Inhibition of TGF-ß Signaling in Tumor Cells by Small Molecule Src Family Kinase Inhibitors.

In a series of studies carried out over the last couple of years in various cell types, it was observed that the experimentally used Src family kinase inhibitors PP1 and PP2 and the clinically used Src/Abl inhibitors AZM475271 and dasatinib are potent inhibitors of TGF-ß mediated cellular responses such as Smad and p38 mitogen-activated protein kinase phosphorylation, Smad-dependent transcriptional activation, growth inhibition, epithelial-mesenchymal transition (EMT), and cell motility. While for PP1/PP2 it was demonstrated that these agents directly inhibit the kinase activity of the TGF-ß type I receptor activin receptor-like kinase 5, the mechanism of the anti-TGF-ß effect of AZM475271 and dasatinib is less clear. In contrast, the anti-TGF-ß effect of yet another Src/Abl inhibitor, bosutinib, is more variable with respect to the type of the TGF-ß response and the cell type affected, and lacks a clear dose-dependency. In the light of their strong anti-activin receptor-like kinase 5 kinase effect, PP1 and PP2 should not be used when studying the role of c-Src as downstream mediators in TGF-ß/activin receptor-like kinase 5 signaling. On the other hand, based upon in vitro findings, it is conceivable that part of the therapeutic effects of AZM475271 and dasatinib seen in preclinical and clinical studies with solid tumors was caused by inhibition of prometastatic TGF-ß rather than Src signaling. If AZM475271 and dasatinib can indeed act as dual Src / TGF-ß inhibitors in vivo, this may be beneficial for prevention of metastatic disease in more advanced tumor stages.

TGF-ß Signal Transduction in Pancreatic Carcinoma Cells is Sensitive to Inhibition by the Src Tyrosine Kinase Inhibitor AZM475271.

BACKGROUND: Earlier results from our group have shown that in pancreatic ductal adenocarcinoma (PDAC)-derived cells transforming growth factor (TGF)-ß1-dependent epithelial-mesenchymal transition (EMT) and cell motility was inhibited by the Src inhibitors PP2 and PP1 both of which targeted the TGF-ß receptors for inhibition. OBJECTIVE: In this study we evaluated the impact of another Src inhibitor, AZM475271, on various TGF-ß responses in PDAC cells. METHOD: The effect of AZM475271 on TGF-ß1-induced random cell migration (chemokinesis), the expression of EMT and migration/invasion-associated genes, TGF-ß-induced luciferase activity, and C-terminal phosphorylation of Smad2 and Smad3 was measured in the PDAC-derived Panc-1 and Colo357 cell lines using real-time cell migration assays, quantitative real-time PCR, luciferase reporter gene assays and phosphoimmunoblotting, respectively. RESULTS: AZM475271 effectively blocked TGF-ß1-induced chemokinesis of Panc-1 cells in a dose-dependent fashion and inhibited the high chemokinetic activity of Panc-1 cells with ectopic expression of a constitutively active ALK5T204D mutant. AZM475271 but not another Src inhibitor, SU6656, partially relieved the suppressive effect of TGF-ß1 on E-cadherin and inhibited TGF-ß1-induced upregulation of the MMP2, MMP9, N-cadherin and vimentin genes, activity of a TGF-ß1-dependent reporter gene, and activation of Smad2 and Smad3. CONCLUSION: Our data suggest that AZM475271 cross-inhibits tumor-promoting TGF-ß signaling and may thus function as an inhibitor of both TGF-ß and Src in both experimental and clinical therapies against metastatic dissemination in late-stage PDAC.

Dasatinib blocks transcriptional and promigratory responses to transforming growth factor-beta in pancreatic adenocarcinoma cells through inhibition of Smad signalling: implications for in vivo mode of action.

BACKGROUND: We have previously shown in pancreatic ductal adenocarcinoma (PDAC) cells that the SRC inhibitors PP2 and PP1 effectively inhibited TGF-ß1-mediated cellular responses by blocking the kinase function of the TGF-ß type I receptor ALK5 rather than SRC. Here, we investigated the ability of the clinically utilised SRC/ABL inhibitor dasatinib to mimic the PP2/PP1 effect. METHODS: The effect of dasatinib on TGF-ß1-dependent Smad2/3 phosphorylation, general transcriptional activity, gene expression, cell motility, and the generation of tumour stem cells was measured in Panc-1 and Colo-357 cells using immunoblotting, reporter gene assays, RT-PCR, impedance-based real-time measurement of cell migration, and colony formation assays, respectively. RESULTS: In both PDAC cell lines, dasatinib effectively blocked TGF-ß1-induced Smad phosphorylation, activity of 3TPlux and pCAGA(12)-luc reporter genes, cell migration, and expression of individual TGF-ß1 target genes associated with epithelial-mesenchymal transition and invasion. Moreover, dasatinib strongly interfered with the TGF-ß1-induced generation of tumour stem cells as demonstrated by gene expression analysis and single cell colony formation. Dasatinib also inhibited the high constitutive migratory activity conferred on Panc-1 cells by ectopic expression of kinase-active ALK5. CONCLUSIONS: Our data suggest that the clinical efficiency of dasatinib may in part be due to cross-inhibition of tumour-promoting TGF-ß signalling. Dasatinib may be useful as a dual TGF-ß/SRC inhibitor in experimental and clinical therapeutics to prevent metastatic spread in late-stage PDAC and other tumours.