Proteomics study of medullary thyroid carcinomas expressing RET germ-line mutations: identification of new signaling
L Gorla1, P Mondellini1, G Cuccuru2
1Proteomics Laboratory, Department of Experimental Oncology and Laboratories, Fondazione IRCCS Istituto Nazionale Tumori, Milan, Italy.
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.
Insights
Proteomics identified key signaling molecules in medullary thyroid carcinoma (MTC) cell lines. Targeting both RET and epidermal growth factor receptor (EGFR) may offer new therapeutic strategies for MTC.
Area of Science:
- Oncology
- Molecular Biology
- Proteomics
Background:
- Proteomics aids in understanding signaling networks and identifying therapeutic targets.
- Multiple Endocrine Neoplasia (MEN) syndromes, including MEN2A and MEN2B, are linked to RET tyrosine kinase receptor mutations.
- Medullary Thyroid Carcinoma (MTC) is a neuroendocrine tumor often associated with RET alterations.
Purpose of the Study:
- To elucidate differential signaling networks in MTC cell lines expressing RET oncoproteins.
- To identify novel therapeutic targets for MTC by analyzing phosphotyrosine signaling.
- To investigate the role of epidermal growth factor receptor (EGFR) signaling in MTC.
Main Methods:
- Proteomic-multiplexed analysis of phosphotyrosine signaling.
- Antibody-based validation techniques.
- Treatment of MTC cell lines (TT and MZ-CRC-1) with the tyrosine kinase inhibitor RPI-1.
- Analysis of epidermal growth factor (EGF) stimulation and Gefitinib treatment.
- Examination of EGFR mRNA expression in archival MTC specimens.
Main Results:
- Identified 23 and 18 phosphotyrosine proteins in TT and MZ-CRC-1 cells, respectively, many sensitive to RPI-1.
- Revealed distinct signaling features of RET-MEN2A and RET-MEN2B oncogenic pathways.
- Detected high-level expression of minimally phosphorylated EGFR in both cell lines, suggesting its relevance.
- EGFR mRNA was expressed in all 14 analyzed MTC specimens.
Conclusions:
- Proteomic analysis successfully identified signaling molecules downstream of RET oncoproteins in MTC.
- EGFR signaling plays a significant role in MTC, alongside RET signaling.
- Combined RET/EGFR targeted inhibitors show promise as a therapeutic strategy for MTC.
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