Studies on potentially druggable pathways that regulate stress-induced TrkAIII mitochondrial internalization, cleavage and activation in human neuroblastoma cells

BACKGROUND-AIM In aggressive pediatric neuroblastomas (NBs), neurotrophin receptor TrkA alternative splicing, resulting in TrkAIII variant expression, positively correlates with posttherapeutic relapse and advanced stage metastatic disease. In NB models, the TrkAIII variant promotes primary and metastatic tumorigenesis consistent with oncogenic function. Alternative TrkAIII splicing in NB cells is promoted by hypoxia, nutrient deprivation, DTT and A23187 in association with unfolded protein response (UPR) activation, implicating the tumour microenvironment in converting fully spliced TrkA tumour suppressing signals to TrkAIII oncogenic signals. UPR activation, induced by DTT and A23187, also promotes mitochondrial TrkAIII internalization, OMI-mediated TrkAIII cleavage and activation, resulting in PDK1-dependent glycolytic adaptation and enhanced stress-resistance. Here, we have investigated potential druggable pathways involved in this novel stress-induced mitochondrial protective mechanism. METHODS RT-PCR, mitochondria purification, regular and co-immuno-precipitation Western blotting, Incucyte cytotoxicity assays, and Student’s t-test. RESULTS In TrkAIII expressing SH-SY5Y NB cells, DTT-induced mitochondrial TrkAIII internalization, cleavage and activation was associated with UPR activation, enhanced TrkAIII complexing with Grp78 and calmodulin, and was abrogated by Grp78 (HA15), calmodulin (W7) and Hsp90 (geldanamycin) inhibitors. DTT-induced intramitochondrial TrkAIII activation but not cleavage was also prevented by Trk (entrectinib), Ca 2+ uniporter (DS16570511), TRAP-1 (Honokiol DCA) inhibitors and by the ROS scavenger Resveratrol. The clinically approved Trk inhibitor entrectinib significantly enhanced DTT-induce cytotoxicity in TrkAIII SH-SY5Y cells. CONCLUSIONS Under ER stress conditions, misfolded N-glycosylated TrkAIII exhibits Grp78, Ca 2+ calmodulin and Hsp90-dependent mitochondrial internalization, where it is cleaved prior to being activated by a Ca 2+ uniporter and oxidation-dependent mechanism, resulting in enhanced survival. This improved understanding adds and extends potential therapeutic inhibition of this novel pro-survival mechanism from Trk to GRP78, calmodulin, HSP90/TRAP1 and Ca 2+ uniporter inhibitors.