TrkAIII signals endoplasmic reticulum stress to the mitochondria in neuroblastoma cells, resulting in glycolytic metabolic adaptation

Abstract

Alternative TrkAIII splicing characterises advanced stage metastatic disease and post-therapeutic relapse in neuroblastoma (NB), and in NB models TrkAIII exhibits oncogenic activity. In this study, we report a novel role for TrkAIII in signaling ER stress to the mitochondria in SH-SY5Y NB cells that results in glycolytic metabolic adaptation. The ER stress-inducing agents DTT, A23187 and thapsigargin activated the ER stress-response in control pcDNA SH-SY5Y and TrkAIII expressing SH-SY5Y cells and in TrkAIII SH-SY5Y cells increased TrkAIII targeting to mitochondria and internalisation into inner-mitochondrial membranes. Within inner-mitochondrial membranes, TrkAIII was subjected to Omi/HtrA2-dependent cleavage to tyrosine phosphorylated 45–48kDa carboxyl terminal active fragments, localised predominantly in tyrosine kinase-domain mitochondrial matrix orientation. This stress-induced activation of mitochondrial TrkAIII was associated with increased ROS production, prevented by the ROS scavenger Resveratrol and underpinned by changes in Ca2+ movement, implicating ROS/Ca2+ interplay in overcoming the mitochondrial TrkAIII activation threshold. Stress-induced, cleavage-activation of mitochondrial TrkAIII resulted in mitochondrial PDHK1 tyrosine phosphorylation, leading to glycolytic metabolic adaptation. This novel mitochondrial role for TrkAIII provides a potential self-perpetuating, drug reversible way through which tumour microenvironmental stress may maintain the metastasis promoting “Warburg effect” in TrkAIII expressing NBs.

The oncogenic neurotrophin receptor tropomyosin-related kinase variant, TrkAIII

“…TrkAIII intracellular re-localisation and oncogenic behaviour supports the growing concept that mislocalization of TrkA-derived oncogenes underpins oncogenic signaling [ 3 , 7 , 19 , 26 , 32 , 40 , 50 ]. In contrast to fully-spliced cell surface TrkA receptors that associate with caveolin in low density membrane fractions [ 23 ] and exhibit tumour suppressing activity in NB [ 1 , 19 ], TrkAIII receptors do not associate with caveolin in low density membrane fractions [ 23 ] and re-localise to intracellular pre-Golgi membranes, in which they exhibits spontaneous activation and self-perpetual recycling between the ER and ERGIC-COPI compartments [ 19 , 40 , 50 ].…”

“…2 ) (manuscript in preparation). Spontaneous TrkAIII activation could also result from down-regulating the expression and activity of TrkA de-phosphorylating PTPases, such as Shp1 and PTP1B [ 19 , 32 ], in a manner analogous to PTP1B regulation of the NB-associated oncogene Alk [ 33 , 34 ], which may also influence potential interactions between Alk and TrkAIII [ 35 ].…”

“…PI3K/Akt/NF-кB signaling is also important for cell survival, and TrkAIII transfectants exhibit enhanced resistance to genotoxic- (cisplatin), oxidative- (paraquat, rotenone and LY83583) and ER-stress (DTT, A23187 and thapsigargin) [ 19 , 32 , 49 ]. Increased stress-resistance exhibited by TrkAIII transfectants associates with elevated expression and mitochondrial localisation of anti-apoptotic Bcl-2, Bcl-xL and Mcl-1 proteins and also with enhanced expression of the mitochondrial anti-oxidant SOD-2 [ 32 , 49 ].…”

“…PI3K/Akt/NF-кB signaling is also important for cell survival, and TrkAIII transfectants exhibit enhanced resistance to genotoxic- (cisplatin), oxidative- (paraquat, rotenone and LY83583) and ER-stress (DTT, A23187 and thapsigargin) [ 19 , 32 , 49 ]. Increased stress-resistance exhibited by TrkAIII transfectants associates with elevated expression and mitochondrial localisation of anti-apoptotic Bcl-2, Bcl-xL and Mcl-1 proteins and also with enhanced expression of the mitochondrial anti-oxidant SOD-2 [ 32 , 49 ]. Trk tyrosine kinase inhibitors (K252a, CEP-701 and Gö6976), PI3K inhibitor (LY294002) and NF-κB inhibitors (dn-NF-кB and PDTC), all reduce Bcl-2, Bcl-xL and SOD2 expression in TrkAIII transfectants and increase sensitivity to genotoxic-, oxidative- and ER-stress induced death, confirming dependence upon TrkAIII tyrosine kinase, PI3K and NF-кB activity for survival [ 19 , 32 , 49 ].…”

“…In addition to pro-survival and pro-angiogenic PI3K/Akt/NF-кB signaling, TrkAIII expression also results in partial activation of a survival-adapted UPR, characterised by ATF6 but not Ire1a/Xbp-1 activation, and increased expression of the UPR chaperone Grp78/Bip and the apoptosis inhibitor Mcl-1 [ 32 , 40 , 50 , 58 ]. In TrkAIII transfectants, ER-associated TrkAIII is complexed with Grp78/Bip, confirming that TrkAIII has difficulty in satisfying ER quality control and helping to explain partial UPR activation [ 50 ].…”