TRK Overexpression

Gene fusions not withstanding, factors that control over expression of wild type Trk are very complicated and at times confusing. TrkA may be over expressed in neuroblastomas. “Over expression” correlates with a more favorable prognosis (Light 2012, Weber 2003, Pajtler 2013, 2014) to site just a few reports that we will cover on this page and in blogs. The intricacies of Trk over expression as a positive prognosis in neuroblastoma is a good example of the importance of looking beneath the surface.  This observation is ironic because the  Trk family of receptor tyrosine kinases were discovered  as a cancer driving, over expressed fusion protein as described on our sister site NTRKfusions.  Using TRK1 as an example, we will explore some factors that control gene expression via the wild type promoter.

Figure 1. The promoter of TRK1 (green) is shared by neighboring genes: INSRR (deep red), SH2D2A (burnt orange)

Upstream of the TRK1 gene

Does transcription of other genes upstream of TRK1 influence its transcription? The answer seems to be “yes.”  We have known about it for about 20 years. It might be informative to examine this matter from the perspective of the sequenced human genome. We have two genes that overlap the promoter of TRK1 that read in the anti-sense direction. Arrows indicate the direction of transcription.  Boxes are exons.  The narrow horizontal lines are introns.

The first influential neighbor is INSRR, or the insulin receptor like receptor. This is receptor tyrosine kinase is activated by alkali extracellular pH. Even it’s coding regions overlap the promoter of TRK1. Further upstream of the TRK1 promoter is the SH2D2A gene, which codes for the T cell specific adapter protein.  The evidence of TrkA and T-cell adapter protein coexpression is really not that plentiful, if it exists at all. The evidence for coexpression of  TrkA and the pH sensing insulin like receptor is more compelling and interesting.

Transcription Factor binding sites in the promoters of TRK1 and its neighbors

Transcription factors are proteins that bind to specific sequences of DNA upstream of the transcription start site of a gene. These response elements may be far upstream in enhancer regions or closer upstream in the gene’s promoter. Depending on the program, transcription factors may enhance or decrease the ability of RNA polymerase to transcribe the DNA sequence into messenger RNA (mRNA) to be translated into protein by the ribosomes.

The TRK1 promoter

The TRK1 promoter A. Transcription factors and their binding sites relative to the transcription start sites B. Environmental factors that regulate NF-kB and gene transcription it controls

Figure 2. Transcription factors of TRK1 A. There are two transcription start sites for canonical TrkA and TrkAIII. B. NF-kB is a transcription factor that may be regulated by things a tumor may experience.

  • The NFκB transcription factors are regulated by environmental factors a tumor growing faster than its blood supply might see, whether or not there is a TRK gene rearrangement driving the cancer.
  • The negative regulator, p53, has sites close to both transcription start sites. In many cancers at least one allele of the TP53 gene codes for a nonfunctional version of the p53 transcription factor. We need to remember that expression of TrkA is a good prognosis for neuroblastoma patients!
  • Binding sites for the glucacorticoid receptor (GR) seem to be plentiful in the region of the primary transcription start site.
  • Sequencing of the mouse TRK1 gene (Sacristán 1999) revealed proximity to the INSRR gene as well as variably methylated CpG islands in the promoter. Promoter methylation was speculated as a means of controlling the transcription of the NTRK1 gene, and ultimately, the sensitivity of the developing neuron to nerve growth factor.

 

The promoters of TRK1′s upstream neighbors

One interesting nuance of this story is that the H2D2A gene shares many common transcription factors with TRK1 except members of the cyclic AMP binding protein family (CREB) and the peroxisome proliferator activated receptor gamma (PPARgamma). Similarities or differences with TRK1 transcription factors not with standing, the T cell adapter expression seems to be greatest in cells of the immune system ( NCBI). One can only speculate that promoter methylation is a factor.

The InsRR like pH sensor, on the other hand, is coexpressed with TrkA yet has completely different transcription factors controlling the transcription of its gene.

Figure 3. Transcription factors of TRK1 neighbors

Interestingly, N-Myc is a brain related transcription factor controlling the production of a pH sensing receptor tyrosin kinase, but not TrkA. Over-expression of N-Myc is correlated with neuroblastoma.

The TRK1 and INSRR genes are coexpressed in health and disease

In the developing mouse brain

Like the human genes, the murine TRK1 and INSRR genes are in close proximity. Reinhardt and coworkers (1994) used in situ hybridization to demonstrate proximity of the InsRR and TrkA mRNA transcripts in the developing and adult mouse brain. Brain sections were 8 μm thick. Silver grain probes were used to detect hybridization. Red images were taken in bright field to view cells. Dark field field images with a green filter allowed visualization of silver grains as green spots. The authors labeled cells to show regions in which InsRR and TrkA were co-expressed and regions in which they were not. These data remind us that expression of TrkA and other isoforms may depend on other events in the genomic neighborhood.

figure 4. From Reinhardt (1994) Dorsal root sensory sensory neurons of the adult rat brain stained with mRNA hybridization probes for A. InsRR and B TrkA.

In pediatric neuroblastomas

Gene expression in 49 pediatric neuroblastoma patients was examined in an effort to predict long term survival (Weber 2004) .

  • A significant coexpression of TrkA and InsRR transcripts was seen (p<0.001).
  • When expression versus non expression were used as predictors of six year survival, expression of TrkA and InsRR were both favorable predictors of survival (p=0.003, p=0.017, respectively.)
  • The patients with a single copy of N-MYC versus amplification, had a much better probability of survival (p<0.0001). On average the survival was 115 versus 49 months for those with single versus multiple copies of the N-MYC gene.

 

Figure 5. From Weber (2004) Figure 1B. rtPCR results from 7 representative neuroblastomas.

It would be a misrepresentation of Weber (2004) to extract any rigorous statements about the coexpression of IRR/InsRR and TrkA. When one protein is over expressed, one has to wonder if it simply a symptom of other things going on in the genomic neighborhood.

A quick note on the InsRR protein

What InsRR is doing in tumor cells is unknown. Deyev and coworkers (2011) studied its function in bicarbonate secreted cells of the kidney. They reported a pH range of 7.9–9.2. Half maximum levels of InsRR autophosphorylation appeared to be around pH 8.5. Judging from publication graphs, no autophosphorylation (activation) appeared to be present at pH 8.0.  One would think that if the environment of a tumor is acidic due to over reliance on glycolysis and insufficient oxygen to fuel the electron transport chain, that InsRR would be shut down.

Is N-Myc a transcription inhibitor or activator of the TRK1 gene?

Iraci and workers addressed this issue (2011).

  • These authors confirmed earlier reports that transcription of TrkA as well as p75NTR (p75 neurotrophin receptor) were negatively correlated with N-Myc transcription factor expression.
  • N-Myc can form heterodimers with  SP1 and MIZ1.  The formation of the heterodimer was speculated to  interfere with the positive  transactivation functions of SP1 and MIZ1.
  • N-Myc may also repress transcription of the TRK1  gene by recruiting histone deacetylase 1. Histone acetylation is usually associated with enabling of transcription, whereas histone deaceylation tends to shut it off.

Important Information

A clinical trial is open for patients whose cancers are driven by TRK  fusions.  As we are starting to explore on this page, many positive and negative factors can lead to changes in TRK gene expression that may be interpreted as over expression.  Entrectinib was highly effective in a TrkB fusion neuroblastoma xenograph model.  In this case the TrkB kinase domain expression was under control of the promoter of the fusion partner.  An adult patient with a BCAN-TRKA driven glioneuronal tumor responded very well to entrectinib treatment.  This patient was over expressing TrkA because of transcription factors operacting on the BCAN promoter.

References

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Iraci N, Diolaiti D, Papa A, Porro A, Valli E, Gherardi S, Herold S, Eilers M, Bernardoni R, Della Valle G, Perini G.(2010) A SP1/MIZ1/MYCN repression complex recruits HDAC1 at the TRKA and p75NTR promoters and affects neuroblastoma malignancy by inhibiting the cell response to NGF. Cancer Res. 71(2):404-12. PubMed

Light JE, Koyama H, Minturn JE, Ho R, Simpson AM, Iyer R, Mangino JL, Kolla V, London WB, Brodeur GM.(2012)Clinical significance of NTRK family gene expression in neuroblastomas. Pediatr Blood Cancer. 59(2):226-32. PubMed

Moon BF, Jones KM, Chen LQ, Liu P, Randtke EA, Howison CM, Pagel MD (2015)A comparison of iopromide and iopamidol, two acidoCEST MRI contrast media that measure tumor extracellular pH. Contrast Media Mol Imaging. 10(6):446-55. PubMed

Pajtler KW, Rebmann V, Lindemann M, Schulte JH, Schulte S, Stauder M, Leuschner I, Schmid KW, Köhl U, Schramm A, Eggert A.(2013) Expression of NTRK1/TrkA affects immunogenicity of neuroblastoma cells. Int J Cancer.133(4):908-19. PubMed

Pajtler KW, Mahlow E, Odersky A, Lindner S, Stephan H, Bendix I, Eggert A, Schramm A, Schulte JH.(2014) Neuroblastoma in dialog with its stroma: NTRK1 is a regulator of cellular cross-talk with Schwann cells. Oncotarget. 5(22):11180-92. PubMed

Reinhardt RR, Chin E, Zhang B, Roth RA, Bondy CA.(1994) Selective coexpression of insulin receptor-related receptor (IRR) and TRK in NGF-sensitive neurons. J Neurosci. 14(8):4674-83.  PubMed

Sacristán MP, de Diego JG, Bonilla M, Martín-Zanca D.(1999) Molecular cloning and characterization of the 5′ region of the mouse trkA proto-oncogene. Oncogene 18(42):5836-42. PubMed

Weber A, Huesken C, Bergmann E, Kiess W, Christiansen NM, Christiansen H.(2003)Coexpression of insulin receptor-related receptor and insulin-like growth factor 1 receptor correlates with enhanced apoptosis and dedifferentiation in human neuroblastomas. Clin Cancer Res. 2003 Nov 15;9(15):5683-92. PubMed