Deep Phospho- and Phosphotyrosine Proteomics Identified Active Kinases and Phosphorylation Networks in Colorectal Cancer Cell Lines Resistant to Cetuximab

Abnormality in cellular phosphorylation is closely related to oncogenesis. Thus, kinase inhibitors, especially tyrosine kinase inhibitors (TKIs), have been developed as anti-cancer drugs. Genomic analyses have been used in research on TKI sensitivity, but some types of TKI resistance have been unclassifiable by genomic data. Therefore, global proteomic analysis, especially phosphotyrosine (pY) proteomic analysis, could contribute to predict TKI sensitivity and overcome TKI-resistant cancer. In this study, we conducted deep phosphoproteomic analysis to select active kinase candidates in colorectal cancer intrinsically resistant to Cetuximab. The deep phosphoproteomic data were obtained by performing immobilized metal-ion affinity chromatography-based phosphoproteomic and highly sensitive pY proteomic analyses. Comparison between sensitive (LIM1215 and DLD1) and resistant cell lines (HCT116 and HT29) revealed active kinase candidates in the latter, most of which were identified by pY proteomic analysis. Remarkably, genomic mutations were not assigned in most of these kinases. Phosphorylation-based signaling network analysis of the active kinase candidates indicated that SRC-PRKCD cascade was constitutively activated in HCT116 cells. Treatment with an SRC inhibitor significantly inhibited proliferation of HCT116 cells. In summary, our results based on deep phosphoproteomic data led us to propose novel therapeutic targets against cetuximab resistance and showed the potential for anti-cancer therapy.