Ki26894, a novel transforming growth factor‐β type I receptor kinase inhibitor, inhibits in vitro invasion and in vivo bone metastasis of a human breast cancer cell line

Abstract

T ransforming growth factor (TGF)-β is a multifunctional cytokine that regulates a wide range of cellular responses, including cell proliferation, differentiation, adhesion, migration, and apoptosis.(1,2) It is a potent inhibitor of various types of cells, including most epithelial cells, whereas it stimulates deposition of extracellular matrix proteins and induction of epithelialto-mesenchymal transition (EMT). TGF-β thus plays two distinct and opposing roles in cancer progression. In early stages of carcinogenesis, it acts as a tumor suppressor by preventing cell proliferation, although in advanced stages of cancer, tumor cells often become refractory to TGF-β-mediated growth inhibition. TGF-β is often overexpressed in tumor cells, and induces migration, invasion, and EMT of tumor cells and facilitates immunosuppression, deposition of extracellular matrix proteins and angiogenesis. TβR-II is the primary ligand-binding receptor at the cell surface and contains constitutively active kinase. Upon ligand binding, the TβR-II kinase transphosphorylates TβR-I, and TβR-I then transduces intracellular signals through various proteins, of which Smad proteins are the major signaling transducers for TGF-β.(6,7) Activated TβR-I phosphorylates receptor-regulated Smads (R-Smads), i.e. Smad2 and Smad3, which interact with common-mediator Smad (Smad4) and translocate to the nucleus where they regulate transcription of various target genes. Smad7 is an inhibitory Smad that inhibits TGF-β signaling through interaction with activated TβR-I and other mechanisms.TGF-β signaling can be regulated by various molecules, including antisense oligonucleotides to certain TGF-β isoforms, monoclonal antibodies to TGF-βs, soluble forms of TβR-II, and small-molecule compounds that act on TGF-β receptor kinases. (8) Of these, TβR-I kinase is one potential target for blockade of TGF-β signaling pathway. Recently, several small-molecule compounds that bind to and inhibit TβR-I kinase activity have been generated and shown to potently inhibit TGF-β activity in vitro and in vivo.(8) Small-molecule inhibitors of TβR-I kinase are highly specific for TβR-I, although they also inhibit kinase activities of closely related molecules, that is, type I receptors for activin and nodal (activin-receptor like kinase-4; ALK-4 and ALK-7). (9) An in vivo experimental bone metastasis model has been established using an intracardiac injection of cancer cells, including MDA-MB-231 cells. (10,11) In this model, growth of cancer cells arrested in bone is easily observed as the progression of osteolysis on radiographs. This model is thus frequently used to explore the molecular interactions between the cancer cells and bone microenvironment. Several studies using this in vivo model suggested that TGF-β and its transcriptional targets, including parathyroid hormone-related protein (PTHrP) and interleukin-11 (IL-11), are the essential mediators of bone metastasis. (12,13) More recent studies have revealed that Smad signaling is essential for the development of bone metas...

Transforming growth factor-β1 is the predominant isoform required for breast cancer cell outgrowth in bone

“…Stable knockdown of Smad4 expression in these cells further reinforces the importance of Smad-mediated TGF-b signaling for promoting breast cancer metastasis to bone Deckers et al, 2006). Furthermore, kinase inhibitors targeting TbRI reduce the incidence of bone metastases following left cardiac ventricle injection of MDA-MB-435 and MDA-MB-231 breast cancer cells (Bandyopadhyay et al, 2006;Ehata et al, 2007). These animal-based studies support a role for TGF-b signaling within breast tumor cells that enable aggressive metastasis to bone.…”

ZEB1‐regulated inflammatory phenotype in breast cancer cells

“…The MDA‐231‐D human basal‐type breast cancer cell line, a highly bone metastatic clone of MDA‐MB‐231, was described previously (Ehata et al ., 2007) and cultured in Dulbecco's modified Eagle's medium (DMEM; #11965, Thermo Fisher Scientific, Waltham, MA, USA). Hs578T human basal‐type breast cancer cells were obtained for use in this study from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in RPMI1640 (#11875, Thermo Fisher Scientific) with 0.01 mg·mL −1 of insulin (12585‐014, Thermo Fisher Scientific).…”

“…We found that ZEB1 directly upregulated the production of inflammatory cytokines in the basal‐type breast cancer cell lines MDA‐231‐D (a highly metastatic clone of MDA‐MB‐231; Ehata et al ., 2007) and Hs578T. ZEB2 partially showed similar function, including the induction of interleukin (IL)‐6 and IL‐8 production.…”

Transforming growth factor-β decreases the cancer-initiating cell population within diffuse-type gastric carcinoma cells

“…Lentivirus vectors were used to generate 2MLN-dnTbRII and control 2MLN-GFP cells as described . A human keratinocyte cells, HaCaT, and a human breast cancer cells, MDA-MB-231, were cultured as described (Ehata et al, 2007a). Human non-small-cell lung carcinoma cells, A549, human hepatocellular carcinoma cells, HuH7, and human cervical carcinoma cells, HeLa, were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, penicillin/streptomycin.…”

“…Cell proliferation assay Cell proliferation assay was performed as described (Ehata et al, 2007a). Briefly, HaCaT and OCUM-2MLN cells were seeded in duplicate at a density of 2.5 Â 10 4 cells per well in 24-well plates.…”