The neuroblastoma tumour-suppressor TrkAI and its oncogenic alternative TrkAIII splice variant exhibit geldanamycin-sensitive interactions with Hsp90 in human neuroblastoma cells

Abstract

Hsp90 chaperones stabilize many tyrosine kinases including several oncogenes, which are inhibited or induced to degrade by the Hsp90 inhibitor geldanamycin (GA). As a consequence, GA has been developed for future chemotherapeutic use in several tumour types including neuroblastoma (NB). Alternative splicing of the neurotrophin receptor tyrosine kinase TrkA may have a pivotal function in regulating NB behaviour, with reports suggesting that tumour-suppressing signals from TrkA may be converted to oncogenic signals by stress-regulated alternative TrkAIII splicing. Within this context, it is important to know whether Hsp90 interacts with TrkA variants in NB cells and how GA influences this. Here, we report that both TrkAI and TrkAIII are Hsp90 clients in human NB cells. TrkAI exhibits GA-sensitive interaction with Hsp90 required for receptor endoplasmic reticulum export, maturation, cell surface stabilization and ligandmediated activation, whereas TrkAIII exhibits GAsensitive interactions with Hsp90 required for spontaneous activity and to a lesser extent stability. We show that GA inhibits proliferation and induces apoptosis of TrkAI expressing NB cells, whereas TrkAIII reduces the sensitivity of NB cells to GA-induced elimination. Our data suggest that GA-sensitive interactions with Hsp90 are critical for both TrkAI tumour suppressor and TrkAIII oncogenic function in NB and that TrkAIII expression exerts a negative impact on GA-induced NB cell eradication, which can be counteracted by a novel TrkAIII-specific peptide nucleic acid inhibitor.

Alternative TrkA Splicing and Neuroblastoma

“…This is supported by the inverse relationship exhibited by TrkA expression and NB stage (Nakagawara et al, 1992;Nakagawara & Koger, 2000;Nakagawara, 2001) and suggests that the reintroduction of adequate TrkA expression levels and/or the activation of post cell surface TrkA receptor signalling represents an important potential therapeutic goal in NB. Recently, however, a darker side to TrkA involvement in NB has been revealed by the discovery of an alternative TrkA splice variant "TrkAIII" expressed by advanced stage primary human NBs that exhibits oncogenic activity in NB models (Tacconelli et al, 2004;Farina et al, 2009aFarina et al, , 2009b.…”

“…The mature gp140 TrkAI receptor is expressed predominantly at the cell surface with GN accumulation of the immature gp110TrkAI receptor, whereas TrkAIII is not expressed at the cell surface but is retained within intracellular membranes, within which it exhibits relatively equal distribution between the endoplasmic reticulum (ER), endoplasmic reticulum and Golgi intermediate (ERGIC); Golgi network (GN) and associated vesicle compartments (Tacconelli et al, 2004;Farina et al, 2009aFarina et al, , 2009b; c) Differences in spontaneous versus liganddependent activation. TrkAI exhibits ligand-dependent but not spontaneous activity, whereas TrkAIII exhibits spontaneous ligand-independent activation and does not bind neurotrophins; d) Differences in post receptor signal transduction.…”

“…Spontaneous ligand-independent TrkAIII activation most likely depends upon the omission of the extracellular D4 Ig-like domain plus associated N-glycosylation sites, which prevent ligand-independent TrkA activation (Arevalo et al, 2000). Differences in adapter protein binding exhibited by ligand-activated TrkAI and spontaneously-active TrkAIII and subsequent differences in post receptor signalling most likely reflect differences in receptor localisation, with lack of post TrkAIII signalling through Ras/MAPK explained either by dislocation from caveolae-associated Ras/MAPK (Paratcha & Ibanez, 2002;Rajagopal et al, 2004), altered adapter protein binding (Tacconelli et al, 2004;Farina et al, 2009aFarina et al, , 2009b; TrkAIII activity below the Ras/MAPK activation threshold (Hallberg et al, 1998); and/or direct PI3K/Akt inhibition of Raf-MEK-ERK signalling (Moelling et al, 2002), the latter possibility supported by TrkAIII antagonism of NGF/TrkAI-induced ERK phosphorylation (Tacconelli et al, 2004). Interestingly, post TrkAIII receptor signalling through PI3K/Akt but not Ras/MAPK resembles post receptor signalling from GN-associated immature gp110TrkAI transactivated by G-protein associated A 2A adenosine receptors (Rajagopal et al, 2004), indicating that an intracellular location alters post TrkA receptor signalling.…”

“…In response to ligand, activated TrkA receptors exhibit receptor-associated tyrosine kinase and PI3K activity, are phosphorylated on Y490, Y674/675 and Y758 residues, bind Shc, Grb2 and FRS2 adapters and signal through PI3K/Akt and Ras/MAPK, whereas spontaneously active TrkAIII exhibits tyrosine kinase and PI3K activity, is constitutively phosphorylated on Y490, Y674/675 and Y758 residues, binds only low levels of non-phosphorylated Shc, does not bind FRS2 or GRB2 and signals through IP3K/Akt but not Ras/MAPK (Tacconelli et al, 2004); and e) differences in biological activity, with TrkAI exhibiting tumour-suppressing and TrkAIII oncogenic activity in NB models (Tacconelli et al, 2004). These differences depend upon the omission of sequences encoded within exons 6/7 and form the basis of the differential TrkAI/II tumour-suppressing and TrkAIII oncogenic activity observed (Tacconelli et al, 2004;Farina et al, 2009aFarina et al, , 2009b. Intracellular TrkAIII retention versus cell surface TrkAI expression may depend upon differences in extracellular domain N-glycosylation, which regulates cell surface TrkA expression (Watson et al, 1999).…”

“…It is clear that the extracellular D4 domain and associated N-glycosylation sites, encoded within TrkA exons 6/7, are critical for correct TrkA receptor physiological function, cell surface expression, ligand-dependent activation and preventing ligand-independent receptor oncogenic activation (Arevalo et al, 2000;Watson et al, 1999). The loss of this domain confers oncogenic potential to TrkAIII (Tacconelli et al, 2004;Arevalo et al, 2000;Watson et al, 1999;Farina et al, 2009aFarina et al, , 2009b. …”