF1 were significantly enriched in LGR5 , þ þ gesting that niclosamide possesses a therapeutic window for its CD44v6 , and ALDH1A1 CSC populations (Fig. 2F). These data antitumor effects. Next, to determine whether niclosamide inhi- suggest that LEF1 expression is higher in cancer than in normal bits Wnt/b-catenin signaling, we performed TOP/FOP assay at tissue and further enriched in CSC populations. Therefore, we lower concentrations than IC50 (Fig. 1B). Indeed, niclosamide investigated whether LEF1 was critical for regulating CSC func- dose-dependently decreased the transcriptional activity of tions. First, we established LEF1 knockout HCT116 cells using the Wnt/b-catenin signaling by more than 50% at lower concentra- CRISPR-Cas9 genome editing system. We generated multiple tions than IC50. At approximately one-third of IC50 concentration clones of LEF1-knockout cells by using two different gRNA (0.4 mmol/L in HCT116 and 2 mmol/L in HT29), niclosamide sequences. LEF1 knockout clone #1 (sequence I) and clone #3 decreased Wnt/b-catenin transcriptional activity by 74.5% and (sequence II) were validated to lose LEF1 expression (Supple- 63.3% in HCT116 and HT29 cells, respectively. We used this mentary Fig. S2A), thus used for further analysis. We discovered concentration in functional and molecular analyses to minimize that various CSC populations were significantly decreased in both the effect on cellular proliferation. Previously, we have discovered LEF1-knockout clones (Fig. 2G). Moreover, the reduction of that Wnt/b-catenin signaling is activated to a greater extent in tumor sphere forming efficiency was observed in LEF1-knockout tumor cells than in normal cells and that it is further enhanced in cells, convincing the impaired self-renewal activity in the absence CSC populations for regulating self-renewal activity and chemore- of LEF1 (Fig. 2H). Also, we found that high LEF1 expression was sistance (29, 33, 34). Therefore, we hypothesized that niclosa- associated with poor prognosis such as low relapse-free survival mide might target CSCs by inhibiting Wnt/b-catenin signaling. and overall survival in patients with colorectal cancer (Fig. 2I), in Consistently, niclosamide treatment effectively decreased various consistent with previous report that high expression of LEF1 serves þ þ þ CSC populations such as LGR5 , CD44v6 , and Aldefluor CSCs as a poor prognostic marker in patients with colorectal cancer in multiple colorectal cancer cells (Fig. 1C; Supplementary (35). Additionally, we found that LEF1 expression was more Fig. S1B and S1C). Additionally, niclosamide inhibited the self- elevated in metastatic colorectal cancer than in nonmetastatic renewal ability of CSC populations (Fig. 1D; Supplementary colorectal cancer (Fig. 2J), and that chemoresistant colorectal Fig. S1D). Moreover, niclosamide-exposed CSCs could not fully cancer tumors displayed higher expression of LEF1 than chemo- restore their self-renewal activity even under niclosamide-free sensitive colorectal cancer tumors (Fig. 2K). Given that metastasis conditions. These effects were accompanied by a shift of mRNA and chemoresistance are typical features of CSCs resulting in poor patterns in which a set of stemness-related transcription factors prognosis in patients with colorectal cancer (4, 5), these findings was decreased, whereas differentiation marker ANPEP was suggest that LEF1 is positively correlated with cancer stemness in enhanced by niclosamide (Supplementary Fig. S1E). Next, we patients with colorectal cancer. Taken together, we first documen- investigated whether CSC-targeting niclosamide could suppress ted that LEF1, a potent target of niclosamide, is highly expressed in

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A HCT116 cells HT29 cells SW480 cells B HCT116 cells HT29 cells 1.5 1.5 1.5

IC50: 1.224 μmol/L IC50: 5.78 μmol/L IC50: 0.298 μmol/L 1.0 1.0 1.0 6 6 −Wnt activator 0.5 0.5 0.5 −Wnt activator 5 + +Wnt activator Wnt activator 4 4 0.0 0.0 0.0 102 103 104 105 102 103 104 105 102 103 104 105 Cell viability (fold) 3

Concentration (nmol/L) (fold) (fold) 2 2 CCD-18Co cells P#14005083 P#21257113 1.5 1.5 1.5 1 0

Relative luciferase activity luciferase Relative activity luciferase Relative 0 1.0 1.0 1.0 Niclosamide 0 0.2 0.4 0 0.2 0.4 024024 (μmol/L) 0.5 0.5 0.5 TOP FOP TOP FOP μ IC50: No cytotoxicity IC50: 3.647 μmol/L IC50: 5.364 mol/L 0.0 0.0 0.0 2 3 4 5 2 3 4 5 2 3 4 5

Cell viability (fold) 10 10 10 10 10 10 10 10 10 10 10 10 Concentration (nmol/L) Niclosamide treatment Without treatment

CDHCT116 cells First generation Second generation 0.2 μ 100 100 Niclosamide 0 0.4 mol/L 5.0 M 5.0 M 80 80 4.0 M 4.0 M 1.0 60 60 3.0 M 3.0 M 0.8 40 40 2.0 M 2.0 M Count Count First 20 20 1.0 M 1.0 M 0.6 Side scatter 0 0 0 0 5 6 7 8 5 6 7 8 generation 100 100 104 106 101 102 103 104 105 10 10 10 10 10 10 10 10 0.4

LGR5 CD44v6 Aldefluor Mean TSFE 0.2

Vehicle (IgG-stained) Vehicle (IgG-stained) Vehicle (+DEAB) HCT116 cells 0.0 Vehicle (LGR5-stained) Vehicle (CD44v6-stained) Vehicle (−DEAB) Niclosamide 0 0.2 0.4 0 0.2 0.4 Niclosamide (IgG-stained) Niclosamide (IgG-stained) Second μ Niclosamide (+DEAB) generation ( mol/L) Niclosamide (LGR5-stained) Niclosamide(CD44v6-stained) Niclosamide(−DEAB) Scale bar: 100 μm First generation Second generation

Niclosamide 024μmol/L 20 30 30 1.2 15 25 0.8

20 First 10 20 generation population (%) population (%)

population (%) 0.4 +

+ 10 5 + 15 Mean TSFE LGR5 HT29 cells 0.0 0 −+0 − +10 −+ CD44v6 Niclosamide Aldeflour Niclosamide 024 024 (0.4 μmol/L) Second (μmol/L) generation First generation Second generation Scale bar: 100 μm EFHCT116 cells HCT116 cells CD44v6+ population Sphere forming assay

30 80 1.5 1.5

+ + 60 20 1.0 1.0 40 10 0.5 0.5 Mean TFE CD44v6 CD44v6 20 Mean TSFE population (%) population (%) 0 0 0.0 0.0 5-FU − + − + RT − + − + 5-FU − + − + RT − + − + Niclosamide − − + + Niclosamide − − + + Niclosamide − − + + Niclosamide − − + +

HCT116 cells GHI Vehicle Niclosamide HCT116 cells HCT116 cells RT −+ −+ (1 Gy) Oxaliplatin 5-Fluorouracil 150 150 Vehicle Vehicle Niclosamide (0.2 μmol/L) Niclosamide (0.2 μmol/L) 100 100 1.5 −Niclosamide IC = 0.34 μmol/L μ 50 IC50 = 3.86 mol/L +Niclosamide (0.2 μmol/L) IC = 0.15 μmol/L 50 IC50 = 1.77 μmol/L 1.0 50 50 Cell viability (%) Cell viability (%) 0.5

0 0.0 101 102 103 104 102 103 104 105 106

Survival fraction (%) Survival fraction 0124810 Concentration (nmol/L) Concentration (nmol/L) Radiation (Gy)

Figure 1. Niclosamide attenuates stemness and therapy resistance in colorectal cancer cells. A, Proliferation inhibition by niclosamide treatment was determined for 48 hours by the MTT assay in multiple colorectal cancer cells, patient-derived primary cells (P#21257113 and P#14005083), and normal epithelial cells (CCD-18Co). (Continued on the following page.)

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various CSC populations and it is required for maintaining of the type A promoter and it regulates transcription of 47 kDa stemness. DCLK1 (DCLK1-B), which lacks two N-terminal DC domains. Thus, we quantified DCLK1 isoforms in various cells (Fig. 3G). DCLK1 is a target of LEF1 and positively correlated with cancer Western blot analysis determined that all colorectal cancer cells progression and CSCs in patients with colorectal cancer predominantly expressed DCLK1-B rather than DCLK1-A, in To elucidate the LEF1-mediated downstream target of niclosa- accordance with previous report that DCLK1-B is highly expressed mide, we compared gene expression profiles between metastatic in human colorectal cancer cells and tissues (30). Indeed, and nonmetastatic primary colorectal cancer samples DCLK1-B promoter was about 30-fold more activated than that (GSE41258) and applied gene expression data to gene set enrich- of DCLK1-A promoter in HCT116 cells, suggesting that the major ment analysis. In these computations, a group of genes that are expression of DCLK1-B may be derived from its highly active upregulated by LEF1 were significantly enriched in metastatic transcription (Supplementary Fig. S3C). Consistently, we con- colorectal cancers (FDR q-value < 0.005; Fig. 3A). Twenty LEF1 firmed that DCLK1-B was principally expressed in colorectal target genes were determined as the leading-edge subset, which cancer tissues and its expression was elevated in colorectal cancer contributed the most to the LEF1 enrichment signal in metastatic tissues than normal tissues (Fig. 3H; Supplementary Fig. S3D), as colorectal cancers. Next, we compared these genes with a set of described in previous report (30). Then, we enriched CSCs by stemness-related genes (n ¼ 3,401), which were generated by culturing multiple colorectal cancer cells in suspension condition combining multiple gene sets from three categories, namely, adult where CSCs were allowed to survive and proliferate to form stem cell-, embryonic stem cell-, and CSC-related genes. Seven spheres while non-CSCs were not. Then, immunofluorescence LEF1 target genes were related to stemness and upregulated in revealed that CSC-enriched spheres expressed DCLK1 at higher metastasis colorectal cancer versus nonmetastatic colorectal can- levels than did monolayer bulk cancer cells (Fig. 3I). In multiple cer tissues (Fig. 3B). Among them, DCLK1 was decreased the most colorectal cancer cells, we newly discovered that DCLK1-B was in a dose-dependent manner after niclosamide treatment (Fig. far more predominant than DCLK1-A in CSCs. Additionally, 3B). In silico analysis showed that DCLK1 expression was upre- DCLK1-A was also increased in colorectal cancer tissues than in gulated in chemoresistant colorectal cancer cells (Fig. 3C) and normal tissue (Fig. 3H; Supplementary Fig. S3D) and it was clinical evidence strongly suggested that DCLK1 expression is further enhanced in CSCs (Fig. 3J) in accordance with previous positively correlated with LEF1 expression in patients with colo- reports that DCLK1-A marks CSCs contributing to colorectal rectal cancer (GSE37892 and GSE14333), and with poor prog- cancer progression in multiple colorectal cancer animal models nosis in patients with colorectal cancer (GSE17538 and (8, 39). However, in these previous reports, DCLK1-B has been GSE14333; Fig. 3D and E). Additionally, the stem cell transcrip- overlooked, and more recently, overexpression of DCLK1-B has tion factor POU5F1 was positively correlated with DCLK1 in emerged as a prognostic factor in patients with colorectal cancer colorectal cancer patient tumors (GSE17538 and GSE14333; (30). Consistently, we observed the principal expression of Supplementary Fig. S3A). Consistent with genomic analyses, IHC DCLK1-B in colorectal CSCs. Therefore, we tried to figure out results revealed that the number of DCLK1þ cells was markedly whether DCLK1-B is critical for cancer stemness and whether it increased in the epithelial region of cancerous tissues than in that could be a target for niclosamide in colorectal cancer cells. of normal tissues (Fig. 3F). Previously, DCLK1þ colorectal cancer þ cells were determined to be CSCs originating from LGR5 stem Niclosamide effectively inhibits DCLK1-B expression via the cells, suggesting that DCLK1 can be a potential CSC-specific target Wnt/b-catenin-LEF1 axis (8). Our comprehensive genomic analysis indicating DCLK1 as a First, we investigated whether DCLK1-B was regulated by potential target of niclosamide that regulates Wnt-induced stem- Wnt/b-catenin signaling using multiple colorectal cancer cells. like properties in colorectal cancer was thus promising. Therefore, DCLK1-B was increased following Wnt activation at both mRNA we decided to perform further investigations on DCLK1 and its and protein levels (Fig. 4A). Next, because comprehensive geno- regulation by niclosamide. According to UniProt database mic analysis demonstrated that DCLK1 might be a target of LEF1, (https://www.uniprot.org), there are two types of DCLK1 protein we validated the molecular relationship between LEF1 and isoforms generated by two distinct promoter regions (Supple- DCLK1-B using LEF1-knockout HCT116 cells (Fig. 4B). LEF1 was mentary Fig. S3B; refs. 36–38). The A promoter (a-promoter) upregulated by Wnt3a in wild-type and control (empty vector- regulates the transcription of 82 kDa DCLK1 (DCLK1-A), which transfected) cells, whereas it was not in LEF1-knockout cells. contains two N-terminal doublecortin (DC) domains, a C-termi- Interestingly, DCLK1-B expression was significantly diminished nal serine/threonine kinase domain, and a middle serine/proline in LEF1-knockout cells and the upregulation of DCLK1-B by rich domain. The B promoter (b-promoter) exists in downstream Wnt3a was disrupted in the absence of LEF1, suggesting that

(Continued.) B, Both HCT116 cells and HT29 cells were transiently transfected with TOPflash and FOPflash vectors and treated with either niclosamide or a Wnt activator (LiCl, 20 mmol/L) for 18 hours. Luciferase activity was normalized by b-galactosidase activity. C, Reduction in CSC population following niclosamide treatment was determined in HCT116 cells by FACS analysis based on various CSC markers including LGR5, CD44v6, and Aldefluor. D, Self-renewal activity of both HCT116 cells and HT29 cells were assessed by tumorsphere-forming assays. Primary tumorspheres treated with niclosamide were collected and dissociated into single cells. These cells were subsequently replated in culture dishes without additional treatment to form secondary tumorspheres. Scale bar represents 100 mm. E and F, HCT116 cells were treated with 5-FU (100 nmol/L) or exposed to radiation (2 Gy) with or without niclosamide 0.2 mmol/L. After 48 hours, cells were subjected to (E) FACS analysis to estimate the proportion of CD44v6þ CSCs and (F) their self-renewal capacity was determined by sphere-forming assays. G and H, HCT116 cells were treated with various concentrations of (G) oxaliplatin or (H) 5-FU with or without niclosamide treatment. Cell viability was measured by MTT assays to determine whether niclosamide sensitizes cells to anticancer drugs. I, HCT116 cells were exposed to various doses of radiation from 1 to 10 Gy with or without niclosamide treatment. Proportion of surviving cells was determined by counting the number of surviving colonies. Bar graphs represent the mean SD, and statistical analyses were performed by one-way ANOVA with Dunnett's multiple comparison. , ,and indicate P < 0.05, P < 0.01, and P < 0.001, respectively.

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ABHCT116 cells HT29 cells SW480 cells C HCT116 cells LEF1 LEF1 β Niclosamide 0 0.2 0.4 1.5 Wnt/ -catenin-related genes 1.5 1.5 μ ( mol/L) 1.0 MYC 1.0 1.0 LEF1 0.5 β 0.0

-Actin change) (fold BMP4 0.5 0.5 0 0.2 0.4 Relative Relative

Relative expression Relative Niclosamide (μmol/L) TCF4

mRNA expression 0.0 mRNA expression 0.0 HT29 cells BAMBI Niclosamide 024 0 0.05 0.1 μ ( mol/L) Niclosamide 0 2 4 1.5 CTNNB1 (μmol/L) 1.0 P #14005083 P #21257113 CCND1 LEF1 0.5 LEF1 LEF1 β-Actin 0.0 AXIN2 1.5 1.5 change) (fold 0 2 4

Relative expression Relative μ WNT1 Niclosamide ( mol/L) 1.0 1.0

1.00.50 P #14005083 WNT3a

Relative 0.5 Relative 0.5 Niclosamide 0 1 2 LEF1 1.5 (μmol/L)

mRNA expression 1.0 Niclosamide 0 0.2 0.4 0.0 mRNA expression 0.0 LEF1 (μmol/L) Niclosamide 012 024 0.5 (μmol/L) β-Actin 0.0

(fold change) (fold 0 1 2 μ Relative expression Relative Niclosamide ( mol/L)

DENuclear LEF1 levels in HCT116 cells P #31701313 P #6441493 Wnt activator − + − + Niclosamide − − + + LEF1 Merge LEF1 Merge

DAPI tissues Normal

LEF1

Merge tissues Cancerous μ LEF1/DAPI Scale bar : 50 μm LEF1/DAPI Scale bar : 100 m FGHCT116 cells HCT116 cells CSC population LGR5 CD44v6 ALDH1A1 30 10 50

100 100 100 + NS LGR5− CD44v6− − + NS 8 NS 40

ALDH1A1 + + + 20 80 LGR5 80 CD44v6 80 ALDH1A1+ 6 30 60 60 60 10 4 20 LGR5 CD44v6 40 40 40 2 Aldefluor 10 % of 20 20 20 population (%) 0 population (%) 0 population (%) 0 Maximum Empty vector − + −− − + −− − + −− 0 0 0 ++ −−++ −−+− 100 102 104 106 108 100 102 104 106 100 102 104 106 LEF1 KO −− LEF1 #1Clone#3 #1Clone#3 #1Clone#3 100 HCT116 cells H #1Clone #3 1.0 NS 80 Empty vector −+− − 0.8 60 LEF1 KO −++ + 0.6 40 0.1 LEF1 (%)

20 Mean TSFE Expression of 0 0.0 −+−+− + Empty vector − +−− Scale bar: 100 μm − − + + LGR5 CD44v6 ADLH1A1 LEF1 KO #1Clone#3 IJKGSE 14333 Boyer cell line GSE 39582 GSE 17538 12 0 100 100 10 −1 8 −2 50 50 6 −3 LEF1-high (n = 104) LEF1-high (n = 144) 4 LEF1-low (n = 453) LEF1-low (n = 55) − 2 LEF1 mRNA expression 4 Overall survival (%) Overall P value = 0.0056 P value = 0.0015 LEF1 mRNA expression 0 0 A BCD Resistant Sensitive Relapse-free survival (%) 024487296120144168192 0 24 48 72 96 120 (n = 44)(n = 94) (n = 91)(n = 61) (n = 3) (n = 3) , Months Months Dukes stage 5-Fluorouracil response status

Figure 2. Niclosamide inhibits cancer stemness by attenuating LEF1 in colorectal cancer cells. A, Inhibitory effect of niclosamide on the expression of Wnt/b-catenin pathway-related genes was determined by RT-PCR in HCT116 cells after 48-hour treatment of niclosamide. Quantitation was performed by RT-PCR, and data are presented as fold change relative to control (log2). (Continued on the following page.)

OF6 Clin Cancer Res; 2019 Clinical Cancer Research

Niclosamide Targets DCLK1-Mediated Cancer Stem Functions

DCLK1-B was regulated by Wnt/b-catenin signaling and LEF1 targeting DCLK1-B and validated that two siRNA sequences might be involved in DCLK1-B regulation. Next, we discovered (siDCLK1-B #1 and siDCLK1-B #2) efficiently decreased that DCLK1-B was significantly decreased by niclosamide at both DCLK1-B in colorectal cancer cells without affecting DCLK1-A mRNA and protein levels in a dose-dependent manner (Fig. 4C (Supplementary Fig. S5A–S5C). Limiting dilution assay demon- and D). Given that niclosamide treatment led to a significant strated that DCLK1-B-knockdown impaired self-renewal abilities suppression in DCLK1-B promoter activity (Fig. 4E), niclosamide of CSCs (Fig. 5A), resulting in a significant decrease in survival might regulate DCLK1-B at transcription level. Therefore, we potential of CSCs (Fig. 5B; Supplementary Fig. S5D). Moreover, subsequently investigated whether Wnt/b-catenin signaling DCLK1-B-knockdown increased AnnexinVþ apoptotic cells directly regulates the transcription of DCLK1-B on its promoter (Fig. 5C) by activating apoptotic cascades, such as cleaved PARP region. To this end, we searched the LEF1-binding sites on DCLK1- and cleaved caspase 3 (Supplementary Fig. S5E and S5F). In B promoter using a prediction tool, ALLGEN PROMO database addition, CSC inhibitory effects of DCLK1-B-knockdown led to version 3.0.2 (http://alggen.lsi.upc.es/cgi-bin/promo_v3/promo/ sensitization of colorectal cancer cells to anticancer therapy such promoin it.cgi?dir DB¼TF_8.3). We found three potential LEF1- as 5-FU and radiation (Fig. 5D and E). Next, to estimate in vivo binding sites at 1743, 1595, and 1412 bp (Fig. 4F). Then function of DCLK1-B, we established DCLK1-B-knockout chromatin immunoprecipitation assay revealed that LEF1 bound HCT116 cells by CRISPR-Cas9 system. Multiple DCLK1-B- to DCLK1-B promoter between 1895 and 1329 regions. Nota- knockout clones were generated using two different gRNA bly, niclosamide inhibited the binding of LEF1 to DCLK1-B sequences (Supplementary Fig. S5G). Clone #1 (sequence I) and promoter (Fig. 4G). Collectively, we first demonstrated that clone #3 (sequence II) were validated to lose DCLK1-B expression niclosamide could target DCLK1-B through abrogating its tran- without affecting DCLK1-A. In similar with DCLK1-B-knockdown scription on its promoter region. In further investigation, we effects (Fig. 5A), the frequency of self-renewing CSC and found that two siRNAs targeting LEF1 (siLEF1) showed different the proportion of CSC populations were diminished by efficacy on LEF1-knockdown, and that the more the siLEF1 DCLK1-B-knockout (Supplementary Fig. S5H and S5I). Next, we decreased LEF1 expression, the more DCLK1-B expression was evaluated in vivo CSC properties of DCLK1-B-knockout cells reduced (Supplementary Fig. S4A). Similarly, DCLK1-B reduction (clone #1) using two different colorectal cancer mouse models. was far more significant in LEF1-knockout cells than in LEF1- Because growing evidence suggests that CSCs display greater knockdown cells. Thus, these data convinced our theory that tumorigenic and metastatic potential in vitro and in vivo than DCLK1-B expression is dependent on LEF1 expression. In fact, non-CSC cancer cells (4), we estimated both tumor-initiating and LEF1 mediates nuclear responses to Wnt signals by forming the metastatic potential of DCLK1-B-knockout cells. First, we subcu- transcriptional regulatory complexes with other components such taneously transplanted limiting dilutions of cell preparations into as b-catenin and T-cell transcription factor (TCF) families. Indeed, mice and found that the frequency of tumor-initiating cells was we found that silencing of b-catenin or TCF4 reduced DCLK1-B decreased with a statistically significance (Fig. 5F; Supplementary expression (Supplementary Fig. S4B and S4C), proposing that a Fig. S5J). Moreover, DCLK1-B-knockout significantly reduced LEF1/b-catenin/TCF4 complex may be involved in DCLK1-B primary tumor volume (Fig. 5G; Supplementary Fig. S5K). Next, regulation. Also, DCLK1-A was suppressed by silencing of LEF1, we used a tail vein injection model which is a common model for b-catenin, or TCF4 (Supplementary Fig. S4A–S4C). These data were studying lung metastasis. Tail vein injection of HCT116 cells in consistent with previous report that DCLK1-A is transcriptionally resulted primarily in pulmonary metastases. Interestingly, regulated by a direct binding of LEF1/b-catenin/TCF4 complex to its DCLK1-B-knockout diminished both the number and size of DCLK1-A promoter (30). Furthermore, we firstly determined that metastatic colonies on lungs (Fig. 5H and I), suggesting that Wnt3a ligand could upregulate DCLK1-A in multiple colorectal DCLK1-B expression appears to be critical for founding of met- cancer cells (Supplementary Fig. S4D) and provided new evidences astatic colonies and their subsequent robust outgrowth. Finally, that niclosmide could suppress DCLK1-A at transcription level we examined whether the reduction of DCLK1-B mediates CSC- (Supplementary Fig. S4E–S4G). Taken together, our data newly inhibitory effects of niclosamide by using DCLK1-B-overexpres- discovered that DCLK1 is a downstream target of Wnt/b-catenin sing cells (Supplementary Fig. S5L). DCLK1-B-overexpressing signaling, therefore, a potent target of niclosamide. cells displayed the increased CD44v6þ CSC population and self-renewal activities. More importantly, DCLK1-B overexpres- DCLK1-B expression is a critical driver of initiation and sion rescued cancer stemness under niclosamide treatment (Fig. 5J maintenance of colorectal cancer stemness and K), suggesting that suppressive effects of niclosamide on CSCs To examine whether DCLK1-B is critical for regulating cancer are achieved, at least in part, through disruption of DCLK1-B stemness in colorectal CSCs, we designed siRNAs specifically expression. Taken together, our results indicated that DCLK1-B, a

(Continued.) B and C, Inhibitory effect of niclosamide on LEF1 expression was assessed in colorectal cancer cells through (B) real-time PCR and (C) Western blotting in HCT116 cells after 48-hour treatment of niclosamide. D, Stimulatory effect of Wnt activator (LiCl, 20 mmol/L) on nuclear LEF1 proteins was attenuated by niclosamide treatment (0.4 mmol/L). E, LEF1 expression levels were visualized by immunostaining in paired normal tissues and cancer tissues obtained from patients with identical colorectal cancer. They were presented with matched H&E images. Blue indicates nuclei, and red indicates LEF1. Scale bar represents 100 mm. F, FACS analysis revealed LEF1 expression levels in LGR5þ, CD44v6þ, and ALDH1A1þ CSC populations in HCT116 cells. G and H, Reduction in cancer stemness in LEF1-knockout cells were determined by evaluating (G) various CSC populations and (H) their self-renewal activity. G, FACS analysis demonstrated the number of LGR5þ, CD44V6þ, and ALDEFLUORþ CSCs. H, Self-renewal activity of LEF1-knockout cells was determined by sphere-forming assays. I, Kaplan–Meier survival analyses were conducted in patients with CSC based on LEF1 expression in two independent cohorts (GSE39582 and GSE17538). J, LEF1 expression were signiﬁcantly elevated in metastatic colorectal cancer patient tumors (Dukes' stage D) versus early noninvasive colorectal cancer patient tumors (Dukes' stage A; GSE14333). K, Comparison of LEF1 expression between chemoresistant and chemosensitive colorectal cancer cells from Oncomine (Boyer cell line). Bar graphs represent the mean SD. Statistical analyses were performed by one-way ANOVA with Dunnett's multiple comparison. , ,and indicate P < 0.05, P < 0.01, and P < 0.001, respectively.

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ANO1 ABAXL C CBS Gyorffy cell line 1 GSE 41258 3,394 CRABP2 13 DCLK1 7 0 MAOB

Gene symbol OBSL1 −1 MAOB TMOD1 LEF1-target gene Stem-related gene − SLC43A3 (n = 20) (n = 3,401) DCLK1 mRNA expression 2 INHBE Sensitive Resistant NES = 2.605 AKAP12 (n = 20) (n = 6) FDR q-value = 0.000 WASF3 MAP1B PDLIM4 0.4 Doxorubicin response status OBSL1 HEY2 Gyorffy cell line FNBP1 0.2 −1.6 DCLK1 − AXL 2 1.8 ANO1 1 −2.0 TMEM47 0 − CBS 0 2.2 ZEDM2 Niclosamide −2.4 AXL

CRABP2 CBS μ −2.6

NRIP3 ( mol/L) ANO1 MAOB DCLK1 PHLDA3 OBSL1 − DCLK1 mRNA expression 2.8 CRABP2 Sensitive Resistant Stem-related LEF1 target genes (n = 27) (n = 3) Paclitaxel response status D E GSE37892 12 GSE14333 100 GSE 17538 100 GSE 14333 15

10 9 50 50 LEF1 LEF1 5 6 DCLK1-high (n = 25) DCLK1-high (n = 28) P value : 0.0002 P value : 7.4e−06 ∗∗∗P value = 0.00064 ∗∗∗P value = 0.001 DCLK1-low (n = 175) R : 0.3180 R : 0.260 0 0 DCLK1-low (n = 198) 0 3 Relapse-free survival (%) −5 0510 15 0246 810 Disease free survival (%) 0 48 96 144 192 0 482472 96 120 DCLK1 DCLK1 Months Months

2 μg 20 μg F P #31701313 P #6441493 G

DCLK1 Merge DCLK1 Merge Mouse brainCCD-18CoHCT116HT29 SW480 P #27423233P #14005083 100 kDa DCLK1-A tissues Normal (82 kDa)

55 kDa DCLK1-B

tissues (47 kDa) Cancerous β-Actin DCLK1/DAPI Scale bar : 100 μm H I J 2 μg 20 μg HCT116 HT29 P #14005083 20 μg DAPI DCLK1 Merge Mouse brain Mono Sphere Mono Sphere Mono Sphere 07312870 07376895 07499140 07094012 100 kDa NormalTumor NormalTumor NormalTumor NormalTumor Monolayer DCLK1-A DCLK1-A (82 kDa) (82 kDa) DAPI DCLK1 Merge DCLK1-B 55 kDa (47 kDa) DCLK1-B (47 kDa) β-Actin Sphere β-Actin

DCLK1/DAPI Scale bar : 100 μm

Figure 3. DCLK1 is a target of LEF1 and correlates with CSCs and poor prognosis in patients with colorectal cancer. A, GSEA of metastatic colorectal cancer patient tumors showed significant enrichment in LEF1 target genes (FDR q-value < 0.005; GES41258). B, A set of leading-edge subset of LEF1 target genes and a set of stemness-related genes (described in the Supplementary Materials and Methods) were compared, and seven common genes were identified. mRNA levels of the seven genes were quantified by real-time PCR following 48-hour treatment of niclosamid. C, mRNA expression levels of DCLK1 were compared between chemosensitive and chemoresistant colorectal cancer cells from Oncomine (Gyorffy cell line). D, Significant positive correlations between DCLK1 and LEF1 were observed in colorectal cancer patient tumors (GSE37892 and GSE14333). Statistical significance was determined by correlation analysis using GraphPad software. E, Kaplan–Meier survival analyses were conducted based on DCLK1 expression in two independent colorectal cancer patients cohorts (GSE17538 and GSE14333). F, DCLK1 expression was determined by immunofluorescence assay in patient-derived colorectal cancer tissues and the paired normal adjacent to tumor (NAT) tissues from the patients with identical colorectal cancer. G, Protein levels of DCLK1-A (82 kDa) and DCLK1-B (47 kDa) isoforms were determined in normal mouse brain tissues, normal human colon epithelial cells, and human colorectal cancer cells by Western blot assays. Total amount of loaded protein is indicated above the wells. b-Actin was used as a loading control. H, DCLK1-A and DCLK1-B protein levels were determined in colorectal cancer tissues and the normal tissues obtained from the identical patients with colorectal cancer. I, HCT116 cells were cultured under attached monolayer condition or sphere-forming conditions to enrich for CSCs. Immunofluorescence assays were performed to visualize DCLK1 expression. Blue indicates nuclei, and red indicates DCLK1. Scale bar represents 100 mm. J, DCLK1-A and DCLK1-B protein levels were compared between attached monolayer colorectal cancer cells and CSC-enriched sphere colorectal cancer cells by Western blot assays. Bar graphs represent the mean SD, and statistical analyses were performed by one-way ANOVA with Dunnett's multiple comparison. , ,and indicate P < 0.05, P < 0.01, and P < 0.001, respectively.

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A HCT116 cells HT29 Cells P #14005083 1 hours 2 hours 3 hours 1 hours 2 hours 3 hours 1 hours 2 hours 3 hours Wnt3a Wnt3a Wnt3a −+−+−+ − + − + − + −+ − + − + (100 ng/mL) (100 ng/mL) (100 ng/mL) DCLK1-B DCLK1-B DCLK1-B

β-Actin β-Actin β-Actin

DCLK-B DCLK-B DCLK-B 4 4 4 3 3 3

2 2 2 Relative Relative 1 Relative 1 1 mRNA levels mRNA levels mRNA levels 0 0 0 Wnt3a +−+− − + Wnt3a −++ −+− Wnt3a −++ −+− (100 ng/mL) 123 hours (100 ng/mL) 1 23hours (100 ng/mL) 1 23hours

B C HCT116 cells HT29 cells P #14005083 HCT116 cells Clone #1 Clone #3

Wnt3a − + − + − + − + 1.5 1.5 − − + + − −−− 1.5 Empty vector 1.0 LEF1 KO −−−−+ +++ 1.0 1.0

DCLK1-B 0.5 0.5 0.5 Relative mRNA levles Relative Relative mRNA levles Relative LEF1 mRNA levles Relative 0.0 0.0 0.0 Niclosamide 0 0.2 0.4 Niclosamide 0 2 4 Niclosamide 0 1 2 β-Actin (μmol/L) (μmol/L) (μmol/L)

E HCT116 cells D HT29 cells P #14005083 HCT116 cells DCLK1-B promoter Niclosamide 0 0.2 0.4 Niclosamide 0 2 4 Niclosamide 0 0.2 0.4 (μmol/L) (μmol/L) (μmol/L) Vehicle 1.4 μ DCLK1-B DCLK1-B DCLK1-B 0.2 mol/L Niclosamide 0.4 μmol/L Niclosamide β-Actin β-Actin β-Actin 1.2

1.5 1.5 1.5 1.0

1.0 1.0 1.0 Relative RLU Relative

Protein level 0.8 0.5 0.5 0.5 (fold change) (fold (fold change) (fold change) (fold Relative expression Relative Relative expression Relative 0.0 0.0 expression Relative 0.0 0.6 Niclosamide 0 0.2 0.4 Niclosamide 0 24Niclosamide 0 1 2 μ μ μ ( mol/L) ( mol/L) ( mol/L) Niclosamide 0 0.2 0.4 (μmol/L) F G HCT116 cells

Input IgG LEF1 ChIP

Niclosamide − + − + − + 1,000 LEF1 LEF1 LEF1 TATAA 500 bp

−1895 −1329 1.5 − 918 − 1412 − 1743 − 1595 1.0

0.5 Band intensity 0.0 Niclosamide − +−− + +

Input Normal LEF1 ChIP IgG

Figure 4. Niclosamide effectively inhibits DCLK1-B expression via the Wnt/b-catenin-LEF1 axis. A, Time-dependent induction of DCLK1-B protein and mRNA levels following Wnt activation in multiple colorectal cancer cells. Cells were treated with Wnt3a (100 ng/mL) in the absence of serum for indicated times, then they were subjected to real-time PCR or Western blot analysis. B, Protein levels of DCLK1-B and LEF1 were determined in wild-type, control (empty vector-transfected), and LEF1-knockout cells with or without Wnt activation (Wnt3a, 100 ng/mL, 2 hours) by Western blot analysis. C and D, Inhibitory effects of niclosamide on DCLK1-B expression were determined in various human colorectal cancer cells by (C) RT-PCR and (D) Western blot assays. Cells were treated with niclosamide for 48 hours. E, Promoter reporter assay was performed against DCLK1-B promoter in HCT116 cells following 24-hour treatment of niclosamide in dose-dependent manner. F, Predicted binding sites of LEF1 on DCLK1-B promoter region according to ALLGEN PROMO database version 3.0.2. G, ChIP assay was conducted in HCT116 cells with or without niclosamide treatment (0.2 mmol/L). Immunoprecipitation was conducted against LEF1 and LEF1-bound DNA fragments ampliﬁed by PCR. PCR products were conﬁrmed by gel electrophoresis. Bar graphs represent the mean SD (n ¼ 3), and statistical analyses were performed by one-way ANOVA with Dunnett's multiple comparison. , ,and indicate P < 0.05, P < 0.01, and P < 0.001, respectively.

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HCT116 cells HT-29 cells 0 A 0 siDCLK1-B #1 siDCLK1-B 1/stem cell frequency #1 − 1 − 1 1/stem cell frequency Group Lower Estimate Upper P value Group Lower Estimate Upper P value − 2 − 2 siDCLK1-B siCTRL 2500 1518 922 - siCTRL 1600 963 579 - siDCLK1-B #2 siDCLK1-B siDCLK1-B siCTRL 8732 5477 3435 0.000173 4851 3070 1943 0.0011 #1 siCTRL #2 #1 − 3 Log fraction without sphere Log fraction − 3 Log fraction without sphere Log fraction siDCLK1-B siDCLK1-B 6425 4076 2587 0.00387 4 4109 2581 1622 0.00569 0 2 4 6 810 #2 0 2 6 810 #2 Number of cells seeded Number of cells seeded 5 5 (×10 cells/well) (×10 cells/well) B C HCT116 cells HCT116 cells siDCLK1-B siDCLK1-B 250 20 NS WT siCTRL #2 siDCLK1-B siDCLK1-B #1 200 WT siCTRL #1 #2 15 150 NS 10 100

Annexin V+ Annexin 5 50 Annexin V Annexin Number of colonies apoptotic cells (%) 0 0 siCTRL − + −− − + −− PI siCTRL siDCLK1-B − − ++ siDCLK1-B − − ++ Sequence #1 #2 Sequence #1 #2

D HCT116 cells 5-Fluorouracil E HCT116 cells 1.5 siCTRL 120 siCTRL siDCLK1-B siCTRL siDCLK1-B #1 NS 100 siDCLK1-B #1 RT (1 Gy) −+ − + 1.0 80 IC = 4.056 μmol/L 60 50 IC50 = 0.480 μmol/L 0.5 40 Cell viability (%)

20 (%) Survival fraction 0.0 0 0 1 248 10 2 3 4 5 6 10 10 10 10 10 Radiation (Gy) Concentration (nmol/L) 1/stem cell frequency G Cell no. of inoculation: 1 × 106 F Tumor formation monitoring Group Lower Estimate Upper P value DCLK1-B KO 200

) 150

(clone #1) Empty 4 5 3 3 − 10 2.36 × 10 1.0 5× 10 4.71 × 10 - vector 100

0 7 14 21 (Days) (mm DCLK1-B 50 5 5 5 1.06 − 2 × 4.01 × 10 1.97 × 10 −

KO 8.17 10 11 volume Tumor ×10 0 S.C. injection Tumor Empty vector (clone #1) Empty DCLK1-B HCT116 cells incidence (Determined after necropsy) vector KO − 3

(empty vector and DCLK1-B KO) and without tumor Log fraction 0 2 46810 (clone #1) necropsy Number of cells inoculated (×105) H I

300 0 7 14 21 28 (Days) Empty vector Tail vein injection Necropsy 200 HCT116 cells (empty vector and DCLK1-B KO)

in lungs 100 vector Empty

0 Number of metastatic nodules (clone #1)

KO + − Empty vector DCLK1 -B KO (clone#1) DCLK1 -B DCLK1-B KO − + Scale bar: 4 mm Scale bar: 600 μm (clone #1)

J HCT116 cells HCT116 cells K DCLK1-B OE 2.5 − 100 Empty vector + − + DCLK1-B OE − + 80 2.0 + 60 1.5 − 40 1.0 Mean TSFE CD44v6 20 0.5 population (%) Count 0 0.0 Empty vector ++− − Empty vector ++−− Niclosamide DCLK1-B OE − − ++ + DCLK1-B OE −−++ CD44v6 − + − + − + +− Vehicle (IgG-stained) Niclosamide (IgG-stained) Niclosamide Niclosamide Vehicle (CD44v6-stained) Niclosamide (CD44v6-stained) Scale bar : 100 μm

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Niclosamide Targets DCLK1-Mediated Cancer Stem Functions

downstream target of niclosamide, is critical for maintaining CSC niclosamide-treated primary tumors and evaluated their self- populations and CSC properties both in vitro and in vivo. renewal ability through sphere-forming assays (Fig. 6H). HCT116 cells from niclosamide-treated mice did not form tumor spheres Niclosamide exerts a potent in vivo antitumor effect in both as efficiently as HCT116 cells from vehicle-treated mice did. These HCT116 xenografts and AOM/DSS-induced spontaneous in vivo results are consistent with our in vitro data, showing that colorectal cancer models niclosamide-exposed colorectal cancer cells could not restore Following mechanistic studies of niclosamide on CSC regula- their self-renewal ability even under niclosamide-free conditions tion, we investigated whether niclosamide can be a potential (Fig. 1D). Immunohistochemical analyses demonstrated that þ therapeutic agent for colorectal cancer using multiple in vivo CD44v6 CSC populations were decreased in niclosamide-trea- systems. First, to investigate the tumor growth-inhibitory effect ted primary tumors (Fig. 6I), indicating the reduced expression of of niclosamide, we subcutaneously xenografted HCT116 cells and stemness-related transcription factor OCT4 (Fig. 6J). In the same treated mice with niclosamide (10 or 40 mg/kg) when the mean manner, in vivo limiting dilution assay revealed that niclosamide primary tumor volume reached 100 mm3. Compared with vehi- treatment irreversibly reduced the frequency of tumor-initiating cle, niclosamide significantly inhibited primary tumor growth cells in PDX tumors (Supplementary Fig. S6E) resulting in (Fig. 6A–C). In addition, 10 mg/kg seemed to be sufficiently reduction of tumor growth (Supplementary Fig. S6F). According- potent in exerting a maximum effect on primary tumor growth, ly, the molecular targets of niclosamide, LEF1 and DCLK1, were because there was no significant difference between 10 and 40 mg/ decreased in niclosamide-treated primary tumors (Fig. 6K and L; kg niclosamide-treated groups. Also, we evaluated therapeutic Supplementary Fig. S6G and S6H). Thus, CSC-targeting niclosa- effect of niclosamide in patient-derived tumor xenograft (PDX) mide efficiently attenuated both tumor growth and AOM/DSS- model (Supplementary Fig. S6A–S6C), which was authenticated induced colonic dysplasia, suggesting that the inhibition of by short tandem repeat profiling analysis (Supplementary Fig. Wnt/LEF1/DCLK1 axis might be a new therapeutic strategy for S6D). The growth of PDX tumors were also inhibited by niclo- colorectal cancer treatment as well as chemoprevention of colo- samide treatment (40 mg/kg). Second, we used the classical rectal cancer development. model of spontaneous colorectal cancer, AOM/DSS model, to evaluate whether niclosamide can be used as a chemopreventive Discussion agent against colorectal cancer development (Fig. 6D). The niclosamide treatment (40 mg/kg) from week 1 to 13 significantly The use of niclosamide, an anthelmintic family drug, has now blocked colorectal cancer development, indicating the decreased been extended to multiple disease models including Helicobacter number of colorectal polyps than control mice (Fig. 6E). Addi- pylori infection, Parkinson's disease, and severe acute respiratory tionally, AOM/DSS treatment significantly reduced colon length, syndrome (40–42). Recently, this antiparasitic drug has been which is a classical symptom of inflammation; however, co- proposed as a promising anticancer agent in several types of treatment with niclosamide blocked the AOM/DSS-induced cancer due to its remarkable ability to inhibit tumor growth shortening of colon (Fig. 6F). Microscopic observations revealed (20–24). However, the potential mechanism of niclosamide that niclosamide efficiently blocked the AOM/DSS-induced during colorectal cancer initiation and progression is still elusive. development of intermediate- or high-grade dysplasia CSCs are believed to initiate cancer and be resistant to chemor- (Fig. 6G). Immunofluorescence analysis demonstrated that adiation by demonstrating higher survival potential and lower AOM/DSS-induced dysplastic tissues expressed higher levels of apoptosis than non-CSCs, thereby resulting in metastasis and DCLK1 than did normal tissues, and niclosamide reduced the poor prognosis (43). In this study, we reported that niclosamide number of DCLK1þ dysplastic regions. Next, to investigate efficiently decreases therapy resistance in colorectal cancers whether this anticancer effect of niclosamide is derived from its by reducing CSC populations and their self-renewal activity, CSC-targeting effect, we isolated HCT116 cells from vehicle- or thereby attenuating the survival potential of CSCs following

Figure 5. DCLK1-B is a critical driver of the initiation and maintenance of colorectal stemness. A, Limiting dilution assay was performed to compare the tumor-initiating potential of CSCs in control cells and in DCLK1-B-knockdown cells. HCT116 cells (left) or HT29 cells (right) were transfected with nontargeting siRNA (siCTRL) or DCLK1-B-targeting siRNAs (siDCLK1-B). Two different siRNA sequences were used. B, Clonogenic assay was performed to compare the survival potential in control (siCTRL-transfected) and in DCLK1-B-knockdown HCT116 cells. The siRNA-transfected cells were seeded in 12-well plates and cultured for 2 weeks in growth media. The number of surviving colonies were counted per wells. C, The apoptotic cells were visualized by staining Annexin Vþ cells. Cells were transfected with the indicated siRNAs and cultured in serum-free media for 48 hours. Apoptotic cells were measured by FACS analysis. D, Cell viability after 48-hour treatment with 5-FU was measured by MTT assay. IC50 values of 5-FU was evaluated by GraphPad Prism using a nonlinear regression model. E, Cell survival fraction was determined two weeks after the radiation exposure (2 Gy) by clonogenic assay in control or DCLK1-B-knockdown HCT116 cells. F and G, Limiting dilution assay was performed as PDX models which were described above. DCLK1-B knockout or control cells were subcutaneously inoculated with Matrigel into the inguinal folds of the NSG mice at four doses (1 103,1 104,1 105, and 1 106,8–10 replicates/group). Tumor formation was observed for 3 weeks following inoculation. G, Primary tumor volumes were presented as a box and whisker plot. Boxes represent the 25th to 75th percentiles, and horizontal lines within the boxes represent the median (control cells: n ¼ 8, DCLK1B-KO cells: n ¼ 10). H and I, DCLK1-B-knockout HCT116 or control cells were inoculated into mouse tail vein and the number of metastatic nodules were counted (n ¼ 6/group). H, Representative image of Indian ink-stained lungs were provided and the number of metastatic nodules were counted. I, Representative image of H&E-stained lungs shows metastatic colonies on lungs. J and K, Niclosamide-induced cancer stemness was restored by DCLK1-B overexpression. Control (empty vector-transfected) or DCLK1-B-overexpressing HCT116 cells were treated by 0.4 mmol/L niclosamide for 48 hours, and then (J) CD44v6þ CSC populations were measured by FACS analysis and (K) their self-renewal activities were determined by analyzing the tumor sphere-forming efﬁciency. Bar graphs represent the mean SD. Statistical analyses were performed by one-way ANOVA with Dunnett's multiple comparison. , ,and indicate P < 0.05, P < 0.01, and P < 0.001, respectively.

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C 0.4 A 800 B

) Vehicle 3 0.3 600 10 mg/kg Niclosamide

40 mg/kg Niclosamide 0 0.2 400 10

Tumor weight (g) weight Tumor 0.1 200 Tumor volume (mm volume Tumor

40 0.0 0 Niclosamide (mg/kg) Niclosamide 0 10 40 10 30 40 20 (mg/kg) Days after treatment G D AOM/DSS − + + Niclosamide (20 mg/kg, twice a day, i.p.) Niclosamide −− + 2.0% 2.0% 2.0% 0 1 2 4 5 7 8 13 (weeks)

AOM (10 mg/kg, i.p.) Necropsy F E Number of polyps Colon length

12 25

20 10

15 8 10 6 5 Colon length (cm) Number of polyps

0 (from cecum to rectum) 4 + AOM/DSS − + AOM/DSS − + + Niclosamide − − + Niclosamide − −+ DCLK1/DAPI Scale bar : 100 μm H Niclosamide I treatment CD44v6 expression level CSC sphere formation CD44v6 Merge (days) 1.5 Single cells 0 7 HCT116 cells 1.0 1.0 Niclosamide 0 10 40 0.8 (mg/kg) 0.6 0.5 Fluorescence 0.4 intensity (fold) 0.0 TSFE (%) 0.2 − + 0.0 Niclosamide Vehicle Scale bar : 100 μm Scale bar : 100 μm Niclosamide 0 10 40 CD44v6/DAPI Niclosamide (mg/kg) J K L OCT4 expression level LEF1 expression level DCLK1 expression level

OCT4 Merge LEF1 Merge DCLK1 Merge Vehicle Vehicle Vehicle Niclosamide Niclosamide Niclosamide μ μ OCT4/DAPI Scale bar : 100 m LEF1/DAPI Scale bar : 100 μm DCLK1/DAPI Scale bar : 100 m 1.5 1.5 1.5 1.0 1.0 1.0 0.5 0.5 0.5 Fluorescence intensity (fold)

0.0 Fluorescence 0.0

Fluorescence 0.0 intensity (fold) − + − + intensity (fold) −+ Niclosamide Niclosamide Niclosamide

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Niclosamide Targets DCLK1-Mediated Cancer Stem Functions

chemoradiation (Fig. 1C–I). These findings potentially broaden serves as a potent anticancer drug by inhibiting tumor growth the clinical application of niclosamide not only to shrink tumors (Fig. 6A–C). Also, we newly documented that in vivo treatment but also to improve the efficacy of current chemoradiotherapy. with niclosamide irreversibly impaired the self-renewal activity Intensive investigations are currently focusing on targeting of CSCs in primary tumors resulting in a reduction in CSC CSCs; however, therapeutic strategies targeting CSCs are still populations (Fig. 6H and I). In fact, niclosamide treatment has limited because CSC markers are often shared by normal stem been demonstrated to exhibit in vivo antitumor activity in cells (NSC). In 2012, DCLK1 was first identified as a CSC-specific HCT116 subcutaneous tumors when it was treated four days marker through in vivo lineage-tracing experiments in colorectal after tumor cell inoculation (20), suggesting that niclosamide cancer mouse models (39). DCLK1 is expressed in differentiated abrogates primary tumor formation. In this study, we used tuft cells but not in NSCs in the normal intestine, whereas DCLK1 the same HCT116 subcutaneous model, but we started the is detected in CSCs that continuously produce tumor cell progeny niclosamide treatment after the primary tumor volume reached þ in the intestinal polyps in ApcMin/ mice. Currently, accumulating 100 mm3, to mimic clinical circumstance of chemotherapy evidence suggests that long-lived DCLK1þ tuft cells may be (Fig. 6A). Our data showed that niclodsamide treatment inhib- responsible for colorectal cancer development by participating ited primary tumor growth even when it was started after the as tumor-initiating populations with persistent Wnt activation formation of primary tumor burden. Thus, our HCT116 model (8). DCLK1 was determined to be responsible for cancer progres- may provide preclinical evidence that niclosamide helps the sion via the enhancement of survival (44), self-renewal (44), and treatment of patients with colorectal cancer for primary tumor epithelial-mesenchymal transition (45, 46). Moreover, clinical shrinkage. In another colorectal cancer model, we documented studies in patients with colorectal cancer demonstrated that that niclosamide delayed AOM/DSS-induced development of DCLK1 was expressed in low-grade adenomas, and its levels colon dysplasia, suggesting that niclosamide can work as an increased with worsening severity of dysplasia (47). DCLK1 inhibitorofcancerprogression(Fig.6D–G). Under normal expression was highly observed in advanced adenomas, which conditions, DCLK1 is expressed in tuft cells at a relatively low þ have a clinically higher malignant potential in colorectal cancer frequency, whereas the number of DCLK1 cells expands in patients (47). dysplasia region of AOM/DSS-treated mice. This result is con- þ There are two types of DCLK1 protein isoforms generated by sistent with previous reports that DCLK cells give rise to cancer two distinct promoter regions, DCLK1-A and DCLK1-B promoter. by producing cancer cells during colon carcinogenesis. Recent- In this study, we first documented that colorectal CSCs predom- ly, NSAIDs, such as celecoxib, rofecoxib, and valdecoxib, have inantly expressed DCLK1-B rather than DCLK1-A (Fig. 3J), and been reported to be used as cancer chemopreventive agents, validated that deletion of DCLK1-B could abrogate CSC popula- which act by blocking inflammatory signaling that contributes tions and their stem-like functions such as self-renewal, survival, to colon carcinogenesis (50). In this study, we showed that and anti-apoptotic potential (Fig. 5A–I). Given that recent clinical in vivo treatment of niclosamide reduced the number of þ investigations provide more evidences that prognostic character- DCLK1 polyps during AOM/DSS-induced colon carcinogen- istics of DCLK1 may be derived from DCLK1-B rather than esis. Therefore, we propose that similar to NSAIDs, niclosamide DCLK1-A (30, 48, 49), our data suggest that DCLK1-B might be might work as chemopreventive drug for colorectal cancer. a promising target for eliminating CSCs. Moreover, our demon- In summary, we revealed that niclosamide exerts in vivo effects stration of niclosamide targeting Wnt/LEF1/DCLK1-B axis sug- against both colon carcinogenesis and tumor growth by targeting gests a new therapeutic strategy for CSC inhibition, and further the Wnt/LEF1/DCLK1-B axis-mediated CSC properties. Niclosa- investigation on DCLK1-B may be helpful for developing other mide inhibits certain CSC functions including survival, anti- Wnt inhibitors and CSC-targeting drugs. apoptosis, and self-renewal, resulting in a reduction in CSC Several previous reports have shown that niclosamide effec- populations. Moreover, CSC-targeting niclosamide successfully tively reduces tumor burden in various type of cancer including sensitizes colorectal cancer to chemoradiation. These findings breast cancer, melanoma and colorectal cancer (20–24). In provide a preclinical rationale to broaden the clinical evaluation consistent with these reports, we validated that niclosamide of niclosamide for colorectal cancer treatment.

Figure 6. Niclosamide exerts a potent in vivo antitumor effect in both HCT116 xenografts and AOM/DSS-induced spontaneous colorectal cancer models. A–C, Therapeutic effect of niclosamide was evaluated in colorectal cancer xenograft models. HCT116 cells were subcutaneously inoculated into NPG mice. When the volume of primary tumors reached approximately 100 mm3, tumor-bearing mice were treated with niclosamide (10 mg/kg or 40 mg/kg, daily, i.p.) or vehicle (PBS) (n ¼ 6/group). A, Primary tumor volume was measured twice per week until the day of sacrifice. B, On the day of sacrifice, all primary tumors were isolated, and (C) the primary tumor weights were evaluated. D–G, Therapeutic effect of niclosamide was examined in an AOM/DSS-induced colorectal cancer mouse model. D, C57BL/6 mice were intraperitoneally injected with AOM (10 mg/kg). One week later, the mice were given 2.0% DSS in the drinking water for a week. Then, the mice were given drinking water without DSS for 2 weeks. This cycle was repeated three times. After injection with AOM, the mice were intraperitoneally treated with niclosamide (20 mg/kg) or vehicle twice a day for 12 weeks (n ¼ 6/group) and sacrificed on the 13th week. On the day of sacrifice, (E) the number of intestinal polyps and (F) colon length were measured. G, DCLK1 expression levels in intestinal polyps were visualized by immunofluorescence and presented with matched H&E images. Blue indicates nuclei, and red indicates DCLK1. H, Colorectal cancer cells were isolated from vehicle- or niclosamide-treated HCT116 xenografts, and the tumor repopulating potential was evaluated by sphere-forming assay. I, CD44v6þ CSC populations within primary tumors of HCT116 xenografted mice were visualized by immunofluorescence assay, and the intensity of CD44v6 expression was quantified using Image-Pro Plus software. Nuclei were counterstained with DAPI, and a matched region of an H&E-stained section is presented. Scale bar represents 100 mm. J–L, Visualization and quantification were performed as above against various target proteins including (J) OCT4, (K) LEF1, and (L) DCLK1. Bar graphs represent the mean SD. Statistical analyses were performed by one-way ANOVA with Dunnett's multiple comparison among more than three groups or Student t test between two groups. , ,and indicate P < 0.05, P < 0.01, and P < 0.001, respectively.

www.aacrjournals.org Clin Cancer Res; 2019 OF13

Park et al.

Disclosure of Potential Conﬂicts of Interest research was supported by a grant of the Korea Health Technology R&D No potential conﬂicts of interest were disclosed. Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea Authors' Contributions (Grant no.: HI15C2056). Additionally, this work was supported by the National Research Foundation of Korea Grant funded by the Korean Conception and design: J.-S. Nam Government (NRF-2017R1E1A1A01075125), by a grant from the Cell Development of methodology: J.-H. Baek Logistics Research Center of the National Research Foundation of Korea Acquisition of data (provided animals, acquired and managed patients, (NRF-2016R1A5A1007318), and by a Gwangju Institute of Science provided facilities, etc.): S.-Y. Park, J.-Y. Kim, J.-H. Choi, J.-H. Kim, C.-J. Lee, and Technology (GIST) Research Institute (GRI) grant funded by the J.-H. Baek GIST in 2018. Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): S.-Y. Park, J.-Y. Kim, J.-H. Choi, J.-H. Kim Writing, review, and/or revision of the manuscript: S.-Y. Park, J.-Y. Kim, The costs of publication of this article were defrayed in part by the J.-S. Nam payment of page charges. This article must therefore be hereby marked Study supervision: J.-S. Nam advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate Other (material support): P. Singh, S. Sarkar this fact.

Acknowledgments We thank Heo Sukyung for establishing the IHC assay and Kim Received April 20, 2018; revised August 10, 2018; accepted November 13, Hyeseon for assistance with Western blotting and cell maintenance. This 2018; published ﬁrst November 16, 2018.

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Inhibition of LEF1-Mediated DCLK1 by Niclosamide Attenuates Colorectal Cancer Stemness

So-Yeon Park, Ji-Young Kim, Jang-Hyun Choi, et al.

Clin Cancer Res Published OnlineFirst November 16, 2018.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-18-1232

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