Doxorubicin-Induced TrkAIII Activation: A Selection Mechanism for Resistant Dormant Neuroblastoma Cells

Abstract

Patients with advanced neuroblastoma (NB) receive multimodal clinical therapy, including the potent anthracycline chemotherapy drug doxorubicin (Dox). The acquisition of Dox resistance, however, is a major barrier to a sustained response and leads to a poor prognosis in advanced disease states, reinforcing the need to identify and inhibit Dox resistance mechanisms. In this context, we report on the identification and inhibition of a novel Dox resistance mechanism. This mechanism is characterized by the Dox-induced activation of the oncogenic TrkAIII alternative splice variant, resulting in increased Dox resistance, and is blocked by lestaurtinib, entrectinib, and crizotinib tyrosine kinase and LY294002 IP3-K inhibitors. Using time lapse live cell imaging, conventional and co-immunoprecipitation Western blots, RT-PCR, and inhibitor studies, we report that the Dox-induced TrkAIII activation correlates with proliferation inhibition and is CDK1- and Ca2+-uniporter-independent. It is mediated by ryanodine receptors; involves Ca2+-dependent interactions between TrkAIII, calmodulin and Hsp90; requires oxygen and oxidation; occurs within assembled ERGICs; and does not occur with fully spliced TrkA. The inhibitory effects of lestaurtinib, entrectinib, crizotinib, and LY294002 on the Dox-induced TrkAIII and Akt phosphorylation and resistance confirm roles for TrkAIII and IP3-K consistent with Dox-induced, TrkAIII-mediated pro-survival IP3K/Akt signaling. This mechanism has the potential to select resistant dormant TrkAIII-expressing NB cells, supporting the use of Trk inhibitors during Dox therapy in TrkAIII-expressing NBs.

The Alternative TrkAIII Splice Variant, a Targetable Oncogenic Participant in Human Cutaneous Malignant Melanoma

“…ER stress involvement in alternative TrkA mRNA splicing in CMMs was suggested by association with unconventional Xbp-1 splicing, indicating UPR activation [44,45]. This extends previous reports of UPR activation in CMM [29][30][31][32], and is supported by the observation that reductive stress induced by DTT activated the UPR and promoted TrkAIII mRNA expression in A375 melanoma cells (this study), and by previous reports that agents that induce ER stress and activate the UPR promote alternative TrkAIII splicing in SH-SY5Y NB cells [14,15,25]. Alternative TrkA mRNA splicing in CMM may also result from mutations in the spliceosome component SF3B1 shown to promote exon skipping [48] but is unlikely to depend upon BRAF mutation, as alternative TrkA mRNA splicing was detected in both BRAF wild-type and BRAF-mutated CMMs.…”

“…In SH-SY5Y NB cells, alternative TrkAIII splicing is also promoted by SV40 poliomavirus large T-antigen expression [15], suggesting that TrkAIII expression in MCPyV-positive Merkel cell carcinomas may be promoted by MCPyV large T antigen [18,19]. Here, we show that reductive stress promotes TrkAIII expression and activation in melanoma cells, extending reports that TrkAIII mRNA expression is promoted by hypoxia [14,25]. As hypoxia promotes reductive stress, which in turn promotes tumour progression [27,28], conditions that promote reductive stress in CMMs may underpin alternative TrkA splicing, TrkAIII expression and TrkAIII activation.…”

“…Unconventional Xbp-1 splicing, as an index of UPR activation [44,45], was detected in 4 of 5 snap-frozen primary CMMs (4 primary CMMs P.5-8 were not analysed due to limited RNA) and in 16 of 16 (100%) of metastatic CMMs (patients P.16, P.17 and P. 25 were not assessed due to limited RNA). Unconventional Xbp-1 splicing was not detected in uninvolved skin samples (Figure 1, and Supplementary Figures S1-S3).…”

“…TrkA signalling through RAS/MAPK, on the other hand, inhibits NB cell proliferation, promotes neural differentiation and suppresses angiogenesis and tumorigenicity in NB models [14,15,17]. NTRK1, therefore, produces a wider range of TrkA protein isoforms than previously considered, with tumour-suppressing and oncogenic potential [14,15,24,25], and TrkAIII represents the pathological equivalent of a previously reported engineered oncogenic TrkA D4-deletion mutant [19].…”

A Study of Alternative TrkA Splicing Identifies TrkAIII as a Novel Potentially Targetable Participant in PitNET Progression

“…The variant TrkAIII receptor lacks the extracellular D4 IG-C1 domain and several N-glycosylation sites that are required for fully spliced TrkA receptor cell surface expression and prevention of ligand-independent activation [ 30 , 31 , 32 ]. These omissions result in the intracellular re-localization of TrkAIII to pre-Golgi membranes, centrosomes, and mitochondria, where TrkAIII exhibits ligand-independent, cell cycle-regulated, stress-regulated, and doxorubicin-induced intracellular activation [ 25 , 33 , 34 , 35 , 36 ]. The intracellular activation of TrkAIII results in pro-survival phosphoinositide 3-kinase (PI3K)/Akt signaling, increased expression of B-cell lymphoma 2 (Bcl-2), myeloid cell leukemia sequence 1 (Mcl-1) and superoxide dismutase 2 (SOD2), a pro-angiogenic expression equilibrium between matrix metalloproteinase-9 (MMP-9)/vascular endothelial cell growth factor (VEGF)/thrombospondin 1 (Tsp1), centrosome amplification, stress-regulated metabolic adaptation, a modified unfolded protein response (UPR), and a more anaplastic stem cell-like phenotype [ 25 , 30 ].…”