Activating germline RET mutations cause Multiple Endocrine Neoplasia type 2A (MEN2A), type 2B (MEN2B) and Familial Medullary Thyroid Carcinoma (FMTC). Despite clear genotype-phenotype correlations, the molecular mechanisms connecting the mutated RET receptors with the distinct subtypes of MEN2 are far from understood. Here we demonstrate that the SRC kinase-, STAT3- and ERK1/2signaling pathways are differentially activated by specific MEN2-RET receptors and that the activation level of these pathways strongly correlate with the degree of RETTyr981 phosphorylation and phenotypical aggressiveness. In addition, inhibition of ERK1/2 activation by U0126 blocked SRC Tyr416 induced phosphorylation by MEN2B-RET. To further explore the signaling pathways activated downstream of the different RET mutants, oligonucleotide microarray analysis, quantitative real time PCR, promoter analysis and immunohistochemistry were performed. Hierarchical clustering analysis revealed a sub-cluster of up-regulated genes implicated in cytokine signaling (IRAK1, IRF6 and SMAD6) and cell cycle regulation (CDKN3, CCNB1, CCND1, CDC42, CDKN1A and CCNA2) associated with the MEN2B-RET expressing cells. A sub-cluster of enriched genes implicated in the integrin and ~-catenin signaling (ITGR8, VAV2, PA2G4 and APC) was found associated with the MEN2A-RET expressing cells. Finally, a subcluster enriched in genes encoding for transcription factors involved in cell proliferation, differentiation and transformation (FOS, EGR1, JUN, JUNB) was observed in FMTC-RET samples. To understand how RET mutations targeting the kinase domain can alter RET activation and signaling, a model for the MEN2B mutations RETA883F and RETM918T is proposed. Our structural-model analysis predicted that MEN2B-type mutations change the conformation of the RET kinase domain altering both the catalytic activity and substrate specificity of the receptor. Together these data provide novel insights into the signaling and gene expression profiles associated with specific disease phenotype RET mutations and reveal potential mechanisms of disease-phenotype in the cancer syndrome MEN2.
MUTATION-SPECIFIC SIGNALING AND GENE EXPRESSION PROFILES BY ONCOGENIC RET REVEAL NOVEL POTENTIAL MECHANISMS OF DISEASEPHENOTYPE IN MULTIPLE ENDOCRINE NEOPLASIA TYPE II SYNDROMES
1 Department of Genetics, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands, 2Department of System Biology, Centre of Genomic Regulation, Barcelona, Spain, 3DepartmentofPathology, University Medical Center Groningen, University of Groningen, The Netherlands, 4Developmental Genetics, Groningen Biomolecular Science and Biotechnology Institute, Haren, University of Groningen, The Netherlands, 5PamGene International B.V., s'-Hertogenbosh, The Netherlands, 6Breakthrough Breast Cancer Research Centre, Institute of Cancer Research, London, UK, 7Section of Molecular and Cellular Medicine, Imperial College, London, UK
Financial support: This work was supported by GUIDE and the Ubbo Emmius Fundation, Breakthrough Breast Cancer, Medical Research Council, and Ministerio de Educaci6n y Ciencia (MEC) of Spain.