Published OnlineFirst December 8, 2015; DOI: 10.1158/0008-5472.CAN-15-1519 Cancer Therapeutics, Targets, and Chemical Biology Research A Small-Molecule Antagonist of the b-Catenin/ TCF4 Interaction Blocks the Self-Renewal of Cancer Stem Cells and Suppresses Tumorigenesis Liang Fang1, Qionghua Zhu1, Martin Neuenschwander2, Edgar Specker2, Annika Wulf-Goldenberg3, William I. Weis4, Jens P. von Kries2, and Walter Birchmeier1 Abstract Wnt/b-catenin signaling is a highly conserved pathway essen- high Wnt activity. LF3 also suppressed features of cancer cells tial for embryogenesis and tissue homeostasis. However, dereg- related to Wnt signaling, including high cell motility, cell-cycle ulation of this pathway can initiate and promote human malig- progression, and the overexpression of Wnt target genes. How- nancies, especially of the colon and head and neck. Therefore, ever, LF3 did not cause cell death or interfere with cadherin- Wnt/b-catenin signaling represents an attractive target for can- mediated cell–cell adhesion. Remarkably, the self-renewal capac- cer therapy. We performed high-throughput screening using ity of cancer stem cells was blocked by LF3 in concentration- AlphaScreen and ELISA techniques to identify small molecules dependent manners, as examined by sphere formation of colon that disrupt the critical interaction between b-catenin and the and head and neck cancer stem cells under nonadherent condi- transcription factor TCF4 required for signal transduction. We tions. Finally, LF3 reduced tumor growth and induced differen- found that compound LF3, a 4-thioureido-benzenesulfonamide tiation in a mouse xenograft model of colon cancer. Collectively, derivative, robustly inhibited this interaction. Biochemical assays our results strongly suggest that LF3 is a specific inhibitor of revealed clues that the core structure of LF3 was essential for canonical Wnt signaling with anticancer activity that warrants inhibition. LF3 inhibited Wnt/b-catenin signals in cells with further development for preclinical and clinical studies as a novel exogenous reporters and in colon cancer cells with endogenously cancer therapy. Cancer Res; 76(4); 1–11. Ó2015 AACR. Introduction Wnt/b-catenin signaling is a system that is essential for embry- onic development and tissue homeostasis that has been highly Great efforts have been made over the past two decades to conserved throughout evolution (5–7). The key mediator of the develop rational targeted cancer therapies, which are more effec- pathway is b-catenin, a transcriptional coactivator whose level is tive than chemo- and radiotherapies and may cause fewer side tightly controlled by a multiprotein destruction complex in the effects. Successes involve drugs that act as tyrosine kinase inhibi- cytoplasm. This complex is composed of Axin, adenomatous tors; to date, 28 such substances have been approved for clinical polyposis coli (APC), casein kinase 1a (CK1a), glycogen synthase use (1). Drugs that interfere with other fundamental signaling kinase 3b (GSK3b), and b-transducin repeats containing protein systems, such as g-secretase inhibitors that target Notch signals (2) (b-TrCP). The latter phosphorylate b-catenin and induce its ubi- or inhibitors that target Sonic hedgehog (3), have been developed quitination and proteosomal degradation. Canonical Wnt and are being evaluated in preclinical and clinical trials. Other ligands, such as Wnt1 or Wnt3a, stimulate the pathway by inhib- signaling systems whose deregulation is essential in the initiation iting the destruction complex; this results in the stabilization and and maintenance of cancers—such as Wnt/b-catenin signaling— nuclear translocation of b-catenin. b-Catenin interacts with mem- need to be identified and targeted as means of expanding the bers of the TCF/LEF family of transcription factors and recruits a current repertoire of antitumor strategies (4). number of coactivators including B-cell lymphoma 9 (BCL9) and CREB-binding protein (CBP), ensuring efficient activation of Wnt target genes. A comprehensive list of Wnt target genes can 1 € Cancer Research Program, Max Delbruck Center for Molecular be found at web.stanford.edu/group/nusselab/cgi-bin/wnt/. Medicine in the Helmholtz Society, Berlin, Germany. 2Screening Unit, Leibniz-Institut fuer Molekulare Pharmakologie, Berlin, The discovery of an association between b-catenin and the Germany. 3Experimental Pharmacology and Oncology, Berlin, tumor suppressor gene APC led to the recognition of the essential 4 Germany. Department of Structural Biology, Stanford Universi- role of Wnt/b-catenin signaling in various cancers (8–11). Aber- ty, Stanford, California. rant Wnt signaling is frequently caused by mutations in APC, Note: Supplementary data for this article are available at Cancer Research b-catenin, and Axin2. A noted exception is melanoma, where Online (http://cancerres.aacrjournals.org/). activated Wnt/b-catenin signaling appears to be associated with Corresponding Author: Walter Birchmeier, Max-Delbrueck Center for Molecular reduced tumor formation (12). Deregulated Wnt signaling is not Medicine, Robert-Roessle-Str. 10, Berlin 13125, Germany. Phone: 49-30-9406- only important for cancer initiation, but is also crucial in late-stage 3800; Fax: 49-30-9406-2656; E-mail: [email protected] cancers and in metastasis formation (13, 14). Recent studies have doi: 10.1158/0008-5472.CAN-15-1519 shown that cancer stem cells (CSC) in various tumors exhibit Ó2015 American Association for Cancer Research. deregulated Wnt/b-catenin signaling (15–17) and require higher www.aacrjournals.org OF1 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2015 American Association for Cancer Research. Published OnlineFirst December 8, 2015; DOI: 10.1158/0008-5472.CAN-15-1519 Fang et al. Wnt activity than differentiated cancer cells to maintain their ATCC and cultured in DMEM (Invitrogen) supplemented with 10% self-renewal and tumorigenic properties. This feature may be FBS, penicillin, and streptomycin. Mouse salivary gland CSCs were useful in specifically targeting CSCs, a crucial factor in combatting isolated and cultured in DMEM/F12 (Invitrogen) supplemented cancer recurrence (18, 19). with 20% KSR, nonessential minimal amino acids, penicillin, Over the past decade, researchers in academia and industry streptomycin, L-glutamine, and b-mercaptoethanol. The cell iden- have developed various strategies to identify small molecules that tity was tested by PCR phenotyping for b-catenin and Bmpr1a (16). target components involved in Wnt signaling. Wnt inhibitors were initially identified through ELISA-based high-throughput screen- Protein purification ing (HTS) of natural compounds that interfere with b-catenin/ The cDNA encoding residues 134–668 of human b-catenin were TCF4 interactions (20). Cell-based HTS has been applied to inserted into the pGEX-4-T1 vector (GE Healthcare), and the cDNA identify additional small-molecule inhibitors of the pathway, encoding residues 1–79 of human TCF4 were inserted into the pET- some of which target Wnt secretion (the porcupine inhibitor 16b vector (Qiagen). A mutation of residue Asp16 of TCF4 was IWP-2; ref. 21) or the Wnt receptor complex (Dishevelled/Frizzled introduced by PCR mutagenesis with the primers: forward 50- interaction inhibitors NSC668036 and FJ9; refs. 22, 23). Other GACCTAGGCGCCAACGCCGAACTGATTTCCTTCA-30 and reverse compounds have been found that enhance the stability of the 50-TGAAGGAAATCAGTTCGGCGTTGGCGCCTAGGTC-30.Recom- b-catenin destruction complex (tankyrase inhibitors XAV939 and binant fusion proteins were expressed in E. coli BL21 cells and JW55; refs. 24, 25) or disrupt the transcription complex (b-cate- purified through GSTrap FF or HisTrap FF columns (GE; details in nin/CBP interaction inhibitor ICG-001, b-catenin/TCF interaction Supplementary Materials and Methods). Biotinylated human TCF4 inhibitors iCRT3 and CWP232228, and b-catenin/BCL9 interac- peptide (aa 8–53) was purchased from Biosyntan. tion inhibitor carnosic acid; refs. 26–29). These studies allowed the further development of small-molecule Wnt inhibitors that Compound library, compound analogues, high-throughput entered early clinical trials, including PRI-724, CWP232291, and AlphaScreen LGK974 (30). A library containing 16,671 World Drug Index–derived com- In human cancer, activating mutations of Wnt signaling com- pounds was provided by ChemBioNet, designed by the drug ponents are frequently found at the level of the destruction or design and modeling group of the Leibniz-Institut fur€ Molekulare transcription complexes. We reasoned that efforts to develop new Pharmakologie, Berlin, Germany (34). Analogues of LF3 were Wnt inhibitors could therefore be directed to the downstream purchased from ChemDiv and ChemBridge and dissolved in – components of the pathway, in particular to the b-catenin TCF DMSO to a concentration of 50 mmol/L. In the AlphaScreens, transcription complexes. This may require interference with pro- purified GST-hb-catenin was first incubated with nickel chelate – fi tein protein interactions, which is a challenging eld of research acceptor beads and His-hTCF4 incubated with glutathione donor (31). A further concern was the overlap of the binding sites of beads (Perkinelmer) in PBS. They were then combined into 384- b-catenin with TCFs and E-cadherin, as seen in the structural well AlphaPlates in the presence of 20 mmol/L test compounds. analyses (32). Substances interfering with E-cadherin interaction Titration was used to determine the concentration