Proteomics study of medullary thyroid carcinomas expressing RET germ‐line mutations: Identification of new signaling elements

Abstract

Proteomics may help to elucidate differential signaling networks underlying the effects of compounds and to identify new therapeutic targets. Using a proteomic-multiplexed analysis of the phosphotyrosine signaling together with antibody-based validation techniques, we identified several candidate molecules for RET (rearranged during transfection) tyrosine kinase receptor carrying mutations responsible for the multiple endocrine neoplasia type 2A and 2B (MEN2A and MEN2B) syndromes in two human medullary thyroid carcinoma (MTC) cell lines, TT and MZ-CRC-1, which express the RET-MEN2A and RET-MEN2B oncoproteins, respectively. Signaling elements downstream of these oncoproteins were identified after treating cells with the indolinone tyrosine kinase inhibitor RPI-1 to knock down RET phosphorylation activity. We detected 23 and 18 affinity-purified phosphotyrosine proteins in untreated TT and MZ-CRC-1 cells, respectively, most of which were shared and sensitive to RPI-1 treatment. However, our data clearly point to specific signaling features of the RET-MEN2A and RET-MEN2B oncogenic pathways. Moreover, the detection of high-level expression of minimally phosphorylated epidermal growth factor receptor (EGFR) in both TT and MZ-CRC-1 cells, together with our data on the effects of EGF stimulation on the proteomic profiles and the response to Gefitinib treatment, suggest the relevance of EGFR signaling in these cell lines, especially since analysis of 14 archival MTC specimens revealed EGFR mRNA expression in all samples. Together, our data suggest that RET/EGFR multi-target inhibitors might be beneficial for therapy of MTC.