MYO5B Mutations in Pheochromocytoma/Paraganglioma Promote Cancer Progression

Identification of additional cancer-associated genes and secondary mutations driving the metastatic progression in pheochromocytoma and paraganglioma (PPGL) is important for subtyping, and may provide optimization of therapeutic regimens. We recently reported novel recurrent nonsynonymous mutations in the MYO5B gene in metastatic PPGL. Here, we explored the functional impact of these MYO5B mutations, and analyzed MYO5B expression in primary PPGL tumor cases in relation to mutation status. Immunohistochemistry and mRNA expression analysis in 30 primary PPGL tumors revealed an increased MYO5B expression in metastatic compared to non-metastatic tumor cases. In addition, subcellular localization of MYO5B protein was altered from cytoplasmic to membranous in some metastatic tumors, and the strongest and most abnormal expression pattern was observed in a paraganglioma case harboring a MYO5B:p.G1611S mutation. In addition to five previously discovered MYO5B mutations, the present mutation analysis of 30 PPGL also revealed two, and hence recurrent, mutations in the paralog MYO5A. The three MYO5B missense mutations with the highest prediction scores (p.L587P, p.G1611S and p.R1641C) were selected and functionally validated using site directed mutagenesis and stable transfection into human neuroblastoma cells (SK-N-AS) and embryonic kidney cells (HEK293 cell line). In vitro analysis showed a significant increased proliferation rate in all three MYO5B mutated clones. The two somatically derived mutations, p.L587P and p.G1611S, were also found to increase the migration rate. Expression analysis of MYO5B mutants compared to wild type clones, demonstrated a significant enrichment of genes involved in migration, proliferation, cell adhesion, glucose metabolism, and cellular homeostasis. Our study validates the functional role of novel MYO5B mutations in proliferation and migration, and suggest the MYO5-pathway to be involved in the malignant progression in some PPGL tumors. Author summary Up to 25% of pheochromocytoma/paraganglioma (PPGL) cases develop metastatic disease with poor outcome and few treatment options. The disease mechanism is not fully understood, and to date there are no reliable markers to predict malignancy. We have recently discovered novel missense mutations in the non-conventional myosin 5 gene (MYO5B), an endosomal transport protein, which we now show enhances progression and migration in PPGLs. MYO5B mutations were preferentially found in patients with metastatic disease and SDH deficiency (germline SDHB-mutations). Abolished SDH activity result in a metabolic switch to aerobic glycolysis requiring increased glucose consumption. Since the MYO5B mutations were found to drive progression through downstream up-regulation of glucose metabolism genes, e.g. glucagon, we hypothesize that these mutations may fuel the pseudohypoxic state by altering glucose uptake in cancer cells. Our result is the first to link the myosin 5 genes to PPGL tumorigenesis. Further, it shows that the tumor progression route in PPGL is complex, with contribution from several genetic factors. An increasing number of studies show dysregulation and importance of the MYO5-proteins in cancer, but little is still known about the precise role and mechanism of mutations, hence more research in this area is needed.