Background: Ligand independent receptor tyrosine kinase activation can be mediated by activation through other growth factor receptors. Understanding this cross-talk has implications in the use of combination therapies, as growth factor receptors often stimulate overlapping signaling pathways.
Purpose: To examine crosstalk between IGF-R1 and MET in prostate cancer (PCa) cells.
Experimental procedures: IGF was added to PC3 prostate cancer cells and phosphorylation of c-Met and activation of signaling proteins was examined by immunoblotting various times thereafter. Activation of MET was determined by immunoblotting with antibodies to the tyrosine phosphorylation sites, Y1234/Y1235 and Y1349. To determine the requirement of transcription for c-Met activation, experiments were performed in the presence of actinomycin D. To determine the requirement of IGFR and/or Src in c-Met phosphorylation, IGF was added to PC3 cells with stable knockdown of IGFR or Src by expression of an shIGFR or shSrc construct. Rescue experiments were performed by expressing an sh-insensitive mouse c-Src. To determine the biological effect of MET activation, PC3 cells and PC3 cells with stable knockdown of c-Met were stimulated with IGF and allowed to migrate in Boyden chambers for various times thereafter.
Results: We demonstrate that addition of 100ng/ml of IGF to culture media of PC3 cells (IGF was chosen because it is abundant in the sera of prostate cancer patients), induces MET tyrosine kinase activation beginning 18 hours after addition and sustained for a minimum of 6 hrs thereafter. Multiple sites on Y1234/Y1235 and Y1349 are phosphorylated, suggesting full MET activation, a conclusion supported by increased migration of cells after c-Met phosphorylation. Phosphorylation of MET is not due to the presence of HGF, which was undetectable in cells by quantitative RT-PCR and in media by ELISA, but requires IGF-1R activation, as IGFR stable knock down or the addition of the IGFR-1R inhibitor BMS754807 inhibited MET phosphorylation. We demonstrated that transcription is also required for MET activation, as MET was not tyrosine phosphorylated in cells grown in the presence of actinomycin D. As other groups have shown delayed activation of MET by EGF required the tyrosine kinase, Src, we performed the experiments in the presence of the pan Src Family Kinase inhibitor, dasatinib, and demonstrated that MET phosphorylation was abolished. We demonstrate that Src itself is required for MET phosphorylation, as a stable shSrc knockdown cell line inhibited IGF-induced MET activation, whereas expression of mouse Src (insensitive to sh knockdown) restores MET phosphorylation in the presence of IGF.
Conclusion: We demonstrate that IGF induces MET activation through a ligand-independent mechanism requiring gene transcription following activation of IGF-1R and Src.
Citation Format: Andreas S. Varkaris, Sanchaika Gaur, Nila Parikh, Christopher Logothetis, Gary E. Gallick. Crosstalk between IGFR and MET in prostate cancer. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr A53.
- ©2012 American Association for Cancer Research.