Oncogenes derived from the neurotrophin receptor tropomyosin-related kinase TrkA act as drivers in sub-populations of a wide-range of human cancers. This, combined with a recent report that both adult and childhood cancers driven by novel oncogenic TrkA chimeric-fusions exhibit profound, long-lived therapeutic responses to the Trk inhibitor Larotrectinib, highlights the need to improve clinical detection of TrkA oncogene-driven cancers in order to maximise this novel therapeutic potential. Cancers potentially driven by TrkA oncogenes include a proportion of paediatric neuroblastomas (NBs) that express the alternative TrkA splice variant TrkAIII, which exhibits exon 6, 7 and 9 skipping and oncogenic-activity that depends upon deletion of the extracellular D4 Ig-like domain. In contrast to fully spliced TrkA, which exhibits tumour suppressor activity in NB and associates with good prognosis, TrkAIII associates with advanced stage metastatic disease, post therapeutic relapse and worse prognosis, induces malignant transformation of NIH-3T3 cells and exhibits oncogenic activity in NB models. TrkAIII induction in NB cells is stress-regulated by conditions that mimic hypoxia or perturbate the ER with potential to change TrkA tumour-suppressing signals into oncogenic TrkAIII signals within the stressful tumour microenvironment. In contrast to cell surface TrkA, TrkAIII re-localises to intracellular pre-Golgi membranes, centrosomes and mitochondria, within which it exhibits spontaneous ligand-independent activation, triggering a variety of mechanisms that promote tumorigenicity and malignant behaviour, which impact the majority of cancer hallmarks. In this review, we present updates on TrkAIII detection and association with human malignancies, the multiple ways TrkAIII exerts oncogenic activity and potential therapeutic approaches for TrkAIII expressing cancers, with particular reference to NB.

Keywords:Breast cancer Thioredoxin Matrix metalloproteinase-9 MMP-9/TIMP-1 equilibrium NF-jB Invasion a b s t r a c t Increased expression of thioredoxin (Trx)-1 and matrix metalloproteinase (MMP)-9 associates with malignant breast cancer progression. Here, we describe a functional relationship between Trx-1 and MMP-9 in promoting MDA-MB-231 breast cancer cell invasive behaviour. Trx-1 overexpression stimulated MMP-9 expression, de-regulated the MMP-9/TIMP-1 equilibrium and augmented MMP-9 involvement in a more invasive phenotype. Trx-1 augmented MMP-9 transcription through NFjB, AP-1 and SP1 elements; stimulated p50/p65 NF-jB activity and recruitment to the MMP-9 promoter; and facilitated MMP-9 promoter-accessibility to NF-jB by preventing HDAC recruitment and maintaining MMP-9 promoter histone acetylation. Our data provide a functional basis for Trx-1 and MMP-9 association in malignant breast cancer and identify Trx-1 and NF-jB as potentially druggable targets for reducing MMP-9 involvement in malignant behaviour.

In human SH-SY5Y neuroblastoma (NB) cells, nascent immature N-glycosylated 110kDa TrkA moves rapidly from the endoplasmic reticulum (ER) to the Golgi Network (GN), where it matures into the 140kDa receptor prior to being transported to the cell surface, creating GN and cell surface pools of inactive receptor maintained below the spontaneous activation threshold by a full compliment of inhibitory domains and endogenous PTPases. In contrast, the oncogenic alternative TrkAIII splice variant is not expressed at the cell surface but re-localises to intracellular membranes, within which it exhibits spontaneous ERGIC/COPI-associated activation and oncogenic Akt signalling. In this study, we characterise the mechanism responsible for TrkAIII re-localisation. Spontaneous TrkAIII activation, facilitated by D4 IG-like domain and N-glycosylation site omission, increases spontaneous activation potential by altering intracellular trafficking, inhibiting cell surface expression and eliminating an important inhibitory domain. TrkAIII, spontaneously activated within the permissive ERGIC/COPI compartment, rather than moving in an anterograde direction to the GN exhibits retrograde transport back to the ER, where it is inactivated. This sets-up self-perpetuating TrkAIII re-cycling between the ERGIC and ER, that ensures continual accumulation above the spontaneous activation threshold of the ERGIC/COPI compartment. This is reversed by TrkA tyrosine kinase inhibitors, which promote anterograde transport of inactivated TrkAIII to the GN, resulting in GN-associated TrkAIII maturation to a 120kDa species that is degraded at the proteasome.

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.

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.

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a b s t r a c tThe motility, angiogenesis and metastasis-stimulating factor Autotaxin (Atx), over expressed by human neuroblastomas (NB), is constitutively expressed by human Nmyc-amplified SK-N-BE and non-Nmyc-amplified SH-SY5Y NB cells. Here, we characterise a novel Atx transcriptional mechanism, utilised by both cell lines, that is restricted to the first 285 bp of the Atx promoter and involves AP-1 and SP transcription factors, acting through a CRE/AP-1-like element at position À142 to À149 and a GAbox at position À227 to À235 relative to the Atx translational start site. This novel transcriptional mechanism can be inhibited by internally initiated SP-3 and the natural phenol curcumin.

a b s t r a c tIrreversible MMP-9 inhibition is considered a significant therapeutic goal in inflammatory, vascular and tumour pathology. We report that divalent cation chelators Alendronate and EDTA not only directly inhibited MMP-9 but also promoted irreversible plasmin-mediated MMP-9 inactivation by exposing cryptic plasmin-degradation sites within the MMP-9 catalytic-domain and producing an inhibitory hemopexin-domain fragment. This effect was also observed using MDA-MB-231 breast cancer cells, which activated exogenous plasminogen to degrade endogenous proMMP-9 in the presence of Alendronate or EDTA. Degradation-mediated inactivation of proMMP-9 occurred in the absence of transient activation, attesting to the incapacity of plasmin to directly activate proM-MP-9 and direct MMP-9 inhibition by Alendronate and EDTA. Our study provides a novel rational for therapeutic Alendronate use in MMP-9-dependent pathology characterised by plasminogen activation.