Roswitha Pfragner

Luminal regulation of normal and neoplastic human EC cell serotonin release is mediated by bile salts, amines, tastants, and olfactants

Mechanisms by which gut luminal content regulates secretion and motility are ill understood. We evaluated whether neuroendocrine enterochromaffin (EC) cells act as luminal sensors for a wide variety of nutrients and defined the secretory mechanisms of this process. Pure (98-99%) FACS-sorted human EC cells and neoplastic EC cells (KRJ-I) were studied. RT-PCR identified transcripts for T2R1 (bitter), OR1G1 (class II olfactory) and trace amine (TAR1) G protein-coupled receptors (GPCRs) and transporters for glutamine (SNAT1/2), glucose (GLUT1/3/SGLT1), and bile salts (ABST). Glutamine and sodium deoxycholate stimulated 5-HT release (EC(50) = 0.002-0.2 microM; 2-fold release) but were 10-100 times more potent in neoplastic EC cells, which also secreted 6-13 times more 5-HT. Tastants (caffeine, tyramine, octopamine) and olfactants (thymol and eugenol) also stimulated normal and neoplastic EC cell 5-HT secretion (EC(50) = 1.2 nM to 2.1 microM and 0.05 nM to 0.1 microM release, respectively); 2-deoxyglucose and the artificial sweetener sucralose also stimulated (EC(50) = 9.2 and 0.38 nM). 5-HT release was associated with ERK phosphorylation (1.5-fold, P < 0.02) and could be inhibited by a somatostatin analog (IC(50) = 1 pM). Eleven secretory associated genes including the vesicle docking inhibitor STXBP3 were upregulated in response to glutamine and bile salt stimulation in neoplastic EC cells. Targeting STXBP3 expression by use of antisense knockdown significantly (P < 0.05) reduced 5-HT secretion. In conclusion, EC cells express GPCRs and transporters for luminal tastants, olfactants, glutamine, glucose, and bile salts. Activation includes a panel of secretory genes, ERK phosphorylation, and 5-HT secretion. Luminal EC cell regulation is likely to be as important as G cell regulation in gastric acid secretion; development of agents to target EC cell function is therefore a critical therapeutic goal.

Lysosomal pathology associated with α‐synuclein accumulation in transgenic models using an eGFP fusion protein

Disorders with Lewy body (LB) formation, such as Parkinsons disease (PD) and dementia with Lewy bodies (DLB), are characterized by α‐synuclein accumulation in the neuronal cell body. Recent studies have suggested that in addition to LBs, α‐synuclein might accumulate more widely throughout the neurons and their processes, leading to neurodegeneration and functional impairment. The precise patterns of α‐synuclein accumulation in vivo, however, and its relationship with subcellular neuronal alterations such as lysosomal pathology are not completely clear. To this end, we developed transgenic (tg) in vivo and in vitro models expressing a stable enhanced green fluorescent protein (eGFP) tagged in the C‐terminal site of a human (h)α‐synuclein construct under the regulatory control of the platelet‐derived growth factor‐β (PDGFβ) promoter and carried out confocal, ultrastructural, and biochemical studies. In tg mice, confocal studies demonstrated a wide distribution of hα‐synuclein–eGFP in the neuronal cell bodies, axons, and presynaptic terminals. In several neuronal cell bodies and their neurites, hα‐synuclein–eGFP was found not only as inclusions but also as discrete granular structures that in double‐labeling studies colocalized with antibodies against hα‐synuclein and the lysosomal marker cathepsin D. Consistent with these findings, ultrastructural analysis showed that hα‐synuclein–eGFP overexpression resulted in the accumulation of electrodense inclusions and laminated bodies suggestive of lysosomal pathology, and that the hα‐synuclein–eGFP protein was more abundant in the lysosomal fractions of the tg animals. Taken together, these findings support the notion that enhanced visualization of α‐synuclein utilizing a hybrid eGFP molecule reveals a more widespread accumulation of this molecule in several neuronal compartments, promoting lysosomal dysfunction. Furthermore, the PDGFβ–hα‐synuclein–eGFP tg model might be a valuable tool in testing new treatments for LBD in a fast and reliable manner.

Limitations in small intestinal neuroendocrine tumor therapy by mTor kinase inhibition reflect growth factor-mediated PI3K feedback loop activation via ERK1/2 and AKT.

Treatment of small intestinal neuroendocrine tumors (SINETs) with mammalian target of rapamycin (mTOR) inhibitors alone or with somatostatin analogs has been proposed as effective therapy, because both agents have been reported to exhibit antiproliferative activity. Because adenocarcinomas escape mTOR inhibition, we examined whether the escape phenomenon occurred in SINETs and whether usage of somatostatin analogs with mTOR inhibitors surmounted loss of inhibition.

Dendritic cell vaccination in medullary thyroid carcinoma.

Purpose: Prognosis and treatment effectiveness for medullary thyroid carcinoma (MTC) are strictly related to tumor stage. Palliative treatment options show no significant benefit. A promising treatment approach for human cancer is based on the vaccination of autologous dendritic cells (DCs). Experimental Design: The objective of this study was to evaluate the effectiveness of DC vaccines in MTC patients. Therefore, we generated autologous tumor lysate-pulsed DCs from 10 patients suffering from advanced MTC for repeated vaccination. Mature DCs were derived from peripheral blood monocytes by using CD14 magnetic bead selection and subsequent culture in the presence of granulocyte macrophage colony-stimulating factor, interleukin 4, and tumor necrosis factor α with or without addition of IFN-γ. DCs were loaded with tumor lysate and further injected into a groin lymph node. Toxicity, tumor marker profile, immune response, and clinical response were determined. Results: Vaccination was well tolerated and induced a positive immunological response in all of the tested patients as evaluated by in vivo delayed-type hypersensitivity reactivity or in vitro intracytoplasmic IFN-γ detection assay. Three patients had a partial response, 1 patient presented a minor response, and 2 patients showed stable disease. The remaining 4 patients had progressive disease. Conclusions: These data provide strong evidence that vaccination with tumor-lysate pulsed DCs results in the induction of a specific immune response in patients suffering from MTC. Objective clinical responses could be observed even for far-advanced disease. Therefore, we suggest that MTC is particularly suited for DC-based immunotherapy.

The Role of Cdk5 in Neuroendocrine Thyroid Cancer

Medullary thyroid carcinoma (MTC) is a neuroendocrine cancer that originates from calcitonin-secreting parafollicular cells, or C cells. We found that Cdk5 and its cofactors p35 and p25 are highly expressed in human MTC and that Cdk5 activity promotes MTC proliferation. A conditional MTC mouse model was generated and corroborated the role of aberrant Cdk5 activation in MTC. C cell-specific overexpression of p25 caused rapid C cell hyperplasia leading to lethal MTC, which was arrested by repressing p25 overexpression. A comparative phosphoproteomic screen between proliferating and arrested MTC identified the retinoblastoma protein (Rb) as a crucial Cdk5 downstream target. Prevention of Rb phosphorylation at Ser807/Ser811 attenuated MTC proliferation. These findings implicate Cdk5 signaling via Rb as critical to MTC tumorigenesis and progression.

Differential cytotoxicity of novel somatostatin and dopamine chimeric compounds on bronchopulmonary and small intestinal neuroendocrine tumor cell lines

Survival rates for gastrointestinal (GI) and bronchopulmonary (BP) neuroendocrine tumors (NETs) have not significantly altered (overall 67%, 5‐year survival) in 30 years (1973‐2004), whereas the incidence has increased (∼ 1000%) in the same time frame. No effective or specific antineoplastic agent is available for treatment, although somatostatin analogs inhibit tumor secretion. Given the coexistence of somatostatin and dopamine regulatory receptors on NET cells, the antiproliferative efficacy as well as the signaling and transcriptional targets of their ligands were evaluated.

Small bowel carcinoid (enterochromaffin cell) neoplasia exhibits transforming growth factor-β1-mediated regulatory abnormalities including up-regulation of C-myc and MTA1

Although it is known that small intestinal carcinoids are derived from enterochromaffin (EC) cells, these cells remain poorly characterized and little is known of the growth regulatory mechanisms of these neuroendocrine cells. Down‐regulation or loss of the transforming growth factor‐β1 (TGFβ1) cytostatic program and activation of TGFβ‐mediated transcriptional networks is associated with uncontrolled growth and metastasis in other neural tumors, glioblastomas. Whether this phenomenon is common to small intestinal carcinoid tumors was investigated.

The 5-HT2B Receptor Plays a Key Regulatory Role in Both Neuroendocrine Tumor Cell Proliferation and the Modulation of the Fibroblast Component of the Neoplastic Microenvironment

Fibrosis is a cardinal feature of small intestinal neuroendocrine tumors (SI‐NETs) both in local peritumoral tissue and systemic sites (cardiac). 5‐HT, a commonly secreted NET amine, is a known inducer of fibrosis, although the mechanistic basis for it and growth factors regulating fibrosis and proliferation in the tumor microenvironment are unclear. We hypothesized that targeting 5‐HT2B receptors on tumor cells would inhibit SI‐NET 5‐HT release and, thereby, fibroblast activation in the tumor microenvironment.

KRJ-I and BON Cell Lines: Defining an Appropriate Enterochromaffin Cell Neuroendocrine Tumor Model

Background: Neuroendocrine tumors (NETs) of the gastrointestinal (GI) system are increasing in incidence with minimal improvement in prognosis. Although the cell of origin has been identified as the enterochromaffin (EC) cell, its secretory and proliferative regulation has not been defined at a mechanistic level. To date, the BON cell line has been the most widely used in vitro EC cell model despite its pancreatic origin. Using whole-genome mathematical analysis as well as secretory and proliferative studies, we compared the BON cell line to the small intestine (SI) EC cell-derived NET cell line, KRJ-I, to assess individual cell line validity and applicability for the investigation of GI-NET disease. Methods and Results: Principal component analysis and ANOVA of KRJ-I and BON transcriptomes (U133 Plus 2) identified substantially different (<10%) overlap in transcripts with minimal (R2 = 0.24) correlation in gene expression profiles. RT-PCR detected large variability (>12%) in neuroendocrine (NE) marker transcripts in the BON cell line and the absence of Tph-2, DDC, TGFβR2, and M3 transcripts in KRJ-I. The KRJ-I cell line secreted serotonin (5-HT) in response to isoproterenol (EC50 = 100 nM), noradrenaline (EC50 = 1.7 nM), and pituitary adenylate cyclase (PACAP, EC50 = 0.03 nM). Cholecystokinin (IC50 = 430 nM), somatostatin (IC50 = 400 nM), acetylcholine (IC50 = 3.7 nM), and γ-aminobutyric acid A (GABAA, IC50 = 2 nM) all inhibited 5-HT release, while gastrin and bombesin had no effect. 5-HT secretion in the BON cell line was stimulated by isoproterenol (EC50 = 900 nM), noradrenaline (EC50 = 20 nM), cholecystokinin (EC50 = 130 nM), PACAP (EC50 = 0.12 nM), bombesin (EC50 = 15 nM), and acetylcholine (EC50 = 0.2 nM). It was inhibited by somatostatin (IC50 = 300 nM) but not GABAA. KRJ-I responded with proliferation to connective tissue growth factor (CTGF, EC50 = 0.002 ng/ml), transforming growth factor-α (TGFα, EC50 = 0.63 ng/ml) and transforming growth factor-β (TGFβ, EC50 = 0.63 ng/ml). Epidermal growth factor (EGF) and somatostatin had no significant effect. BON cell proliferation was stimulated only by EGF and TGFα (EC50 = 15.8 and 10 ng/ml). TGFβ (IC50 = 0.16 ng/ml), MZ-4-147 (IC50 = 0.5 nM), and BIM23A761 (IC50 = 0.06 nM) all inhibited proliferation. CTGF and somatostatin had no effect. Conclusion: KRJ-I and BON cell lines demonstrate substantial differences in gene level transcripts, inconsistent receptor profile expression, wide variability in NE marker transcript levels, and significantly differential proliferative and secretory responses. Given the EC cell origin of KRJ-I, these results provide evidence that the BON cell line does not represent an EC cell system and is not a valid study model of (carcinoid) EC cell-derived NET.

The role of mechanical forces and adenosine in the regulation of intestinal enterochromaffin cell serotonin secretion

Enterochromaffin (EC) cells of the diffuse neuroendocrine cell system secrete serotonin (5-HT) with activation of gut motility, secretion, and pain. These cells express adenosine (ADORA) receptors and are considered to function as mechanosensors. Physiological pathways mediating mechanosensitivity and adenosine responsiveness remain to be fully elucidated, as do their roles in inflammatory bowel disease (IBD) and neoplasia. Pure (98-99%) FACS-sorted normal and IBD human EC cells and neoplastic EC cells (KRJ-I) were studied. IBD-EC cells and KRJ-I overexpressed ADORA2B. NECA, a general ADORA receptor agonist, stimulated, whereas the A2B receptor antagonist MRS1754 inhibited, 5-HT release (EC50 = 1.8 × 10-6 M; IC50 = 3.7 × 10-8 M), which was associated with corresponding alterations in intracellular cAMP levels and pCREB (Ser133). Mechanical stimulation using a rhythmic flex model induced transcription and activation of Tph1 (tryptophan hydroxylase) and VMAT₁ (vesicular monoamine transporter 1) and the release of 5-HT, which could be inhibited by MRS1754 and amplified by NECA. Secretion was also inhibited by H-89 (PKA inhibitor) while Tph1 and VMAT₁ transcription was regulated by PKA/MAPK and PI₃K-mediated signaling. Normal and IBD-EC cells also responded to NECA and mechanical stimulation with PKA activation, cAMP production, and 5-HT release, effects reversible by MRS1754. EC cells express stimulatory ADORA2B, and rhythmic stretch induces A2B activation, PKA/MAPK/IP3-dependent transcription, and PKA-dependent secretion of 5-HT synthesis and secretion. Receptor expression is amplified in IBD and neoplasia, and 5-HT release is increased. Determination of factors that regulate EC cell function are necessary for understanding its role as a mechanosensory cell and to facilitate the development of agents that can selectively target cell function in EC cell-associated disease.