Cell Carcinoid Research Articles

Double-Detargeted Oncolytic Adenovirus Shows Replication Arrest in Liver Cells and Retains Neuroendocrine Cell Killing Ability

BackgroundWe have previously developed an oncolytic serotype 5 adenovirus (Ad5) with chromogranin-A (CgA) promoter-controlled E1A expression, Ad[CgA-E1A], with the intention to treat neuroendocrine tumors, including carcinoids. Since carcinoids tend to metastasize to the liver it is important to fully repress viral replication in hepatocytes to avoid adenovirus-related liver toxicity. Herein, we explore miRNA-based regulation of E1A expression as a complementary mechanism to promoter-based transcriptional control.Methodology/Principal FindingsAd[CgA-E1A-miR122], where E1A expression is further controlled by six tandem repeats of the target sequence for the liver-specific miR122, was constructed and compared to Ad[CgA-E1A]. We observed E1A suppression and replication arrest of the miR122-detargeted adenovirus in normal hepatocytes, while the two viruses killed carcinoid cells to the same degree. Repeated intravenous injections of Ad[CgA-E1A] induced liver toxicity in mice while Ad[CgA-E1A-miR122] injections did not. Furthermore, a miR122-detargeted adenovirus with the wild-type E1A promoter showed reduced replication in hepatic cells compared to wild-type Ad5 but not to the same extent as the miR122-detargeted adenovirus with the neuroendocrine-selective CgA promoter.Conclusions/SignificanceA combination of transcriptional (promoter) and post-transcriptional (miRNA target) regulation to control virus replication may allow for the use of higher doses of adenovirus for efficient tumors treatment without liver toxicity.

Notch1 Is a Downstream Effector of Raf-1 Signaling in Gastrointestinal Carcinoid Cells

Introduction: The Raf-1 pathway has been shown to be a tumor suppressing pathway in human carcinoid cancer, inhibiting in vitro as well as in vivo growth and the expression of Achaete Scute Complex-Like1 (ASCL1), a transcription factor central in carcinoid oncogenesis. Interestingly, the Notch1 pathway is capable of inducing many of the same anti-carcinoid effects. As the downstream effectors of the Raf-1 pathway have not been well described, we hypothesized that Notch1 is a downstream effector of the Raf-1 pathway.

Hypoxia stimulates CXCR4 signalling in ileal carcinoids

Tumour hypoxia is associated with increased metastatic potential and resistance to radiotherapy and chemotherapy. Ileal carcinoids are usually metastatic at the time of diagnosis and respond poorly to chemotherapy. The aim of this study was to investigate the extent of hypoxia in ileal carcinoids and the response of tumour cells to induced hypoxia. Vascular endothelial growth factor (VEGF), carbonic anhydrase (CA-IX), hypoxia-inducible factor (HIF)-1alpha and HIF-2alpha were studied by immunohistochemistry in biopsies from 24 patients with ileal carcinoids. All hypoxic markers were shown to be highly expressed in localized areas of the tumours irrespective of tumour location or stage. However, HIF-2alpha expression was significantly higher in distant metastases compared to primary tumours in the same patient. Global gene expression profiling of GOT1 carcinoid cells revealed a marked response to hypoxia. Expression of genes related to epithelial-to-mesenchymal transition and development was altered including increased expression of the C-X-C chemokine receptor type 4 (CXCR4), an important regulator of invasive growth and metastasis formation. High expression of CXCR4 was confirmed by immunohistochemistry in tumour biopsies. Stimulation of GOT1 cells by the CXCR4 ligand, CXCL12 (stromal cell-derived factor 1 (SDF-1)), activated the mitogen-activated protein kinase (MAPK) p42/44 signalling pathway and increased tumour cell migration. We conclude that ileal carcinoids contain hypoxic areas expressing HIF-1alpha, HIF-2alpha and CXCR4. Signalling through the CXCL12-CXCR4 axis may contribute to the metastatic potential of ileal carcinoids. Targeting of HIFs and/or the CXCR4 signalling pathway may offer new therapeutic strategies for carcinoid tumour disease.

Tumor compuesto de intestino grueso: adenocarcinoma mucinoso y carcinoma neuroendocrino de célula grande. A propósito de un caso

Los tumores compuestos primarios del tracto intestinal son infrecuentes y representan entre 2,5 – 20% de estas neoplasias. Se caracterizan por la presencia de un componente glandular y otro neuroendocrino, entremezclados en proporción similar. La mayoría de los casos descritos incluyen un componente de estirpe glandular de adenoma o adenocarcinoma convencional y un componente de carcinoide o carcinoma neuroendocrino de célula pequeña. Presentamos un tumor compuesto de intestino grueso con componentes que no han sido descritos hasta ahora: un adenocarcinoma mucinoso con presencia de células en anillo de sello y un carcinoma neuroendocrino de célula grande. El tratamiento de este tipo de tumores es el mismo que el que se emplea para los adenocarcinomas convencionales pero el componente endocrino les confiere un peor pronóstico. Primary composite tumours of the intestine are rare and are characterised by the presence of glandular and neuroendocrine components occurring in almost equal proportions. The majority of reported cases include conventional type adenoma or adenocarcinoma and carcinoid or small cell neuroendocrine carcinoma. A composite tumour of the large intestine with as yet unreported components is described. Mucinous adenocarcinoma with signet-ring cells and large cell neuroendocrine carcinoma were found. Although the treatment of these tumours is similar to that of conventional adenocarcinomas, the presence of a neuroendocrine component worsens the prognosis.

Development of an arganotypic System to Image Metastasis of Carcinoid Tumors in Situ

Patients with neuroendocrine tumors of the small or large bowel commonly called carcinoid often develop liver metastases. Hepatic portal circulation is a predicted route for establishing liver metastases of carcinoid tumors or following intra-splenic injection of cancer cells. However, the cellular and molecular events that mediate this site-specific metastasis are not well understood. We developed a system to study extravasation, migration, invasion and proliferation of a human carcinoid cell line using mouse liver organotypic slice culture as a tractable preparation that more closely resembles the three-dimensional, multi-cellular tumor microenvironment than does a dispersed cell culture system. BON cells stably transfected with GFP were introduced to liver by portal vein injection. Organotypic slices obtained from the liver were monitored using fluorescence macroscopy and confocal/multi-photon microscopy. The seeded cancer cells adopted an elongated morphology and arrested in the lumens of red fluorescently-labeled venules. Seeded cells in the liver slices were monitored out to 14 days in culture. Although liver slices generally remained viable over the experiment, there was a general reduction in the number of seeded cells with the greatest reduction occurring by the 24 hr or 48 hr time points. After 72 hr to 96 hr, there was a detectable increase in GFP-labeled cells indicating that subsets of the remaining seeded cells were proliferating. These cells formed small “tumorlet” or spheroid structures that extended beyond the vasculature and into or on the surface of the parenchymal tissue. These tumorlets continued to increase in size to a maximum diameter of about 300 μm. We provide evidence that this organotypic slice/xenograft model is a promising tool with considerable potential as a means to probe the early events mediating metastasic tumor growth in the liver.

Abstract A215: Hsp90 inhibitor IPI-504 demonstrates antitumor activity against a panel of neuroendocrine/carcinoid cell lines in vitro and in vivo

Abstract Heat shock protein 90 (Hsp90), an emerging target in cancer, is a chaperone protein that contributes to tumorigenesis by maintaining the stability and activity of multiple oncogenic proteins. Infinity Pharmaceuticals is currently developing two Hsp90 inhibitors: IPI-504 (retaspimycin hydrochloride), an intravenous agent that has entered several phase II clinical trials, and IPI-493, an orally administered compound that is currently being tested in a phase I dose-escalation trial. In pre-clinical animal models, IPI-504 and IPI-493 have demonstrated anti-tumor activity against a wide variety of tumor types. Neuroendocrine tumors form a heterogeneous group of malignancies, which differ from each other in their biology, prognosis, and genetics. They mainly occur in the gastrointestinal tract and bronchopulmonary system. Conventional cytotoxic therapy has failed to demonstrate effective treatment of neuroendocrine/carcinoid cancers over the past 4 decades, resulting in an unmet need for improved pharmacological treatment of these tumors. To date, no data have been reported on the activity of Hsp90 inhibitors against carcinoid cancer cells. Herein we describe the activity of the Hsp90 inhibitors IPI-504 and IPI-493 against a panel of neuroendocrine tumors including: BON-1, a cell line derived from a metastasis of a primary pancreatic neuroendocrine tumor, NCI-H720, a lung carcinoid, QGP-1, a pancreatic carcinoma of islet cells and HC-45, an ileal carcinoid. In vitro, most of the carcinoid cell lines are sensitive to the Hsp90 inhibitors with EC50 values between 10 and 189 nM for IPI-493 and 49 and 930 nM for IPI-504. In vivo, IPI-504 dosed twice weekly inhibits the tumor growth of BON-1 cells in mice. To identify the potential mechanism of action of IPI-504 in BON-1 cells we have determined that IGF-1R, a tyrosine kinase receptor and an Hsp90 client protein, is constitutively activated in these cells suggesting that it could participate in the unregulated growth of these tumor cells. Upon treatment of BON-1 cells with IPI-504, phospho-IGF-1R is degraded in a dose-dependent manner. The EC50 of the protein degradation and the in vitro growth inhibitory activity of IPI-504 are similar (∼50 nM), suggesting that the anti-tumor activity of IPI-504 could be due, in part, to the inactivation of this growth factor receptor. In conclusion, we demonstrated that the Hsp90 inhibitors IPI-504 and IPI-493 potently inhibit the growth of neuroendocrine tumor cell lines both in vitro and in vivo and identified IGF-1R as a potential client protein. These data suggest further exploring IPI-504 for the possible treatment of neuroendocrine tumors in the clinic. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A215.

Abstract A34: VEGFR-2 expression in carcinoid cancer cells and its role in tumor growth and metastasis

Abstract Carcinoid tumors are slow growing and highly vascular neuroendocrine neoplasms that have been increasing in incidence since the 1970s. Carcinoid tumors express vascular endothelial growth factor receptor 2 (VEGFR-2); however, its role is not completely understood. The aims of this study were 1) to assess the expression and regulation of VEGFR-2 in carcinoid tumors and cells and 2) to evaluate the effect of VEGFR-2 on carcinoid growth and metastasis. Methods: 1) The expression of VEGFR-2 in carcinoid tumor sections and in the novel carcinoid cell line BON, which was established and characterized in our laboratory, was assessed by immunohistochemistry andWestern blot, respectively. As VEGFR-2 signals through the PI3K/Akt pathway, signaling through Akt was adjusted chemically and through RNAi to delineate its effect on VEGFR-2 expression. 2) BON cell proliferation was measured following treatment with the VEGFR-2 inhibitor sunitinib malate to assess the effect of VEGF signaling on cell growth. A BON cell line with stably decreased VEGFR-2 expression, designated as BON shVEGFR-2, was generated by transfecting cells with shRNA to VEGFR-2. Boyden chamber and scratch assays were performed on BON shVEGFR-2 cells to assess the effect of VEGFR-2 on invasion and migration. Results: 1) Immunhistochemical analysis confirmed positive expression of VEGFR-2 in the endothelial and epithelial components of carcinoid tumor sections. Consistent with this finding, BON cells also expressed VEGFR-2. Interestingly, we noted that the regulation of VEGFR-2 expression is inversely related to PI3K/Akt signaling. Inhibition of PI3K/Akt signaling with wortmannin upregulated VEGFR-2 expression, as noted with western blot and mRNA analysis. shRNA against PTEN increased phospho-Akt, resulting in decreased expression of VEGFR-2 in BON cells. BON shPTEN cells injected into the pancreas of nude mice formed increased liver metastases, compared to BON shControl cells, and the liver metastatic cells retained decreased VEGFR-2 expression. 2) Treatment with sunitinib malate had only a modest effect on BON cell inhibition. Consistent with our in vivo studies, BON shVEGFR-2 cells have decreased expression of E-cadherin compared to BON shControl cells. Additionally, the migratory and invasive potential of BON shVEGFR-2 cells is approximately four and 1.75 times that of control BON cells, respectively, at 48 h. Conclusions: We demonstrate the expression and activity of VEGFR-2 in BON cells. The expression of VEGFR-2 on the epithelial component of carcinoid tumors and in the BON cell line, suggests an alternate role for VEGFR-2, in addition to its well-defined role in angiogenesis. The modest inhibition of BON cell growth with sunitinib malate suggests that VEGFR-2 on carcinoid tumor cells has a limited role in cell proliferation. Interestingly, we show that the level of VEGFR-2 expression in BON cells is inversely proportional to the degree of total Akt signaling, suggesting a negative feedback loop in the regulation of VEGFR-2. The finding that decreased VEGFR-2 expression results in increased invasion and migration suggests that VEGFR-2 may inhibit carcinoid metastasis. Thus, while VEGFR-2 inhibition prevents angiogenesis, it may also increase the metastatic potential of surviving carcinoid cells. Citation Information: Cancer Res 2009;69(23 Suppl):A34.

Primary, combined, atypical carcinoid and squamous cell carcinoma of the larynx: a new variety of composite tumour

We report the first case of a laryngeal composite tumour consisting of a squamous cell carcinoma combined with an atypical carcinoid. Case report and review of the literature concerning laryngeal composite tumours. Primary laryngeal carcinoma is the most common malignancy of the upper aerodigestive tract. The vast majority are of the squamous cell type. Primary neuroendocrine neoplasms represent a rare, heterogeneous subset of laryngeal malignancies, comprising typical carcinoid, atypical carcinoid, small cell carcinoma and paraganglioma. Primary combined neuroendocrine and squamous cell carcinoma of the larynx is even more rarely encountered, with only 14 publications of this so-called composite tumour to date. In each case, the neuroendocrine component has been small cell carcinoma. The treatment of primary neoplasms comprising more than one histological type is tailored to the most biologically aggressive tumour. Accurate diagnosis of the histological nature of laryngeal composite tumours is imperative to ensure optimal therapy.

A Study of Human Calcitonin in an Ovarian Carcinoid and Ovarian Cancers

High concentration of human calcitonin (hCT) was found in an ovarian carcinoid by radioimmunoassay. The hCT value was not affected by the presence of protease inhibitors. To confirm the presence of hCT in an ovarian carcinoid, hCT was isolated by the Baghdiantz method. The molecular weight of the ovarian hCT was determined using Sephadex G-75 gel filtration. Though the molecular weight of the ovarian hCT was variable, 90% corresponded to that of the authentic hCT. The carcinoid cells were examined by immunoperoxidase techniques. Those composed of strumal and trabecular structure were all argyrophilic, but hCT was only found in strumal structure. Significant concentrations of hCT were also found in ovarian cancers.

Inhibition of Phosphatidylinositol 3-Kinase/Akt Signaling Suppresses Tumor Cell Proliferation and Neuroendocrine Marker Expression in GI Carcinoid Tumors

Overactivation of PI3K/Akt signaling facilitates tumor proliferation in several cancers. We have shown that various signal transduction pathways promote tumorigenesis in carcinoid tumors, which exhibit endogenously high levels of active, phosphorylated Akt. Therefore, we hypothesized that inhibition of the PI3K/Akt pathway would suppress carcinoid tumor cell growth and neuroendocrine (NE) marker production. Human carcinoid BON cells were treated in vitro with LY294002, a PI3-kinase inhibitor, or transfected with Akt1 siRNA. Tumor cell proliferation was measured by MTT for 6 days. The effect of LY294002 or Akt1 siRNA treatment was assessed by Western analysis. We examined the levels of phosphorylated Akt, total Akt, Akt1, and the NE markers human achaete-scute homolog1 (ASCL1) and chromogranin A (CgA). Treatment of BON cells with LY294002 reduced tumor cell proliferation (76%) in a dose-dependent manner. Growth also decreased in Akt1 siRNA transfected cells (26%). Levels of active, phosphorylated Akt and the NE tumor markers, ASCL1 and CgA, were diminished with both LY294002 and Akt1 siRNA treatments proportional to the degree of Akt inhibition. Total Akt, Akt2, and Akt3 levels were unaffected by these experiments. These data indicate that PI3K/Akt signaling performs a critical role in human carcinoid tumor cell survival and NE hormone generation. Furthermore, the development of novel therapeutics targeting Akt1 or components of the PI3K/Akt pathway may enhance the management of carcinoid disease.

MG-132 Inhibits Carcinoid Growth and Alters the Neuroendocrine Phenotype

Carcinoid cancers are the most common neuroendocrine (NE) tumors, and limited treatment options exist. The inhibition of glycogen synthase kinase-3beta (GSK-3beta) has been shown to be a potential therapeutic target for the treatment of carcinoid disease. In this study, we investigate the ability of MG-132, a proteasome inhibitor, to inhibit carcinoid growth, the neuroendocrine phenotype, and its association with GSK-3beta. Human pulmonary (NCI-H727) and gastrointestinal (BON) carcinoid cells were treated with MG-132 (0-4microM). Cellular growth was measured by the 3-[4,5-dimethylthiazole-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay. Levels of total and phosphorylated GSK-3beta and the NE markers chromogranin A (CgA), Achaete-Scute complex-like 1 (ASCL1), as well as the apoptotic markers poly (ADP-ribose), polymerase (PARP), and cleaved caspase-3 were determined by Western blot. Treating carcinoid cells with MG-132 resulted in growth inhibition, a dose-dependent inhibition of CgA and ASCL1, as well as an increase in the levels of cleaved PARP and cleaved caspase-3. Additionally, an increase in the level of phosphorylated GSK-3beta was observed. MG-132 inhibits cellular growth and the neuroendocrine phenotype. This proteasome inhibitor warrants further preclinical investigation as a possible therapeutic strategy for intractable carcinoid disease.

Gene set enrichment analysis to evaluate expression of autoantigens in lung cancer

e22048 Background: There is a well-established association between certain autoimmune diseases and the development of specific malignancies. It has been demonstrated that myositis-specific autoantigens are expressed at higher levels in tumors associated with myositis compared to normal tissue, suggesting that immune responses to antigens expressed in nascent tumors may contribute to the autoimmune process. Whether this observation is a general feature of autoantigen expression in tumor tissue, and whether the relative expression of these antigens is enriched in relation to the rest of the tumor transcriptome is currently unknown. Methods: Tumor tissue from ten lung cancer biopsies (adenocarcinoma (4), carcinoid (4), and squamous cell carcinoma (2)) and normal lung from the same patients were obtained. Total RNA was extracted and hybridized to Illumina Sentrix BeadChips. Hiearchical clustering was used to visualize the expression levels of 146 known autoantigens. Gene Set Enrichment Analysis was performed using 20 disease-specific autoantigen gene sets and 1892 gene sets from the Molecular Signatures Database. Protein levels of selected autoantigens were assessed by immunoblotting detergent tissue lysates. Results: Single-linkage hierarchical clustering analysis reveals groups of autoantigens that are differentially expressed between normal lung tissue, carcinoid tumors, and adenocarcinomas. Adenocarcinoma tumor samples were significantly enriched for myositis (nominal p-value <0.001, false discovery rate (FDR) q-value 0.009) and SLE autoantigens (p-value 0.004, FDR 0.029). Scleroderma autoantigens were enriched in carcinoid tumors (p-value 0.003, FDR 0.053). Increased protein expression of the autoantigens Mi-2 and topoisomerase-1 in carcinoid tumors was confirmed by immunoblotting. Conclusions: This study reveals that autoantigens targeted in several autoimmune diseases are both transcribed and expressed at high levels in malignancies known to associate with those disorders. While autoantigens comprise only a small fraction of the total transcriptome, they are disproportionately expressed in tumors known to associate with autoimmunity, supporting the hyporthesis that autoimmunity to these proteins may arise via nascent anti-tumor responses. No significant financial relationships to disclose.

A phase II clinical and biological study of lithium carbonate (Li) in patients with low-grade neuroendocrine tumors

e15662 Background: Low-grade neuroendocrine tumors (NETs), such as carcinoid, islet cell tumors, and medullary thyroid carcinomas, respond poorly to chemotherapy, and effective, less toxic therapies are needed. Glycogen Synthase Kinase (GSK)-3β, a multifunctional protein kinase, has been shown to regulate growth and hormone production in NETs. Use of lithium carbonate (Li) in murine models suppressed carcinoid cell growth, reduced GSK-3β levels and reduced expression of chromogranin A. This study assessed the efficacy of Li in patients with NETs. Methods: Eligible pts had pathologically-proven, measurable low-grade NETs. Prior treatment was allowed if completed >4wks prior to registration. Standard eligibility criteria were used, and use of medications affecting Li metabolism or levels were prohibited. A single-arm, open-label, two-stage Phase II design was used. Li was dosed at 300mg orally TID with meals, titrated to a target serum level of 0.8–1.0mmol/L. The primary endpoint was objective tumor response by RECIST. Secondary endpoints included overall survival, progression-free survival, decrease of serum tumor markers, toxicity, and quality of life. Results: 15 pts were enrolled between 10/3/07and 7/17/08; 6 men, 9 women. The median age was 58 (range 47–74). Patients’ diagnoses were carcinoid tumor for 8 subjects, islet cell tumor for 5 subjects, and 2 unknown primary sites. ECOG PS was 0 (6 patients) or 1 (9 patients). Two pts came off study due to side effects (tremor, dizziness/abdominal pain). There were no radiographic responses. Due to an early stopping rule requiring at least 1 objective response in the first 13 evaluable pts, the study was closed to further accrual. 13 patients had pre- and post-therapy biopsies. Evaluation of quality of life and GSK-3β levels in tumor tissue is ongoing. Conclusions: Li was ineffective at obtaining a radiographic response in our 13 evaluable patients who were treated as part of this study. We will determine from tumor biopsies whether Li was effective at phosphorylating GSK-3β in order to make conclusions about GSK-3β as a therapeutic target for future NET treatment strategies. Funded through NIH grant R21CA117117- 01A2 and CTRC grant 1ULRR025011. No significant financial relationships to disclose.

Analysis of ErbB Receptors in Pulmonary Carcinoid Tumors

This study aimed to investigate the expression of the ErbB family of receptor tyrosine kinases in pulmonary typical carcinoid and atypical carcinoid tumors and to understand the role of epidermal growth factor receptor (EGFR) signaling in pulmonary carcinoid tumor proliferation. Surgically resected typical carcinoid (n = 24) and atypical carcinoid (n = 7) tumor tissues were analyzed by immunohistochemical staining for EGFR, ErbB2, ErbB3, and ErbB4. Sequencing of tumor DNA of exons 18 to 21 of the EGFR gene and the KRAS gene was carried out. Biochemical analysis of lung carcinoid cell lines was used to investigate EGFR signal transduction and response to erlotinib inhibition. The analysis showed that 45.8% of typical carcinoid and 28.6% of atypical carcinoid tumors express EGFR, 100% of the tumors lack expression of ErbB2, and 100% have moderate to intense staining for ErbB3 and ErbB4. Sequencing of tumor DNA of exons 18 to 21 of the EGFR gene revealed the absence of tyrosine kinase domain mutations in these tumors. Instead, 80.6% tumors harbored a synonymous single nucleotide polymorphism in exon 20. Because EGFR and KRAS mutations tend not to be present at the same time, we sequenced the KRAS gene from pulmonary carcinoid tumor DNA and found that 100% were wild-type. Using a lung carcinoid cell line that expresses EGFR, we found that erlotinib reduced proliferation by inhibiting EGFR signal transduction. Our findings suggest clinical potential for the use of EGFR inhibitors in the treatment of patients with pulmonary carcinoid tumors, particularly for patients with EGFR-positive pulmonary carcinoid tumors not amenable to surgical resection.

W1950 Novel Expression and Activation of the Vascular Endothelial Growth Factor Receptor-2 in Human Carcinoid Cells

W1950 Novel Expression and Activation of the Vascular Endothelial Growth Factor Receptor-2 in Human Carcinoid Cells

Phosphatidylinositol 3-Kinase-Akt Signaling in Pulmonary Carcinoid Cells

In several types of cancer, upregulation of phosphatidylinositol 3-kinase (PI3K)-Akt signaling facilitates tumor cell growth and inhibits apoptosis. Previous reports demonstrated that this pathway promotes growth, survival, and chemotherapy resistance in non-small cell and small cell lung cancer cells. But the importance of PI3K-Akt signaling has not been explored in pulmonary carcinoids. In this study, our objective was to establish the role of the PI3K-Akt signal transduction pathway in pulmonary carcinoid cells. Human pulmonary carcinoid NCI-H727 cells were treated with LY294002 (0 to 100 microM), a well-known PI3K inhibitor, or transfected with Akt1 small interfering RNA (75 nM). Cellular proliferation was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay for up to 8 days. Western blot analysis was performed for expression of active, phosphorylated Akt (pAkt), total Akt, Akt1, and the neuroendocrine markers chromogranin A and achaete-scute complex-like1. Treatment of NCI-H727 cells with LY294002 significantly reduced tumor cell growth (85.3%). Similarly, Akt1 small interfering RNA transfection led to diminished tumor cell proliferation (31.3%). A dose-dependent decrease in chromogranin A and achaete-scute complex-like1 production was observed with both PI3K inhibition and Akt1 RNA interference. Expression of Akt1 was reduced at all time points by transient Akt1 small interfering RNA transfection. The PI3K-Akt pathway plays a role in both tumor cell growth and neuroendocrine hormone secretion in human pulmonary carcinoid cells. Inhibition of Akt1, PI3K-Akt signaling, or a downstream mediator of this pathway may provide therapeutic approaches for patients with pulmonary carcinoid tumors.

W1583 Modulation of Neurotensin Secretion Through mTOR-Mediated AKT Inhibition in the Human Carcinoid Cell Line BON

W1583 Modulation of Neurotensin Secretion Through mTOR-Mediated AKT Inhibition in the Human Carcinoid Cell Line BON

Combination analyses of anti-cancer drugs on human neuroendocrine tumor cell lines

There is a large need for better pharmacological treatment of neuroendocrine tumors. The aim of this study was to investigate and quantify the cytotoxic potentiating effects resulting from a combination of five substances, NSC 95397, emetine, CGP-74514A hydrochloride, Brefeldin A and sanguinarine chloride, chosen from a previous screening of 1,280 pharmacologically active agents on neuroendocrine tumor cells, with standard cytotoxic agents currently used in the treatment of neuroendocrine tumors. The human pancreatic carcinoid cell line BON-1, human typical bronchial carcinoid cell line NCI-H727 and the human atypical bronchial carcinoid cell line NCI-H720 were used. Combinations between doxorubicin, etoposide, oxaliplatin, docetaxel, and each one of the five agents were studied and simultaneous exposures were explored using the median-effect method. Most of the combinations of NSC-95397 and emetine with doxorubicin, etoposide, docetaxel, and oxaliplatin showed synergism, and their remaining combinations were additive. Almost all of the CGP-74514A hydrochloride interactions were additive, while brefeldin A and sanguinarine displayed less synergy but more additive and antagonistic interactions in combination with the standard drugs. The synergistic and additive interactions make NSC-95397, emetine, and CGP-74514A hydrochloride potential candidates for incorporation into combination chemotherapy regimens and these drugs might be the suitable candidates for further clinical studies in patients with bronchial carcinoids and pancreatic endocrine tumors.

Development and Characterization of a Novel In vivo Model of Carcinoid Syndrome

Carcinoid syndrome, characterized by flushing, diarrhea, and valvular heart disease, can occur following carcinoid tumor metastasis to the liver and systemic release of bioactive hormones into the systemic circulation. Treatment of this devastating disease is hampered by the lack of an in vivo model that recapitulates the clinical syndrome. Here, we have injected BON cells, a novel human carcinoid cell line established in our laboratory, into the spleens of athymic nude mice to establish liver metastases. The majority of mice injected intrasplenically with BON cells developed significant increases in plasma serotonin and urine 5-hydroxyindoleacetic acid, and several mice exhibited mesenteric fibrosis, diarrhea, and fibrotic cardiac valvular disease reminiscent of carcinoid syndrome by both echocardiographic and histopathologic evaluation. Mice pretreated with octreotide, a long-acting somatostatin analogue, or bevacizumab, a vascular endothelial growth factor inhibitor, developed fewer liver metastases and manifestations of carcinoid syndrome, including valvular heart disease. We have provided an important in vivo model to further delineate novel treatment modalities for carcinoid syndrome that will also be useful to elucidate the factors contributing to the sequelae of carcinoid disease (e.g., mesenteric fibrosis and valvular heart disease).

LYVE-1 immunocytochemical staining for gastrointestinal carcinoids

By immunocytochemical staining for lymphatic vessels using anti-lymphatic vessel hyaluronan receptor (LYVE-1) antibody, pancreatic islets and some pancreatic endocrine tumours (PETs) were positively stained for the cytoplasm in addition to lymphatic vessels. The current study was extended to investigate possible immunostaining of gastrointestinal carcinoids using goat antihuman LYVE-1 antibody. Lymphatic vessels were immunostained by LYVE-1 and blood vessels were immunostained by factor 8 (F-8). Among 27 gastrointestinal carcinoids, six cases (22%) including five primary and one metastatic carcinoids were positive in the cytoplasm for LYVE-1 in addition to lymphatic vessels and there were abundant lymphatic and blood vessels at the margin of the carcinoids. These six LYVE-1 positive cases consisted of three of 10 small intestinal, one of five appendiceal and two of seven large intestinal carcinoids. There was no LYVE-1 positive staining in the adjacent normal gastro-intestinal mucosa. The presence of LYVE-1 immunostaining in some gastrointestinal carcinoids may support a structure-function relationship of lymphatic vessels/hyaluronan receptor for modulating synthesis and secretion of hormones and amines by carcinoid tumour cells.