Enhanced Src activation has been implicated in many cancers, however single-agent therapies targeting Src have achieved limited success in the clinical setting. It remains unclear what regulatory influence Src has at the kinomic level, where many anti-cancer drug targets are found. This study aims to define the Src-regulated kinome by using a cutting-edge chemical proteomics strategy, and to combine this with functional genomics in order to identify kinase signaling networks that are important for Src-induced oncogenesis.
We first developed a novel kinase capture reagent termed CTx-0294885, that is capable of purifying 2/3 of the expressed kinome. This was used in combination with 3 other kinase capture reagents for kinase enrichment from control MCF-10A and MCF-10A cells expressing constitutively active Src. The global activation kinase profiles of these two cell lines were identified and compared by quantitative mass spectrometry. Nearly 300 protein kinases and 1500 kinase-derived phosphosites were identified from the kinome profiling. Importantly, over 100 kinases respond to Src activation with changes at the protein expression or phosphorylation level. This Src-regulated kinome contains: several well characterized kinases that are already being targeted in the clinical setting, such as Egfr and Braf; proto-oncogenes implicated in breast cancer but with no current functional link to Src, such as Ikke; kinases involved in tumour suppression such as Last1/2 and the Tao family kinases; and many poorly characterized kinases that may represent novel targets for drug discovery. In addition, approximately 70 other proteins co-purified with protein kinases also exhibited changes upon Src activation. These include several lipid kinases and numerous proteins involved in RNA metabolism. All of these Src-regulated proteins are currently being functionally characterized by a focused RNAi screen, specifically regarding their roles in cell viability, migration and morphology.
This study will provide novel insights into the global impact of Src-induced transformation on the expressed kinome and may lead to identification of novel therapeutic targets and combination treatment strategies.