Small Molecule Therapeutics EW-7197, a Novel ALK-5 Kinase Inhibitor, Potently Inhibits Breast to Lung Metastasis

Advanced tumors produce an excessive amount of transforming growth factor b (TGFb), which promotes tumor progression at late stages of malignancy. The purpose of this study was to develop anti-TGFb therapeutics for cancer. We synthesized a novel small-molecule TGFb receptor I kinase (activin receptor– like kinase 5) inhibitor termedN-[[4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-1H-imidazol2-yl]methyl]-2-fluoroaniline (EW-7197), and we investigated its potential antimetastatic efficacy in mouse mammary tumor virus (MMTV)/c-Neu mice and 4T1 orthotopic–grafted mice. EW-7197 inhibited Smad/ TGFb signaling, cell migration, invasion, and lung metastasis in MMTV/c-Neu mice and 4T1 orthotopic– grafted mice. EW-7197 also inhibited the epithelial-to-mesenchymal transition (EMT) in both TGFb-treated breast cancer cells and 4T1 orthotopic–grafted mice. Furthermore, EW-7197 enhanced cytotoxic T lymphocyte activity in 4T1 orthotopic–grafted mice and increased the survival time of 4T1-Luc and 4T1 breast tumor– bearing mice. In summary, EW-7197 showed potent in vivo antimetastatic activity, indicating its potential for use as an anticancer therapy. Mol Cancer Ther; 13(7); 1704–16. 2014 AACR. Introduction Transforming growth factor b (TGFb) is a multifunctional cytokine that plays a central role in a variety of cellular processes, including cell growth, differentiation, cell adhesion, migration, and extracellular matrix deposition (1). The binding of TGFb to a heteromeric complex containing the TGFb receptor facilitates activation of activin receptor–like kinase 5 (ALK5), which phosphorylates Smad2/3 (2). Phosphorylated Smad2/3 then forms a heteromeric complex with Smad4 and is translocated into the nucleus (3), resulting in altered gene expression (4, 5). One of the main functions of TGFb signaling is preservation of the homeostasis of epithelial, endothelial, and hematopoietic cells. However, in pathologic circumstances, the homeostatic action of TGFb is diverted to alternative roles. During cancer progression, TGFb signaling plays a dual role. At the early stages of tumorigenesis, TGFb signaling elicits a preventive or tumorsuppressing effect, and epithelial cells retain delicate growth sensitivity to TGFb. However, at later stages, when carcinoma cells become resistant to TGFb-mediated growth inhibition, the intracellular signaling circuitry of cells is altered and leads to the progression of tumors (6). TGFb ligands are often enriched in the breast tumor microenvironment and can be produced by tumor cells or tumor-associated stromal and immune cells (4, 7). In addition, TGFb has been shown to play a critical role in breast cancer metastasis to the lung and in the maintenance of cancer stem cells (CSC) in breast carcinomas (8– 11). TGFb is also a potent inducer of the epithelial-tomesenchymal transition (EMT) in mammary cells (12), and this transformation has been associated with the acquisition of tumor stem-like properties (13). Indeed, the TGFb receptor I/II kinase inhibitor has been shown to reverse EMT and induce the mesenchymal-to-epithelial transition in CD44þ mammary epithelial cells (7). Studies with triple-negative breast cancer cells further suggest that CSCs with self-renewing and tumor-initiating capacities are responsible for chemotherapy resistance and relapse after treatment (14). Therefore, inhibition of the TGFb signaling pathway offers a rational approach to cancer therapy. Thus far, the following three approaches have been used to inhibit TGFb signaling: (i) inhibition of TGFb signaling at a translational level using antisense oligonucleotides (15, 16); (ii) inhibition of the ligand– receptor interaction using monoclonal antibodies (mAb; refs. 17–20); and (iii) inhibition of the receptor-mediated signaling cascade using inhibitors of TGFb receptor kinases (21). Small-molecule inhibitors of TGFb/ALK5 Authors' Affiliations: College of Pharmacy, Ewha Womans University, Seodaemun-gu, Seoul; and Laboratory of Tumor Suppressor, Lee Gil Ya Cancer and Diabetes Institute, Gachon University, Incheon, South Korea Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). J.Y. Son and S.-Y. Park contributed equally to this work. Corresponding Authors:Yhun Yhong Sheen, College of Pharmacy, Ewha WomansUniversity, Seodaemun-gu, Seoul, 120-750, SouthKorea. Phone: 82-2-3277-3028; Fax: 822-3277-2851; E-mail: [email protected]; and Jeong-SeokNam, Laboratory of Tumor Suppressor, LeeGil YaCancer and Diabetes Institute, Gachon University, 7-45, Songdo-dong, Yeonsu-ku, Incheon, 406-840, South Korea, [email protected]. doi: 10.1158/1535-7163.MCT-13-0903 2014 American Association for Cancer Research. Molecular Cancer Therapeutics Mol Cancer Ther; 13(7) July 2014 1704 on June 20, 2017. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from Published OnlineFirst May 9, 2014; DOI: 10.1158/1535-7163.MCT-13-0903