Wednesday, February 3, 2016

pro-NPY and PCa

We continue to find new markers for the detection of and prognostication of prostate cancer. We herein examine one of the somewhat newer one the pro-NPY protein. There are always two questions that arise. The first is diagnostic; namely, does this patient have PCa? We have examined several of the recent methodologies in this area[1]. The second is prognostic tests, where we are looking for markers of aggressive PCa. Just because a patient is diagnosed with PCa it is well known that very few will die of the disease. Thus the issue of identifying that group and using aggressive treatments is an imperative.

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.
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