ABSTRACT
TGF-ß signaling plays an important role in breast cancer progression and metastasis. Epithelial-mesenchymal transition (EMT) is an important step in the progression of solid tumors to metastatic disease. We previously reported that IN-1130, a novel transforming growth factor-ß type I receptor kinase (ALK5) inhibitor, suppressed renal fibrosis in obstructive nephropathy (Moon et al., 2006). Here, we show that IN-1130 suppressed EMT and the lung metastasis of mammary tumors in mouse models. Treating human and mouse cell lines with IN-1130 inhibited TGF-ß-mediated transcriptional activation, the phosphorylation and nuclear translocation of Smad2, and TGF-ß-induced-EMT, which induces morphological changes in epithelial cells. Additionally, we demonstrated that IN-1130 blocked TGF-ß-induced 4T1 mammary cancer cell migration and invasion. The TGF-ß-mediated increase in matrix metalloproteinase (MMP)-2 and MMP-9 expression was restored by IN-1130 co-treatment with TGF-ß in human epithelial cells and in 4T1 cells. Furthermore, we found that lung metastasis from primary breast cancer was inhibited by IN-1130 in both 4T1-xenografted BALB/c mice and MMTV/c-Neu transgenic mice without any change in primary tumor volume. IN-1130 prolonged the life span of tumor-bearing mice. In summary, this study indicated that IN-1130 has therapeutic potential for preventing breast cancer metastasis to the lung.
Subject(s)
Epithelial-Mesenchymal Transition/drug effects , Imidazoles/pharmacology , Lung Neoplasms/prevention & control , Mammary Neoplasms, Experimental/drug therapy , Protein Serine-Threonine Kinases/antagonists & inhibitors , Quinoxalines/pharmacology , Receptors, Transforming Growth Factor beta/antagonists & inhibitors , Animals , Cell Line, Tumor , Cell Movement , Cell Nucleus/metabolism , Drug Screening Assays, Antitumor , Female , Humans , Lung/pathology , Lung Neoplasms/metabolism , Lung Neoplasms/secondary , Mammary Neoplasms, Experimental/metabolism , Mammary Neoplasms, Experimental/pathology , Mice , Mice, Inbred BALB C , Neoplasm Invasiveness , Neoplasm Transplantation , Protein Serine-Threonine Kinases/metabolism , Protein Transport , Receptor, Transforming Growth Factor-beta Type I , Receptors, Transforming Growth Factor beta/metabolism , Transforming Growth Factor beta/physiology , Tumor Burden/drug effectsABSTRACT
A series of 1-substituted-3-(6-methylpyridin-2-yl)-4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)pyrazoles 14a-ae, 16a, 16b, and 21a-c has been prepared and evaluated for their ALK5 inhibitory activity in an enzyme assay and in a cell-based luciferase reporter assay. The 4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)-N-(4-methoxyphenyl)-3-(6-methylpyridin-2-yl)-1H-pyrazole-1-carbothioamide (14n) inhibited ALK5 phosphorylation with IC(50) value of 0.57 nM and showed 94% inhibition at 100 nM in a luciferase reporter assay using HaCaT cells permanently transfected with p3TP-luc reporter construct.
Subject(s)
Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Pyrazoles/chemistry , Pyrazoles/pharmacology , Receptors, Transforming Growth Factor beta/antagonists & inhibitors , Animals , Cell Line , Humans , Insecta/cytology , Luciferases/analysis , Protein Kinase Inhibitors/chemical synthesis , Protein Serine-Threonine Kinases/metabolism , Pyrazoles/chemical synthesis , Receptor, Transforming Growth Factor-beta Type I , Receptors, Transforming Growth Factor beta/metabolism , Recombinant Fusion Proteins/antagonists & inhibitors , Recombinant Fusion Proteins/metabolismABSTRACT
Recently, researchers are actively pursuing efforts to develop potent and selective ALK5 (TßRI) 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-ß-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-ß(1)-stimulated transcriptional activations of p3TP-Lux and pCAGA(12)-Luc. In addition, EW-7195 decreased phosphorylated Smad2 levels and the nuclear translocation of Smad2 increased by TGF-ß(1). In addition, EW-7195 inhibited TGF-ß(1)-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-ß(1)-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.
Subject(s)
Antineoplastic Agents/therapeutic use , Breast Neoplasms/drug therapy , Lung Neoplasms/prevention & control , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Protein Serine-Threonine Kinases/antagonists & inhibitors , Pyridines/therapeutic use , Receptors, Transforming Growth Factor beta/antagonists & inhibitors , Triazoles/therapeutic use , Animals , Cell Line, Tumor , Cell Movement/drug effects , Cell Transformation, Neoplastic/drug effects , Epithelial-Mesenchymal Transition/drug effects , Female , Hep G2 Cells/enzymology , Humans , Luciferases/metabolism , Lung Neoplasms/secondary , Mice , Mice, Transgenic , Neoplasm Invasiveness , Phosphorylation/drug effects , Receptor, Transforming Growth Factor-beta Type I , Smad2 Protein/metabolismABSTRACT
A series of 1-substituted-3(5)-(6-methylpyridin-2-yl)-4-(quinoxalin-6-yl)pyrazoles 14a-d, 15a-d, 17a, 17b, 18a-d, 19a, and 19b has been synthesized and evaluated for their ALK5 inhibitory activity in an enzyme assay and in a cell-based luciferase reporter assay. The 2-[3-(6-methylpyridin-2-yl)-4-(quinoxalin-6-yl)-1H-pyrazol-1-yl]-N-phenylethanethioamide (18a) inhibited ALK5 phosphorylation with an IC(50) value of 0.013 µM and showed 80% inhibition at 0.1 µM in a luciferase reporter assay using HaCaT cells permanently transfected with p3TP-luc reporter construct.
Subject(s)
Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Pyrazoles/chemical synthesis , Pyrazoles/pharmacology , Receptors, Transforming Growth Factor beta/antagonists & inhibitors , Animals , Cell Line , Humans , Magnetic Resonance Spectroscopy , Models, Molecular , Phosphorylation , Protein Kinase Inhibitors/chemistry , Pyrazoles/chemistry , Receptor, Transforming Growth Factor-beta Type I , Spectrometry, Mass, Electrospray Ionization , X-Ray DiffractionABSTRACT
A series of 1-substituted-3(5)-(6-methylpyridin-2-yl)-4-(quinolin-6-yl)pyrazoles 14a-e, 15a-e, 17a-c, and 18a-d have been synthesized and evaluated for their ALK5 inhibitory activity in an enzyme assay and in a cell-based luciferase reporter assay. The 6-quinolinyl pyrazole analogue 14b inhibited ALK5 phosphorylation with IC(50) value of 0.022 µM and showed 84% inhibition at 0.1 µM in a luciferase reporter assay using HaCaT cells permanently transfected with p3TP-luc reporter construct.
