RESUMO
Endothelins (EDN) are peptide hormones that activate a GPCR signalling system and contribute to several diseases, including hypertension and cancer. Current knowledge about EDN signalling is fragmentary, and no systems level understanding is available. We investigated phosphoproteomic changes caused by endothelin B receptor (ENDRB) activation in the melanoma cell lines UACC257 and A2058 and built an integrated model of EDNRB signalling from the phosphoproteomics data. More than 5,000 unique phosphopeptides were quantified. EDN induced quantitative changes in more than 800 phosphopeptides, which were all strictly dependent on EDNRB. Activated kinases were identified based on high confidence EDN target sites and validated by Western blot. The data were combined with prior knowledge to construct the first comprehensive logic model of EDN signalling. Among the kinases predicted by the signalling model, AKT, JNK, PKC and AMP could be functionally linked to EDN-induced cell migration. The model contributes to the system-level understanding of the mechanisms underlying the pleiotropic effects of EDN signalling and supports the rational selection of kinase inhibitors for combination treatments with EDN receptor antagonists.
Assuntos
Endotelinas/farmacologia , Regulação Neoplásica da Expressão Gênica , Melanócitos/metabolismo , Fosfoproteínas/genética , Processamento de Proteína Pós-Traducional , Transdução de Sinais , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Endotelinas/genética , Endotelinas/metabolismo , Redes Reguladoras de Genes , Humanos , MAP Quinase Quinase 4/genética , MAP Quinase Quinase 4/metabolismo , Melanócitos/efeitos dos fármacos , Melanócitos/patologia , Fosfoproteínas/metabolismo , Fosforilação , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Proteômica/métodos , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor de Endotelina B/genética , Receptor de Endotelina B/metabolismoRESUMO
The clinical benefit of MAPK pathway inhibition in melanoma patients carrying BRAF mutations is temporal. After the initial response to treatment, the majority of tumors will develop resistance and patients will relapse. Here we demonstrate that the endothelin-endothelin receptor B (ETBR) signaling pathway confers resistance to MAPK pathway inhibitors in BRAF mutated melanoma. MAPK blockade, in addition to being anti-proliferative, induces a phenotypic change which is characterized by increased expression of melanocyte-specific genes including ETBR. In the presence of MAPK inhibitors, activation of ETBR by endothelin enables the sustained proliferation of melanoma cells. In mouse models of melanoma, including patient-derived xenograft models, concurrent inhibition of the MAPK pathway and ETBR signaling resulted in a more effective anti-tumor response compared to MAPK pathway inhibition alone. The combination treatment significantly reduced tumor growth and prolonged survival compared to therapies with MAPK pathway inhibitors alone. The phosphoproteomic analysis revealed that ETBR signaling did not induce resistance towards MAPK pathway inhibitors by restoring MAPK activity, but instead via multiple alternative signaling pathways downstream of the small G proteins GNAq/11. Together these data indicate that a combination of MAPK pathway inhibitors with ETBR antagonists could have a synergistically beneficial effect in melanoma patients with hyperactivated MAPK signaling pathways.