Cell Carcinoid Tumors Research Articles

Regulation of Neuroendocrine Differentiation in Gastrointestinal Carcinoid Tumor Cells by Notch Signaling

Gastrointestinal (GI) carcinoid tumors elaborate serotonin and other vasoactive substances, causing the carcinoid syndrome. Based on developmental biology data, we hypothesized that basic helix-loop-helix transcription factors, including achaete-scute complex homolog-like 1 (Ascl1)/hASH1, and the Notch signaling pathway might regulate the neuroendocrine phenotype in GI carcinoids. The aim of this study was to evaluate expression of developmental transcription factors and Notch signaling components in GI carcinoids and model their interaction in a relevant GI carcinoid cell line. Fourteen GI carcinoid tumor specimens, five paired adjacent normal tissues, fetal tissues, and tumor cell lines were analyzed by RT-PCR and immunoblot. BON carcinoid cells were further analyzed after Notch overexpression for neuroendocrine marker expression, serotonin production, and growth. The study was conducted in an academic referral center. Deidentified archival pathology specimens were examined. Among a panel of six developmental transcription factors tested, only Ascl1 mRNA was overexpressed compared with surrounding normal tissue (seven of 10 GI carcinoid tumors and in BON cells, none of five normal tissues). Ascl1 protein was also expressed in four of four carcinoid tumors and BON cells). Notch pathway ligands, receptors, and downstream effectors were widely expressed in tumor and normal specimens. Overexpression of activated Notch1 in BON cells led to induction of the Notch effector hairy and enhancer of split 1 (Hes1), loss of Ascl1, reductions in neuron-specific enolase, synaptophysin, and chromogranin A, and most significantly, an 89% decrease in serotonin concentration and equivalent reductions in serotonin-reactive cells and repression of tryptophan hydroxylase 1 mRNA. The Notch signaling pathway is a significant regulator of neuroendocrine differentiation and serotonin production in GI carcinoid tumors.

Different expression levels of lumican in human carcinoid tumor and neuroendocrine cell carcinoma

Lumican is a member of the small leucine-rich proteoglycan (SLRP) family and participates in the maintenance of tissue structures and tumor growth. Neuroendocrine cell tumors (NETs) including carcinoid tumors and NE cell carcinomas (NECs) possess numerous neuroendocrine (NE) granules, and differences between these tumors are in terms of their biological and metastatic aggressiveness during tumor progression. The purpose of this study was to examine the expression of lumican in NETs, and to determine whether the presence of lumican may be associated with the growth of NETs by comparing its expression between carcinoid tumors and NECs. Immunohistochemically, the positivity rates of lumican expression in the cytoplasm of the tumor cells were 87.5% in carcinoid tumors and 37.5% in NECs. Those of lumican expression in the stroma adjacent to the tumors were 90.1% in carcinoid tumors and 79.2% in NECs. In situ hybridization analysis revealed the lumican mRNA expression in the cytoplasm of carcinoid tumor and NEC cells. Ultrastructurally, the lumican protein was observed in the rough endoplasmic reticulum and NE granules of NETs and interspaces of collagen fibers. Furthermore, RT-PCR analysis revealed the presence of lumican mRNA in NEC cell lines. These results indicate that the higher expression level of cytoplasmic lumican in carcinoid tumors than in NECs may play a role in the slow growth of these tumors.

A Mouse Model of Carcinoid Syndrome and Heart Disease

Carcinoid heart disease occurs in over 65% of patients with the carcinoid syndrome and is characterized by fibrous thickening of cardiac valves, leading to heart failure. Whether serotonin is directly responsible for these cardiac abnormalities is unknown. Therefore, to further understand the etiology and pathophysiology of carcinoid heart disease, we developed an animal model of the carcinoid syndrome. Seventeen nude mice underwent intrasplenic injection of human pancreatic carcinoid BON cells (10(7)) and then were euthanized 9 weeks later. Murine livers were analyzed by immunohistochemistry. Murine hearts were sectioned and the surface area of the right heart valves determined. Blood was also collected and analyzed for platelet serotonin by ELISA. Sixty-five percent of the mice developed gross carcinoid liver metastases demonstrated by chromogranin-A-staining lesions within the liver. Mice with carcinoid liver metastases had elevated platelet serotonin levels (1058 +/- 529 ng/ml versus 123 +/- 52 ng/ml, P = 0.002) when compared to the controls. Animals with carcinoid liver metastases also had a trend toward greater tricuspid valvular surface areas (242 +/- 24 versus 174 +/- 25 microm, P = 0.08). On histological examination, this increase in tricuspid surface area in mice with liver metastases appeared to be due to fibrosis of the valvular tissues, consistent with the pathologic findings of carcinoid heart disease. Using this novel animal model of carcinoid syndrome, the tricuspid valve thickening resembling carcinoid heart disease could be due to exposure to factors such as serotonin secreted by carcinoid tumor cells.

Fine-Needle Aspiration of Pulmonary Hamartoma: A Common Source of False-Positive Diagnoses in the College of American Pathologists Interlaboratory Comparison Program in Nongynecologic Cytology

We use data from the College of American Pathologists Interlaboratory Comparison Program in Nongynecologic Cytology to evaluate the accuracy of fine-needle aspiration (FNA) biopsy for diagnosing pulmonary hamartoma (PH). To use the performance characteristics of the PH cases in the Nongynecologic Cytology Program to determine the accuracy of FNA for identifying these lesions and to determine potential sources of interpretative errors. A retrospective review of the College of American Pathologists Nongynecologic Cytology cumulative data from 1997 to 2003 was performed to identify the overall accuracy of FNA for diagnosing PH and to determine the most common interpretative pitfalls. The slides from each of the cases of PH in the Nongynecologic Cytology Program were then reviewed in an effort to identify the cytologic characteristics that contributed to the poor performance of these cases. A total of 766 participant responses for 19 PH FNA specimens were reviewed. The specificity of FNA for making the correct general reference interpretation of benign was 78%. The false-positive rate was 22%, with the most common false-positive diagnoses being carcinoid tumor, adenocarcinoma, and small cell carcinoma. The overall accuracy for making the correct specific reference diagnosis of PH was 26%. Microscopic review of the individual cases revealed possible explanations for some of the interpretative errors and the most frequent false-positive interpretations. Cytologists should be aware of the potential false-positive interpretations that can occur in FNAs of PH and the potential reasons for these inaccuracies in order to minimize clinically significant diagnostic errors.

Insulin-like growth factor 1 signaling in human gastrointestinal carcinoid tumor cells

Background Insulin-like growth factor 1 (IGF-1) is an autocrine regulator of carcinoid tumors. Blockade of IGF-1 signaling has been proposed as a therapeutic target in the treatment of patients with carcinoid syndrome. We hypothesized that the induction of parallel raf-1/MEK1 pathways will block IGF-1-mediated chromogranin A (CgA) maintenance. Methods Human gastrointestinal carcinoid tumor cells (BON) were treated with IGF-1 (0-500 ng/mL). Raf-1/MEK1 activation was achieved with an estrogen-inducible raf-1 vector that was transduced into BON cells. Activation of IGF-1/raf-1 pathways was determined by phosphorylation of downstream targets p70s6 and ERK1/2. The secreted and intercellular levels of CgA were measured in conditioned media and whole cell extracts by Western and enzyme-linked immunosorbent assay analysis. Results IGF-1 and raf-1 pathways were activated successfully in BON cells, as shown by high levels of phosphorylated p70s6 and phosphorylated ERK1/2, respectively. Treatment of BON cells with IGF-1 stimulated the release of CgA, while high intracellular CgA levels were maintained. The activation of raf-1/MEK1 reversed the effect of IGF-1 treatment by the depletion of intracellular CgA. Conclusions The induction of the raf-1/MEK1 pathway blocks IGF-1-mediated intracellular neuroendocrine hormone regulation. Therefore, raf-1/MEK1 activation may be a viable method to block IGF-1-mediated cellular effects and serve as a therapeutic target in gastrointestinal carcinoid tumors.

ZM336372, a novel raf-1 activating compound, suppresses cellular growth, induces cell-cycle inhibitors, and reduces hormone production in carcinoid tumor cells

Introduction. Neuroendocrine (NE) tumors such as carcinoids are highly metastatic neoplasms that produce numerous bioactive hormones resulting in significant symptomatology. We have previously demonstrated that activation of the raf-1/MEK/ERK signaling pathway can suppress cellular growth and hormone production in NE tumor cells in vitro. Thus, we were interested in developing pharmacologic methods to activate the raf-1 signaling pathway. In this study, we investigated a novel therapy for NE tumors based on pharmacologic raf-1 activation using the compound ZM336372. Methods. Pulmonary carcinoid H727 tumor cells were treated with ZM336372 (20–100 μM) or carrier (DMSO). Raf-1/MEK/ERK pathway activation, cellular bioactive hormone production, and levels of cell-cycle inhibitors were determined by Western Blot for phosphorylated MEK1/2, chromogranin A (CgA), and p21, and INK4c, respectively. Cellular growth was assessed by MTT assay. Results. H727 cells did not have any detectable phosphorylated MEK1/2 at baseline. Escalating doses of ZM336372 in H727 cells resulted in increasing raf-1 activation as measured by phosphorylation of MEK1/2. Importantly, treatment of H727 cells with ZM336372 led to a marked suppression of cellular proliferation and induction of the cell-cycle inhibitors p21 and INK4c. Furthermore, treatment with ZM336372 resulted in a progressive reduction of bioactive hormone production (CgA) by the tumor cells. Conclusions. ZM336372 is an effective pharmacologic activator of raf-1/MEK/ERK signaling in carcinoid tumor cells. Raf-1 pathway induction in NE tumor cells by ZM336372 suppressed cellular proliferation and reduced bioactive hormone production. Therefore, this novel signaling-based therapy may be a viable therapeutic and palliative treatment for patients with NE tumors.

Ossifying gastric carcinoid tumor containing bone morphogenetic protein, osteopontin and osteonectin

We report a case of gastric carcinoid tumor with ossification. A 47-yr-old man complaining of abdominal discomfort underwent gastrointestinal endoscopic examination, which revealed a submucosal tumor in his stomach. The tumor was extirpated by endoscopic enucleation. Histologically, the tumor was widely occupied by mature bone tissues, where scattered carcinoid tumor cell nests surrounded bone tissues or located in stromal areas. Immunohistochemically, the tumor cells were strongly positive for cytokeratin, chromogranin A, synaptophysin, neurofilaments and neuron-specific enolase, underscoring the diagnosis of carcinoid tumor. They also stained positive with markers of bone formation and differentiation, such as bone morphogenetic protein, osteopontin and osteonectin. There are only four cases in the world literature, including a current case of ossifying gastric carcinoid tumor, in which the excessive production of peptides promoting ossification was considered to be implicated in the unusual appearance of the bone.

A mouse model of carcinoid syndrome and heart disease

Abstract Introduction: Carcinoid heart disease occurs in over 65% of patients with the carcinoid syndrome and is characterized by fibrous thickening of cardiac valves, leading to heart failure. Whether serotonin is directly responsible for these cardiac abnormalities is unknown. Therefore, in order to further understand the etiology and pathophysiology of carcinoid heart disease, we developed an animal model of the carcinoid syndrome. Methods: Seventeen nude mice underwent intrasplenic injection of human pancreatic carcinoid BON cells (107), and then sacrificed 9 weeks later. Murine livers were analyzed by immunohistochemistry. Murine hearts were sectioned and the surface area of the right heart values determined. Blood also was collected and analyzed for platelet serotonin by ELISA. Results: Sixty-five percent of the mice developed gross carcinoid liver metastases demonstrated by chromogranin A-staining lesions within the liver. Mice with carcinoid liver metastases had elevated platelet serotonin levels (1058 ± 529 vs. 123 ± 52 ng/ml, p=0.002) when compared to the controls. Animals with carcinoid liver metastases also had a trend towards greater tricuspid valvular surface areas (242 ± 24 vs. 174 ± 25 microns, p=0.08). On histologic examination, this increase in tricuspid surface area in mice with liver metastases appeared to be due to fibrosis of the valvular tissues, consistent with the pathologic findings of carcinoid heart disease. Conclusions: Using this novel animal model of carcinoid syndrome, the tricuspid value thickening resembling carcinoid heart disease appears to be due to exposure to factors such as serotonin secreted by carcinoid tumor cells.

Large cell neuroendocrine carcinoma of the lung: a comparison with large cell carcinoma with neuroendocrine morphology and small cell carcinoma

Large cell neuroendocrine carcinoma (LCNEC) of the lung is a malignant neuroendocrine tumor clinicopathologically similar to and falling in-between atypical carcinoid tumor and small cell lung carcinoma (SCLC). The diagnosis of LCNEC is based mainly on a characteristic neuroendocrine morphology and biological neuroendocrine differentiation. In order to know the discrepancy between morphological and biological neuroendocrine differentiation, LCNEC was immunohistochemically and molecular biologically compared with large cell carcinoma with neuroendocrine morphology (LCCNM), which lacked only biological neuroendocrine differentiation among the criteria of LCNEC. Immunohistochemically, disruption of the RB pathway, namely a lack of RB expression and simultaneous overexpression of p16 protein, was characteristic of LCNEC, but not LCCNM. In G2/M cell cycle regulation, 14-3-3 sigma expression was markedly reduced in LCNEC. Moreover, the antibody 34 beta E12 recognizing a set of large-sized keratin gave a different staining pattern between LCNEC and LCCNM. The immunohistochemical data suggested that LCNEC has a similar biological marker profile to SCLC and different from LCCNM. However, a loss of heterozygosity (LOH) analysis using microsatellite markers showed a high frequency of LOH at 3p in both LCNEC and LCCNM as well as in SCLC. Morphological neuroendocrine differentiation might not be identical to biological neuroendocrine differentiation in large cell carcinoma of the lung.

Expression of occludin in human rectal carcinoid tumours as a possible marker for glandular differentiation.

To examine whether or not the tight junction-associated transmembrane protein occludin is expressed in rosette or gland-like structures in human rectal carcinoid tumours. The tight junction is crucial for the formation and maintenance of organized tubular structures in glandular epithelia. Previous studies have reported the presence of glandular structures in carcinoid tumours, though they are not believed to arise from glandular epithelium. The expression profiles of occludin in 40 carcinoid tumours were examined immunohistochemically, using an anti-occludin monoclonal antibody. In eight (20%) samples of typical carcinoid tumours, a small number of rosette-like tubular structures outlined by occludin were detected. Tight junction-associated molecules, including occludin, are thought to be one of the most characteristic structural markers of polarized glandular structures. The results of the present study provide supportive evidence that carcinoid tumour cells are capable of glandular differentiation.

CT findings of surgically resected large cell neuroendocrine carcinoma of the lung in 38 patients.

We sought to assess the CT features of surgically resected large cell neuroendocrine carcinoma of the lung. The cases of all patients who underwent surgical resection for primary lung cancer in a single institution from 1993 to 2000 and who received an initial diagnosis of poorly differentiated non-small cell lung carcinoma, small cell carcinoma, carcinoid tumor, and large cell neuroendocrine carcinoma were histologically reviewed. The findings for 43 patients were histologically reclassified and confirmed as large cell neuroendocrine carcinoma. The CT scans available for 38 patients were evaluated by two observers. In the 38 patients, six central tumors and 32 peripheral tumors, with diameters ranging from 12 to 92 mm (mean +/- SD, 32 +/- 19 mm), were identified. None of the tumors had air bronchograms or calcification in the mass or nodule. Of the 19 patients with thin-section CT scans, 14 (74%) showed the tumor-lung interface as well defined and five (26%) showed the interface to be ill defined. Lobulation was identified on 15 scans (79%) and spiculation was evident on six scans (32%). On contrast-enhanced CT scans, inhomogeneously enhanced tumors appeared to be larger (51 +/- 18 mm) than homogeneously enhanced tumors (25 +/- 10 mm; p < 0.001). At histopathologic examination, gross necrosis was noted in 20 of 28 patients who had undergone contrast-enhanced CT, and the cause of inhomogeneous enhancement on CT scans was determined to be intratumoral necrosis. Multiple microscopic necroses were present in all 28 patients. Large cell neuroendocrine carcinoma usually appears as a well-defined and lobulated tumor with no air bronchograms or calcification. The inhomogeneous enhancement (caused by necrosis) seen in large cell neuroendocrine carcinomas with large diameters is not necessarily apparent in small-diameter (< 33 mm) large cell neuroendocrine carcinomas, even if the tumor contains necrosis.

Chondrosarcoma arising in the trachea: a case report and review of the literature

Chondrosarcoma arising in the trachea: a case report and review of the literature

Isolated Tricuspid Stenosis and Heart Failure: a Focus on Carcinoid Heart Disease

A rare clinical occurrence, heart failure in the setting of tricuspid stenosis should immediately trigger a search for underlying systemic carcinoid disease. Carcinoid tumor cells can secrete a variety of vasoactive substances that result in skin erythema, excretory diarrhea, bronchospasm, and hemodynamic instability, but these manifestations are noted only in a few patients. Right heart valvular disease is common since the vasoactive noxious substances pass through the right heart unaffected and undergo metabolism in the pulmonary circulation, thereby decreasing involvement of the left-sided valves. Localization of the carcinoid tumor followed by surgically directed valvular treatment is mandatory for relief of symptoms. In nonoperative candidates, cytotoxic chemotherapy or long-term symptomatic drug treatment with somatostatin is indicated.

Activation of the ras/raf-1 signal transduction pathway in carcinoid tumor cells results in morphologic transdifferentiation

Background. Recent studies of neuroendocrine tumor cell lines suggest that ras/raf-1 activation could be detrimental to tumorigenesis. The mechanism by which it alters neuroendocrine tumor cells is unclear. We hypothesize that activation of the ras/raf signal transduction pathway may alter gastrointestinal carcinoid cells by inducing morphologic transdifferentiation. Methods. Pancreatic carcinoid (BON) cells were transduced in a stable manner with an estrogen inducible raf-1 fusion protein (creating “BON-raf cells”). BON and BON-raf cells were then treated with either control or 1 μmol/L estradiol (E2). Western blots were used to confirm the phosphorylation of extracellular signal-regulated kinase 1/2. Morphologic changes were evaluated using light and electron microscopy. Results. Western blots using antibodies against phosphorylated and unphosphorylated extracellular signal-regulated kinase 1/2. confirmed that phosphorylation was only present in the BON-raf E2 cells. BON cells treated with control and E2 and BON-raf cells treated with control all looked identical in culture. After treatment with E2 to induce raf-1, the BON-raf cells underwent dramatic morphologic changes. Under light and electron microscopy the cells became flatter and developed much sharper cellular borders mimicking cellular differentiation. Conclusions. Activation of the ras/raf-1 signal transduction pathway leads to prominent phenotypic changes that resemble differentiation of gastrointestinal carcinoid cells in vitro. (Surgery 2002;132:1035-9.)

Cystic fibrosis transmembrane conductance regulator modulates neurosecretory function in pulmonary neuroendocrine cell-related tumor cell line models.

The pulmonary neuroendocrine cell (PNEC) system consists of solitary cells and distinctive cell clusters termed neuroepithelial bodies (NEB) localized in the airway epithelium. PNEC/NEB express a variety of bioactive substances, including amine (serotonin, 5HT) and neuropeptides. We have previously shown that NEB cells are O(2) sensors expressing nicotinamide adenine diphosphate oxidase complex and O(2) sensitive K(+) channel. Recently, we demonstrated expression of functional cystic fibrosis transmembrane conductance regulator (CFTR) and Cl(-) conductances in NEB cells of rabbit neonatal lung. Because PNEC/NEB are sparsely distributed and difficult to study in native lung, we investigated small-cell lung carcinoma (SCLC) and carcinoid tumor cell lines (tumor counterparts of normal PNEC/NEB) as models for PNEC/NEB. SCLC (H146, H345) and carcinoid (H727) cell lines express neuroendocrine cell markers, including chromogranin A, neural cell adhesion molecule (N-CAM), 5HT, and tryptophan hydroxylase. We report that H146, H345, and H727 express CFTR messenger RNA (reverse transcription polymerase chain reaction) and protein (immunoblotting) and possess functional CFTR Cl(-) conductance, demonstrated by an iodide efflux assay inhibitable by transfection with antisense CFTR. Using an immunoassay to quantitate 5HT secretion, we also show that downregulation of CFTR abolishes hypoxia-induced 5HT release, and reduces secretory response to high potassium. Our findings suggest that CFTR may modulate neurosecretory activity of PNEC/NEB possessing O(2) sensor function. We propose that these tumor cell lines may be useful models for investigating the role of CFTR in PNEC/NEB functions in health and disease.

Effects of Interferon Alpha on the Expression of p21cip1/waf1 and Cell Cycle Distribution in Carcinoid Tumors

Interferon alpha (IFN-α) has been shown to produce antitumor effects in 50–80% of carcinoid tumor patients and has demonstrated anti-proliferative effects in carcinoid tumor cells, but the mechanism is not well established. This study presents evidence that in a carcinoid tumor cell line, Bon1, IFN-α increases the expression of p21 and promotes nuclear translocation of endogenous p21. Furthermore, immunoprecipitation experiments demonstrated that p21 formed immuno-complexes with Stat1 and Stat2 in the nucleus of cells. Interferon alpha can decrease G1- and G2-phase cells, but increase S-phase population. The p21 mRNA expression is inversely correlated to the G1 population (r=−0.933, P < 0.05) and positively correlated to the S-phase population (r=0.901, P < 0.05). In addition, IFN-α inhibited cyclin dependent kinases (CDK), CDK2-, CDK3-, CDK4-, and cyclin E- but not cyclin A-associated kinase activities. Immunodepletion of p21 resulted in a significant enhancement of CDK3 kinase activity (∼1.6-fold increase). These results suggest that the mechanism of antitumor and cell cycle regulation of IFN-α in carcinoid tumors may, at least in part, be p21-dependent. Based on these results, we conclude that IFN-α exerts antitumor effects by increased p21 expression in neuroendocrine tumors.

Microtubule-Associated Protein-2: A New Sensitive and Specific Marker for Pulmonary Carcinoid Tumor and Small Cell Carcinoma

Microtubule-associated proteins (MAPs) are a major component of cytoskeleton family proteins associated with microtubule assembly. MAP-2 has been shown to be specifically expressed in neuronally differentiated cells. Pulmonary neuroendocrine carcinomas such as carcinoid tumors and small cell carcinomas are derived from neuroendocrine cells. We hypothesize that neuroendocrine cells may also express MAP-2, and therefore, MAP-2 may be used as a marker for pulmonary carcinomas of neuroendocrine differentiation. To investigate the utility of using MAP-2 expression to separate pulmonary neuroendocrine from non-neuroendocrine tumors, we examined the expression of MAP-2 immunohistochemically in 100 cases of pulmonary carcinomas. The immunoperoxidase method with antigen retrieval was used to characterize the expression of MAP-2, chromogranin, synaptophysin, and neuron-specific enolase in 25 small cell carcinomas, 25 carcinoid tumors, 25 adenocarcinomas, and 25 squamous cell carcinomas. All tumors were lung primaries. All 25 cases of carcinoid tumors (100%) as well as 23 of 25 cases (92%) of small cell carcinomas were positive for MAP-2. Four of 25 cases (16%) of adenocarcinomas were positive for MAP-2 and synaptophysin. Among the 25 squamous carcinomas, 4 cases (16%) were positive for MAP-2, 2 cases (8%) were positive for synaptophysin, 11 cases (44%) were positive for neuron-specific enolase, and none was positive for chromogranin. In conclusion, MAP-2 is a new sensitive and specific marker for the pulmonary tumors of neuroendocrine differentiation. We recommend that MAP-2 be added to immunohistochemical panels to separate non-neuroendocrine from neuroendocrine lung tumors.

Interferon Alpha Induction of Stat1 and Stat2 and Their Prognostic Significance in Carcinoid Tumors

IFN-α has been shown to elicit antitumor activity in carcinoid tumors. In the present study the effects of IFN-α on the protein expression involved in the IFN-α signaling, which were recognized as signal transducer and activators of transcription (Stats), were investigated. Archive specimens from 45 carcinoid patients, 33 before IFN-α and 45 during treatment, were studied. The tissues were immunostained for protein expression by using monoclonal anti-Stat1 and anti-Stat2 antibodies. Results showed that Stat1 and Stat2 immunostaining were significantly increased during IFN-α treatment. Stats scores were significantly increased only in patients with objective response as well as those with stable disease but not in those with progressive disease. The induction of Stats correlated with the survival rates of the patients. In addition, both Stat scores significantly correlated with the p68 protein expression. In a carcinoid tumor cell line, Bon1, IFN-α dose-dependently increased the Stat expression. Analysis of cell fractions showed that IFN-α increased Stat protein both in cytoplasm and nucleoplasm of the cells. Furthermore, IFN-α enhanced tyrosine phosphorylation of Stat1 and Stat2. Thus, the antitumor effect, in vivo and in vitro, in IFN-α-treated carcinoid tumors seems to be mediated via upregulation of Stat proteins and enhancement of phosphorylation of these proteins.

Regulation of the neuroendocrine phenotype in pancreatic carcinoid tumor cells

Regulation of the neuroendocrine phenotype in pancreatic carcinoid tumor cells

Regulation of the neuroendocrine phenotype in pancreatic carcinoid tumor cells

Gastrointestinal carcinoid tumors exhibit characteristic neuroendocrine (NE) features capable of promoting tumor growth and virulence via autocrine growth loops and ectopic hormone production. However, the genetic pathways underlying NE differentiation in 61 carcinoid tumors remain unknown. Previously, we have shown that hASH1, a proneural transcription factor, is expressed in small cell lung cancers (SCLC) and is required for maintenance of the NE phenotype. In SCLC, hASH1 expression is regulated by the Notch signaling pathway, involving the transcription factor HES-I. We hypothesized that hASH1 and Notch signaling might also function to regulate the NE phenotype in GI carcinoid tumors. Methods: Expression of hASH1 and HES-1 as well as the NE markers neuron-specific enolase (NSE) and synaptophysin (Syn) were evaluated in the human pancreatic carcinoid cell line BON by Western blot analysis. Recombinant adenoviruses were used to overexpress Notch1 or ~galactosidase (control) in BON cells. Results: High constitutive expression of hASH1 protein was detected in BON cells but not in two pancreatic adenocarcinoma cell lines (BxPC-3, Capan-2). Overexprassion of Notch1 in BON cells led to induction of HES-1 protein, a negative regulator of hASH1 expression. Increased HES-1 levels were associated with a complete loss of hASH1 protein by one day post-infection with Notch1. By two days after Notch1 infection, expression of Syn protein was dramatically reduced. Similarly, NSE protein levels in BON cells progressively declined over the course of three days following Notch1 infection. Overexpression of ~galactosidase in dON cells had no effect on HES-1, hASHf, Syn, or NSE protein expression. Conclusions: We show that hASH1, a transcription factor essential for NE differentiation in certain contexts, is highly expressed in the pancreatic carcinoid cell line BON. Significantly, hASH1 was not detected in two pancreatic adenocarcinoma cell lines. We also demonstrate that the Notch signaling pathway is intact in dON cells, leading to rapid extinguishing of hASH1 expression and subsequent depletion of NE markers. Our studies provide a molecular basis for potential strategies aimed toward manipulating Notch signaling in GI carcinoid tumors as a means to regulate the prominent NE phenotype that is central_to their pathogenesis.