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Department of Surgery (E.K.N.), University of California, San Francisco, California 94115; The Sidney Kimmel Comprehensive Cancer Center (V.R.S., K.E.S., M.W.B., N.J., B.J.C., B.D.N., D.W.B.) and Departments of Medicine (E.C.H., D.W.B.), and Surgery (B.J.C.), The Johns Hopkins University School of Medicine, Baltimore, Maryland 21231; and Department of Surgery (M.K., H.C.), University of Wisconsin Medical School, and the University of Wisconsin Comprehensive Cancer Center, Madison, Wisconsin 53792
Address all correspondence and requests for reprints to: Eric K. Nakakura, 1600 Divisadero Street, Room A-724, San Francisco, California 94115. E-mail: NakakuraE{at}surgery.ucsf.edu.
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Abstract |
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Objective: 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.
Design: 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.
Setting: The study was conducted in an academic referral center.
Patients or Other Participants: Deidentified archival pathology specimens were examined.
Results: 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.
Conclusions: The Notch signaling pathway is a significant regulator of neuroendocrine differentiation and serotonin production in GI carcinoid tumors.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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GI carcinoids are defined by their NE phenotype that appears to contribute directly to tumor pathogenesis and progression. The elaboration of serotonin and its metabolites, histamine, and other hormones are implicated in causing the carcinoid syndrome, which manifests as diarrhea, facial flushing, wheezing, and right-sided valvular heart disease (3). Moreover, serotonin and IGF-I stimulate carcinoid growth in an autocrine or paracrine fashion (4, 5). Current therapy, including somatostatin analogs, interferon 
, and cytotoxic chemotherapy, has been moderately effective in palliating symptoms of the carcinoid syndrome but has little activity in preventing tumor progression (6). Elucidating the signal transduction pathways underlying NE differentiation in GI carcinoid tumors has the potential to provide novel targets for diagnosis and treatment.
GI carcinoid tumors are thought to arise from endocrine cells of the GI tract, the enteroendocrine system (7). Enteroendocrine cells share a common origin, from an endodermal progenitor cell, with other cells of the digestive tract (8, 9). Members of the basic helix-loop-helix (bHLH) transcription factor family regulate endocrine cell differentiation in mouse endoderm. For example, in the absence of one of these bHLH factors, Math1, the intestinal secretory lineage, including enteroendocrine, goblet, and Paneth cells, fails to develop (10). Loss of neurogenin 3 (Ngn3) results in the absence of all pancreatic and intestinal endocrine cells, whereas gastric cells expressing serotonin, histamine, and ghrelin are still present (11, 12). Another bHLH factor, neurogenic differentiation 1 (neuroD1), is necessary for pancreatic islet morphogenesis and terminal differentiation of certain enteroendocrine cell types (13). Finally, in the foregut, disruption of achaete-scute complex homolog-like 1 (Ascl1/Mash1) leads to a lack of pulmonary endocrine cells (14), but the gut endocrine cells in these mutants have not been analyzed in detail.
In the developing endoderm, Notch signaling inhibits endocrine differentiation by repressing the expression of bHLH transcription factors. Forced Notch activation during pancreas development expands a pool of undifferentiated precursors and prevents the initial emergence of endocrine cells and subsequent exocrine differentiation (15, 16). Conversely, mice lacking the Notch effector hairy and enhancer of split 1 (Hes1) display excessive endocrine differentiation in the lung, stomach, and intestine (17, 18). In the pancreas, disruption of Notch signaling components results in precocious endocrine differentiation (19). Although Notch activation blocks the differentiation of endocrine precursors, fully differentiated endocrine cells are resistant to Notch signaling (15).
Because bHLH transcription factors and Notch signaling are known to play key opposing roles in GI endocrine differentiation, we first surveyed the expression of a panel of developmental transcription factors and Notch pathway components in human GI carcinoid tumors and in the BON GI carcinoid tumor cell line (20, 21). Compared with surrounding normal tissues, the bHLH factor Ascl1 was most frequently overexpressed in carcinoid tumors. Using the BON cell in vitro model, we show that NE differentiation, serotonin production, and growth of carcinoid tumor cells are all susceptible to activation of the Notch signaling pathway.
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Materials and Methods |
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Use of human carcinoid tumor samples with corresponding normal tissue specimens was approved by the Johns Hopkins Hospital Joint Committee on Clinical Investigation (10 primary tumors) and the University of Wisconsin Medical School Human Subjects Committee (three primary tumors and one metastasis). Histological sections were reviewed by a pathologist, and in five cases, matched tumor and surrounding normal specimens were available. All samples were made anonymous by removal of all identifiers before analysis. Total RNA was prepared from frozen human specimens and cell lines using Trizol Reagent (Life Technologies, Rockville, MD). cDNA for RT-PCR was made from total RNA using Superscript II Rnase H– reverse transcriptase (Life Technologies) according to the manufacturer’s instructions and amplified for 30 cycles for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or 25–35 cycles for all others. Primers used for RT-PCR spanned an intron and can be obtained from the authors upon request. Ribonuclease protection assay (RPA) was performed by using the RPA III kit (Ambion, Austin, TX) with 5 µg total RNA. A tryptophan hydroxylase 1 probe yielded a 342-bp protected fragment (nucleotides 561–902; GenBank accession no. NM004179). Probes for Ascl1 and GAPDH have been described (22).
Cell culture
BON cells, a gift from Dr. B. Mark Evers (Galveston, TX), were established from a lymph node containing a metastasis from a human pancreatic carcinoid tumor and were cultured in DMEM and F-12K (1:1) media supplemented with 10% fetal bovine serum and 100 U penicillin and 100 µg streptomycin per ml (20, 23). Passage numbers were 10–50. The bronchial carcinoid NCI-H727 and the pancreatic adenocarcinoma BxPC-3 and MIA PaCa-2 cells were maintained as described (14, 24).
Recombinant adenovirus infection
Recombinant adenoviral constructs containing a constitutively active form of human Notch1 (Ad.Notch1) and Escherichia coli ß-galactosidase (Ad.ßGal) linked to green fluorescent protein (GFP) were generated and carefully titered as described (22). Each experiment used matched titers of control and Notch1 adenovirus and was verified by using GFP fluorescence. BON cells were infected at a multiplicity of infection (moi) of 10–30 pfu/cell, which led to approximately 70% of cells expressing GFP at 48 h post infection with minimal evidence of cytotoxicity.
Immunoblot analysis
After being rinsed with PBS, cells were harvested in sodium dodecyl sulfate sample buffer (62.5 mM Tris, 2% sodium dodecyl sulfate, 10% glycerol) with 1x protease inhibitor cocktail (Sigma Chemical Co., St. Louis, MO) and sonicated. The protein concentration for each cell lysate was determined by using the DC protein assay (Bio-Rad, Hercules, CA). Fifty micrograms of protein were loaded in each lane for gel electrophoresis. Fast Green (Fisher Scientific, Pittsburgh, PA) staining of filters was used to verify equivalent loading and transfer. Filter blocking and antibody dilutions were done in 0.1 M Tris (pH 7.5), 0.9% NaCl, 0.05% Tween 20 with 5% nonfat dry milk. Primary antibody dilutions were as follows: Ascl1, 1:1000 (BD PharMingen, San Diego, CA); Notch1, 1:1000 (Santa Cruz Biotechnology, Santa Cruz, CA); Hes1, 1:10,000 (a gift from T. Sudo, Toray Industries, Inc., Kanagawa, Japan); and neuron-specific enolase (NSE) (Dako, Carpinteria, CA), synaptophysin (Syn) (Chemicon, Temecula, CA), chromogranin A (CgA) (NeoMarkers, Fremont, CA), and GAPDH (Trevigen, Gaithersburg, MD) all at 1:5000. Secondary horseradish peroxidase-conjugated antibodies were used at 1:5000 dilution except for Ascl1 (1:50,000) and GAPDH (1:10,000). Filters were processed using the Supersignal West Pico or Femto (for Ascl1) chemiluminescence kit (Pierce, Rockford, IL).
Serotonin measurement
Supernatant from BON cells analyzed for growth rate was removed and stored at –20 C before adding 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent. Concentration of serotonin was determined using an ELISA kit purchased from Research Diagnostics Inc. (Flanders, NJ) according to the manufacturer’s instructions. Supernatants from two independent experiments were analyzed.
Serotonin immunofluorescence
A total of 105 BON cells were plated onto glass coverslips in six-well plates 1 d before infection at an moi of 10 with Ad.Notch1 or Ad.ßGal. BON cells were processed 4 d after infection and analyzed by confocal microscopy as described (25). Briefly, cells were fixed for 20 min with 3% paraformaldehyde in PBS and then permeabilized with 0.5% Triton X-100 for 10 min. The primary antibody for serotonin (1:100) was obtained from Dako; a secondary Cy5-conjugated donkey antimouse antibody (1:500) was purchased from Jackson ImmunoResearch (West Grove, PA). Imaging was performed using an Olympus 1X70 inverted light microscope equipped with a Noran confocal laser scanning system. Phase contrast and Cy5 fluorescent signals were acquired simultaneously and processed using Noran Intervision software. Images were arranged using Adobe Photoshop (version 5.5).
Growth assays
The MTT assay (Sigma) was performed as described (26). The MTT assay allows for the spectrophotometric determination of cell number based on the mitochondrial dehydrogenase activity of living cells, which cleaves the MTT substrate, generating a purple solution. Briefly, BON cells were grown in phenol red-free DMEM and F-12 media (1:1) with 10% fetal bovine serum and antibiotics and seeded into 24-well plates at 104 cells per well. Twenty-four hours later, adenoviruses were added at a final moi of 30 pfu/well. We titered the adenoviral stocks and used a viral dose that resulted in approximately 70% of cells expressing GFP at 48 h post infection and had minimal cytopathic effect. At various time points, medium was removed and replaced with 250 µl medium containing MTT (0.5 mg/ml) and incubated at 37 C for 2 h. Then 750 µl of dimethylsulfoxide (Sigma) was added to each well and plates were placed on an orbital shaker for 5 min. Absorbance of triplicate or quadruplicate samples was measured at 540 nm. Experiments were performed twice.
Statistics
Statistical significance was performed by one-way ANOVA followed by the Bonferroni test or Mann-Whitney U test as appropriate. A two-sided P value < 0.05 was considered significant.
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Results |
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We obtained frozen tumor specimens with matched adjacent normal tissue from five patients with GI carcinoid tumors. An additional nine carcinoid specimens lacked corresponding adjacent normal tissue. We used RT-PCR to screen for expression of a panel of developmental transcription factors implicated in GI or NE differentiation and looked for factors differentially expressed in tumor and normal tissues. We also assayed for expression of ligands, receptors, and downstream effectors of the Notch signaling pathway, known to negatively regulate GI endocrine differentiation in fetal development. Transcripts for the bHLH factor Ascl1 were detected in seven of 10 carcinoid tumor samples by RT-PCR in a variety of foregut and midgut primary sites (Fig. 1A
, top line). ASCL1 protein was detected by immunoblot in four of four GI carcinoid specimens (Fig. 1B). In contrast to Ascl1, other proneural bHLH transcription factors, NeuroD1, Ngn1, Ngn2, and Ngn3 as well as the LIM homeodomain transcription factor islet-1 (Isl1), were not specifically expressed in primary carcinoid tumors (Fig. 1A). RT-PCR sensitivity was evaluated using human fetal brain cDNA, (or human fetal pancreas cDNA in the case of Ngn3) as positive controls for bHLH gene expression. In addition, we used the human pancreatic cancer cell line BxPC-3 as a positive control for Notch pathway component expression. We conclude that Ascl1 is specifically over-expressed in a majority of GI carcinoid tumors compared with surrounding normal tissues, and that NeuroD1, Ngn1, -2, and -3, and Isl1 are not.
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). Dll3 was expressed strongly in fetal brain and weakly in four of 10 carcinoid tumor specimens. To screen for Notch pathway signaling activity in carcinoid tumors, we analyzed transcripts for the four Hes-related Notch effectors. We found evidence for Hes1 expression, suggesting active Notch signaling, in all of the tested samples. Hey1, Hey2, and HeyL also appeared widely expressed.
These expression data suggest that the proneural transcription factor Ascl1 is differentially expressed in human GI carcinoid tumors compared with normal gut and that the counterregulating Notch pathway also may be active in these tumors and surrounding tissues. To model the interactions of Ascl1 and Notch in carcinoid tumors, we first tested expression levels in the BON pancreatic carcinoid cell line. We found that BON cells had detectable mRNA for Ascl1, NeuroD1, and Ngn2 but trace or negative levels for Ngn1, Ngn3, and Isl1 (Fig. 1A). The levels of ASCL1 protein in BON cells by immunoblot were similar to those seen in the bronchial carcinoid cell line NCI-H727, whereas two pancreatic adenocarcinoma cell lines, MIA PaCa-2 and BxPC-3, were negative (Fig. 1B). In addition, BON cells expressed detectable levels of the tested Notch components at the mRNA level.
Activated Notch1 inhibits Ascl1 mRNA and protein in BON cells
We explored the effect of Notch pathway activation on Ascl1 expression. Endogenous Notch1 protein levels in BON cells was not readily detectable by immunoblot (Fig. 2A). Infection with an adenovirus expressing activated (cleaved) Notch1, termed Ad.Notch1, resulted in the accumulation of Notch protein levels over 4 d (Fig. 2A). Whereas low-level Hes1 expression could be observed in untreated BON cells, this Notch effector was strongly induced by Ad.Notch1 (Fig. 2A). Impressively, Ad.Notch1 also led to a complete loss of Ascl1 mRNA and protein by 1 d after infection (Fig. 2, A and B). Thus, BON cells are competent to respond to activated Notch, which caused a loss of ASCL1 protein reminiscent of the action of Notch in repressing the expression of bHLH transcription factors during pancreatic and gut endocrine cell differentiation (15, 18, 19).
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We examined whether Notch could inhibit the expression of general NE markers in BON cells. NSE protein levels in BON cells progressively declined over the course of 4 d after Notch1 infection (Fig. 3). More strikingly, by 1 d after Notch1 infection, expression of Syn and CgA were dramatically reduced with near complete absence of signal at 3 d post infection compared with infection with a control ß-galactosidase adenovirus (Fig. 3). Thus, it appears that active Notch signaling may decrease NE differentiation in this carcinoid tumor cell model.
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Because serotonin is an autocrine growth factor for BON cells and is implicated in the pathogenesis of the carcinoid syndrome, we evaluated the effect of Notch signaling on serotonin production in BON cells. Serotonin concentration was dramatically decreased in the supernatant of Notch1-infected cells compared with cells infected with the control virus (Fig. 4A). On d 7 after infection, overexpression of Notch1 led to an 89% decrease in serotonin concentration measured in the supernatant of BON cells by ELISA. In addition, indirect immunofluorescence showed that Notch1 infection reduced the number of serotonin-reactive cells by 86% (Fig. 4B). The decrease in production of serotonin in BON cells overexpressing Notch1 was associated with a 1.8- to 2.6-fold repression in tryptophan hydroxylase 1 mRNA expression (Fig. 4C). We conclude that Notch signaling can reduce serotonin production in this carcinoid model system, at least in part mediated by a reduction in steady-state mRNA expression of the rate-limiting enzyme in serotonin biosynthesis.
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We examined the effect of Notch activation on BON cell growth. Mock- and ß-galactosidase-infected BON cells had logarithmic growth over the course of 7 d (Fig. 5). In contrast, overexpression of Notch1 moderately inhibited BON cell growth. On d 5 and 7 after infection, cells overexpressing Notch1 were reduced by 60 and 40%, respectively, compared with mock- or ß-galactosidase-infected BON cells.
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Discussion |
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The evolutionarily conserved Notch signaling pathway negatively regulates proneural bHLH genes during Drosophila and mammalian development (27). Moreover, a process resembling Notch-mediated lateral inhibition operates in endodermal endocrine differentiation in mammals, keeping the endocrine cell fate in check and allowing normal GI organogenesis to proceed (18, 19). In this study, we have shown that the bHLH transcription factor Ascl1, which plays critical roles in mammalian neural development (28) and NE differentiation of normal as well as neoplastic lung (14), is specifically expressed in human foregut and midgut carcinoid tumors and in GI carcinoid BON cells. In addition, we have found that overexpression of an active form of Notch1 led to up-regulation of Hes1, silencing of Ascl1, and down-regulation of both general and cell-type-specific NE markers in GI carcinoid tumor cells (Fig. 6). We observed significant reductions in serotonin accumulation into the culture medium and a corresponding decline in BON cell serotonin immunofluorescent staining. Although this effect may be mediated in part by down-regulation of tryptophan hydroxylase 1 mRNA, multiple levels of regulation of serotonin biosynthesis and secretion could be altered by Notch signaling. Because we have detected the expression of multiple components of the Notch signaling pathway in primary GI carcinoid tumors, it is possible that these tumors may also be susceptible to negative regulation of NE differentiation by Notch activation.
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Similar growth-inhibitory effects caused by Notch1 overexpression have been reported in other contexts. Like BON cells, small-cell lung cancer (SCLC) cell lines constitutively express Ascl1 and display characteristic NE features (14, 35). Moreover, Ascl1 is essential for maintenance of the NE phenotype in SCLC cells. Overexpression of Notch1 in SCLC cell lines led to the extinction of Ascl1 expression as well as a G1 cell-cycle block and growth arrest mediated by increased p21 and p27 (36). Indistinguishable growth curves for mock- and Ad.ßGal-infected BON cells argues against a nonspecific viral effect on BON cell proliferation.
Others have shown that truncated forms of Notch1 and Notch4 formed by chromosomal translocations or insertion of mouse mammary tumor virus, respectively, have been associated with T-cell acute lymphoblastic leukemias and lymphomas (Notch1) (37, 38) and mouse mammary tumors (Notch4) (39). Interestingly, Notch promotes growth arrest and apoptosis of B cell precursors but proliferation of T cell lineages during lymphoid development (40). Thus, the effects of Notch signaling on cell growth are highly context dependent. However, it appears that in the setting of cancers with NE features in which proneural bHLH transcription factors may figure prominently in growth, Notch signaling frequently leads to growth inhibition.
Approximately 10–18% of patients with carcinoid tumors will develop the carcinoid syndrome (3). The presence of the carcinoid syndrome typically reflects advanced disease, with a heavy tumor burden, active synthesis of serotonin and its precursors, and access to the systemic circulation, often via liver metastases. Although aggressive surgical resection of regional spread and limited hepatic metastases may prolong survival in carefully selected patients, the results of cytotoxic chemotherapy and somatostatin analogs have been relatively disappointing (6). Our findings could provide a molecular basis for potential strategies that activate Notch signaling in GI carcinoid tumors to treat the carcinoid syndrome.
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Acknowledgments |
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Footnotes |
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First Published Online May 3, 2005
1 H.C. and D.W.B. share senior authorship. 
Abbreviations: Ad., Adenoviral construct; Ascl1, achaete-scute complex homolog-like 1; bHLH, basic helix-loop-helix; CgA, chromogranin A; Dll1, delta-like 1; ßGal, ß-galactosidase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; GI, gastrointestinal; Hes1, hairy and enhancer of split 1; Hey1, hairy/enhancer-of-split related with YRPW motif 1; Isl1, islet-1; moi, multiplicity of infection; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NE, neuroendocrine; NeuroD1, neurogenic differentiation 1; Ngn, neurogenin; NSE, neuron-specific enolase; RPA, ribonuclease protection assay; SCLC, small-cell lung cancer; Syn, synaptophysin.
Received March 10, 2005.
Accepted April 26, 2005.
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q signaling pathways and 5-hydroxytryptamine release from human carcinoid BON cells. J Clin Invest 108:1051–1059[CrossRef][Medline]
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