Targeting metastatic tumor cell functions by inhibition of Src signaling using the small molecule Src inhibitor, AZD0530 Free

B187

Introduction: Src is a non-receptor tyrosine kinase that is frequently overexpressed in malignances, and increased Src activity (regulated by Src phosphorylation) may be associated with poor patient prognosis. Src is involved in many signaling pathways, including mitogen-activated protein kinase (MAPK), focal adhesion kinase (FAK), and Met that regulate cell proliferation, survival, and migration. Since these cellular functions are key components of the metastatic cascade, Src may be a novel target for cancer therapy. In the present study, Src inhibition by AZD0530, a potent, selective and orally available Src inhibitor with Bcr-Abl activity, was evaluated in the highly metastatic rodent KHT sarcoma cell line.
 Methods: The effect of AZD0530 treatment on expression of total and phosphorylated Src, FAK, extracellular signal-regulated kinase (ERK), and Met protein was determined by Western blot. Src and FAK cellular distribution were evaluated by immunofluorescence using confocal microscopy. Tumor cell functions, including viability, cell cycle, and migration, were examined by trypan blue exclusion, flow cytometry, and migration assays (wound healing and transwell), respectively.
 Results: At the molecular level, AZD0530 doses greater than 2.5 µM significantly reduced Src phosphorylation at tyrosine 418 in KHT cells. Reductions in Src phosphorylation occurred within 2 hours of treatment (~80% at 10 µM). Met and phosphorylated ERK were also decreased at this dose. AZD0530 treatment also altered the localization of phosphorylated Src and phosphorylated FAK within the tumor cells from focal adhesion to cytoplasm. Functionally, AZD0530 significantly reduced the ability of KHT sarcoma cells to migrate into a denuded area or through a transwell membrane (0.5-2.5 µM), and increased the proportion of cells in the G₁ phase of the cell cycle.
 Conclusions: These results indicate that AZD0530 treatment inhibits functional tumor cell characteristics associated with a metastatic phenotype in the KHT sarcoma cell line. These data serve as a basis for future studies aiming to evaluate the in vivo antimetastatic efficacy of this Src inhibitor.