EW-7195, a novel inhibitor of ALK5 kinase inhibits EMT and breast cancer metastasis to lung

Abstract

Recently, researchers are actively pursuing efforts to develop potent and selective ALK5 (TβRI) kinase inhibitors for clinical development. In this study, the authors examined a novel small molecule inhibitor of ALK5, 3-((4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-1H-imidazol-2-yl)methylamino)benzonitrile (EW-7195) to determine if it has potential for cancer treatment. The inhibitory effects of EW-7195 on TGF-β-induced Smad signaling and epithelial-to-mesenchymal transition (EMT) were investigated in mammary epithelial cells using luciferase reporter assays, immunoblotting, confocal microscopy and wound healing assays. In addition, the suppressive effects of EW-7195 on mammary cancer metastasis to lung were examined using a Balb/c xenograft and MMTV/cNeu transgenic mice model system. The novel ALK5 inhibitor, EW-7195, inhibited the TGF-β₁-stimulated transcriptional activations of p3TP-Lux and pCAGA₁₂-Luc. In addition, EW-7195 decreased phosphorylated Smad2 levels and the nuclear translocation of Smad2 increased by TGF-β₁. In addition, EW-7195 inhibited TGF-β₁-induced EMT and wound healing of NMuMG cells. Furthermore, in xenografted Balb/c and MMTV/cNeu mice, EW-7195 inhibited metastasis to lung from breast tumours. The novel ALK5 inhibitor, EW-7195, efficiently inhibited TGF-β₁-induced Smad signaling, EMT and breast tumour metastasis to the lung in vivo, demonstrating that EW-7195 has therapeutic potential for the breast cancer metastasis to the lung.

Introduction

Breast cancer is the first leading cause of cancer mortality among women in Korea and England, and the second leading cause of mortality among women in the United States of America. Invasion and metastasis are the most lethal characteristics of breast cancer.1, 2 In normal mammary epithelial cells, TGF-β signaling maintains homeostasis and elicits tumour-suppressive responses such as growth inhibition and apoptosis.³ However, during the course of breast cancer progression, TGF-β signaling loses its tumour-suppressive function and acquires the ability to drive tumour promoting activities such as migration, invasion and metastasis.⁴ The underlying TGF-β signaling mechanism which accounts for the switch in TGF-β signaling activities are not well known. The TGF-β₁ ligand preferentially signals through TGF-β type II receptor (TβRII) and TGF-β type I receptor (TβRI), one of which is an ALK5 kinase. Upon binding the ligand, activated heterotetrameric receptor complexes propagate canonical TGF-β signaling through phosphorylation of receptor associated Smads at the carboxyterminal.5, 6

Once phosphorylated, the Smads are shuttled into the nucleus and associated with Smad4 to regulate gene expression. The tumour promoting role of TGF-β₁ is linked to its ability to induce an epithelial-to-mesenchymal transition (EMT) in late stage cancers. EMT is characterised by a decrease in cell–cell adhesion, an increase in cell motility, and the activation of proteolysis, properties that are associated with tumour cell invasion and metastasis.7, 8, 9 In mouse mammary epithelial NMuMG cells, treatment with TGF-β dramatically induced morphological changes polarised epithelial types to motile mesenchymal types, and down-regulated an epithelial gene E-cadherin.¹⁰ The overexpression of TGF-β ligands has been reported in most tumour types, and elevated levels of these ligands in tumour tissues or in patient serum correlate with more metastatic phenotypes or poorer patient outcome.11, 12, 13, 14, 15 Development of TβRI kinase inhibitors has become an attractive target for drug development. To date, several selective inhibitors of TβRI nase have been described.16, 17, 18, 19, 20, 21, 22 Several pharmaceutical and biotechnology companies are actively pursuing efforts to develop potent and selective TβRI kinase inhibitors for clinical development. SD-093 and LY-580276 have been shown to block EMT and tumour cell migration in pancreatic cancer and mouse mammary epithelial cells, respectively.23, 24

In this study, we have characterised 3-((4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-1H-imidazol-2-yl)methylamino)benzonitrile (EW-7195), a novel synthesized small molecule as a specific inhibitor of ALK5 kinase. EW-7195 inhibited the EMT, motility, and invasiveness of breast cancer cells in vitro and lung metastasis development in both 4T1 orthotopic xenograft and MMTV/cNeu transgenic mice in vivo.