TrkA Overexpression Enhances Growth and Metastasis of Breast Cancer Cells, and Ku86 Is Crucial for TrkA Overexpression-Induced Breast Cancer Cell Invasion.

Abstract

Abstract The Trk family of neurotrophin tyrosine kinase receptors is emerging as an important player in carcinogenic progression in non-neuronal tissues. Here, we show that breast tumors present high levels of TrkA and phospho-TrkA compared to normal breast tissues. To further evaluate the precise functions of TrkA overexpression in breast cancer development, we have performed a series of biological tests using breast cancer cells that stably overexpress TrkA. We show that (1) TrkA overexpression promoted cell growth, migration and invasion in vitro; (2) overexpression of TrkA per se conferred constitutive activation of its tyrosine kinase activity; (3) signal pathways including PI3K-Akt and ERK/p38 MAP kinases were activated by TrkA overexpression and were required for the maintenance of a more aggressive cellular phenotype; and (4) TrkA overexpression enhanced tumor growth, angiogenesis and metastasis of xenografted breast cancer cells in immunodeficient mice. Moreover, recovered metastatic cells from the lungs exhibited enhanced anoikis resistance that was abolished by the pharmacological inhibitor K252a, suggesting that TrkA-promoted breast tumor metastasis could be mediated at least in part by enhancing anoikis resistance. Together, these results provide the first direct evidence that TrkA overexpression enhances the tumorigenic properties of breast cancer cells and point to TrkA as a potential target in breast cancer therapy.In a second step, we used a proteomic-based approach to identify proteins involved in TrkA overexpression-stimulated invasion of MDA-MB-231 breast cancer cells. Proteins from control and TrkA overexpressing cells were separated using a cup-loading two-dimensional electrophoresis system before MALDI and LC-MS/MS mass spectrometry analysis. Among several putative modified proteins, Ku86 was found to be the major protein which was increased in TrkA overexpressing cells. Moreover, Ku86 was co-immunoprecipitated with TrkA and its level at the cell surface was increased in TrkA overexpressing cells. Interestingly, inhibition with small-interfering RNA and neutralizing antibodies showed that Ku86 was required for TrkA-stimulated cell invasion. Together, these data allowed the identification of Ku86 as a new player involved in metastasis of breast cancer cells. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4169.