GFAP-directed Inactivation of Men1 Exploits Glial Cell Plasticity in Favor of Neuroendocrine Reprogramming.

BACKGROUND & AIMS: Efforts to characterize the signaling mechanisms that underlie gastroenteropancreatic neoplasms (GEP-NENs) are precluded by a lack of comprehensive models that recapitulate pathogenesis. Investigation into a potential cell-of-origin for gastrin-secreting NENs revealed a non-cell autonomous role for loss of menin in neuroendocrine cell specification, resulting in an induction of gastrin in enteric glia. Here, we investigated the hypothesis that cell autonomous Men1 inactivation in glial fibrillary acidic protein (GFAP)-expressing cells induced neuroendocrine differentiation and tumorigenesis. METHODS: Transgenic GFAPΔMen1 mice were generated by conditional GFAP-directed Men1 deletion in GFAP-expressing cells. Cre specificity was confirmed using a tdTomato reporter. GFAPΔMen1 mice were evaluated for GEP-NEN development and neuroendocrine cell hyperplasia. Small interfering RNA-mediated Men1 silencing in a rat enteric glial cell line was performed in parallel. RESULTS: GFAPΔMen1 mice developed pancreatic NENs, in addition to pituitary prolactinomas that phenocopied the human MEN1 syndrome. GFAPΔMen1 mice exhibited gastric neuroendocrine hyperplasia that coincided with a significant loss of GFAP expression. Men1 deletion induced loss of glial-restricted progenitor lineage markers and an increase in neuroendocrine genes, suggesting a reprogramming of GFAP+ cells. Deleting Kif3a, a mediator of Hedgehog signaling, in GFAP-expressing cells attenuated neuroendocrine hyperplasia by restricting the neuroendocrine cell fate. Similar results in the pancreas were observed when Sox10 was used to delete Men1. CONCLUSIONS: GFAP-directed Men1 inactivation exploits glial cell plasticity in favor of neuroendocrine differentiation.

A unique neuroendocrine cell model derived from the human foetal neural crest.

PURPOSE: Nowadays, no human neuroendocrine cell models derived from the neural crest are available. In this study, we present non-transformed long-term primary Neural Crest Cells (NCCs) isolated from the trunk region of the neural crest at VIII-XII gestational weeks of human foetuses obtained from voluntary legal abortion. METHODS AND RESULTS: In NCC, quantitative real-time RT PCR demonstrated the expression of neural crest specifier genes, such as Snail1, Snail2/SLUG, Sox10, FoxD3, c-Myc, and p75NTR. Moreover, these cell populations expressed stemness markers (such as Nanog and nestin), as well as markers of motility and invasion (TAGLN, MMP9, CXCR4, and CXCR7), and of neuronal/glial differentiation (MAP2, GFAP, SYP, and TAU). Functional analysis demonstrated that these cells not only possessed high migration properties, but most importantly, they expressed markers of sympatho-adrenal lineage, such as ASCL1 and tyrosine hydroxylase (TH). Moreover, the expression of TH increased after the induction with two different protocols of differentiation towards neuronal and sympatho-adrenal phenotypes. Finally, exposure to conditioned culture media from NCC induced a mature phenotype in a neuronal cell model (namely SH-SY5Y), suggesting that NCC may also act like Schwann precursors. CONCLUSION: This unique human cell model provides a solid tool for future studies addressing the bases of human neural crest-derived neuroendocrine tumours.

Remote CB1 receptor antagonist administration reveals multiple sites of tonic and phasic endocannabinoid neuroendocrine regulation.

Endogenous cannabinoids (endocannabinoids, eCB) are expressed throughout the body and contribute to regulation of the hypothalamo-pituitary-adrenal (HPA) axis and general stress reactivity. This study assessed the contributions of CB1 receptors (CB1R) in the modulation of basal and stress-induced neural and HPA axis activities. Catheterized adult male rats were placed in chambers to acclimate overnight, with their catheters connected and exteriorized from the chambers for relatively stress-free remote injections. The next morning, the CB1R antagonist AM251 (1 or 2 mg/kg) or vehicle was administered, and 30 min later, rats were exposed to loud noise stress (30 min) or no noise (basal condition). Blood, brains, pituitary and adrenal glands were collected immediately after the procedures for analysis of c-fos and CB1R mRNAs, corticosterone (CORT) and adrenocorticotropin hormone (ACTH) plasma levels. Basally, CB1R antagonism induced c-fos mRNA in the basolateral amygdala (BLA) and auditory cortex (AUD) and elevated plasma CORT, indicating disruption of eCB-mediated constitutive inhibition of activity. CB1R blockade also potentiated stress-induced hormone levels and c-fos mRNA in several regions such as the bed nucleus of the stria terminalis (BST), lateral septum (LS), and basolateral amygdala (BLA) and the paraventricular nucleus of the hypothalamus (PVN). CB1R mRNA was detected in all central tissues investigated, and the adrenal cortex, but at very low levels in the anterior pituitary gland. Interestingly, CB1R mRNA was rapidly and bidirectionally regulated in response to stress and/or antagonist treatment in some regions. eCBs therefore modulate the HPA axis by regulating both constitutive and activity-dependent inhibition at multiple levels.

Neurodevelopmental effects of natural and synthetic ligands of estrogen and progesterone receptors in zebrafish eleutheroembryos.

Natural and synthetic estrogens and progestins are widely used in human and veterinary medicine and are detected in waste and surface waters. Our previous studies have clearly shown that a number of these substances targets the brain to induce the estrogen-regulated brain aromatase expression but the consequences on brain development remain virtually unexplored. The aim of the present study was therefore to investigate the effect of estradiol (E2), progesterone (P4) and norethindrone (NOR), a 19-nortestosterone progestin, on zebrafish larval neurogenesis. We first demonstrated using real-time quantitative PCR that nuclear estrogen and progesterone receptor brain expression is impacted by E2, P4 and NOR. We brought evidence that brain proliferative and apoptotic activities were differentially affected depending on the steroidal hormone studied, the concentration of steroids and the region investigated. Our findings demonstrate for the first time that steroid compounds released in aquatic environment have the capacity to disrupt key cellular events involved in brain development in zebrafish embryos further questioning the short- and long-term consequences of this disruption on the physiology and behavior of organisms.

Neurotensin and its receptors mediate neuroendocrine transdifferentiation in prostate cancer.

Castration-resistant prostate cancer (CRPC) with neuroendocrine differentiation (NED) is a lethal disease for which effective therapies are urgently needed. The mechanism underlying development of CRPC with NED, however, remains largely uncharacterized. In this study, we explored and characterized the functional role of neurotensin (NTS) in cell line and animal models of CRPC with NED. NTS was acutely induced by androgen deprivation in animal models of prostate cancer (PCa) and activated downstream signaling leading to NED through activation of neurotensin receptor 1 (NTSR1) and neurotensin receptor 3 (NTSR3), but not neurotensin receptor 2 (NTSR2). Our findings also revealed the existence of a CK8+/CK14+ subpopulation in the LNCaP cell line that expresses high levels of both NTSR1 and NTSR3, and displays an enhanced susceptibility to develop neuroendocrine-like phenotypes upon treatment with NTS. More importantly, NTSR1 pathway inhibition prevented the development of NED and castration resistance in vivo. We propose a novel role of NTS in the development of CRPC with NED, and a possible strategy to prevent the onset of NED by targeting the NTS signaling pathway.

[Infiltrating mast cells promote neuroendocrine differentiation and increase docetaxel resistance of prostate cancer cells by up-regulating p21].

OBJECTIVE: To investigate the effect of infiltrating mast cells on neuroendocrine differentiation (NED) and docetaxel sensitivity of prostate cancer (PCa) cells in vitro. METHODS: Human PCa cell lines (LNCaP and C4-2) were co-cultured with human mast cell line (HMC-1) in Transwell chambers. Androgen receptor (AR) was silenced in C4-2 cells using sh-AR lentivirus, and p21 was knocked down and overexpressed by transfecting C4-2 cells with pLKO.1-sh-p21 and pCMV-p21, respectively. The morphological changes of LNCaP and C4-2 cells were observed. MTT assay and colony formation assay were used to assess the proliferation of LNCaP and C4-2 cells. CCK8 assay was used to detect the cell viability of C4-2 cells following docetaxel trreatment. RT-qPCR and Western blotting were performed to determine the mRNA and protein expressions of neuroendocrine markers, AR and p21 in the cells. RESULTS: Co-culture with HMC-1 cells enhanced the neuroendocrine phenotypes, inhibited the proliferation and up-regulated the expression of p21 in LNCaP and C4-2 cells. P21 positively regulated NED through a non-AR-dependent signaling pathway, while p21 knockdown partially reversed NED promoted by the mast cells. PCa cells co-cultured with HMC-1 cells showed increased resistance to docetaxel, and silencing p21 partially reversed docetaxel resistance in PCa cells. CONCLUSION: Infiltrating mast cells up-regulates p21 to promote NED and increase docetaxel resistance in PCa cells in vitro.

GnRH in the Human Female Reproductive Axis.

Gonadotropin-releasing hormone (GnRH) is recognized as the central regulator of the functions of the pituitary-gonadal axis. The increasing knowledge on the mechanisms controlling the development and the function of GnRH-producing neurons is leading to a better diagnostic and therapeutic approach for hypogonadotropic hypogonadisms and for alterations of the puberty onset. During female life span, the function of the GnRH pulse generator may be affected by a number of inputs from other neuronal systems, offering alternative strategies for diagnostic and therapeutic interventions. Moreover, the identification of a GnRH/GnRH receptor system in both human ovary and endometrium has widened the spectrum of action of the peptide outside its hypothalamic functions. The pharmacological use of GnRH itself or its synthetic analogs (agonists and antagonists) provides a valid tool to either stimulate or block gonadotropin secretion and to modulate the female fertility in several reproductive disorders and in assisted reproduction technology. The use of GnRH agonists in young female patients undergoing chemotherapy is also considered a promising therapeutic approach to counteract iatrogenic ovarian failure.

The Dual Function of the Polybasic Juxtamembrane Region of Syntaxin 1A in Clamping Spontaneous Release and Stimulating Ca2+-Triggered Release in Neuroendocrine Cells.

The exact function of the polybasic juxtamembrane region (5RK) of the plasma membrane neuronal SNARE, syntaxin 1A (Syx), in vesicle exocytosis, although widely studied, is currently not clear. Here, we addressed the role of 5RK in Ca2+-triggered release, using our Syx-based intramolecular fluorescence resonance energy transfer (FRET) probe, which previously allowed us to resolve a depolarization-induced Ca2+-dependent close-to-open transition (CDO) of Syx that occurs concomitant with evoked release, both in PC12 cells and hippocampal neurons and was abolished upon charge neutralization of 5RK. First, using dynamic FRET analysis in PC12 cells, we show that CDO occurs following assembly of SNARE complexes that include the vesicular SNARE, synaptobrevin 2, and that the participation of 5RK in CDO goes beyond its participation in the final zippering of the complex, because mutations of residues adjacent to 5RK, believed to be crucial for final zippering, do not abolish this transition. In addition, we show that CDO is contingent on membrane phosphatidylinositol 4,5-bisphosphate (PIP2), which is fundamental for maintaining regulated exocytosis, as depletion of membranal PIP2 abolishes CDO. Prompted by these results, which underscore a potentially significant role of 5RK in exocytosis, we next amperometrically analyzed catecholamine release from PC12 cells, revealing that charge neutralization of 5RK promotes spontaneous and inhibits Ca2+-triggered release events. Namely, 5RK acts as a fusion clamp, making release dependent on stimulation by Ca2+SIGNIFICANCE STATEMENT Syntaxin 1A (Syx) is a central protein component of the SNARE complex, which underlies neurotransmitter release. Although widely studied in relation to its participation in SNARE complex formation and its interaction with phosphoinositides, the function of Syx's polybasic juxtamembrane region (5RK) remains unclear. Previously, we showed that a conformational transition of Syx, related to calcium-triggered release, reported by a Syx-based FRET probe, is abolished upon charge neutralization of 5RK (5RK/A). Here we show that this conformational transition is dependent on phosphatidylinositol 4,5-bisphosphate (PIP2) and is related to SNARE complex formation. Subsequently, we show that the 5RK/A mutation enhances spontaneous release and inhibits calcium-triggered release in neuroendocrine cells, indicating a previously unrecognized role of 5RK in neurotransmitter release.

Treatment of Diabetes and Obesity by Rationally Designed Peptide Agonists Functioning at Multiple Metabolic Receptors.

Obesity and its comorbidities such as type 2 diabetes constitute major worldwide health threats, and the identification of an effective medical intervention has emerged as a global priority. The limited effectiveness of historical, anti-obesity treatments is commonly attributed to the complexity of the disease and the redundancy of metabolic regulatory mechanisms that sustain body weight. At the forefront of obesity research is the development of combinational drug therapies that simultaneously target multiple regulatory pathways, which promote dysfunctional metabolism. Recently, molecularly crafted unimolecular "multi-agonism" of balanced activity at 3 key receptors involved in metabolism and specifically the glucagon-like peptide (GLP)-1 receptor, glucose-dependent insulinotropic polypeptide (GIP) receptor and glucagon receptor was reported as superior to conventional monoagonist therapy. These mixed peptide agonists are designed to pharmacologically integrate the insulinotropic and anorexigenic effects of GLP-1, the thermogenic and lipolytic activities of glucagon, and the insulinotropic and insulin sensitizing properties of GIP. The molecular mechanism of these purposefully promiscuous ligands is not completely understood, however, recent studies in pancreatic beta cells point to the prospect of a complex signaling network that can magnify the signaling of multi-agonist ligands. The activation of this signalosome might explain the additional therapeutic benefit inherent to simultaneous cellular activation through multiple metabolic receptors.

FOXA1 inhibits prostate cancer neuroendocrine differentiation.

Neuroendocrine prostate cancer (NEPC) has increasingly become a clinical challenge. The mechanisms by which neuroendocrine (NE) cells arises from prostate adenocarcinoma cells are poorly understood. FOXA1 is a transcription factor of the forkhead family that is required for prostate epithelial differentiation. In this study, we demonstrated that FOXA1 loss drives NE differentiation, demarcated by phenotypical changes and NEPC marker expressions. Mechanistically, this is mediated by FOXA1 binding to the promoter of interleukin 8 (IL-8), a chemokine previously shown elevated in NEPC, to directly inhibit its expression. Further, IL-8 upregulation activates the MAPK/ERK pathway, leading to ERK phosphorylation and enolase 2 (ENO2) expression. IL-8 knockdown or ERK inhibition, on the other hand, abolished FOXA1 loss-induced NE differentiation. Analysis of xenograft mouse models confirmed FOXA1 loss in NEPC tumors relative to its adenocarcinoma counterparts. Importantly, FOXA1 is downregulated in human NEPC tumors compared to primary and castration-resistant prostate cancers, and its expression is negatively correlated with that of ENO2. These findings indicate that FOXA1 transcriptionally suppresses IL-8, the expression of which would otherwise stimulate the MAPK/ERK pathway to promote NE differentiation of prostate cancer cells. Our data strongly suggest that FOXA1 loss may play a significant role in enabling prostate cancer progression to NEPC, whereas IL-8 and MAPK/ERK pathways may be promising targets for therapeutic intervention.

SRRM4 Drives Neuroendocrine Transdifferentiation of Prostate Adenocarcinoma Under Androgen Receptor Pathway Inhibition.

BACKGROUND: Neuroendocrine prostate cancer (NEPC) is an aggressive subtype of castration-resistant prostate cancer that typically does not respond to androgen receptor pathway inhibition (ARPI), and its diagnosis is increasing. OBJECTIVE: To understand how NEPC develops and to identify driver genes to inform therapy for NEPC prevention. DESIGN, SETTING, AND PARTICIPANTS: Whole-transcriptome sequencing data were extracted from prostate tumors from two independent cohorts: The Beltran cohort contained 27 adenocarcinoma and five NEPC patient samples, and the Vancouver Prostate Centre cohort contained three patient samples and nine patient-derived xenografts. INTERVENTION: A novel bioinformatics tool, comparative alternative splicing detection (COMPAS), was invented to analyze alternative RNA splicing on RNA-sequencing data. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: COMPAS identified potential driver genes for NEPC development. Biochemical and biological validations were performed in both prostate cell and tumor models. RESULTS AND LIMITATION: More than 66% of the splice events were predicted to be regulated by the RNA splicing factor serine/arginine repetitive matrix 4 (SRRM4). In vitro and in vivo evidence confirmed that one SRRM4 target gene was the RE1 silencing transcription factor (REST), a master regulator of neurogenesis. Moreover, SRRM4 strongly stimulated adenocarcinoma cells to express NEPC biomarkers, and this effect was exacerbated by ARPI. ARPI combined with a gain of SRRM4-induced adenocarcinoma cells to assume multicellular spheroid morphology and was essential in establishing progressive NEPC xenografts. These SRRM4 actions were further enhanced by loss of function of TP53. CONCLUSIONS: SRRM4 drives NEPC progression. This knowledge may guide the development of novel therapeutics aimed at NEPC. PATIENT SUMMARY: Using next-generation RNA sequencing and our newly developed bioinformatics tool, we identified a neuroendocrine prostate cancer (NEPC)-specific RNA splicing signature that is predominantly controlled by serine/arginine repetitive matrix 4 (SRRM4). We confirmed that SRRM4 drives NEPC progression, and we propose SRRM4 as a potential therapeutic target for NEPC.

Nitric Oxide Modulates HCN Channels in Magnocellular Neurons of the Supraoptic Nucleus of Rats by an S-Nitrosylation-Dependent Mechanism.

The control of the excitability in magnocellular neurosecretory cells (MNCs) of the supraoptic nucleus has been attributed mainly to synaptic inputs from circunventricular organs. However, nitric oxide (NO), a gaseous messenger produced in this nucleus during isotonic and short-term hypertonic conditions, is an example of a modulator that can act directly on MNCs to modulate their firing rate. NO inhibits the electrical excitability of MNCs, leading to a decrease in the release of vasopressin and oxytocin. Although the effects of NO on MNCs are well established, the mechanism by which this gas produces its effect is, so far, unknown. Because NO acts independently of synaptic inputs, we hypothesized that ion channels present in MNCs are the targets of NO. To investigate this hypothesis, we used the patch-clamp technique in vitro and in situ to measure currents carried by hyperpolarization-activated and nucleotide-gated cation (HCN) channels and establish their role in determining the electrical excitability of MNCs in rats. Our results show that blockade of HCN channels by ZD7288 decreases MNC firing rate with significant consequences on the release of OT and VP, measured by radioimmunoassay. NO induced a significant reduction in HCN currents by binding to cysteine residues and forming S-nitrosothiol complexes. These findings shed new light on the mechanisms that control the electrical excitability of MNCs via the nitrergic system and strengthen the importance of HCN channels in the control of hydroelectrolyte homeostasis. SIGNIFICANCE STATEMENT: Cells in our organism live in a liquid environment whose composition and osmolality are maintained within tight limits. Magnocellular neurons (MNCs) of the supra optic nucleus can sense osmolality and control the synthesis and secretion of vasopressin (VP) and oxytocin (OT) by the neurohypophysis. OT and VP act on the kidneys controlling the excretion of water and sodium to maintain homeostasis. Here we combined electrophysiology, molecular biology, and radioimmunoassay to show that the electrical activity of MNCs can be controlled by nitric oxide (NO), a gaseous messenger. NO reacts with cysteine residues (S-nitrosylation) on hyperpolarization-activated and nucleotide-gated cation channels decreasing the firing rate of MNCs and the consequent secretion of VP and OT.

Resident CAPS on dense-core vesicles docks and primes vesicles for fusion.

The Ca(2+)-dependent exocytosis of dense-core vesicles in neuroendocrine cells requires a priming step during which SNARE protein complexes assemble. CAPS (aka CADPS) is one of several factors required for vesicle priming; however, the localization and dynamics of CAPS at sites of exocytosis in live neuroendocrine cells has not been determined. We imaged CAPS before, during, and after single-vesicle fusion events in PC12 cells by TIRF micro-scopy. In addition to being a resident on cytoplasmic dense-core vesicles, CAPS was present in clusters of approximately nine molecules near the plasma membrane that corresponded to docked/tethered vesicles. CAPS accompanied vesicles to the plasma membrane and was present at all vesicle exocytic events. The knockdown of CAPS by shRNA eliminated the VAMP-2-dependent docking and evoked exocytosis of fusion-competent vesicles. A CAPS(ΔC135) protein that does not localize to vesicles failed to rescue vesicle docking and evoked exocytosis in CAPS-depleted cells, showing that CAPS residence on vesicles is essential. Our results indicate that dense-core vesicles carry CAPS to sites of exocytosis, where CAPS promotes vesicle docking and fusion competence, probably by initiating SNARE complex assembly.

Hypothalamus as an endocrine organ.

The endocrine hypothalamus constitutes those cells which project to the median eminence and secrete neurohormones into the hypophysial portal blood to act on cells of the anterior pituitary gland. The entire endocrine system is controlled by these peptides. In turn, the hypothalamic neuroendocrine cells are regulated by feedback signals from the endocrine glands and other circulating factors. The neuroendocrine cells are found in specific regions of the hypothalamus and are regulated by afferents from higher brain centers. Integrated function is clearly complex and the networks between and amongst the neuroendocrine cells allows fine control to achieve homeostasis. The entry of hormones and other factors into the brain, either via the cerebrospinal fluid or through fenestrated capillaries (in the basal hypothalamus) is important because it influences the extent to which feedback regulation may be imposed. Recent evidence of the passage of factors from the pars tuberalis and the median eminence casts a new layer in our understanding of neuroendocrine regulation. The function of neuroendocrine cells and the means by which pulsatile secretion is achieved is best understood for the close relationship between gonadotropin releasing hormone and luteinizing hormone, which is reviewed in detail. The secretion of other neurohormones is less rigid, so the relationship between hypothalamic secretion and the relevant pituitary hormones is more complex.

Anticancer effects of metformin on neuroendocrine tumor cells in vitro.

Metformin is a widely used oral antidiabetic drug with good tolerability. Recent studies suggest that it also possesses adjuvant potent anticancer properties in a variety of tumors. Neuroendocrine tumors (NETs) of the gastro-entero-pancreatic system (GEP) comprise a heterogeneous group of tumors with increasing incidence and limited effective therapeutic options. Here we report the antiproliferative effects of metformin in neuroendocrine tumor cells in vitro. Treatment of human pancreatic BON1, bronchopulmonary NCI-H727, and midgut GOT1 neuroendocrine tumor cells with increasing concentrations of metformin (0.1-10 mM) dose-dependently suppressed cell viability and cell counts. Metformin induced AMPK phosphorylation in pancreatic BON1 and midgut GOT1 but suppressed AMPK activity in bronchopulmonary NCI-H727. Thus, AMPK-dependent and AMPK-independent properties may be operative in NETs of different origin. Metformin suppressed mTORC1 signaling in all three tumor cell types, evidenced by suppression of 4EBP1, pP70S6K, and S6 phosphorylation, and was associated with compensatory AKT activity. We observed induction of ERK phosphorylation in BON1 and NCI-H727 and inhibition of ERK in midgut GOT1 cells, while all three tumor cell types responded with induction of GSK3 phosphorylation. This suggests a central role for GSK3 in metformin-mediated signal transduction. Inhibition of cell proliferation by metformin was associated with apoptosis induction only in midgut GOT1, evidenced by increased subG0/1 fraction and PARP cleavage. These results suggest a potential role of metformin as a (adjuvant) therapeutic for patients with NETs.

A potentially novel nicotinic receptor in Aplysia neuroendocrine cells.

Nicotinic receptors form a diverse group of ligand-gated ionotropic receptors with roles in both synaptic transmission and the control of excitability. In the bag cell neurons of Aplysia, acetylcholine activates an ionotropic receptor, which passes inward current to produce a long-lasting afterdischarge and hormone release, leading to reproduction. While testing the agonist profile of the cholinergic response, we observed a second current that appeared to be gated only by nicotine and not acetylcholine. The peak nicotine-evoked current was markedly smaller in magnitude than the acetylcholine-induced current, cooperative (Hill value of 2.7), had an EC50 near 500 µM, readily recovered from desensitization, showed Ca(2+) permeability, and was blocked by mecamylamine, dihydro-ß-erythroidine, or strychnine, but not by α-conotoxin ImI, methyllycaconitine, or hexamethonium. Aplysia transcriptome analysis followed by PCR yielded 20 full-length potential nicotinic receptor subunits. Sixteen of these were predicted to be cation selective, and real-time PCR suggested that 15 of the 16 subunits were expressed to varying degrees in the bag cell neurons. The acetylcholine-induced current, but not the nicotine current, was reduced by double-strand RNA treatment targeted to both subunits ApAChR-C and -E. Conversely, the nicotine-evoked current, but not the acetylcholine current, was lessened by targeting both subunits ApAChR-H and -P. To the best of our knowledge, this is the first report suggesting that a nicotinic receptor is not gated by acetylcholine. Separate receptors may serve as a means to differentially trigger plasticity or safeguard propagation by assuring that only acetylcholine, the endogenous agonist, initiates large enough responses to trigger reproduction.

Prognostic significance of neuroendocrine differentiation in colorectal adenocarcinoma after radical operation: a meta-analysis.

BACKGROUND: The phenomenon of neuroendocrine differentiation has been observed in colorectal adenocarcinoma. However, the ability of neuroendocrine differentiation to predict the outcome of colorectal adenocarcinoma remains controversial. METHODS: We conducted an extensive search of research studies related to neuroendocrine differentiation using scientific databases, including the PubMed, Embase, OVID, BIOSIS Previews, and Cochrane Central Register of Controlled Trials (up to July, 2013), according to the established search terms. RevMan version 5.2 statistical program was used to analyze the data. An odds ratio (OR) with a 95% confidence interval (CI) was used for the dichotomous data. RESULTS: Eleven studies with a total of 1,587 patients were included. Patients with neuroendocrine differentiation who underwent a radical operation had a lower 5-year survival rate (pooled OR 0.60, 95% CI 0.37-0.97) compared with those without neuroendocrine differentiation, with evidence of moderate heterogeneity (I (2) = 37%, p = 0.10). A sensitivity analysis and meta-regression showed that the different classification criteria of neuroendocrine differentiation used in these studies were the main source of heterogeneity. When the strong positive rates of neuroendocrine differentiation indicators between the higher (stage III + IV) and the lower (stage I + II) clinical stages were compared, the pooled OR was 1.84 (703 patients; 95% CI 0.98-3.43) without evidence of heterogeneity (I (2) = 0 %, p = 0.89). However, comparisons between consecutive stages showed different ORs: stage II vs. I (203 patients; OR = 0.52, 95% CI 0.17-1.56), stage III vs. II (569 patients; OR = 2.27, 95% CI 1.03-4.98), and stage IV vs. III (375 patients; OR = 1.81, 95% CI 1.00-3.29). CONCLUSION: The patients with strong positive indicators of neuroendocrine differentiation had a lower 5-year survival rate. The ability to detect neuroendocrine indicators using conventional methods could improve the prognosis judgment of colorectal adenocarcinoma.

Voltage-gated Ca2+ influx and mitochondrial Ca2+ initiate secretion from Aplysia neuroendocrine cells.

Neuroendocrine secretion often requires prolonged voltage-gated Ca(2+) entry; however, the ability of Ca(2+) from intracellular stores, such as endoplasmic reticulum or mitochondria, to elicit secretion is less clear. We examined this using the bag cell neurons, which trigger ovulation in Aplysia by releasing egg-laying hormone (ELH) peptide. Secretion from cultured bag cell neurons was observed as an increase in plasma membrane capacitance following Ca(2+) influx evoked by a 5-Hz, 1-min train of depolarizing steps under voltage-clamp. The response was similar for step durations of ≥ 50 ms, but fell off sharply with shorter stimuli. The capacitance change was attenuated by replacing external Ca(2+) with Ba(2+), blocking Ca(2+) channels, buffering intracellular Ca(2+) with EGTA, disrupting synaptic protein recycling, or genetic knock-down of ELH. Regarding intracellular stores, liberating mitochondrial Ca(2+) with the protonophore, carbonyl cyanide-p-trifluoromethoxyphenyl-hydrazone (FCCP), brought about an EGTA-sensitive elevation of capacitance. Conversely, no change was observed to Ca(2+) released from the endoplasmic reticulum or acidic stores. Prior exposure to FCCP lessened the train-induced capacitance increase, suggesting overlap in the pool of releasable vesicles. Employing GTP-γ-S to interfere with endocytosis delayed recovery (presumed membrane retrieval) of the capacitance change following FCCP, but not the train. Finally, secretion was correlated with reproductive behavior, in that neurons isolated from animals engaged in egg-laying presented a greater train-induced capacitance elevation vs quiescent animals. The bag cell neuron capacitance increase is consistent with peptide secretion requiring high Ca(2+), either from influx or stores, and may reflect the all-or-none nature of reproduction.

Stem cells and cancer stem-like cells in endocrine tissues.

Cancer stem-like cells are a subpopulation of self-renewing cells that are more resistant to chemotherapy and radiation therapy than the other surrounding cancer cells. The cancer stem cell model predicts that only a subset of cancer cells possess the ability to self-renew and produce progenitor cells that can reconstitute and sustain tumor growth. Evidence supporting the existence of cancer stem-like cells in the thyroid, pituitary, and in other endocrine tissues is rapidly accumulating. These cells have been studied using specific biomarkers including: CD133, CD44, Nestin, Nanog, and aldehyde dehydrogenase enzyme. Putative cancer stem-like cells can be studied in vitro using serum-free media supplemented with basic fibroblast growth factor and epidermal growth factor grown in low attachment plates or in extracellular matrix leading to sphere formation in vitro. Cancer stem-like cells can also be separated by fluorescent cell sorting and used for in vitro or in vivo studies. Injection of enriched populations of cancer stem-like cells (also referred to as tumor initiating cells) into immunodeficient mice results in growth of xenografts which express cancer stem-like biomarkers. Human cancer stem-like cells have been identified in thyroid cancer cell lines, in primary thyroid cancers, in normal pituitary, and in pituitary tumors. Other recent studies suggest the existence of stem cells and cancer stem-like cells in endocrine tumors of the gastrointestinal tract, pancreas, lungs, adrenal, parathyroid, and skin. New discoveries in this field may lead to more effective therapies for highly aggressive and lethal endocrine cancers.

The DNA methylation landscape of small cell lung cancer suggests a differentiation defect of neuroendocrine cells.

Small cell lung cancer (SCLC) is a disease characterized by aggressive clinical behavior and lack of effective therapy. Owing to its tendency for early dissemination, only a third of patients have limited-stage disease at the time of diagnosis. SCLC is thought to derive from pulmonary neuroendocrine cells. Although several molecular abnormalities in SCLC have been described, there are relatively few studies on epigenetic alterations in this type of tumor. Here, we have used methylation profiling with the methylated-CpG island recovery assay in combination with microarrays and conducted the first genome-scale analysis of methylation changes that occur in primary SCLC and SCLC cell lines. Among the hundreds of tumor-specifically methylated genes discovered, we identified 73 gene targets that are methylated in >77% of primary SCLC tumors, most of which have never been linked to aberrant methylation in tumors. These methylated targets have potential for biomarker development for early detection and therapeutic management of SCLC. SCLC cell lines had a greater number of hypermethylated genes than primary tumors. Gene ontology analysis indicated a significant enrichment of methylated genes functioning as transcription factors and in processes of neuronal differentiation. Motif analysis of tumor-specific methylated regions identified enrichment of binding sites for several neural cell fate-specifying transcription factors including NEUROD1, HAND1, ZNF423 and REST. We hypothesize that two potential mechanisms, loss of cell fate-determining transcription factors by methylation of their promoters and functional inactivation of their corresponding genomic-binding sites by DNA methylation, can promote a differentiation defect of neuroendocrine cells thus enhancing the ability of tumor progenitor cells to transition toward SCLC.