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The gastrointestinal tract and pancreas exhibit ~17 different neuroendocrine cell types, but neither the cell of origin nor the biological basis of GEP-NETs is understood. This review examines GEP-NETs from the cellular and molecular perspective and addresses the distinct patterns of functional tumor biology pertinent to clinicians. Although grouped as a neoplastic entity (NETs), each lesion is derived from distinct cell precursors, produces specific bioactive products, exhibits distinct chromosomal abnormalities and somatic mutation events and has uniquely dissimilar clinical presentations. GEP-NETs demonstrate very different survival rates reflecting the intrinsic differences in malignant potential and variations in proliferative regulation. Apart from the identification of the inhibitory role of the somatostatin receptors, there is limited biological knowledge of the key regulators of proliferation and hence a paucity of successful targeted therapeutic agents. IGF-I, TGFβ and a variety of tyrosine kinases have been postulated as key regulatory elements; rigorous data is still required to define predictably effective and rational therapeutic strategy in an individual tumor. A critical issue in the clinical management of GEP-NETs is the need to appreciate both the neuroendocrine commonalities of the disease as well as the unique characteristics of each tumor. The further acquisition of a detailed biological and molecular appreciation of GEP-NETs is vital to the development of effective management strategy.

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BACKGROUND: 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. METHODS: The effects of the somatostatin analog octreotide (OCT), the mTOR inhibitor RAD001 (RAD), or the combination were evaluated in SINET cell lines (KRJ‐I, H‐STS) using cell viability assays, western blotting, enzyme‐linked immunosorbent assay, and reverse‐transcription polymerase chain reaction to assess antiproliferative signaling pathways and feedback regulation. RESULTS: RAD (10−9 M) incompletely decreased cell viability (−40% to +15%); growth escape (P < .001) was noted at 72 hours in both cell lines. Phosphorylated (p)mTOR/mTOR and pp70S6K/p70S6K ratios were decreased but were associated with increases in phosphorylated extracellular signal‐regulated kinase (pERK)/ERK and pAKT/AKT in both cell lines, whereas phosphorylated insulin‐like growth factor 1 receptor (pIGF‐1R)/IGF‐1R levels were elevated only in H‐STS cells. Increased (P < .05) transcript levels for AKT1, MAPK, mTOR, IGF‐1R, IGF‐1, and TGFβ1 were evident. OCT (10−6 M) itself had no significant effect on growth signaling in either cell line. An antiproliferative effect (66 ± 5%) using OCT+RAD was only noted in the KRJ‐I cells (P < .05). CONCLUSIONS: SINET treatment with the mTOR inhibitor RAD had no antiproliferative effect based on activation of pAKT and pERK1/2. A combinatorial approach using OCT and RAD failed to overcome this escape phenomenon. However, differences in RAD response rates in individual NET cell lines suggested that pretreatment identification of different tumor sensitivity to mTOR inhibitors could provide the basis for individualized treatment. Cancer 2011;. © 2011 American Cancer Society.

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.

BACKGROUND: 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-HT 2B receptors on tumor cells would inhibit SI-NET 5-HT release and, thereby, fibroblast activation in the tumor microenvironment. METHODS: We studied the 5-HT 2B receptor antagonist PRX-08066 in NET cell lines (KRJ-I, H720) and in the coculture system (KRJ-I cells: fibroblastic HEK293 cells) using real time polymerase chain reaction, ELISA, Ki67 immunostaining, and flow cytometry-based caspase 3 assays to assess antiproliferative and profibrotic signaling pathways. RESULTS: In the 5-HT 2B expressing SI-NET cell line, KRJ-I, PRX-08066 inhibited proliferation (IC 50 4.6x10 À9 M) and 5-HT secretion (6.9 Â 10 À9 M) and decreased ERK1/2 phosphorylation and profibrotic growth factor synthesis and secretion (transforming growth factor beta 1 [TGFb1], connective tissue growth factor [CTGF] and fibroblast growth factor [FGF2]). In the KRJ-I:HEK293 coculture system, PRX-08066 significantly decreased 5-HT release (>60%), Ki67 (transcript and immunostaining: 20%-80%), TGFb1, CTGF, and FGF2 transcription (20%-50%) in the KRJ-I cell line. 5-HT itself stimulated HEK293 proliferation (25%) and synthesis of TGFb1, CTGF and FGF2. PRX-08066 inhibition of KRJ-I function reversed these effects in the coculture system. CONCLUSIONS: Targeting the 5-HT 2B receptor may be an effective antiproliferative and antifibrotic strategy for SI-NETs because it inhibits tumor microenvironment fibroblasts as well as NET cells. Fibrosis and proliferation appear to be biologically interfaced neuroendocrine neoplasia domains. Cancer 2010;116:2902-12.

Neuroendocrine tumors (NETs) are a heterogeneous group of cancers of which the commonest site is the small intestine (SI). Most information available to determine tumor behavior reflects univariate assessment of factors or is anecdotal or experience based. There currently exists no objective multivariate analysis of indices that defines SI NET prognosis. A key unmet need is the lack of a rigorous mathematical-based tool – a nomogram – for the assessment of parameters that define progress, determine prognosis and can guide therapy. Since prediction of NET behavior is a critical criterion in determining clinical strategy, we constructed a NET nomogram (Modlin Score) for prognosis prediction, patient group comparisons and a guide for stratification of treatment and surveillance. We used hazard ratio (HR), Cox analysis and Kaplan-Meier analysis of published data and the current Surveillance, Epidemiology and End Results (SEER) database (approx. 20,000 patients) to develop a nomogram from 15 variables demonstrated to provide significant multivariate HRs. These included age, gender, ethnicity, symptoms, urinary 5-hydroxyindoleacetic acid, plasma chromogranin A, liver function tests, tumor size, invasion, metastasis, histology, Ki-67 index, carcinoid heart disease and therapy (surgery or long-acting somatostatin analogs). Internal validation was assessed using 33 SI NET patients. A NET nomoscore (Modlin Score) was developed by HR weighting and stratification into low (<75), medium (75–95) and high risk (>95). This identified significant differences (p <0.03, Kaplan-Meier) in survival (15.5 ± 4.3, 9.7 ± 2.5 and 6.4 ± 1.1 years, respectively). The Modlin Score was significantly elevated (p <0.01) in deceased compared to alive patients. This nomogram represents an optimized construct based upon currently analyzable data, and application will facilitate accurate stratification for comparison in clinical trials. External validation and amplification by identification of additional indices, e.g. molecular biomarkers, are necessary. The development of a mathematically validated nomogram provides a platform for objective assessment of SI NET disease, a finite basis for precise prognostication and a tool to guide management strategy.

Introduction-Survival rates for gastrointestinal (GI) and bronchopulmonary (BP) neuroendocrine tumors (NETs) have not significantly altered (5yr survival: 64.1% and 87-89%) in thirty years (1973-2004). No effective or specific anti-neoplastic agent(s) is available although somatostatin analogs inhibit NET serotonin (5-HT) secretion. Given the expression of 5-HT receptors on NETs, we hypothesized that 5-HT autoregulated NET proliferation. Methods-Proliferation was evaluated in three NET cell lines using MTT uptake while real-time PCR and ELISA studies were performed to delineate 5-HT-mediated signaling pathways. To determine the receptor and role of endogenous 5-HT production, the effects of ketanserin (5-HT 2A/C receptor antagonist), ondansetron (5-HT 3 antagonist) and the suicide inhibitor 7-Hydroxytryptophan (7-HTP) were investigated. Results-Exogenously added 5-HT stimulated proliferation in the atypical BP-NET, NCI-H720 (+50%, EC 50 =10nM), the typical BP-NET, NCI-H727 (+40%, EC 50 =0.01nM), and the GI-NET, KRJ-I (+60%, EC 50 =25nM). In NCI-H720, proliferation was inhibited by ketanserin (IC 50 =0.06nM) and ondansetron (IC 50 =0.4nM) as well as 7-HTP (IC 50 =0.3nM). In NCI-H727, ketanserin and 7-HTP inhibited proliferation (IC 50 =0.3nM and 2.3nM respectively) while ondansetron had no effect. In KRJ-I, ketanserin (IC 50 =0.1nM) and 7-HTP (IC 50 =0.6nM) but not ondansetron inhibited proliferation. In all cell lines, 5-HT activated proliferation through ERK1/2 phosphorylation and JNK-mediated pathways (c-JUN and Ki67 transcription). An auto-regulatory effect was indicated by 7-HTP-mediated inhibition of extracellular 5-HT and downstream effects on NET proliferation. Conclusions-Lung and GI-NET proliferation is autoregulated by 5-HT through alterations in ERK and JNK signaling. Targeting NET cells with 5-HT2 receptor antagonists and 7-HTP reversed proliferation. 5-HT 2 receptor subtype-specific antagonists may represent a viable antiproliferative therapeutic strategy.