Caratterizzazione molecolare del ruolo di protezione mitocondriale della proteina “misfolded” TrkAIII in cellule di Neuroblastoma

Pediatric neuroblastomas (NBs) are heterogeneous, aggressive, therapy-resistant embryonal tumours that originate from cells of neural crest origin committed to the sympathoadrenal progenitor cell lineage. Stress and drug resistance mechanisms drive post therapeutic relapse and metastatic progression, the characterization and inhibition of which are major goals in improving therapeutic responses. Stress and drug resistance mechanisms in NBs include alternative TrkAIII splicing of the NTRK1/TrkA neurotrophin receptor tropomyosin-related kinase, which correlates with post therapeutic relapse and advanced stage metastatic disease. The TrkAIII receptor variant exerts oncogenic activity in NB models by mechanisms that include stress-induced mitochondrial importation and activation. In this study, we characterize novel targetable and non-targetable participants in this pro-survival mechanism in TrkAIII expressing SH-SY5Y NB cells, using dithiothreitol (DTT) as an activator and a variety of inhibitors by regular and immunoprecipitation Western blotting of purified mitochondria and Incucyte cytotoxicity assays. We report that stress-induced TrkAIII misfolding initiates this mechanism, resulting in Grp78, Ca2+-calmodulin, adenosine ribosylating factor 27 (Arf) and Hsp90-regulated mitochondrial importation. TrkAIII imported into inner mitochondrial membranes is cleaved by OmiHtrA2 then activated by a mechanism dependent upon CaMKII/Akt, mitochondrial Ca2+ uniporter and ROS, involving inhibitory mitochondrial PTPase oxidation, resulting in PI3K activation of mitochondrial Akt, which enhances stress resistance. This novel prosurvival function for misfolded TrkAIII mitigates the cytotoxicity of mitochondrial Ca2+ homeostasis disrupted during integrated stress responses, and is prevented by clinically approved Trk and Akt inhibitors, and also by inhibitors of Grp78, Hsp90, Ca2+-calmodulin and PI3K. This identifies Grp78, Ca2+-calmodulin, Hsp90, PI3K and Akt as novel targetable participants in this mechanism, in addition to TrkAIII, the inhibition of which has potential to enhance the stress-induced elimination of TrkAIII expressing NB cells, with potential to improve therapeutic outcomes in NBs that exhibit TrkAIII expression and activation.