Purpose: ​ Use questions to guide your research on the different aspects of

the trkA receptor and summarize your findings following the format below.

Alexis Nolin-Lapalme (if any problem with my info contact me!)

Ryszard Kubinski
Reilly Pidgeon
Luca Cuccia

Please Write The Result Of Your Research Here

Use APA style parenthetical citations (First Author Last Name (et al.), Year
of Publication).

Questions to Answer​- Add some if you can!

trkA​- tropomyosin receptor kinase A (Bertrand et al, 2012)

What is the ​
pathway​that involves trkA in cancer?

PI3K-Akt, ERK/p38 Map kinase are both found activated but also
necessary for the overactive phenotype of late tumors allowing increase
growth, angiogenesis and metastasis in xenographed cancer cells in
immunodepressed mice (Lagatec et al. 2009). In various cancer, but more
frequently in prostate and and thyroid cancers, to find constitutively active
versions of trka (Nakagawara, 2001).

Sequence of activation (for NGF): NGF binds to each monomer ->

dimerization -> autophosphorylation of the intracellular ​ tails​-> this
sollicitates the binding of adaptor proteins that include: Shc, growth
factor-receptor bound protein-2 (Grb2), and Gbr2-associated Binder-1
(GAB1). These then activate two main cellular pathways: ) the
phosphatidylinositol-3 kinase (PI3K) and 2) Akt kinase. Alternatively, the
TrkA phosphorylation results in recruitment of a guanine nucleotide
exchange factor (GEF) that allows GTP phosphorylation and activation of
the membrane-associated G protein Ras . Activated Ras in turn
phosphorylated Raf that then activates the mitogen-activated protein kinase
(MAPK) and subsequently the ribosomal s6 kinase (RSK). The activation of
Akt or RSK results in phosphorylation of the cyclic AMP response element
binding protein (CREB) transcription factor that then translocates into the
nucleus and mediates the trophic and anti-apoptotic effects of NGF. When
there is deficiency of NGF, the Akt or MAPK pathways cannot suppress the
activity of cell death-promoting transcription factors such as c-Jun, resulting
in apoptosis.(Demir et al, 2016)

On the other hand, signaling via p75NTR can have contrasting effects in a
cell. Upon binding of NGF or its uncleaved precursor pro-NGF, recruited
cytoplasmic adaptor proteins recruit the tumor necrosis factor receptor
member TRAF6, which can in turn activate the inhibitor of the kappaB
kinase (IKK) and thus the canonical NFkappaB signaling. RelA/p65 in the
NFkappaB pathway acts as regulator of nuclear gene transcription and
primarily promotes cell survival. Alternatively, TRAF6 and neurotrophin
receptor interacting factor (NRIF) can activate c-Jun N-terminal kinase
(JNK), which phosphorylates the transcription factor c-Jun. Activated c-Jun
subsequently mediates apoptosis by upregulating the expression of
pro-apoptotic genes.

p75NTR can 1) act as a “helper” for TrkA and 2) enhance TrkA

phosphorylation in a variety of cells by increasing the ability of TrkA to bind
NGF and promote cell death. Like other members of the tumor necrosis
factor superfamily, p75NTR contains a cysteine-rich extracellular domain
repeated two to six times and intracellular motif termed “death domain”.
Importantly, the cytoplasmic domain of p75NTR and not the “death domain”
was shown to be sufficient to mediate death signals via its interaction with
the MAGE homologue (NRAGE), and FAP-1. Moreover, ​​on the role of
NGF in cancer progression have discovered novel aspects of NGF
signaling within cancer cells that go beyond our classical knowledge on the
NGF-TrkA-p75NTR interactions (Demir et al, 2016).

p75NTR seems to actually be important for the binding & activation of TrkA
by NGF. (Barker et Al, 1994)

TrkA can also act as an ​

apoptosis-INDUCING ​receptor. In
pheochromocytoma cells, TrkA can inhibit cell proliferation by activating
p21. Rats with glioma cells containing TrkA survived longer than those
without. High expression of TrkA correlates with increased survival in
neuroblastoma. (kaplan, miller ,1997)
Known interactome for trka (illustrated here with its gene name:NTRK1). Retrieved from STRINGS-db:
https://string-db.org/cgi/network.pl?taskId=68G2Ulfn6cUY
trkA is involved in what ​
cancer​
?

Trka overexpression both in its phosporylated and unphosporylated

form is found overexpressed in ​ breast cancer​tumors (Lagatec et al. 2009)
It has also been found to play a critical role in ​
thyroid cancer​(McGregor et
al, 1999) ​
lung cancer​(Ricci et al, 2001), p​ ancreatic​(Zhu et al. 1999)
prostatic​(Weeraratna et al., 2000) and ​ ovarian​cancer (Davidson et al.,
2003). ​Colorectal​and ​
acute myeloid leukaemia​(Demir et al., 2016).

Nucleophosmin‐anaplastic lymphoma kinase‐expressing (NPM‐ALK

+) T‐cell lymphoma has been shown to be dependent on the mutant ALK’s
interactions with TrkA. TrkA signalling inhibition was associated with
anti-tumor effects in vivo. (Shi, Wenyu et al.,2017) -> they used an
anti-TrkA ​
antibody​to inhibit the signalling.

The colorectal carcinoma cell line KM12 is strictly dependent on

activated TRKA due to the presence of a chromosomal rearrangement that
leads to the expression of oncogenic fusion protein TPM3–TRKA. (inhibitor
used listed under “any other inhibitors” (Ardini et al, 2016)
trkA​encodes the high-affinity receptor of nerve growth factor (NGF)
and is predominantly expressed in ​ neuroblastomas ​
with favorable
prognosis...data suggests that ​ trkA​may have more impact on prognosis of
the patients than previously reported by studying the expression of ​ trkA​I
and II, but not ​
trkA​III (Hiskiki et al, 2010)

The overexpression of PTP-MEG2, a pTyr-phosphatase, inhibits

neurite outgrowth and differentiation in cultured cells (PC12) and cortical
neurons ​via ​
the inhibition of NGF/TrkA signal transduction. (Zhang et al.,
2016)
Compiled information of the protein signalling pathway involved in tumergenic growth in thyroid cancers.
Retreived from Kegg: ​http://www.genome.jp/kegg-bin/show_pathway?hsa05216+4914

What are the ​

genes affected​by trkA?

The gene for TrkA is NRTK1, it codes for the receptor tyrosine
kinase. Mutations include: TPM3, TPR, TFG genes which attach as
overactive kinase domains to the receptor. They will phosphorylate even
when there is no ligand binding to the receptor. (Genetics Home
Reference)
-important to note that these mutations occur intracellularly so we
cannot really target them.

What are the ​

ligands​of trkA?

Nerve growth factor (NGF) is a common ligand of trk family receptors.

Aside from trk it also bonds p75. NGF has long been known to play a role
in the neural development however new research has been unveiling its
role as a growth factor in the periphery. Moreover, NGF seems to act as an
autocrine factor in cancerous lesions whereby the cell releases to cause
self-growth (lagadec et al, 2009) Using anti NGF antibodies and RNAi KO
have shown to diminish the breast cancer growth (Adriaenssens et al.,
2008). Trka also binds with high avidity to neurotrophin 7 and to a lesser
extent neurotrophin 6 (Bertrand et al, 2012).

Neurotrophin 3, NGF 4,5 are good ligands for TrkA as well. (D O

Clary, L F Reichardt, 1994). Neurotrophin-3 is mostly expressed in ovaries.

Important to note that neurotrophin signalling is involved also in p75

receptor signalling which activates TNF. (Patapoutian, Ardem, and Louis F
Reichardt., 2001)

How is TrkA ​transported ​

inside the cell
and how is this ​
regulated​
?

TrkA transport to the cell surface,

where it can bind NGF and activate
downstream effectors, requires MEG2
(PTP-MEG2), a phospho-tyrosine
phosphatase, activity.
Anterograde transport of newly
synthesized TrkA to the cell surface is
blocked by the inhibition of PTP-MEG2,
though this does not occur in catalytically
inactive mutant TrkA receptors (point mutation in the kinase domain).
(Zhang et al., 2016)

Are there any key ​

mutations sites​or ​
hotspots​or ​
alternative splicing​?
 Alternatively spliced TrkA receptor has been shown to be more
sensitive to Neurotrophin-3 than its other version (which lacks an exon).
From the paper’s findings, it seems that pheochromocytoma cells express
this alternatively spliced TrkA while fibroblasts do not. (D O Clary, L F
Reichardt, 1994)

a novel splice variant of ​ trkA​

, in which exons 6, 7, and 9 are deleted, yields
a product that lacks the functional extracellular IG-C1 and N-glycosylation
domains, and exhibits spontaneous tyrosine kinase activity that leads to
oncogenic signals. In contrast to the previously known isoforms ​ trkA​I and
trkA​II, this newly identified product, named ​trkA​III, is unresponsive to NGF,
and antagonizes the differentiation/regression signals of NGF/TrkA. (Hiskiki
et al, 2010)

Genomic rearrangement (e.g. fusion genes like LMNA-NTRK1) is the

most common mechanism of oncogenic activation for the Trk family of
receptors, leading to cell proliferation ​
via​the activation of MAPK and
AKT(PKB) pathways. These rearrangements can confer resistance to first,
second, and third-line therapies (FOLFOX, FOLFIRI/cetuximab, and
irinotecan, respectively) (Russo et al., 2015).

Mutation sites in NTRK1 can confer secondary resistance to drug

therapy (even the newer drugs like entrectinib). These mutations can occur
after the start of drug therapy, and higher/lower doses appear to select for
different mutations (​p.G595R​→ blocks binding and p.G667C → reduces
affinity). Resistance is dependent on TrkA expression. These NTRK1
mutations are present in the kinase domain of TrkA and are conserved
among 6 clinically relevant tyrosine kinases (i.e. these alterations are
common when targeting other tyrosine kinases) (Russo et al., 2015).

What proteins ​
regulates​its ​
expression​or ​
degradation​
?
It is frequently found co-overexpressed with p75 (lagatec, 2009). The
receptor is marked for degradation by the Cbl-b E3 ubiquitin ligase that
promotes its ubiquitination and thus its degradation. Another ubiquitin,
Nedd4-2​​ligase is mutated in cases of mechanical allodynia whereby their
inactivation leads to hypersensitivity pathways to be overactive (Demir et
al, 2016)

What ​
tissue​expresses TRKA?

Tissue is widely seen in key parts of the nervous system including the
trigeminal, dorsal root and sympathetic nervous system. The salivary
glands, stomach, intestines, pancreas, bone marrow, adrenal glands,
prostate, ovary, uterus, skeletal muscle and skin are all TrkA and/or TrkB
producers. (Bertrand et al., 2012)

Transcripts of NGF and TrkA were identified in all adult and fetal
ovaries tested. Transcripts of NGF and TrkA were identified in all adult and
fetal ovaries tested. TrkA can influence the overall binding affinities for
NGF. (Abir et al, 2005)

Is there any ​
environmental factor​ s that are correlated with its involvement
in cancer?
the expression of NGF, TrkA and p75 in the uterus of the wild ground
squirrel was highest in the breeding season, when estradiol and
progesterone production were greatest. the expression levels of NGF and
its receptors, TrkA and p75 were significantly higher in the breeding season
as compared to the nonbreeding season, implying that NGF system may
be involved in the regulation of ovarian function change. (Li et al, 2014)
-Yes this might be pushing it, but I thought it was interesting and could
serve as a jumping off point for further literature review!
Relevant ​
structural components​of the receptor

The domain 5, region containing the binding site for NGF has been a
great target for possible inhibitors as it mostly specific to the trka receptor.
However the tyrosine kinase domains are roughly 70% homologous
between all three receptors and very similar to other tyrosine domain in
other protomeric family. (Bertrad et al, 2012).

Domain 5 has been shown to have very similar activation as that of

the natural receptor. This means that the hotspot for inhibition is in this
domain. (Wiesmann, 1999)

Any ​
other diseases​linked with TRKA mutations?

Trka inhibition has shown to be a potential target for analgesia and

allow pain inhibition. (Indo et al, 1996)

Congenital insensitivity to pain and anhydrosis have been shown to

be related to two different TrkA mutations. Moderate to severe mental
retardation is also present. This occurs in Israel-Bedouin populations. The
defect in the TrkA kinase domain disrupts the signalling cascade following
NGF activation this may lead to impaired development of C-fibers. It is
therefore suggested that TrkA is important to the development of the
nociceptive system.(Shatzky, Sharon et Al., 2000)

What is the ​
epidemiological​data on TRKA mutation?

Israel-Bedouin Populations with inactive kinase domain show lack of

developed nociceptive system. (Shatzky, Sharon et Al., 2000).

Any ​
other inhibitors​
?
 k252a with a low IC50 of 10-30nM for trk family but nonbinding to
other tyrosine kinase receptor. (Binding site still to be determined)(tampley
et al, 1992)

Entrectinib ​
abolishes autophosphorylation of TPM3–TRKA after 2
hours treatment, concomitant with complete inhibition of the
phosphorylation of PLCγ1, AKT, and MAPK. TRKA inhibition may
represent an innovative opportunity for the therapy of selected patients
whose tumors harbor ​ NTRK1​gene rearrangements and this has supported
the enrollment of TRKA-positive patients in ongoing clinical trials of
entrectinib (Ardini et al, 2016)

Entrectinib is a pan-Trk inhibitor that is currently in clinical trials for

colorectal cancer and other types of tumors. It makes H-bonds and
hydrophobic interactions with TrkA in the ATP pocket where p.G595 and
p.G667 residues are located. These residues can be mutated (secondary
resistance), conferring resistance to entrectinib due to steric hindrance
(p.G595R → blocks binding and p.G667C → reduces affinity). So far,
these are the only mechanisms of resistance to this drug (Russo et al.,
2015).

There is a need for next generation TrkA inhibitors that do not bind
near the G595 region, thus allowing us to overcome the p.G595R
resistance mechanism (Russo et al., 2015).