TRK Gene Amplification

Under normal conditions humans are diploid for 22 pairs of homologous chromosomes:  one from the father and the other from the mother.  The 23rd pair is  of course the XX or XY pair, for women or men, respectively. Gene amplification may involve duplication of a whole chromosome such that the number of the chromosome in question exceeds two, i.e. polyploid.

Gene amplification  may also involve duplication of a specific gene or region. Once the gene is duplicated, smaller changes can result  within each duplicated gene.  The outcome may be  isoform diversity and the ability of the same basic protein to serve different functions.

TRK1 is in a region of gene amplification

Note the TRK1 gene in panel A of figure 1.  Taking a journey towards the centromer we see the first cluster of S100 family members.

S100 is a family of calcium binding proteins. In mammals, S100A proteins are all located on the same chromosome, in humans, chromosome 1.  Subgroup 1 (S100A1-9, S100A12-16) are clustered in a locus to one side of the TPM3 gene, and subgroup 2 (S100A10 and S100A11) to the other side. There are at least seven copies of S100A7 (Shang 2008). Figure 1B and 1D show the gene locations of sub groups 1 and 2 of the S100A genes on human chromosome 1.

More gene amplification between two S100 loci are shown in Figure 1C. A protein known as cornulin as well as many isoforms of late cornified envelope protein (LCE) reside in this region.   Cabral and coworkers (2001) have reviewed differences in the promoters of the  small proline rich repeat (SPRR) protein genes that reside in this region.

Chromosome 1 showing amplified regions of various genes.

Figure 1. Gene amplification  in several regions in the A. vicinity of the TRK1 gene on chromsome 1.  B. Subgroup 1 S100A genes occupy a closely packed locus separated by other loci C. of late cornified envelope (LCE) and small proline rich repeat (SPRR) proteins. The CRNN gene product, cornulin, is like S100, a high affinity Ca2+ binding protein. D. Subgroup 2 S100A proteins have their own locus on chromosome 1.

 

 

Gene Amplification:  Remember that genes are more than their protein coding regions!

Gene amplification has always been a normal part of life on this planet. Amplified genes may be subject to modification to create different models of a protein that can serve slightly different functions.   This tweaking not only includes the protein coding regions but also the gene promoters. Cabral and coworkers (2001) took a closer look at the small proline rich repeat (SPRR) locus of chromosome 1 (Figure 1C) They made note of changes in the direction of the reading frames in the family members (Figure 2A)

Chromosome 1 showing two different regions of SPRR genes with different response elements in their promoters. Inset C, shows PCR products of different isoforms under different UV light conditions.

Figure 2. from Cabral 2001. A. The SPRR genes fall into two groups on their chromosome 1 locus. B. Some select genes have different response elements in their promoters. C. Isoform specific PCR reveals that these genes are transcribed differently in response to UV light in two separate experiments

 

Note the sense and anti-sense orientation of these genes. Slight differences were observed in the response elements of select promoters (Figure 2B). In two experiments keratinocytes were exposed or not exposed to UV light. The mRNA was extracted and subjected to isofrom specific PCR (Figure 2C).

Cabral and coworkers, back in 2001, did not consider variation in the 3′-UTR as a mechanism of regulating protein production. Their work remains truly thought provoking. One must always back up gene copy number data with proof that an increase in functional protein has also occurred. Interesting nuances to the story could involve changes in the promoters of these duplicated genes as well.

TRK1 amplification in malignant melanoma

Pasini and coworkers (2015) used comparative genome hybridization, as validated by genomic qPCR, to identify gene amplifications associated with progression in 64 cases of malignant melanoma.  This publication was focused on signaling ramifications of TRK1 gene amplification.  For now, our discussion will focus on copy number changes in the TRK1 gene in malignant melanoma.

Diagram showing how comparitive gene copy number is predicted using red and green fluorescence.

Figure 3 Techniques used by Pasini (2015) CGH relies on the principle of red fluorescence plus green fluorescence producing yellow. If copy numbers are equal, the array spot should be yellow. qPCR is also based on fluorescence. Log base 2 is often used to describe copy number

  • In 12 target chromosomes, 40 common amplification hot spots were detected.
  • TRK1‘s locus, 1q23.1, was amplified in 16% of the malignant melanoma patients’ tumors.
  • This particular amplification had the strongest statistical association with melanoma thickness.
  • Median thickness of melanomas was broken into three strata for the 1q23.1 region.
  1. diploid, 2.3 mm (range 1.5–3.0 mm)
  2. duplication, 3.0 mm (range 2.5–12.0 mm)
  3. amplification 4.7 mm (range 1.6–20.0 mm)

Genes in the 1q23.1  and 1q21.3 loci

Table showing top scoring amplified loci and their correlation to melanoma.

Table 1 Top scoring amplified loci as they correlate with melanoma thickness, Pasini 2015.

  • INSRR is an insulin  receptor like tyrosine kinase that is regulated by extracellular alkalinity.  We have discussed this gene and protein on another page.
  • The PEAR1 gene product, platelet endothelial aggregation receptor, is a membrane protein with a wide tissue distribution.
  • There is not a lot of information on the leucine rich repeat protein 71 product of the LLRC71 gene.
  • The ARHGEF11 gene codes for a Rho guanidine nucleotide exchange factor 11.  This protein plays a role in neurotrophin induced neurite outgrowth.  One might anticipate that it could share regulatory elements with a gene coding for one of the neurotrophin receptors.  Perhaps, when we look at amplified genes, we also need to take into account neighboring genes that might be co-regulated.
  • ETV3 codes for a  a transcription repressor.
  • The micro RNA MiR765 suppresses  the translation of the TrkC transcript by binding to the 3′-UTR of the mRNA.

TRK1 amplification break point occurs between two genes

Pasini and coworkers presented the observation that a duplication/amplification breakpoint occurred between two genes that read in opposite directions: INSRR and TRK1.  Brief mention was made of protein coding exons occurring 5′ to the break point.

Comparison of Chromosome 1 region containing NTRK1 breaking point hot spots from publications of Pasini, 2015 and Luberg, 2015.

Figure 4.  Two different views of the TRK1/INSRR region Top from Pasini 2015, a break point in NTRK1 gene amplication occurs 5′ to non-tranditional TRK1 exons. Bottom Luberg (2015) reviewed isoforms of TrkA, traditional and canonical exons, and different transcription start sites.  It wasn’t really clear in Pasini 2015 if all of the TRK1 duplication/amplification melanoma patients exhibited break points in the same region between INSRR and TRK1.

 

Additional, complicating factors

Increases in gene copy number do not necessarily correlate with increases in mRNA transcripts.  For that matter, increases in transcripts do not always translate into more  protein.  Pasini and coworkers electronically published supplemental data from  the  Broad-Novartis Cancer Cell Line Encyclopedia (CCLE).  In this supplemental data they compared gene copy number and mRNA transcripts of TrkA in several malignant melanoma cell lines from a variety of research centers.   Their supplemental data included

Graphs depicting TrkA mRNA expression levels against gene copy numbers in various cell lines.

Figure 5 Relating mRNA levels to gene copy numbers

 

Even though the TrkA mRNA levels in the cell lines are really low compared to the tumors, there seems to be some hints of patterns within cell lines from different sources.

Colorado  A really weak linear relationship seems to exist once the log2 N/2 copy number exceeds 0., i.e. that there there are the expected diploid two copies and maybe a bit more.

HS  Not sure where this series of melanoma cell lines came from.  There appear to be two groups in these cell lines.  In diploid cell lines, there is no predicting the mRNA concentration.  Above diploid, there seems to be a weak linear relationship.

Sloan-Kettering melanoma tumors seem to have an inverse relationship between mRNA concentration and copy number above the diploid state.

We do not know if these tumor cells lines were processed the same way or even if the mRNA was extracted under the same culture conditions.

Concluding remarks

Gene amplification seems to be a fairly common event in biology.  Amplification of some genes like the SPRR genes allows for additional modification of the promoters such that their expression can occur in response to different environmental stimuli.  UV light was was addressed by Cabral and coworkers.  Perhaps there is a yet to be discovered skin damaging agent that may stimulate SPRR transcription via interferon stimulated response elements (Fig 2B).     If the growth regulating TRK1 gene were to be duplicated or amplified, the next question would be what controls the promoter in duplicated genes.  TRK1 gene amplification can occur without an increase in TrkA mRNA  transcripts.   Overexpressed TrkA has been explored as an actionable target of a small molecule Trk inhibitor.  The jury is still out as to whether an amplified TRK gene is indication of an actionable target.

References

Cabral A, Voskamp P, Cleton-Jansen AM, South A, Nizetic D, Backendorf C. (2001)Structural organization and regulation of the small proline-rich family of cornified envelope precursors suggest a role in adaptive barrier function. J Biol Chem. 276(22):19231-7. PubMed

Luberg K, Park R, Aleksejeva E, TimmuskT (2015) Novel transcripts reveal a complex structure of the human TRKA gene and imply the presence of multiple protein isoforms. BMC Neurosci. 16:78. PubMed

Pasini L, Re A, Tebaldi T, Ricci G, Boi S, Adami V, Barbareschi M, Quattrone A.(2015)TrkA is amplified in malignant melanoma patients and induces an anti-proliferative response in cell lines. BMC Cancer. 15:777. PubMed