This search for the markers has been going on for several
decades. For example from Walker et al (1999) we have the following:
We wish to identify genes associated with disease. To do
so, we look for novel genes whose expression patterns mimic those of known
disease-associated genes, using a method we call Guilt-by-Association (GBA), on
the basis of a combinatoric measure of association. Using GBA, we have examined
the expression of 40,000 human genes in 522 cDNA libraries, and have discovered
several hundred previously unidentified genes associated with cancer,
inflammation, steroid-synthesis, insulin-synthesis, neurotransmitter
processing, matrix remodeling, and other disease processes.
The majority of the genes, thus discovered, show no
sequence similarity to known genes, and thus could not have been identified by
homology searches. We present here an example of the discovery of eight genes
associated with prostate cancer. Of the 40,000 most-abundant human genes, these
8 are the most closely linked to the known diagnostic genes, and thus are prime
targets for pharmaceutical research.... PSA,
the most closely coexpressed genes are glandular kallikrein, three novel genes,
prostate seminal protein, PAP, a fourth novel gene, prostate transglutaminase,
a fifth novel gene, and neuropeptide Y. (IPCA-9, IPCA-10 and IPCA-11 are
coexpressed with PSA but appear to be 38 untranslated sequences.) ….Neuropeptide
Y is coexpressed with PSA. It has been reported to be associated with prostate cancer
The neuropeptide Y, NPY, has thus
been on the list as a putative marker for almost two decades. This specific
one, namely pro-NPY, the predecessor of NPY, is the target of recent interest. A
recent paper purports to establish a basis for pro-NPY. Namely from MedicalNews[2] we
have the following:
Researchers at the University of Copenhagen have identified
a new prognostic biomarker: the neuropeptide pro-NPY, which may help determine
the risk of dying from prostate cancer. This particular type of protein is very
specific to prostate cancer cells and could help identify whether newly
diagnosed patients require radical prostatectomy surgery or if it is safe to
delay surgery. The research has been published in the journal, European Urology[3].
Using mass spectrometry, the researchers measured
concentration changes in thousands of proteins in both normal and tumour tissue
from prostate cancer. They discovered that in comparison to normal tissue, the
prostate tumors exhibit numerous metabolic alterations including exacerbated
activity of mitochondria.
Among the 9000 proteins identified, one protein, the
neuropeptide, pro-NPY, was demonstrated to exhibit high levels in a subgroup of
prostate cancer samples. Pro-NPY was analyzed in 750 patients with prostate
cancer to show that pro-NPY levels correlate with increased risk of prostate
cancer death.
"Our research shows that high pro-NPY levels are
very specific to prostate cancer and can serve to predict prostate cancer
related death among diagnosed patients who have not received surgical
treatment," says Professor Amilcar Flores-Morales from the Department of
Veterinary Disease Biology, University of Copenhagen.
"So identifying the biomarker pro-NPY could help us
identify patients who would benefit from early active treatment, whereby we
would also reduce unnecessary treatment of patients who undergo surgery when
they have low-grade tumors that for the most part do not put their lives at
risk. In the end, due to side effects, this could prove more harmful than
beneficial to patients," adds Amilcar Flores-Morales.
Proteins are key effectors of cellular functions.
Therefore, a better understanding of the protein signaling pathways deregulated
in prostate cancer could lead to better preventive and therapeutic strategies
for the treatment of this disease. Specifically, it is possible that metabolic
alterations such as the increase in mitochondria activity could be targeted in
the treatment of prostate cancer.
"We hope to contribute to the advance of
translational cancer research and the implementation of precision medicine in
the field of prostate cancer by providing a unique insight into the protein
level alterations associated with tumor tissue in clinical samples," adds
Flores-Morales.
This specific gene product pro-NPY has been studied by many,
either directly or as its product NPY.
From Shay and Mangian we have a history of the
identification and understanding of the function of NPY. Specifically they
state:
In the 1970s, it was determined by Wurtman, Fernstrom and
colleagues that dietary concentrations of tyrosine and tryptophan could affect
the synthesis and concentrations of the neurotransmitters norepinephrine and
serotonin. In turn, the diet-affected central concentrations of these
neurotransmitters could affect the relative appetite/satiety state of an
individual. These findings spurred nutrition researchers to connect zinc
deficiency, dietary amino acid intake and anorexia. In 1980 it was reported
that norepinephrine had a profound influence on feeding behavior within
specific sites in the hypothalamus. Leibowitz and Brown (1980) reported that
the predominantly inhibitory neurotransmitter norepinephrine had a strong
stimulatory effect on food intake. When exogenous norepinephrine was delivered
to the paraventricular nucleus (PVN) of the hypothalamus, short-term food
intake increased. Later in the 1980s, neuropeptides were also discovered to
have a profound impact on feeding behavior.
In 1982, neuropeptide Y (NPY) was first isolated from
neural tissue within the porcine intestine. Soon after, NPY was found to have
significant stimulatory effect on food intake (Clark et al. 1984). Although NPY
may be synthesized by all neurons within the body, it is synthesized at very
high levels within cell bodies derived in the arcuate nucleus of the
hypothalamus. A high percentage of these neurons project to the PVN of the
hypothalamus. Within the PVN, exogenously administered NPY has been
demonstrated to stimulate appetite to a greater degree than any other agent yet
tested, when considered on a molar basis. Interestingly, it was also found that
the administration of NPY to the PVN specifically stimulated carbohydrate
intake when rats were allowed to freely select from a three-choice
macronutrient diet system.
Some investigators have also suggested that the results
demonstrating an effect of NPY on macronutrient preference may be influenced by
the past history or dietary preferences of rats chosen for study (Welch et al.
1994). Even specifics of the diet ingredients used in macronutrient choice
studies may influence the results obtained (Glass et al. 1997). Because of its
very potent effect on food intake, NPY has been investigated very vigorously at
many laboratories. Targets of research have included the effects of NPY, the
development of agonists and antagonists of NPY and the identification and study
of NPY receptors. The development of an NPY antagonist with an
appetite-modulating activity is of interest to pharmaceutical concerns.
Consistent with the complex nature of the appetite
regulation system, NPY has proved to be a difficult target to study.
First, it has been found that there are a family of NPY
receptors, and it is still unclear whether a single NPY receptor or a subset of
a few receptors mediate the appetite-generating effect of NPY.
Second, the NPY knockout mouse regulates food intake in a
relatively normal fashion.
This has led some to suggest that the large set of
physiological studies investigating the effect of NPY on food intake may need
to be reconsidered. A possible explanation for normal appetite in the NPY
knockout mouse is that NPY action may be accommodated for by other
neuropeptides during development. The paradox between physiological data and
the NPY knockout results is of great interest and is likely to be further
investigated.
Finally in the current Inglesias-Gato et al paper they
conclude:
The evaluation of pro-NPY as a biomarker of disease
progression in historic TURP-detected watchful-waiting cohorts has some
limitations as PSA levels are not available and also as tumor tissue obtained
through TURP could be different from small cancers located in the peripheral
zone. However, it also has some strength. Watchful-waiting cohorts are superior
for identifying patients with an excellent outcome also in the absence of
treatment and for identifying tumors that will progress when left in situ.
Markers for long-term indolent tumors, or tumors that
will eventually progress when left in situ are difficult to identify in cohorts
where patients are treated at an early stage. However, in order to implement
pro-NPY measurements into current practice, the prognostic potential of pro-NPY
should be addresses in modern, PSA tested cohorts, to evaluate its performance
relative to current standard of care.
Furthermore, pro-NPY’s nature as a secreted peptide and
its high specificity for PCa would support additional studies to validate
pro-NPY as a prognostic blood biomarker for disease progression in PSA screened
patients and patients on active surveillance.
Neuropeptides are a class of molecules that have the
capability of establishing communications between neurons. They are most common
therefore amongst the nerve cells. Thus their presence in the prostate and
especially their over-expression is of some interest. One of the key questions
one may ask is; why do we see such an overexpression of neuropeptides in
aggressive PCa? We will examine also the presence of neuroendocrine cells in
the prostate as well. Neuroendocrine cells are a special class of cells that
receive signals from nerve cells in the form of neurotransmitters, such as the
neuropeptides, and then release various hormones[4].
Neuroendocrine PCa is a highly aggressive and androgen receptive negative form
of PCa with associated fatal results[5].
We shall focus on this collection of relationships to interpret some of the
results of the paper in discussion.
Specifically we focus on pro-NPY and not its successor NPY. As
Wulff et al noted the following about NPY, a successor to pro-NPY:
Peptide hormones, neuropeptides, and most other
biologically active peptides are generated from larger precursors through
proteolytic processing at dibasic or monobasic sites . In recent years a series
of enzymes have been characterized that appear to be involved in this
maturation process.
The two so-called precursor convertases, PC2, cloned from
a human insulinoma and from mouse pituitary, and PC3, cloned from mouse
pituitary and from AtT-20 cells , both have the expected specificity for
certain pairs of basic residues. These enzymes are also expressed exclusively
in peptide producing neuronal and endocrine cells . Coexpression of
proopiomelanocortin and the two precursor convertases indicate that the balance
between the expression of PC2 and PC3 probably can explain certain cases of tissue
specific processing of precursors .
Furthermore, the processing of proopiomelanocortin in
AtT-20 cells has been suppressed by antisense constructs of PC3 . Thus PC2 and
PC3 appear to constitute key enzymes of the precursor processing machinery in
the neuroendocrine system.
PP and NPY belong to the so-called PP-fold family of
peptides, which have relatively simple precursors with a single dibasic
processing site, plus, in pro-PP, an additional mono basic processing site.
The overall homology of the secreted products, PP and
NPY, is 45%, and this homology is mainly restricted to residues that are
important in the stabilization of the PP-fold structure and to the C-terminal
part of the molecules, which is involved in receptor recognition.
From NCBI they discuss the NPY gene and its product:[6]
This gene encodes a neuropeptide that is widely expressed
in the central nervous system and influences many physiological processes,
including cortical excitability, stress response, food intake, circadian rhythms,
and cardiovascular function. The neuropeptide functions through G
protein-coupled receptors to inhibit adenylyl cyclase, activate
mitogen-activated protein kinase (MAPK), regulate intracellular calcium levels,
and activate potassium channels. A polymorphism in this gene resulting in a
change of leucine 7 to proline in the signal peptide is associated with
elevated cholesterol levels, higher alcohol consumption, and may be a risk
factor for various metabolic and cardiovascular diseases. The protein also
exhibits antimicrobial activity against bacteria and fungi.
First we examine NPY, a 36 amino acid molecule derived from
pro-NPY which as we will discuss is 69 amino acids in size. From Silva et al we
have the following discussion on NPY:
NPY (neuropeptide Y) is a 36-amino-acid peptide involved
in the regulation of the cardiovascular system. It has vasopressor effects and
potentiates the effect of other vasoconstrictor molecules such as noradrenaline
or histamine. When used at low, non-vasoconstrictive doses on cultured vascular
SMCs (smooth muscle cells), NPY stimulates SMC proliferation, an effect
potentiated by noradrenaline.
NPY also acts on vascular ECs (endothelial cells). The
potentiating effect of NPY on noradrenaline-induced vasoconstriction has been
shown to be endothelium dependent on human saphenous veins.
NPY is capable of promoting EC proliferation, migration
and adhesion on the extracellular matrix. It also stimulates capillary tube
formation in vitro and angiogenesis in vivo. Similar to other secreted
peptides, NPY is produced as a pre-pro-peptide. After removal of the signal
peptide in the endoplasmic reticulum, pro-NPY is further cleaved by successive
enzymes to generate the biologically active amidated NPY.
In neuroendocrine cells, mature NPY is localized in
secretory granules, e.g. in neurons, chromaffin cells or in the pituitary.
Immunoreactive NPY has been detected in HUVEC (human umbilical-vein endothelial
cells), but NPY synthesis, storage and secretion have not been studied.
The potential for NPY to promote endothelial cell
proliferation may be one of the factors in its excess and the growth of PCa
beyond AR inhibition. It may facilitate angiogenesis via its endothelial
action. Again this is speculative. The identification in neuroendocrine cells
may also be significant in view of the aggressiveness of neuroendocrine based
PCa. Again we are speculating here as well.
But pro-NPY is a more complex molecule as described by Eggelkraut-Gottanka:
Similar to many other hormones and neurotransmitters,
neuropeptide Y (NPY) is derived from a larger precursor molecule, the 69 amino
acid pro-neuropeptide Y (pro-NPY). Precursor proteins undergo a highly specific
conversion process to yield their biologically active products. As part of a
finely tuned regulation network, the biosynthesis of hormones and neuropeptides
plays a major role in many physiological and pathophysiological processes.
Diseases such as diabetes, obesity and diverse sorts of
cancer could be associated with dysfunctions in the biosynthetic pathways.
From Silva et al who connect pro-NPY and NPY as follows:
The present study shows the expression of NPY and its
precursor pro-NPY in HUVEC at the mRNA and protein levels as demonstrated by
RT–PCR and ELISA. NPY expression has previously been determined in HUVEC only
in a small number of HUVEC cultures. In the present study, NPY expression was
evidenced in all cell preparations tested, independent of the medium used for
cell culture ...The difference between the results of these two studies
may be due to a different initial amount of RNA used for RT–PCR and the number
of passages, since we used cells at passage 1, whereas …The presence of NPY and
pro-NPY in HUVEC was also assessed by immunofluorescence. NPY immunoreactivity
appeared as small punctate granular structures disseminated in the cytoplasm...The antibody NPY02 used in our study is directed against
an epitope borne by both NPY and pro-NPY, thus leading to the labelling of both
NPY and its precursor. The small punctate granular appearance of endocytic
vesicles …. However, it is unlikely that intracellular NPY derives from
cell-culture medium since NPY staining is not lost or altered in HUVEC
incubated in the absence of serum. Furthermore, labelling of EEA1, an early
endosome marker, showed a staining completely different from the
immunolabelling of NPY.
From the work of Magni and Motta (2001) we know that:
By showing that NPY receptors are expressed in the
androgen-independent cell line PC-3 and that their activation results in cell
proliferation, the present date suggest that NPY-related mechanisms might be
relevant in certain stages of CaP, such as the progression of the disease
during the androgen-independent stage.
They continue to state:
Prostate cancer (CaP) is initially often androgen
dependent, and it may progress to androgen independence in later stages. In
this condition, hormonal therapy is no longer useful and the prognosis becomes
worse. It is believed that the molecular basis underlying this transition
includes a host of factors, some of which are now being identified as peptidic
molecules, such as growth factors and neurohormones. Several studies suggest
that neuroendocrine mechanisms play an important role in the control of the
development and the function of the normal prostate, as well as of the
progression of CaP to androgen independence.
Few data, however, are presently available about one of
these neuroendocrine modulators, neuropeptide Y (NPY), and on the related
receptors in the normal as well as in the tumoral prostate. NPY, a peptide of
36 aminoacids, is abundantly distributed through the nervous system, and
activates specific membrane receptors that exist in at least five different
isoforms .
NPY participates to the regulation of a variety of
physiological functions, including regulation of neuroendocrine mechanisms,
cognitive functions, eating behavior and cardiovascular activity, and has also
been shown to stimulate cell proliferation . In the context of the normal human
prostate, NPY is mainly localized in the nerve fibers, and in the
neuroendocrine (NE) cells .
In conclusion, the present study, together with other
data present in the literature , suggests that the prostatic NPY neuroendocrine
system might participate in the modulation of the proliferation of CaP cells.
Moreover, the presence and the activation of NPY receptors might represent a
marker of CaP progression toward a stage sensitive to non-androgenic trophic
and proliferative agents. Further studies in this field might also give
indications about possible novel future lines for the treatment of CaP,
especially when this disease has progressed to the androgen-independent stage.
The regulation of neuroendocrine regulation may be a
significant factor in the presence of pro-NPY in PCa and its aggressive forms.
Again we have examined also the neuroendocrine types and these are driven by
neuropeptides and are AR independent. From the recent paper (2015) which we are
examining by Inglesias et al[7]:
Clinical management of the prostate needs improved
prognostic tests and treatment strategies. Because proteins are the ultimate
effectors of most cellular reactions, are targets for drug actions and
constitute potential biomarkers; a quantitative systemic overview of the
proteome changes occurring during prostate cancer (PCa) initiation and
progression can result in clinically relevant discoveries. To study cellular
processes altered in PCa using system-wide quantitative analysis of changes in
protein expression in clinical samples and to identify prognostic biomarkers
for disease aggressiveness. Over 9000 proteins were identified as expressed in the
human prostate. Tumor tissue exhibited elevated expression of proteins involved
in multiple anabolic processes including fatty acid and protein synthesis,
ribosomal biogenesis and protein secretion but no overt evidence of increased
proliferation was observed. Tumors also showed increased levels of
mitochondrial proteins, which was associated with elevated oxidative phosphorylation
capacity measured in situ. This study represents the first system-wide quantitative
analysis of proteome changes associated to localized prostate cancer and as
such constitutes a valuable resource for understanding the complex metabolic
changes occurring in this disease. We also demonstrated that pro-NPY, a protein
that showed differential expression between high and low risk tumors in our
proteomic analysis, is also a PCa specific prognostic biomarker associated with
increased risk for disease specific death in patients carrying low risk tumors.
The identification of proteins whose expression change in
prostate cancer provides novel mechanistic information related to the disease
etiology. We hope that future studies will prove the value of this proteome
dataset for development of novel therapies and biomarkers. Deep and
quantitative proteomic profiling was obtained from formalin-fixed
paraffin-embedded prostate cancer specimens and revealed that: prostate cancer cells preferably use
oxidative phosphorylation for energy production; and proneuropeptide-Y expression defines a
subgroup of prostate cancer patients with worsened prognosis, who might benefit
from active intervention.
As with many such markers this results is not a causative
result but a fortuitous result from observation. It appears to provide a
prognostic marker. From Science Daily[8]:
A new prognostic biomarker has been identified by
researchers: the neuropeptide pro-NPY, which may help determine the risk of
dying from prostate cancer. This particular type of protein is very specific to
prostate cancer cells and could help identify whether newly diagnosed patients
require radical prostatectomy surgery or if it is safe to delay surgery.
Researchers at the University of Copenhagen have
identified a new prognostic biomarker: the neuropeptide pro-NPY, which may help
determine the risk of dying from prostate cancer. This particular type of
protein is very specific to prostate cancer cells and could help identify
whether newly diagnosed patients require radical prostatectomy surgery or if it
is safe to delay surgery.
Using mass spectrometry, the researchers measured
concentration changes in thousands of proteins in both normal and tumour tissue
from prostate cancer. They discovered that in comparison to normal tissue, the
prostate tumors exhibit numerous metabolic alterations including exacerbated
activity of mitochondria. Among the 9000 proteins identified, one protein, the
neuropeptide, pro-NPY, was demonstrated to exhibit high levels in a subgroup of
prostate cancer samples. Pro-NPY was analyzed in 750 patients with prostate
cancer to show that pro-NPY levels correlate with increased risk of prostate
cancer death.
"Our research shows that high pro-NPY levels are
very specific to prostate cancer and can serve to predict prostate cancer
related death among diagnosed patients who have not received surgical
treatment," says Professor Amilcar Flores-Morales from the Department of
Veterinary Disease Biology, University of Copenhagen.
"So identifying the biomarker pro-NPY could help us
identify patients who would benefit from early active treatment, whereby we
would also reduce unnecessary treatment of patients who undergo surgery when
they have low-grade tumors that for the most part do not put their lives at
risk. In the end, due to side effects, this could prove more harmful than
beneficial to patients," adds Amilcar Flores-Morales.
Proteins are key effectors of cellular functions.
Therefore, a better understanding of the protein signaling pathways deregulated
in prostate cancer could lead to better preventive and therapeutic strategies
for the treatment of this disease. Specifically, it is possible that metabolic
alterations such as the increase in mitochondria activity could be targeted in
the treatment of prostate cancer.
"We hope to contribute to the advance of
translational cancer research and the implementation of precision medicine in
the field of prostate cancer by providing a unique insight into the protein
level alterations associated with tumor tissue in clinical samples," adds
Flores-Morales.
This work is the result of collaborations between the
research groups of Professor Flores-Morales at IVS, Professor Matthias Mann at
Novo Nordisk Foundation Center for Protein Research both from the Faculty of
Health and Medical Sciences together with the Danish Cancer Society Research
Center and Associate Professor Pernilla Wikström from the Umeå University,
Sweden. The validation of pro-NPY as a biomarker was possible due to the
contribution of patients and clinical researchers from several institutions in
Sweden.
The creation of NPY from pro-NPY is discussed in Brakch et
al as noted below:
Proneuropeptide Y (ProNPY) undergoes cleavage at a single
dibasic site Lys38-Arg39 resulting in the formation of 1-39 amino acid NPY which
is further processed successively by carboxypeptidase-like and peptidylglycine
alpha-amidating monooxygenase enzymes.
To investigate whether prohormone convertases are
involved in ProNPY processing, a vaccinia virus derived expression system was
used to coexpress recombinant ProNPY with each of the prohormone convertases
PC1/3, PC2, furin, and PACE4 in Neuro2A and NIH 3T3 cell lines as regulated
neuroendocrine and constitutive prototype cell lines, respectively. The
analysis of processed products shows that only PC1/3 generates NPY in NIH 3T3
cells while both PC1/3 and PC2 are able to generate NPY in Neuro2A cells.
The convertases furin and PACE4 are unable to process Pro-NPY
in either cell line.
Moreover, comparative in vitro cleavage of recombinant
NPY precursor by the enzymes PC1/3, PC2 and furin shows that only PC1/3 and PC2
are involved in specific cleavage of the dibasic site.
Kinetic studies demonstrate that PC1/3 cleaves ProNPY
more efficiently than PC2. The main difference between the cleavage efficiency
is observed in the Vmax values whereas no major difference is observed in Km
values.
In addition the cleavage by PC1/3 and PC2 of two peptides
reproducing the dibasic cleavage site with different amino acid sequence
lengths namely (20-49)-Pro-NPY and (28-43)-Pro-NPY was studied. These shortened
Pro-NPY substrates, when recognized by the enzymes, are more efficiently
cleaved than Pro-NPY itself.
The shortest peptide is not cleaved by PC2 while it is by
PC1/3.
On the basis of these observations it is proposed,
first, that the
constitutive secreted NPY does not result from the cleavage carried out by
ubiquitously expressed enzymes furin and PACE4;
second, that PC1/3 and PC2 are not equipotent in the
cleavage of Pro-NPY; and
third, substrate peptide length might discriminate
PC1/3 and PC2 processing activity.
From Khan et al:
A total of 80 proteins was found to be elevated in PCA
compared with Benign. Included among these were previously known alterations
for prostate cancer, namely GOLM1 , transcription elongation factor B (SIII),
polypeptide 1 (15 kDa; elongin C or TCEB1) , neuropeptide Y , Parkinson disease
(autosomal recessive, early onset) 7 (PARK7 or DJ-1) , anterior gradient
homolog-2 (AGR2) , growth differentiation factor 15 (GDF15, MIC-1, or NAG-1) ,
ferritin heavy chain (FTH1) , tumor necrosis factor, -induced protein 9 (STAMP2
or STEAP4) , fatty acid-binding protein (FABP5) , and VIM .
A similar analysis for down-regulated proteins revealed
81 proteins whose expression was decreased in PCA compared with Benign.
Prominent among these were lactotransferrin , 2-glycoprotein (AZGP1) ,
microseminoprotein (prostatic secretory
protein of 94 amino acids, PSP94, or MSMB) , isoforms of glutathione transferase
(GSTP1 and GSTM3) (58–60), lactate dehydrogenase B , and N-myc downstream
regulated gene (NDRG1) , all of which have been reported earlier to be
down-regulated in organ-confined disease.
From Inglesias-Gato et al:
Over 9000 proteins were identified as expressed in the
human prostate. Tumor tissue exhibited elevated expression of proteins involved
in multiple anabolic processes including fatty acid and protein synthesis,
ribosomal biogenesis and protein secretion but no overt evidence of increased
proliferation was observed. Tumors also showed increased levels of
mitochondrial proteins, which was associated with elevated oxidative
phosphorylation capacity measured in situ. ...Pro-NPY expression, alone or in combination with the ERG status of the tumor,
was associated with an increased risk of PCa specific mortality, especially in
patients with Gleason score 7 tumors.
They continue at length:
Pro-NPY as a novel biomarker of disease progression
Patients with primary Gleason grade at diagnosis have a more aggressive disease
course …..In order to further select candidate biomarkers for
clinical validation, we also employed a supervised learning approach, support
vector machines, and combined it with feature selection...The pro-NPY derived tryptic peptide most commonly identified
by MS corresponded to a portion of the protein C terminal end, which is
normally proteolytically removed to generate mature NPY. This specificity for PCa was further confirmed in a panel
of tumors available from the human protein atlas database.
PCa has several modes or presentation, the most common is
the adenocarcinoma type of the gland, basal and luminal, and also a
neuroendocrine variety, less well known but highly aggressive. Both Staibano
and Mydlo and Godec provide an extensive discussion of this form. We briefly examine
it here since it relates to neuropeptide presence. Neuroendocrine
Differentiation, NED, is considered a normal and ultimately lethal step in PCa
progression. The neuroendocrine, NE, are a small part of the prostate cell
mass. They tend to infiltrate the basal layers and at times may even penetrate
into the lumen. They get signals from nerve cells and in turn emit stimulants
to the surrounding cells. The set of surrounding cells impacted by this
signalling also includes the blood network and their endothelial structures[9].
The NE have some specific characteristics of note here:
1. They are AR negative. Namely they androgen receptors are
non-functional
2. They are PSA negative
3. They emit NPY to the surrounding cells. This is the nexus
we have been examining. Perhaps it is the NE PCa via the NED that is the reason
we have the nexus between lethal PCa and the presence of pro-NPY
From PCF we have a brief description of neuroendocrine PCa[10]:
Over 90 % of malignant prostate cancers occur in the form
of adenocarcinoma, which is characterized by uncontrolled growth of the
prostate cells that secrete the prostate specific antigen (PSA). This is why
many men with malignant adenocarcinoma of the prostate have elevated PSA
levels. Generally adenocarcinoma is highly treatable with excellent cure rates,
even though every prostate cancer of this type has the subtype neuroendocrine
cancer cells scattered throughout the tumor. (Even benign, normal prostate
glands have a tiny population, roughly about 0.1 %, of neuroendocrine cells,
nested throughout the gland. See Figure One. It is thought these neuroendocrine
cells normally play a role in early prostate development or perhaps function.)…
The neuroendocrine cells scattered throughout
adenocarcinoma of the prostate generally make up about 1% or less of the total
tumor. Neuroendocrine prostate cancer (NEPC) is diagnosed when vast numbers of
neuroendocrine cells are found in a tumor. “Neuroendocrine prostate cancer
cells look small under the microscope,” …“And they tend to metastasize not just
to bone, as is common in adenocarcinoma, but to liver or other abdominal
visceral organs.” There are also a number of biochemical markers for NEPC that
can be detected by tissue-staining lab tests, which aids in diagnosis of this
disease.
Very rarely are men newly diagnosed with prostate cancer
that is the neuroendocrine subtype. When this does occur it is called de novo
NEPC, referring to the thought that this subtype of cancer has been there de
novo, or from the beginning. Far more commonly, NEPC is a result of treatment
with hormone therapy, and is known as treatment-related NEPC, or t-NEPC.
From Aggarwal et al:
Neuroendocrine prostate cancer (NEPC) encompasses various
clinical contexts, ranging from the de novo presentation of small cell
prostatic carcinoma to a treatment-emergent transformed phenotype that arises
from typical adenocarcinoma of the prostate. The development of resistance to
potent androgen receptor signaling inhibition may be associated with the
emergence of aggressive phenotype, advanced castration-resistant NEPC.
Clinically, small cell prostate cancer and NEPC are often
manifested by the presence of visceral or large soft tissue metastatic disease,
a disproportionately low serum prostate-specific antigen level relative to the
overall burden of disease, and a limited response to targeting of the androgen
signaling axis. These tumors are often characterized by loss of androgen
receptor expression, loss of retinoblastoma tumor suppressor copy number or
expression, amplification of Aurora kinase A and N-Myc, and activation of the
PI3K pathway.
As Parimi et al state:
Neuroendocrine cells are one of the epithelial
populations in the prostate. Neuroendocrine differentiation (NED) has been
observed in prostate cancer. In addition to small cell neuroendocrine carcinomas
and carcinoid tumors of the prostate, prostatic adenocarcinomas may have NED.
The incidence and clinical relevance of NED in prostatic adenocarcinoma is not
clearly understood because of conflicting results in the reported studies, and
evaluation of NED is not routinely performed in clinical practice. …we are
stratifying these lesions into separate subtypes based on histologic parameters
such as tumor morphology, neuroendocrine cell density and distribution and
clinical parameters.
They continue to describe NE as follows and then details
some of the specifics in type:
Even though the definition of neuroendocrine prostatic
carcinomas is still emerging, in this review from a morphologic standpoint,
neuroendocrine prostatic carcinomas is considered as a special type of
neuroendocrine differentiation of prostatic epithelial neoplasms (Table 2).
Neuroendocrine carcinoma of the prostate may represent a subset of prostate
cancer phenotypes which may be linked to resistance to androgen receptor (AR)
signaling inhibition with aggressive tumor characteristics and a largely dismal
prognosis. Neuroendocrine prostatic carcinomas (NEPC) are often diagnosed on
primary prostate needle biopsy or on biopsies of metastatic lesions with
negative or low PSA levels.
Thus the existence of NED and the relationship to NPY and
pro-NPY is worthy of further examination. As noted we continue to see the reporting of many new
markers for both detection of and prognosis of PCa. However in many cases these
markers are not always causative but adventitious. They just happen to be there
and may or may not reflect a process, one which may allow for a therapeutic
approach.As we have noted in Inglesias-Gato et al where they
conclude:
The combined assessment of pro-NPY levels and ERG status
improves prediction of PCa related death High NPY protein expression was
recently demonstrated in prostate tumors harboring the TMPRSS2-ERG fusion gene .
Previous evaluation of ERG expression in the … cohort
showed it to be related to increased risk of disease- related mortality.
Therefore we analyzed whether pro- NPY expression correlated with ERG
expression in our sample cohort. Forty-four percent of the tumors included in
the analysis were positive for ERG expression and of these, 52% showed high
pro-NPY levels.
Patients with tumors expressing high levels of pro-NPY
with positive ERG protein expression have a significant increase in the
relative risk of PCa related death, especially within the low GS group.
Accordingly, multivariate regression analysis shows that high expression of
both ERG and NPY increases the risk for PCa mortality independently of GS
We use this to examine a few issues.
Causative Results: The above indicate the ERG expression which we know has
causative effect, especially when we see the ERG: TMPRSS merge. However the
relationship and systemic details on the pro-NPY effect are missing. One would
like to see this in some detail.
Therapeutic Targets: It is not clear if NPY or pro-NPY can be therapeutic targets
or even more so the underlying gene. Since the causative effect does not seem
apparent one wonders if having a prognostic marker is of substantial value.
Metastasis is most likely an existing fact, albeit on a micro scale. Thus the
question can be stated: of what clinical value do we have measuring pro-NPY?
Process Control: What are the
pathways, receptors, ligand, and the like that are involved in pro-NPY and NPY
generation, communication, and activation? The details appear to be missing in
this overall analysis.
Neuroendocrine Differentiation: When we examined the NED type of progression we saw the presence of NPY as a secretion from the NE cells to the remainder of the prostate. We also understand the evolution of NED in most PCas and the question posed would be; what is the driver for NED? Also does the NE communications somehow relate to stress activation via the nerve cells communicating with the NE calls. Also is it the endothelial enhancement of the NE cells that facilitate the metastatic growth.
1. Aggarwal R1, Zhang T, Small EJ, Armstrong AJ., Neuroendocrine
prostate cancer: subtypes, biology, and clinical outcomes. J Natl Compr Canc
Netw. 2014 May;12(5):719-26.
2. Brakch , N., et al, Role of prohormone convertases in pro-neuropeptide
Y processing: coexpression and in vitro kinetic investigations, Biochemistry.
1997 Dec 23;36:16309-20.
3. Eggelkraut-Gottanka. R., Synthesis and Characterization of the
Precursor of Neuropeptide Y, Doctor of Natural Sciences, Swiss Federal Institute
of Technology Zurich, 2003.
4. Iglesias, D., et al, The Proteome of Primary Prostate Cancer, European
Urology, December 2015
5. Kahn, A., et al, Quantitative Proteomic Profiling of Prostate
Cancer Reveals a Role for miR-128 in Prostate Cancer, Molecular & Cellular
Proteomics, 2010.
6. Magni, P., M. Motta, Expression of neuropeptide Y receptors in
human prostate cancer cells, Annals of Oncology 12 (Suppl 2): S27-S29, 2001
7. Mydlo, J., C. Godec, Prostate Cancer, 2nd Ed,
Academic Press, (Boston) 2016.
8. NCCN, NCCN Guidelines Version 2.2015 Updates Prostate Cancer
Early Detection,
9. NCCN, Prostate Cancer, 2016, https://www.nccn.org/store/login/login.aspx?ReturnURL=http://www.nccn.org/professionals/physician_gls/pdf/prostate.pdf
10. Parimi, V., et al, Neuroendocrine differentiation of prostate
cancer: a review, Am J Clin Exp Urol 2014;2(4):273-285
11. RayBiotech, RayBio® Human/Mouse/Rat Pro-Neuropeptide Y/CPON
Enzyme Immunoassay Kit, www.RayBiotech.com
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14. Silva, A., et al, Neuropeptide Y expression, localization and
cellular transducing effects in HUVEC, Biol. Cell (2005) 97, 457–467
15. Staibano, S., Prostate Cancer: Shifting from Morphology to
Biology, Springer, (New York) 2013.
16. Tatemoto, K., Neuropeptide Y: History and Overview, Department
of Molecular Physiology, Institute for Molecular and Cellular Regulation, Gunma
University, 371-8512 Maebashi, Japan
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in Prostate Cancer: Implications for New Treatment Modalities, European Urology
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Expression Analysis: Prostate Cancer-Associated Genes, Genome Research
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Pro-neuropeptide Y and Pro-pancreatic Polypeptide, in Transfected Cell Lines
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[1]
See the list of White Papers attached for details. They are part of and an
addition to the Draft book, Prostate Cancer
[3]
See References Inglesias-Gato et al.
[4]
See Mydlo & Godec, pp 149-155.
[5]
See Staibano, pp 87-109.
[7] http://www.europeanurology.com/article/S0302-2838%2815%2901087-8/abstract/the-proteome-of-primary-prostate-cancer
[9]
See Mydlo and Godec p 150.
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