Ghrelin system comprises a complex family of peptides, receptors (GHSRs), and modifying enzymes [e.g. ghrelin-O-acyl-transferase (GOAT)] that control multiple pathophysiological processes. Aberrant alternative splicing is an emerging cancer hallmark that generates altered proteins with tumorigenic capacity. Indeed, In1-ghrelin and truncated-GHSR1b splicing variants can promote development/progression of certain endocrine-related cancers. Here, we determined the expression levels of key ghrelin system components in neuroendocrine tumor (NETs) and explored their potential functional role. Twenty-six patients with NETs were prospectively/retrospectively studied [72 samples from primary and metastatic tissues (30 normal/42 tumors)] and clinical data were obtained. The role of In1-ghrelin in aggressiveness was studied in vitro using NET cell lines (BON-1/QGP-1). In1-ghrelin, GOAT and GHSR1a/1b expression levels were elevated in tumoral compared to normal/adjacent tissues. Moreover, In1-ghrelin, GOAT, and GHSR1b expression levels were positively correlated within tumoral, but not within normal/adjacent samples, and were higher in patients with progressive vs. with stable/cured disease. Finally, In1-ghrelin increased aggressiveness (e.g. proliferation/migration) of NET cells. Altogether, our data strongly suggests a potential implication of ghrelin system in the pathogenesis and/or clinical outcome of NETs, and warrant further studies on their possible value for the future development of molecular biomarkers with diagnostic/prognostic/therapeutic value.

The treatment landscape and biologic understanding of neuroendocrine tumors (NET) has shifted dramatically in recent years. Recent studies have shown that somatostatin analogues have the potential not only to control symptoms of hormone hypersecretion but also have the ability to slow tumor growth in patients with advanced carcinoid. The results of clinical trials have further shown that the VEGF pathway inhibitor sunitinib and the mTOR inhibitor everolimus have efficacy in patients with advanced pancreatic NETs. The efficacy of these targeted therapies in NET suggests that the molecular characterization of NETs may provide an avenue to predict both which patients may benefit most from the treatment and to overcome potential drug resistance. Recent genomic studies of NETs have further suggested that pathways regulating chromatin remodeling and epigenetic modification may play a key role in regulating NET growth. These observations offer the potential for new therapeutic and diagnostic advances for patients with NET.

Progress in understanding the dynamics of gene expression has been hampered by lack of a strategy for continuously monitoring this process within normal, living cells. Here, we employed a modification of conventional luciferase technology to make single and repeated real-time measurements of PRL gene expression from individual, living lactotropes from nursing rats. Cells were individually transfected by microinjection with a PRL promoter/luciferase reporter construct. Levels of PRL gene transcription were quantified by photonic imaging in the same cells before and after 24 h of culture in the presence or absence of the dopamine agonist bromocryptine or epidermal growth factor, two well known regulators of PRL gene transcription. We found these cells to be remarkably heterogeneous with respect to basal PRL gene expression and that the degree of activity within a single cell could fluctuate greatly over time under basal culture conditions. Treatment with bromocryptine or epidermal growth factor induced predictable and reversible changes in the average responses observed, yet individual cells displayed marked differences in response to these agents. These findings demonstrate the utility of this paradigm for monitoring dynamics of gene expression within normal, living cells of any type. Moreover, they provide a molecular basis for the secretory heterogeneity and plasticity that have come to be known as hallmarks of lactotrope cell function.

Previous results demonstrate that porcine somatotropes can be separated by density gradient centrifugation into low density (LD) and high density (HD) subpopulations. In rat, two analog somatotrope subpopulations differ morphologically and functionally. In an attempt to determine whether morphological differences were also present within LD and HD porcine somatotropes, we undertook a quantitative electron microscope study of the subcellular organelles of immunoidentified LD and HD somatotropes. In addition, to test for the existence of functional differences, cultures of separated HD and LD subpopulations were treated for 4 h with or without 10 microM GRF-(1-29) and/or 100 microM somatostatin (SRIF), and porcine GH release and intracellular content were evaluated using a homologous enzyme immunoassay. Morphometric results demonstrate that LD somatotropes are smaller in size (P < 0.05) and contain fewer secretory granules (P < 0.05) and more rough endoplasmic reticulum (P < 0.05) than HD somatotropes. In terms of secretion, LD somatotropes showed a classical response; GRF increased GH release 1.7-fold (n = 6; P < 0.05) over the control value, whereas treatment with SRIF alone did not affect basal GH release in this subpopulation, but partially blocked GRF-induced GH release. HD somatotropes responded to GRF with a similar 1.7-fold increase in GH release. However, SRIF administered alone or in combination with GRF exerted a paradoxical stimulatory effect on HD somatotropes (2.15- and 2.12-fold over control value, respectively; n = 6; P < 0.05). These results demonstrate that the porcine somatotrope population is composed of two major subpopulations that display a distinctive pattern of ultrastructural organization and a markedly divergent secretory response to in vitro SRIF treatment.

Purpose: Somatostatin analogues (SSA) are efficacious and safe treatments for a variety of neuroendocrine tumors, especially pituitary neuroendocrine tumors (PitNET). Their therapeutic effects are mainly mediated by somatostatin receptors SST 2 and SST 5 . Most SSAs, such as octreotide/lanreotide/pasireotide, are either nonselective or activate mainly SST 2 . However, nonfunctioning pituitary tumors (NFPTs), the most common PitNET type, mainly express SST 3 and finding peptides that activate this particular somatostatin receptor has been very challenging. Therefore, the main objective of this study was to identify SST 3 -agonists and characterize their effects on experimental NFPT models.Experimental Design: Binding to SSTs and cAMP level determinations were used to screen a peptide library and identify SST 3agonists. Key functional parameters (cell viability/caspase activity/chromogranin-A secretion/mRNA expression/intracellular signaling pathways) were assessed on NFPT primary cell cultures in response to SST 3 -agonists. Tumor growth was assessed in a preclinical PitNET mouse model treated with a SST 3 -agonist.Results: We successfully identified the first SST 3 -agonist peptides. SST 3 -agonists lowered cell viability and chromogranin-A secretion, increased apoptosis in vitro, and reduced tumor growth in a preclinical PitNET model. As expected, inhibition of cell viability in response to SST 3 -agonists defined two NFPT populations: responsive and unresponsive, wherein responsive NFPTs expressed more SST 3 than unresponsive NFPTs and exhibited a profound reduction of MAPK, PI3K-AKT/mTOR, and JAK/STAT signaling pathways upon SST 3 -agonist treatments. Concurrently, SSTR3 silencing increased cell viability in a subset of NFPTs.Conclusions: This study demonstrates that SST 3 -agonists activate signaling mechanisms that reduce NFPT cell viability and inhibit pituitary tumor growth in experimental models that expresses SST 3 , suggesting that targeting this receptor could be an efficacious treatment for NFPTs.

Somatostatin (SRIF) inhibits GH release from rat somatotropes by reducing adenylate cyclase (AC) activity and the free cytosolic calcium concentration ([Ca(2+)](i)). In contrast, we have reported that SRIF can stimulate GH release in vitro from pig somatotropes. Specifically, 10(-7) and 10(-15) M SRIF stimulate GH release from a subpopulation of high density (HD) somatotropes isolated by Percoll gradient centrifugation, whereas in low density (LD) somatotropes only 10(-15) M SRIF induces such an effect. To ascertain the signaling pathways underlying this phenomenon, we assessed SRIF effects on second messengers in cultured LD and HD cells by measuring cAMP, IP turnover, and [Ca(2+)](i). Likewise, contribution of the corresponding signaling pathways to SRIF-induced GH release was evaluated by blocking AC, PLC, extracellular Ca(2+) influx, or intracellular Ca(2+) mobilization. Both 10(-7) and 10(-15) M SRIF increased cAMP, IP turnover, and [Ca(2+)](i) in HD cells. Conversely, in LD cells 10(-7) M SRIF reduced [Ca(2+)](i), but did not alter cAMP or IP, and 10(-15) M SRIF was without effect. Interestingly, SRIF-stimulated GH release was abolished in both subpopulations by AC blockade, but not by PLC inhibition. Furthermore, SRIF-induced GH release was not reduced by blockade of extracellular Ca(2+) influx through voltage-sensitive channels or by depletion of thapsigargin-sensitive intracellular Ca(2+) stores. Therefore, SRIF stimulates GH secretion from cultured porcine somatotrope subpopulations through an AC/cAMP pathway-dependent mechanism that is seemingly independent of net increases in IP turnover or [Ca(2+)](i). These novel actions challenge classic views of SRIF as a mere inhibitor for somatotropes and suggest that it may exert a more complex, dual function in the control of porcine GH release, wherein molecular heterogeneity of somatotropes would play a critical role.

The adipokine resistin is an insulin-antagonizing factor that also plays a regulatory role in inflammation, immunity, food intake, and gonadal function. Although adipose tissue is the primary source of resistin, it is also expressed in other tissues and organs, including the pituitary. However, there is no information on whether resistin, as described previously for other adipokines such as leptin and adiponectin, could regulate this gland. Likewise, the molecular basis of resistin actions remains largely unexplored. Here we show that administration of resistin to dispersed rat anterior pituitary cells increased GH release in both the short (4 h) and long (24 h) term, decreased mRNA levels of the receptor of the somatotrope regulator ghrelin, and increased free cytosolic Ca(2+) concentration in single somatotropes. By means of a pharmacological approach, we found that the stimulatory action of resistin occurs through a Gs protein-dependent mechanism and that the adenylate cyclase/cAMP/protein kinase A pathway, the phosphatidylinositol 3-kinase/Akt pathway, protein kinase C, and extracellular Ca(2+) entry through L-type voltage-sensitive Ca(2+) channels are essential players in mediating the effects of resistin on somatotropes. Taken together, our results demonstrate for the first time a regulatory role for resistin on somatotrope function and provide novel insights on the intracellular mechanisms activated by this protein.

Progress in understanding the dynamics of gene expression has been hampered by lack of a strategy for continuously monitoring this process within normal, living cells. Here, we employed a modification of conventional luciferase technology to make single and repeated real-time measurements of PRL gene expression from individual, living lactotropes from nursing rats. Cells were individually transfected by microinjection with a PRL promoter/luciferase reporter construct. Levels of PRL gene transcription were quantified by photonic imaging in the same cells before and after 24 h of culture in the presence or absence of the dopamine agonist bromocryptine or epidermal growth factor, two well known regulators of PRL gene transcription. We found these cells to be remarkably heterogeneous with respect to basal PRL gene expression and that the degree of activity within a single cell could fluctuate greatly over time under basal culture conditions. Treatment with bromocryptine or epidermal growth factor induced predictable and reversible changes in the average responses observed, yet individual cells displayed marked differences in response to these agents. These findings demonstrate the utility of this paradigm for monitoring dynamics of gene expression within normal, living cells of any type. Moreover, they provide a molecular basis for the secretory heterogeneity and plasticity that have come to be known as hallmarks of lactotrope cell function.