The focus on pathways, receptors, ligands and promoters as
control elements for cancer has seen a great deal of development in the past
decade. One key approach is the development and identification of inhibitors,
molecules which can block an over excited pathway. We examine here a specific
recent such example as relates to melanoma. It is already well known that BRAF
suppression is an effective approach albeit often of limited duration. The
development of inhibitors for a selection of evolving pathway aberrations will
most likely be the way to turn a deadly disease into a chronic but manageable
problem, assuming that one can get permission to use such molecules, a process
which not is costly and lengthy.
We use a recent paper by Schlegel et al and use MER as a prototypical
example of pathway control via inhibitor blockage.
MER is a tyrosine kinase (“TK”) receptor (“TKR”)[1].
As Marks et al state there are 85 members in the TK family and 58 of these are
receptors. The receptors are divided into various families based upon their
structures and one family contains Axl, Sky and MER, also known as the TAM
family[2].
This family, as we shall see, has immunoglobulin like regions on the outside of
the cell surface and kinase domains on the inner surface. The family also has a
dual fibronectin III-like domain on the outside just below the immunoglobulin
domains, of which there are two.
Generally the receptors are activated by ligands which in
turn result in the phosphorlyation of the kinase region and associated area and
then commence the activation of the related pathways. Now these pathways are
the ones that result in proliferation and loss of localization and thus result
ultimately in metastasis.
We use this example for two reasons: (i) it is a good
example to demonstrate the activation of pathways and metastatic growth; (ii)
it also is a good example of how inhibitors can function on receptors and thus
can inhibit metastatic growth.
As Schlegel et al state:
Receptor tyrosine kinases (RTKs) are frequently
ectopically expressed, overexpressed, or hyperactivated in tumor cells and are therefore
attractive targets for cancer therapy. C-MER proto-oncogene tyrosine kinase
(MERTK), a member of the TAM (TYRO, AXL, MERTK) family of RTKs, has been
characterized as a therapeutic target in hematopoietic malignancies and several
solid tumors including lung, prostate, and brain
There is a subtle question posed but not answered here. Is
it over-expression, and if so by what ligand, or is it an excess production of
MER and thus an over-expression. What is the status of the benign cell, and is
this the dominant pathway. Clearly by having too active or too many MER
receptors, actually any TAM like receptor will do, leads to proliferation. This
goal of blocking the receptor so that it does not start the process is a valid
approach.
The authors clearly state:
Stimulation of melanoma cells with the MERTK ligand GAS6
resulted in the activation of several downstream signaling pathways including MAPK/ERK,
PI3K/AKT, and JAK/STAT. MERTK inhibition via shRNA reduced MERTK-mediated
downstream signaling, reduced colony formation by up to 59%, and diminished
tumor volume by 60% in a human melanoma murine xenograft model.
Namely we have a ligand, GAS6, which activates the MER
pathway. Is that ligand over expressed. On the other hand the molecule shRNA
reduced the activation.
They specifically state:
In addition, Sensi et al. found that melanoma cells often
secrete GAS6, a ligand of TAM receptors, indicating a mechanism of TAM
autocrine signaling in melanoma…. The mechanism of MERTK activation in melanoma
cells is not clear, but Sensi et al. have previously described melanoma cell
expression and secretion of GAS6, the common ligand for all members of the TAM family
of proteins, suggesting a method of autocrine and/or paracrine activation of MERTK.
Since expression of MERTK by melanoma cells increases during progression from
primary to metastatic melanoma, it would be interesting to determine whether
corresponding increases in GAS6 levels occur in serum from patients with
metastatic melanoma, implicating serum GAS6 levels as a potential early marker
of melanoma progression, as in other cancers.
Thus possibly inhibiting GAS6 may be profitable as well[3].
However the focus here is receptor inhibition.
1.1
MER and Melanoma
Let us consider a recent development in understanding MER
and melanoma. We return to the recent paper by Schlegel et al where the author’s
state:
C-MER proto-oncogene tyrosine kinase (MERTK) is a
receptor tyrosine kinase with oncogenic properties that is often overexpressed
or activated in various malignancies. Using both protein immunohistochemistry
and microarray analyses, we demonstrate that MERTK expression correlates with
disease progression.
MERTK expression was highest in metastatic melanomas,
followed by primary melanomas, while the lowest expression was observed in
nevi. Additionally, over half of melanoma cell lines overexpressed MERTK
compared with normal human melanocytes; however, overexpression did not
correlate with mutations in BRAF or RAS.
Stimulation of melanoma cells with the MERTK ligand GAS6
resulted in the activation of several downstream signaling pathways including
MAPK/ERK, PI3K/AKT, and JAK/STAT. MERTK inhibition via shRNA reduced
MERTK-mediated downstream signaling, reduced colony formation by up to 59%, and
diminished tumor volume by 60% in a human melanoma murine xenograft model.
Treatment of melanoma cells with UNC1062, a novel
MERTK-selective small-molecule tyrosine kinase inhibitor, reduced activation of
MERTK-mediated downstream signaling, induced apoptosis in culture, reduced
colony formation in soft agar, and inhibited invasion of melanoma cells. This
work establishes MERTK as a therapeutic target in melanoma and provides a
rationale for the continued development of MERTK-targeted therapies.
Thus, like to work that led to BRAF V600 inhibitors, we see
MER TK is another interesting target. The authors also provide an inhibitor
molecule as well.
1.2
Tyrosine Kinases and MER
Tyrosine Kinases receptors have received a great deal of
attention especially in the area of cancer metastasis and in cancer control.
They are as Verma et al state:
Receptor tyrosine kinases (RTK) are a large family of transmembrane
proteins exhibiting great diversity in their extracellular regions, although
sharing in common a highly conserved intracellular tyrosine kinase domain. They
function as sensors for extracellular ligands, the binding of which triggers
receptor dimerization and activation of the receptor’s kinase activity. This
activation leads to the recruitment, phosphorylation, and activation of
multiple downstream signaling proteins, which ultimately change the physiology
of the cell. RTKs regulate cellular processes, including survival, growth,
differentiation, adhesion, proliferation, and motility. Fifty-eight known RTKs
in the human genome are classified into 20 families by amino acid sequence
identity within the kinase domain and structural similarities within their extracellular
regions.
There are many such tyrosine kinase receptors. One class is
the TAM family and as Verma et al state:
One subfamily is referred to as the TAM family,
identified in 1991, comprising Tyro-3 (also called Sky), Axl, and Mer. The TAM
receptors are characterized by a combination of 2 immunoglobin-like domains and
dual fibronectin type III repeats in the extracellular region and a cytoplasmic
kinase domain. The primary ligand for TAM receptors is growth arrest-specific 6
(Gas 6), a fairly large (75 kDa) vitamin K–dependent protein known to activate
downstream signaling
We depict a simple structure below containing the elements
specified above.
Let us consider a simple development of MER controlled
pathways. The Figure below shows two separate and un-activated MERTK molecules
with the immunoglobulin terminals on the outside and the kinase areas on the
inside.
Now along comes a GAS6 ligand, and it attaches to and
connects the MERTK molecules at the immunoglobulin ends and this activates the
kinase tails inside the cell.
Once activated the kinase ends commence pathway activation
via the phosphorylation process. The pathways are depicted below.
It is the activation of these pathways by the excess GAS6
production or the excess MERTK production or both that results in excess
proliferation and metastasis.
As Verma et al relate about the pathway:
Studies using chimeric Mer receptors expressed in NIH3T3
fibroblasts linked downstream signaling pathways, such as PI3K, phospholipase
C-g (PLCg), and ERK, to Mer activation. Gas 6–dependent activation of Mer stimulates
phosphorylation of ERK1/2, leading to cellular transformation and increased
proliferation and DNA synthesis.
The ultimate downstream targets of the pathway differ
according to cell type and tissue microenvironment. In leukemia cells,
ligand-dependent activation of EGF receptor (EGFR)–Mer chimeric receptor
stimulates phosphorylation of Akt, ERK 1/2, and p38 mitogenactivated protein
kinases (MAPK), which results in decreased apoptosis but no change in
proliferation (30). Expression of CD8-Mer chimera in pro-B cells results in
transcriptional activation of NF-kB via PI3K/Akt.
Additional activation of p38/MAPK and meiosis-specific serine/threonine
protein kinase 1 (MEK1) occurs via CD8-Mer, leading to protection from
apoptosis. Some atypical signaling pathways involved in cell survival have been
studied as a link between Mer and the actin cytoskeleton via growth factor
receptor-bound protein 2 (Grb2), Shc, and Vav1. Downregulation of the proapoptotic
tumor suppressor WW domain-containing
We depict below how one can inhibit this process. We depict
an inhibitor molecule which binds to the sites as before but now does not
activate the TK pathways. The inhibitor must be stronger in affinity than the
GAS6 which most likely is still in ECM abundance.
Note above the RAs to RAF (especially BRAF) to MEK to MAPK
pathways flow. We have examined this in details elsewhere[4].
The implication is that by targeting the TK Receptor, one targets all elements
of the pathway. It should be noted however that the separate pathway elements
may be activated and over expressed via other factors such as epigenetic ones.
Thus the suggestions of Schlegel et al are of great merit but should be
balanced by understanding the epigenetic issues as well.
An example of a pathway and its control with BRAF
functionality is depicted below:
This simple explanation is also a paradigm for many other
such pathway activations and especially for those of the tyrosine kinase
verity.
1.3
MER and miRNA
There are other dimensions of interest here as well. In
cancers there unfortunately is not just a single point of failure. There often
are multiple. We show here just another example where MER and miRNA play an
interesting role. This is an essential point to make because all too often the
initial observers may all too often jump at a simple solution leaving behind a
complexity of other factors which take control.
Let us consider a miRNA control using MER. As Halberg et al
state:
Tumours require the establishment of vasculature for
their increasing nutrient, energy, and oxygen requirements as well as for
removal of metabolic waste. Cancer cells within a tumour generate such
pathologic vasculature by recruiting endothelial cells to the tumour site. This
is accomplished by secreting molecular factors, such as the well-known vascular
endothelial growth factor (VEGF, into the extracellular space.
VEGF binding to VEGF receptors on endothelial cells
results in the migration and recruitment of endothelial cells. In this way,
proteins expressed by cancer cells can regulate the cellular and structural
content of tumours—giving rise to continued tumour growth. Recent work has
revealed a major role for another class of genes— known as small non-coding
RNAs (microRNAs)—in the regulation of endothelial recruitment and tumour
angiogenesis.
One member of this family (miR-126) was recently found to
inhibit endothelial recruitment by suppressing a set of cancer genes that
activate endothelial migration. In this way, a non-coding RNA expressed by
cancer cells could shape the tumour and metastatic microenvironment.
This is thus depicted below from the Halberg paper. Here we
have two cells, the top cell is a cancer cell where miR126 is blocking IGBP2
and blocking the MERTK receptor which in turn would have blocked the entrance
of GAS6. But since miR126 has blocked the blocker, we have excess GAS6. Thus we
have a problem, namely the GAS6 “overproduction” is really a failure to block
resulting from the cancer cell miR126 production.
It is critical always therefore to look across all paths,
direct as well as epigenetic.
As Zhuang et al state:
Angiogenesis plays a crucial role during tumorigenesis
and much progress has been recently made in elucidating the role of VEGF and
other growth factors in the regulation of angiogenesis. Recently, microRNAs
(miRNAs) have been shown to modulate a variety of physiological and
pathological processes.
We identified a set of differentially expressed miRNAs in
microvascular endothelial cells co-cultured with tumour cells. Unexpectedly,
most miRNAs were derived from tumour cells, packaged into microvesicles (MVs), and
then directly delivered to endothelial cells.
Among these miRNAs, we focused on miR-9 due to the strong
morphological changes induced in cultured endothelial cells. We found that
exogenous miR-9 effectively reduced SOCS5 levels, leading to activated JAK-STAT
pathway. This signalling cascade promoted endothelial cell migration and tumour
angiogenesis.
Remarkably, administration of anti-miR-9 or JAK
inhibitors suppressed MV-induced cell migration in vitro and decreased tumour
burden in vivo. Collectively, these observations suggest that tumour-secreted
miRNAs participate in intercellular communication and function as a novel
pro-angiogenic mechanism.
1.4
Inhibitors
Inhibitors of pathways are being developed at a rapid rate.
Knowing the pathway and molecular structure of the receptors it is somewhat
readily possible to develop a strong inhibitor, a molecule that interferes with
the normal ligand.
Schlegel et al have developed and tested an inhibitor of the
MERTK receptor and it is shown below.
Schlegel et al characterize this molecule as follows:
A novel MERTK tyrosine kinase inhibitor, UNC1062,
inhibits MERTK mediated signaling, promotes apoptosis, and inhibits colony
formation in melanoma cells. While activating mutations in BRAF and NRAS occur
in melanoma at rates of 41% and 18%, respectively, lower mutation frequency or
gene amplifications in other signaling molecules, such as RTKs, can also
contribute to melanoma pathogenesis.
UNC1062 was
developed as a MERTK-selective tyrosine kinase inhibitor. Its structure is based
on a previously published pyrazolopyrimidine scaffold, and it has an improved
affinity and specificity profile compared with its parent compound, UNC569 .
UNC1062 potently inhibits MERTK kinase activity in vitro
and exhibits specificity within the TAM family. Treatment of HMCB and G361
cells with increasing concentrations of UNC1062 resulted in a potent
dose-dependent reduction in MERTK phosphorylation
In the work of Verma et al they present an interesting
collection of molecules which exhibit inhibitor characteristics (see their
Figure 2). This is an expansion of what Schlegel et al have presented.
Again from Schlegel et al we have:
MAPK/ERK and PI3K/AKT are 2 of the most frequently dysregulated
pathways in melanoma. These 2 pathways not only play a role in melanoma
development and progression, but are also involved in primary and secondary
resistance to BRAF inhibitors.
The observation that MERTK signals via both pathways, as
well as through others whose roles in melanoma biology are currently unclear
(e.g., STAT6), not only highlights the complex regulation of these pathways by
membrane receptors, such as MERTK, but may also provide a therapeutic
advantage, since targeting MERTK may disrupt signaling in multiple pathways.
These observations and the data presented here suggest
that MERTK-targeted therapies could potentially be considered for patients,
irrespective of BRAF and NRAS status and/or prior treatment with BRAF
inhibitors.
The latter observation is of possible significant merit. Namely
the MERTK targeting allows for alternative pathway blocking, namely doing so at
the source of pathway activation.
1.5
Observations
We conclude with some general and specific observations.
This work by Schlegel et al is of significant importance for reasons already
indicated.
1. MERTK presents an attractive target for metastatic
diseases.
To best summarize, we use the words directly from the paper.
Schlegel et al conclude:
We believe this work has led to several novel insights.
First, MERTK expression is significantly elevated in
distant metastatic tumors compared with primary melanomas.
Second, MERTK is overexpressed in approximately half of
melanoma cell lines, irrespective of BRAF and NRAS status, and is an active
receptor.
Third, targeting MERTK suppresses prosurvival pathways
such as STAT6, AKT, and ERK1/2.
Fourth, targeting MERTK suppresses colony-forming
potential and migration.
And fifth, targeting MERTK in vivo retards tumor growth
in a human melanoma xenograft model.
The finding that MERTK expression is highest in distant
metastatic melanomas compared with primary melanomas and the roles of MERTK in
colony formation, migration, and invasion suggest that MERTK plays a role in
the progression of primary melanomas and the development of distant metastases.
Similar to the observations in this report, the migratory
nature of glioblastoma cells could be reduced by MERTK inhibition with either
shRNA knockdown or a MERTK monoclonal antibody, suggesting that increased MERTK
expression may contribute to outgrowth of the metastatic tumor.
2. MER and other TAM receptors show significant impact across
broad areas of cancer activity.
Now Verma et al present an interesting summary table as show
below which recounts what cancer types are also upregulated TAM pathways. The
breath of such upregulation is significant. It also may present significant
opportunities for blockage molecules, namely inhibitors of the total pathway.
3. GAS6 Inhibition on MERTK by inhibitors is an
attractive approach to metastatic melanoma
Developing receptor inhibitors is a powerful approach to
controlling metastatic growth and proliferation.
As Verma et al state:
A potential ability of sAxl to serve as a natural
antagonist of Gas 6 could have clinical relevance. Similarly, the
membrane-bound Mer protein is cleaved in the extracellular domain via a
metalloproteinase (38). Further studies are needed to establish sAxl and sMer
as important biomarkers for correlation with disease stage and predicting
prognosis.
As Segal et al state:
A subset of genes, including the small monomeric GTPase
RABB33, the proto-oncogene MERTK, the glycopeptide hormone STC1, and the
neuropeptide GAL were shown to discriminate CCS/MSP from both STS and melanoma.
We further surveyed specific genes of interest and found
melanoma differentiation antigens TYRP1, TYRP2/DCT, and MART-1 to be expressed
at varying levels in the CCS/MSP specimens. PMEL17 was most consistently
expressed in all four tumors in a similar distribution to that of MITF.
Interestingly, SOX10, which induces MITF expression, was expressed in all
CCS/MSP and most melanoma specimens
Thus it appears that this approach and ones like it are
useful for thorough examination as attractive and effective means of metastatic
control and management.
However there is still a long way from this point to
approved therapeutics.
1.6
References
1. Halberg, N., et al, microRNA regulation of cancer–endothelial
interactions: vesicular microRNAs on the move, The EMBO Journal (2012) 31,
3509–3510.
2. Marks, F., et al, Cellular Signal Processing, Garland (NY) 2009.
3. Park, H. et al, The TAM-family receptor Mer mediates production
of HGF through the RhoA-dependent pathway in response to apoptotic cells, Molecular
Biology of the Cell, Volume 23 August 15, 2012.
4. Schlegel, J., et al, MERTK receptor tyrosine kinase is a
therapeutic target in melanoma, The Journal of Clinical Investigation, http://www.jci.org,
Volume 123, Number 5, May 2013 p.2257.
5. Segal, N., et al, Classification of Clear-Cell Sarcoma as a
Subtype of Melanoma by Genomic Profiling, J Clin Oncol 21:1775-1781, 2003.
6. Verma, A., et al, Targeting Axl and Mer Kinases in Cancer, Mol
Cancer Ther 2011; 10: 1763-1773.
7. Zhuang, G., et al, Tumour-secreted miR-9 promotes endothelial
cell migration and angiogenesis by activating the JAK-STAT pathway, The EMBO
Journal 31, 3513 - 3523 (6 July 2012) http://www.nature.com/emboj/journal/v31/n17/full/emboj2012183a.html
[1] From
NCBI we have (2q14.1): This gene is a member of the MER/AXL/TYRO3 receptor
kinase family and encodes a transmembrane protein with two fibronectin type-III
domains, two Ig-like C2-type (immunoglobulin-like) domains, and one tyrosine
kinase domain.
[2]
TAM, (TYRO, AXL, MERTK)
[3] As
NCBI states: This gene product is a gamma-carboxyglutamic acid
(Gla)-containing protein thought to be involved in the stimulation of cell
proliferation, and may play a role in thrombosis. Alternatively spliced
transcript variants encoding different isoforms have been found for this gene.
Located at 13q34. http://www.ncbi.nlm.nih.gov/gene/2621
[4]
See McGarty, Melanoma Genomics, DRAFT, 2013.
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