Abstract
The developmental and stress-regulated alternative TrkAIII splice variant of the NGF receptor TrkA is expressed by advanced stage human neuroblastomas (NBs), correlates with worse outcome in high TrkA expressing unfavourable tumours and exhibits oncogenic activity in NB models. In the present study, we report that constitutive TrkAIII expression in human SH-SY5Y NB cells inhibits Rotenone, Paraquat and LY83583-induced mitochondrial free radical reactive oxygen species (ROS)-mediated death by stimulating SOD2 expression, increasing mitochondrial SOD2 activity and attenuating mitochondrial free radical ROS production, in association with increased mitochondrial capacity to produce H2O2, within the context of a more tumour stem cell-like phenotype. This effect can be reversed by the specific TrkA tyrosine kinase inhibitor GW441756, by the multi-kinase TrkA inhibitors K252a, CEP-701 and Gö6976, which inhibit SOD2 expression, and by siRNA knockdown of SOD2 expression, which restores the sensitivity of TrkAIII expressing SH-SY5Y cells to Rotenone, Paraquat and LY83583-induced mitochondrial free radical ROS production and ROS-mediated death. The data implicate the novel TrkAIII/SOD2 axis in promoting NB resistance to mitochondrial free radical-mediated death and staminality, and suggest that the combined use of TrkAIII and/or SOD2 inhibitors together with agents that induce mitochondrial free radical ROS-mediated death could provide a therapeutic advantage that may also target the stem cell niche in high TrkA expressing unfavourable NB.
Highlights
The alternative TrkAIII splice variant (UniProtKB/Swiss-Prot: P04629-4) of the Nerve growth factor (NGF) receptor TrkA (NCBI: NM_0010122331.1; GenBank: AB019488.2; UniProtKB/Swiss-Prot: P04629) is expressed by advanced stage human neuroblastoma (NB), is associated with poor outcome in high TrkA expressing unfavourable tumours and exhibits oncogenic activity in NB models [1,2,3,4,5,6,7]
We report that constitutive expression of the TrkAIII oncogene in human SH-SY5Y NB cells inhibits mitochondrial free radical reactive oxygen species (ROS)-mediated death induced by Rotenone, Paraquat and LY83583, by stimulating SOD2 expression, increasing mitochondrial SOD2 activity and attenuating mitochondrial free radical ROS production
These effects associate with increased mitochondrial capacity to produce H2O2, occur within the context of a more tumour stem cell-like phenotype, and can be reversed by the specific TrkA tyrosine kinase inhibitor GW441756 [51], by the multi-kinase TrkA inhibitors CEP-701 [59], K252a [49] and Go6976 [50] and by siRNA knockdown of SOD2 expression, all of which restore the sensitivity of TrkAIII SH-SY5Y cells to Rotenone, Paraquat and LY83583-induced mitochondrial free radical accumulation and free radical-mediated death
Summary
The alternative TrkAIII splice variant (UniProtKB/Swiss-Prot: P04629-4) of the NGF receptor TrkA (NCBI: NM_0010122331.1; GenBank: AB019488.2; UniProtKB/Swiss-Prot: P04629) is expressed by advanced stage human neuroblastoma (NB), is associated with poor outcome in high TrkA expressing unfavourable tumours and exhibits oncogenic activity in NB models [1,2,3,4,5,6,7]. Alternative TrkAIII splicing is stress-regulated, providing a mechanism through which tumour suppressing signals from fully spliced TrkA receptors can be converted to oncogenic signals from the alternative spliced TrkAIII variant within the tumour microenvironment. We consider this to potentially represent the conservation and pathological subversion of a physiological developmental and stress-regulated, neural stem/progenitor cell stress-protection mechanism [1,8]. Alternative TrkAIII splicing is characterised by exon 6,7 and 9 skipping and produces a TrkAIII protein that is devoid of the extracellular D4 Ig-like domain and related N-glycosylation sites required for cell surface receptor expression and prevention of ligand-independent activation [9,10]. TrkAIII exerts its ‘‘oncogenic’’ activity in NB cells by: protective IP3K/Akt/NF-kB signalling; induction of a pro-angiogenic pattern of gene expression; interacting with the centrosome, promoting centrosome amplification, peri-nuclear microtubule assembly and genetic instability; increasing the level of sister chromatid exchange; and modulating the unfolded protein response, pre-conditioning and adapting cells to stress [1,2,3,4,5]
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