Gene Network Inference and Biochemical Assessment Delineates GPCR Pathways and CREB Targets in Small Intestinal Neuroendocrine Neoplasia

Ignat Drozdov,Shrikant Mane,Roswitha Pfragner,Bjorn I Gustafsson,Mark Kidd,Bernhard Svejda,Irvin M Modlin,Hava Karsenty Avraham

doi:10.1371/journal.pone.0022457

Ignat Drozdov, Shrikant Mane + Show 6 more

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https://doi.org/10.1371/journal.pone.0022457

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Abstract

Small intestinal (SI) neuroendocrine tumors (NET) are increasing in incidence, however little is known about their biology. High throughput techniques such as inference of gene regulatory networks from microarray experiments can objectively define signaling machinery in this disease. Genome-wide co-expression analysis was used to infer gene relevance network in SI-NETs. The network was confirmed to be non-random, scale-free, and highly modular. Functional analysis of gene co-expression modules revealed processes including ‘Nervous system development’, ‘Immune response’, and ‘Cell-cycle’. Importantly, gene network topology and differential expression analysis identified over-expression of the GPCR signaling regulators, the cAMP synthetase, ADCY2, and the protein kinase A, PRKAR1A. Seven CREB response element (CRE) transcripts associated with proliferation and secretion: BEX1, BICD1, CHGB, CPE, GABRB3, SCG2 and SCG3 as well as ADCY2 and PRKAR1A were measured in an independent SI dataset (n = 10 NETs; n = 8 normal preparations). All were up-regulated (p<0.035) with the exception of SCG3 which was not differently expressed. Forskolin (a direct cAMP activator, 10−5 M) significantly stimulated transcription of pCREB and 3/7 CREB targets, isoproterenol (a selective ß-adrenergic receptor agonist and cAMP activator, 10−5 M) stimulated pCREB and 4/7 targets while BIM-53061 (a dopamine D2 and Serotonin [5-HT2] receptor agonist, 10−6 M) stimulated 100% of targets as well as pCREB; CRE transcription correlated with the levels of cAMP accumulation and PKA activity; BIM-53061 stimulated the highest levels of cAMP and PKA (2.8-fold and 2.5-fold vs. 1.8–2-fold for isoproterenol and forskolin). Gene network inference and graph topology analysis in SI NETs suggests that SI NETs express neural GPCRs that activate different CRE targets associated with proliferation and secretion. In vitro studies, in a model NET cell system, confirmed that transcriptional effects are signaled through the cAMP/PKA/pCREB signaling pathway and that a SI NET cell line was most sensitive to a D2 and 5-HT2 receptor agonist BIM-53061.

Highlights

Neuroendocrine or ‘‘carcinoid’’ tumors of the gut, usually misperceived as a rare, indolent neoplasia, have not rigorously been studied, are poorly understood and often misdiagnosed [1]
Since little is known about neoplastic EC cell transcription and proliferation or secretion, we considered that delineation of the molecular basis of GPCRmediated transcription through cAMP/protein kinase A (PKA)/cyclic AMP responsive element-binding (CREB) would provide novel information regarding the mechanistic basis of these processes and facilitate the identification of new therapeutic targets that might be used to inhibit neuroendocrine tumors (NET) function
Our results provide novel information regarding the transcription of CREB response elements (CREs) known to be relevant to tumor proliferation and secretion that are activated by G-protein coupled receptors (GPCRs) regulation of intracellular cAMP

Summary

Introduction

Neuroendocrine or ‘‘carcinoid’’ tumors of the gut, usually misperceived as a rare, indolent neoplasia, have not rigorously been studied, are poorly understood and often misdiagnosed [1]. The perception that these tumors are rare has been altered by introduction of diagnostic strategies including endoscopy, the measurement of plasma biochemical markers such as Chromogranin A, and nuclear medicine techniques, including somatostatin receptor scintigraphy (SRS) [2]. In the event of liver metastases, bioactive tumor products enter the systemic circulation, bypassing hepatic inactivation, and engender a ‘carcinoid syndrome’ This consists of a variety of symptoms including episodic skin flushing, diarrhea, bronchoconstriction, sweating and abdominal cramping, and as many as 30–50% of individuals may have cardiac valvular disease [5]. The most successful therapy, to date, has been somatostatin analogs which activate inhibitory G-protein coupled receptors (GPCRs) and result in decreased secretion of bioactive products with concomitant amelioration of symptoms [10,11,12]

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