TrkAIII and SOD2 in neuroblastoma:

When a splice variant like TrkAIII behaves differently than the canonical TrkAII variant, one has to ask what this could mean for Trk  fusion proteins.

Pierdomenico Ruggeri and coworkers tested the hypothesis that not only are neuroblastoma associated proteins TrkAIII and mitochondrial superoxide dismutase 2 (SOD2) causally related, but that combination therapy might offer an  advantage for the treatment of neuroblastoma.  Their reasoning was based on published literature that they reviewed in their introduction.

Cartoon of how SOD catalyzes the breakdown of super oxide into hydrogen peroxide and water

Superoxide dismutase (SOD) catalyzes the breakdown of super oxide into hydrogen peroxide and water. SOD2 resides in the matrix of the mitochondria.

TrkAIII

    • The TrkAIII transcript has been associated with neuroblastoma
    • Canonical wild type TrkA’s ligand, nerve growth factor, has been shown to be associated with SOD2 expression.
    • TrkAII is expressed on the cell membrane. TrkAIII is restricted to intracellular membranes.
    • TrkAIII lacks one Ig repeat and a small intracellular region between the transmembrane domain and the kinase domain.
  • In many sister sites we cite references that claim Trk signaling through RAS, PI3K, and PLCγ pathways. Ruggeri cite references that claim that TrkAIII does not signal through the RAS/MAPK pathway. The main pathways for TrkAIII are PI3K, Akt, NF-kB.

SOD2

    • is the major mitochondrial superoxide dismutase.
    • promotes cell survival by scavenging of superoxide and production of the  signaling molecule H2O2, hydrogen peroxide.
    • is expressed as 1.5 kb and 4.2 kb mRNAs that code for the same protein but differ in the 3’-UTR.
    • has four isoforms as a translated protein.
    •  is regulated by CpG promoter methylation,  histone hyper-acetylation, and DNA damage and the cell cycle signals.
  • has the following transcription factors: SP1, NF-kB, AP-1, AP-2, CREB, C/EBP, p53, FoxO, and STAT3.

The 4.2 kb SOD2 transcript

  • is found in undifferentiated, proliferating cells.
  • is negatively regulated by small inhibitory RNAs of Alu/7SL origin
  • is positively regulated by the SBP1 binding protein

TrkAIII and the SOD2 transcripts

SH-Sy5Y neuroblastoma cells were transfected with the empty pcDNA expression vector, and the vector loaded with a DNA constructs coding for TrkAII and TrkAIII.

cartoon of splicing and domains of TrkA. Bar Graph showing Splice variant TrkAIII increases the presence of the 4.2 kb SOD2 transcript and protein.

Figure 1. Splice variant TrkAIII increases the presence of the 4.2 kb SOD2 transcript and protein.  A. splicing and domains of TrkA B. Select results Ruggeri (2014)

Use of inhibitors to block TrkAIII induced SOD2 expression

Bar graphs showing how Inhibition of TrkA pathways decreases SOD2 protein expression. NFkB inhibitors are in green, MAPK inhibitor, PD098 is yellow.

Figure 2 Inhibition of TrkA pathways decreases SOD2 protein expression. Specific and quasi-specific TrkA kinase inhibitors are in shades of blue. NFkB inhibitors are colored in shades of green. The MAPK inhibitor, PD098 , is yellow.

Inhibitors used

  • 1 μM CEP-701 (CEP) TrkA
  • 1 μM K252a (K2) TrkA
  • 25 μM LY294002 (LY294) PI3K
  • 1 μM GÖ6976 (GÖ69) TrkA
  • 100 μM GW441756 (GW44) TrkA specific
  • 500 μM PDTC (PDTC) NFκB
  • 100 μM Bay 11–7082 (BAY11)  NFκB
  • 30 μM PD098059 (PD098) MAPK

for 24 hours at 37oC.

Trx2 is an enzyme that reduces protein disulfide bonds.  Like SOD2, it is part of the cell’s oxidative stress defense arsenal.  The TrkA pathway inhibitors were  effective in reducing the 4.2 and the 1.5 kb SOD2 transcript, not shown.

TrkAIII protects against the reactive oxygen species superoxide

Three methods were used to generate reactive oxygen species. The cell and mitochondrial permeable superoxide indicator Mitosox was used to detect the production of superoxide in transfected neuroblastoma cells.

LY83583 is a small molecule that has been reported in the literature to generate reactive oxygen species.

rotenone transfers electrons from from complex I in the mitochondria to molecular oxygen

paraquat may transfer electrons from NADPH to molecular oxygen

In situ fluorescence pictures showing in red color detection of TrkA in neuroblastoma cells

Figure 3. TrkA and control transfected neuroblastoma cells were treated with agents to generate super oxide. Superoxide was detected as red fluorescence using the probe Mitosox that becomes fluorescent when it reacts with super oxide.

Ruggeri P, Farina AR, Di Ianni N, Cappabianca L, Ragone M, Ianni G, Gulino A, Mackay AR. (2014) The TrkAIII oncoprotein inhibits mitochondrial free radical ROS-induced death of SH-SY5Y neuroblastoma cells by augmenting SOD2 expression and activity at the mitochondria, within the context of a tumour stem cell-like phenotype. PLoS One. 9(4):e94568. PubMed 

Could the  TrkAIII story give us insight into TRK fusion proteins?

Why isn’t the MAPK pathway activated in TrkAIII transfected neuroblastoma cells?

    • MAPK is part of the NGF/TrkAII system.
    • Except for a small part of the juxta membrane region, the intracellular domain is the same in the two isoforms.
    • While there is evidence of dimerization of the LRR and Ig-like domains dimerizing on their own in other proteins, they don’t seem to do so in absence of ligand in canonical TrkAII
    • Examples of TrkA extracellular domains dimerization can be found in Ig-like repeats of nestin and leucine rich domains of decorin.
  • Perhaps two Ig-like domains inhibit some other dimerization in absence of ligand. This could explain constitutive activity of TrkAIII but not the lack of MAPK activity.
Cartoon showing dimerization of LLR and Ig-like domains

Figure 4. The dimerization of LLR and Ig-like domains A. TrkAIII lacks the first Ig-like domain present in TrkAII. B. The LLR domain of decorin is able to dimerize C. The Ig-like domain of cell adhesion protein can dimerize.

If slight differences in the extracellular domain between TrkAII and TrkAIII and the region between the transmembrane (TM) domain and kinase domain result in different kinase, we might expect the same for TrkA fusion proteins.

Comparative horizontal bar diagram showing TrkA fusion proteins in cancer

Figure 5. TrkA fusion proteins in cancer

Important information

TrkA fusion driven cancers might differ from one another in subtle ways like induction of SOD2 and lack of MAPK activation. Their commonality is the same actionable target: the kinase domain of TrkA. There is an open clinical trial for solid tumor cancer patient with TRK fusions. The small molecule being tested is a Trk inhibitor.