Erythropoietin-producing hepatocellular receptor B6 is highly expressed in non-functioning pituitary neuroendocrine tumors and its expression correlates with tumor size.

BACKGROUND: Erythropoietin-producing hepatocellular (EPH) receptors are the largest known family of receptor tyrosine kinases characterized in humans. These proteins are involved in tissue organization, synaptic plasticity, vascular development and the progression of various diseases including cancer. The Erythropoietin-producing hepatocellular receptor tyrosine kinase member EphB6 is a pseudokinase which has not attracted an equivalent amount of interest as its enzymatically-active counterparts. The aim of this study was to assess the expression of EphB6 in pituitary tumors. METHODS AND RESULTS: Human normal pituitaries and pituitary tumors were examined for EphB6 mRNA expression using real-time PCR and for EphB6 protein by immunohistochemistry and Western blotting. EphB6 was highly expressed in non-functioning pituitary neuroendocrine tumors (NF-PitNETs) versus the normal pituitary and GH-secreting PitNETs. EphB6 mRNA expression was correlated with tumor size. CONCLUSIONS: Our results suggest EphB6 aberrant expression in NF-PitNETs. Future studies are warranted to determine the role and significance of EphB6 in NF-PitNETs tumorigenesis.

Antibody array strategy for human growth factor secretome profiling of GH-secreting adenomas.

PURPOSES: To test if the antibody array strategy could be utilized to simultaneously detect the secretion of multiple growth factors by human pituitary GH-adenomas and to measure octreotide-induced alterations. METHODS: Specimens of human pituitary adenomas were cultured and incubated with or without octreotide for 24 h. Conditional media were analyzed by human growth factor antibody array and VEGF concentrations were measured by ELISA. Media were also analyzed for GH concentrations. p21 expression levels were examined by Western blot of the specimens lysates. RESULTS: The antibody arrays successfully identified growth factors secreted by GH-adenomas in vitro. Octreotide treatment induced both elevations and reductions in growth factors secretion. GH response to octreotide was measured, and in this small-sized study resistant and sensitive GH-adenomas presented with no unique secretome pattern of each of the groups. Octreotide-induced VEGF alterations analyzed by the antibody array and by ELISA were not fully matched. CONCLUSIONS: This study suggests that the broad proteomic strategy of antibody arrays may be utilized to study the growth factors secretion pattern of GH-adenomas and its regulation by somatostatin analogs or other compounds.

Combination of mTOR Inhibitors Augments Potency while Activating PI3K Signaling in Pituitary Tumors.

BACKGROUND: Despite the success in treating some cancers, the efficacy of the mTOR inhibitors rapalogs as anti-cancer therapeutics has been limited. AIMS: We undertook to examine the effects of Torin1, a second-generation selective ATP-competitive mTOR inhibitor, in non-functioning pituitary tumor cells. During characterization of the molecular mechanisms that mediate Torin1 actions, there seemed to be a rationale for combining it with rapalogs. METHODS: Proliferation assays, flow cytometry and Western blotting were applied to assess the effects of Torin1, RAD001 and their combination on an MtT/E pituitary cell line and human-derived non-functioning pituitary tumor cells. RESULTS: Combined long treatments of Torin1 and RAD001 induced a pronounced reduction in cell growth and viability of both MtT/E pituitary cells and human-derived non-functioning pituitary tumor cells, superior to each drug alone. This was remarkable in the 10 nM combination and was reflected in a triggered decrease of cyclin D3 and p21/CIP expression. Interestingly, Akt-Thr308 and SIN1-Thr86 phosphorylations were robustly elevated in the combined treatment, accompanied by a reduction in PTEN expression. Phosphorylation of p70S6K was abolished in all individual and combined treatments. Akt-Ser473 phosphorylation, induced by RAD001, was reduced by the combined treatment to the same extent as when treated by Torin1 alone. CONCLUSIONS: Our results suggest that the differential signaling mechanisms induced by these compounds eventually converge to lead to an efficient blockade of the PI3K/Akt/mTOR pathway in pituitary tumor cells and may facilitate a reduction in treatment dosage.

The aging suppressor klotho: a potential regulator of growth hormone secretion.

Klotho is a transmembranal protein highly expressed in the kidneys, choroid plexus, and anterior pituitary. Klotho can also be cleaved and shed and acts as a circulating hormone. Klotho-deficient mice (kl/kl mice) develop a phenotype resembling early aging. Several lines of evidence suggest a role for klotho in the regulation of growth hormone (GH) secretion. The kl/kl mice are smaller compared with their wild-type counterparts, and their somatotropes show reduced numbers of secretory granules. Moreover, klotho is a potent inhibitor of the IGF-I pathway, a negative regulator of GH secretion. Therefore, we hypothesized that klotho may enhance GH secretion. The effect of klotho on GH secretion was examined in GH3 rat somatotrophs, cultured rat pituitaries, and cultured human GH-secreting adenomas. In all three models, klotho treatment increased GH secretion. Prolonged treatment of mice with intraperitoneal klotho injections increased mRNA levels of IGF-I and IGF-I-binding protein-3 mRNA in the liver, reflecting increased serum GH levels. In accord with its ability to inhibit the IGF-I pathway, klotho partially restored the inhibitory effect of IGF-I on GH secretion. Klotho is known to be a positive regulator of basic bFGF signaling. We studied rat pituitaries and human adenoma cultures and noted that bFGF increased GH secretion and stimulated ERK1/2 phosphorylation. Both effects were augmented following treatment with klotho. Taken together, our data indicate for the first time that klotho is a positive regulator of GH secretion and suggest the IGF-I and bFGF pathways as potential mediators of this effect.

The role of cell lines in the study of neuroendocrine tumors.

Cell lines originating from neuroendocrine tumors (NETs) represent useful experimental models to assess the control of synthesis and release of different hormones and hormone-like peptides, to evaluate the mechanisms of action of these agents in target tissues at the cellular and subcellular levels, and to study cell proliferation and tumor development, as well as the effect of different drugs on these complex processes. To date, the understanding of NET biology (with regard to their mechanisms of hormone secretion, cell proliferation and metastatic spread) has been hampered by the lack of appropriate animal models or cell lines for their study. In the present review, we aim to summarize the recent in vitro/in vivo data regarding cell lines derived from NETs which are most frequently employed in experimental neuroendocrinology.

PICT-1 regulates p53 splicing and sensitivity of medullary thyroid carcinoma cells to everolimus.

Protein interacting with carboxyl terminus 1 (PICT-1) is a nucleolar protein shown to act as a tumor suppressor that interacts with PTEN, or in a contrasting manner to facilitate the accessibility of p53 to ubiquitination and degradation, thus to function as an oncogene. The aim of the study was to examine the potential role of PICT-1 in neuroendocrine neoplasm (NEN) tumorigenesis and response to mTOR inhibitor treatment. PICT-1 was overexpressed in medullary thyroid (TT) and pancreatic (BON1) NEN cell lines using lentiviral vector. Whereas in BON1 cells PICT-1 overexpression exhibited no significant impact, in TT cells it induced the appearance of p53ß lacking the C-terminus end. This was accompanied by a robust decrease in p21 expression and elevation of cell viability. Remarkably, PICT-1 overexpression completely reversed the reduction in cell viability of medullary thyroid neoplasm cells induced by everolimus, a therapeutic option for patients with progressive NENs. mTOR pathway investigations revealed that PICT-1 overexpression induced a reduction in PTEN expression and a robust increase in the expression level of phospho-Akt-Ser47 only partially inhibited by everolimus. These findings suggest a possible role of PICT-1 in the spliceosome machinery and provide functional involvement of PICT-1 in the complex network of mTOR.

Cortistatin inhibits growth hormone release from human fetal and adenoma pituitary cells and prolactin secretion from cultured prolactinomas.

CONTEXT: Cortistatin (CST) is a neuropeptide that shares high homology with somatostatin and binds with high affinity to all somatostatin receptor (SSTR) subtypes. Many of its endocrine and biological activities overlap with those of somatostatin. OBJECTIVE/DESIGN: The objective of the study was to assess the direct in vitro effects of CST on human pituitary hormone secretion. SETTING: This study was performed in the endocrine laboratory of a tertiary academic medical center. MATERIALS: Primary cell cultures of human fetal (21-25 wk gestation) pituitary tissues and cultured hormone-secreting adenoma cells were used in this study. INTERVENTIONS: Cell cultures were incubated with CST-14 or CST-17, somatostatin, GHRH, SSTR analogs, and ghrelin analogs, and hormone secretion was analyzed. OUTCOME MEASURES: GH and prolactin (PRL) medium concentrations were tested by hormone assay, and SSTR mRNA was tested by RT-PCR. RESULTS: CST-14 (10 nm) inhibited GH secretion by up to 65% in all fetal pituitary specimens after 4-h incubation (P < 0.05). CST-14 or CST-17 (10 nm) inhibited basal GH secretion in six of the 13 GH-cell adenomas and two of the three GH-PRL mixed adenomas. CST-17 (100 nm) suppressed the GH response to GHRH and ghrelin analog (10 nm each) by 30-50% in adenomas (P < 0.05). Three PRL-adenomas treated with CST-17 (10 nm) showed a 20-40% inhibition of PRL release (P < 0.05), whereas in three others no suppression or mild response was achieved at this concentration. A comparable inhibition of PRL secretion was obtained with SSTR5-selective analog but significantly less with SSTR2-preferential compounds. RT-PCR revealed the expression of both SSTR2 and SSTR5 in all GH-cell and mixed adenomas studied and all PRL-secreting adenomas studied, except for two of the CST-resistant prolactinomas, in which SSTR5 was absent. CONCLUSIONS: This is the first report of in vitro CST suppression of human GH and PRL in cultured pituitary tissues. The regulation of PRL release from cultured adenomas appears to be primarily mediated by SSTR5.

Cortistatin - a new neuroendocrine hormone?

Cortistatin (CST) is a recently described neuropeptide. Although the expression of CST was initially reported to be restricted to the rat cerebral cortex and hippocampus, it is currently known to have wide distribution in many human organs. CST shares high homology with somatostatin and binds with high affinity to all somatostatin receptor (SSTR) subtypes. Indeed, many of its endocrine and biological activities overlap with somatostatin. However, CST and somatostatin are expressed in only partially overlapping cortical neuron populations. They are regulated by different stimuli, their cortical actions are diverse and CST, at variance with somatostatin, was shown to have a possible role in the human immune system. The activities specific to cortistatin may be attributable to its unique binding to the GH secretagogue (GHS) receptor (GHS-R) and the orphan G-protein-coupled receptor, MrgX2, which is not shared by somatostatin. The potential biological roles and therapeutic use of CST and the receptors involved in its action require further investigation.

pNET co-secreting GHRH and calcitonin: ex vivo hormonal studies in human pituitary cells.

UNLABELLED: Acromegaly due to ectopic GHRH secretion from a neuroendocrine tumor (NET) is rare and comprises <1% of all acromegaly cases. Herein we present a 57-year-old woman with clinical and biochemical features of acromegaly and a 6 cm pancreatic NET (pNET), secreting GHRH and calcitonin. Following surgical resection of the pancreatic tumor, IGF1, GH and calcitonin normalized, and the clinical features of acromegaly improved. In vitro studies confirmed that the tumor secreted large amounts of both GHRH and calcitonin, and incubation of pNET culture-derived conditioned media stimulated GH release from a cultured human pituitary adenoma. This is a unique case of pNET secreting both GHRH and calcitonin. The ability of the pNET-derived medium to stimulate in vitro GH release from a human pituitary-cell culture, combined with the clinical and hormonal remission following tumor resection, confirmed the ectopic source of acromegaly in this patient. LEARNING POINTS: Signs, symptoms and initial work-up of acromegaly due to ectopic GHRH secretion are similar to pituitary-dependent acromegaly. However, if no identifiable pituitary lesion is found, somatostatin receptor scan and further imaging (CT, MRI) should be performed.Detection of GHRH in the blood and in the tumor-derived medium supports the diagnosis of ectopic GHRH secretion.Functional bioactivity of pNET-secreted GHRH can be proved in vitro by releasing GH from human pituitary cells.

The ERK cascade: a prototype of MAPK signaling.

Sequential activation of protein kinases within the mitogen-activated protein kinase (MAPK) cascades is a common mechanism of signal transduction in many cellular processes. Four such cascades have been elucidated thus far, and named according to their MAPK tier component as the ERK1/2, JNK, p38MAPK, and ERK5 cascades. These cascades cooperate in transmitting various extracellular signals, and thus control cellular processes such as proliferation, differentiation, development, stress response, and apoptosis. Here we describe the classic ERK1/2 cascade, and concentrate mainly on the properties of MEK1/2 and ERK1/2, including their mode of regulation and their role in various cellular processes and in oncogenesis. This cascade may serve as a prototype of the other MAPK cascades, and the study of this cascade is likely to contribute to the understanding of mitogenic and other processes in many cell lines and tissues.

IGF1 induces cell proliferation in human pituitary tumors - functional blockade of IGF1 receptor as a novel therapeutic approach in non-functioning tumors.

Insulin-like growth factor (IGF1) and its receptor display potent proliferative and antiapoptotic activities and are considered key players in malignancy. The objective of the study was to explore the role of IGF1 and its downstream pathways in the proliferation of non-functioning pituitary tumor cells and to develop a targeted therapeutic approach for the treatment of these tumors. Cultures of human non-functioning pituitary adenomas and the non-secreting immortalized rat pituitary tumor cell line MtT/E were incubated with IGF1, IGF1 receptor inhibitor or both, and cell viability, proliferation and signaling were examined. Our results show that IGF1 elevated cell proliferation and enhanced cell cycle progression as well as the expression of cyclins D1 and D3. IGF1 also induced the phosphorylation of ERK, Akt and p70S6K. On the other hand, the selective IGF1R inhibitor NVP-AEW541 abrogated IGF1-induced cell proliferation as well as IGF1 receptor phosphorylation and downstream signaling.

PI3K/Akt/mTOR and Raf/MEK/ERK signaling pathways perturbations in non-functioning pituitary adenomas.

Non-functioning pituitary adenomas (NFPAs) comprise a heterogeneous group, which are considered the most common pituitary tumor. As no clinically hormone hypersecretion is apparent, non-functioning pituitary adenomas are often diagnosed only when they are large enough to cause tumor mass effects, such as hypopituitarism, visual field defects or headaches. Efficient medical therapy for NFPAs is currently unavailable and surgical treatment of these tumors is not always satisfactory. Characterization of signaling regulatory events in the context of NFPAs may enable the development of new attractive novel strategies. Although data regarding gene expression profiling of signaling pathways in NFPAs have accumulated, studies aimed at fine-classification of NFPAs-specific signaling regulatory mechanisms and feedback loops are scarce.

Heparanase is highly expressed and regulates proliferation in GH-secreting pituitary tumor cells.

Pituitary tumorigenesis involves remodeling of the extracellular matrix (ECM). Heparanase, an endoglycosidase capable of degrading heparan sulfate, a major polysaccharide constituent of the ECM, is implicated in diverse processes associated with ECM remodeling, such as morphogenesis, angiogenesis, and tumor invasion. The aim of this study was to investigate the possible role of heparanase in pituitary tumorigenesis. Human normal pituitaries and pituitary tumors were examined for heparanase mRNA and protein expression using real-time PCR and immunohistochemistry, respectively. Cell proliferation was assessed by colony formation after heparanase overexpression in GH3 and MtT/S cells. Cell viability and cell cycle progression were evaluated after heparanase gene silencing. Higher heparanase mRNA and protein expression was noted in GH tumors as compared with normal pituitaries. Heparanase overexpression in GH3 and MtT/S cells resulted in a 2- to 3-fold increase in colony number, compared with control cells. Cell viability decreased by 50% after heparanase gene silencing due to induced apoptosis reflected by increased fraction of cleaved poly-ADP-ribose polymerase and sub-G1 events. Notably, exogenously added heparanase enhanced epidermal growth factor receptor, Src, Akt, ERK, and p38 phosphorylation in pituitary tumor cells. Our results indicate that heparanase enhances pituitary cell viability and proliferation and may thus contribute to pituitary tumor development and progression.

The rapamycin-derivative RAD001 (everolimus) inhibits cell viability and interacts with the Akt-mTOR-p70S6K pathway in human medullary thyroid carcinoma cells.

Over-expression of the proto-oncogene Akt/PKB has been demonstrated in some neuroendocrine tumor models. Akt may activate downstream proteins such as mTOR and p70S6K, inducing tumor proliferation. The rapamycin-derivative RAD001, everolimus, interacts with this pathway by antagonizing mTOR, but its effects on neuroendocrine tumors are largely unknown. We explored the mechanism of action of RAD001 on cell proliferation, hormonal secretion and on Akt/mTOR/p70S6K pathway activation, in a human medullary thyroid carcinoma (MTC) cell-line (TT) and in cells derived from human MTCs. Treatment with RAD001 significantly inhibited cell viability in a dose- and time-dependent fashion, and diminished phosphorylation of Akt downstream targets, mTOR and p70S6K, in both TT cell-line and cultured human MTCs. Akt phosphorylation was not affected by RAD001. RAD001 induced cell-cycle arrest in the G(0)/G(1) phase in TT cells, but had no effect on apoptosis. Moreover, RAD001 did not affect calcitonin and carcinoembryonic antigen secretion in TT cells and in human MTCs. RAD001 seems to have potent anti-proliferative effect in human MTC cells, which suggest that clinical trials of this agent are of considerable interest.

The somatostatin analogue octreotide confers sensitivity to rapamycin treatment on pituitary tumor cells.

Rapamycin and its analogues have significant antiproliferative action against a variety of tumors. However, sensitivity to rapamycin is reduced by Akt activation that results from the ablative effects of rapamycin on a p70 S6K-induced negative feedback loop that blunts phosphoinositide 3-kinase (PI3K)-mediated support for Akt activity. Thus, sensitivity to rapamycin might be increased by imposing an upstream blockade to the PI3K/Akt pathway. Here, we investigated this model using the somatostatin analogue octreotide as a tool to decrease levels of activated Ser(473)-phosphorylated Akt (pAkt-Ser(473)) in pituitary tumor cells that express somatostatin receptors. Octreotide increased levels of phosphorylated insulin receptor substrate-1 that were suppressed by rapamycin, subsequently decreasing levels of pAkt-Ser(473) through effects on phosphotyrosine phosphatase SHP-1. Octreotide potentiated the antiproliferative effects of rapamycin in immortalized pituitary tumor cells or human nonfunctioning pituitary adenoma cells in primary cell culture, sensitizing tumor cells even to low rapamycin concentrations. Combined treatment of octreotide and rapamycin triggered G(1) cell cycle arrest, decreasing E2F transcriptional activity and cyclin E levels by increasing levels of p27/Kip1. These findings show that adjuvant treatment with a somatostatin analogue can sensitize pituitary tumor cells to the antiproliferative effects of rapamycin.

Mammalian target of rapamycin inhibitors rapamycin and RAD001 (everolimus) induce anti-proliferative effects in GH-secreting pituitary tumor cells in vitro.

The effect of mammalian target of rapamycin (mTOR) inhibitors on pituitary tumors is unknown. Akt overexpression was demonstrated in pituitary adenomas, which may render them sensitive to the anti-proliferative effects of these drugs. The objective of the study was to evaluate the anti-proliferative efficacy of the mTOR inhibitor, rapamycin, and its orally bioavailable analog RAD001 on the GH-secreting pituitary tumor GH3 and MtT/S cells and in human GH-secreting pituitary adenomas (GH-omas) in primary cell cultures. Treatment with rapamycin or RAD001 significantly decreased the number of viable cells and cell proliferation in a dose- and time-dependent manner. This was reflected by decreased phosphorylation levels of the downstream mTOR target p70S6K. Rapamycin treatment of GH3 cells induced G0/G1 cell cycle arrest. In other tumor cell types, this was attributed to a decrease in cyclin D1 levels. However, rapamycin did not affect cyclin D1 protein levels in GH3 cells. By contrast, it decreased cyclin D3 and p21/CIP, which stabilizes cyclin D/cyclin-dependent kinase 4 (cdk4) complexes. Rapamycin inhibited FCS-induced retinoblastoma phosphorylation and subsequent E2F-transcriptional activity. In response to decreased E2F activity, the expression of the E2F-regulated genes cyclin E and cdk2 was reduced. Our results showed that mTOR inhibitors potently inhibit pituitary cell proliferation, suggesting that mTOR inhibition may be a promising anti-proliferative therapy for pituitary adenomas. This therapeutic manipulation may have beneficial effects particularly for patients harboring invasive pituitary tumors resistant to current treatments.

Melanin-concentrating hormone stimulates human growth hormone secretion: a novel effect of MCH on the hypothalamic-pituitary axis.

Melanin-concentrating hormone (MCH), a 19-amino acid orexigenic (appetite-stimulating) hypothalamic peptide, is an important regulator of energy homeostasis. It is cleaved from its precursor prepro-MCH (ppMCH) along with several other neuropeptides whose roles are not fully defined. Because pituitary hormones such as growth hormone (GH), ACTH, and thyroid-stimulating hormone affect body weight and composition, appetite, insulin sensitivity, and lipoprotein metabolism, we investigated whether MCH exerts direct effects on the human pituitary to regulate energy balance using dispersed human fetal pituitaries (21-22 wk gestation) and cultured GH-secreting adenomas. We found that MCH receptor-1 (MCH-R1), but not MCH receptor-2, is expressed in both normal (fetal and adult) human pituitary tissues and in GH cell adenomas. MCH (10 nM) stimulated GH release from human fetal pituitary cultures by up to 62% during a 4-h incubation (P < 0.05). Interestingly, neuropeptide EI (10 nM), which is also cleaved from ppMCH, increased human GH secretion by up to 124% in fetal pituitaries. A milder, albeit significant, induction of GH secretion by MCH (20%) was seen in cultured GH-secreting pituitary adenomas. A comparable stimulation of GH secretion was seen when cultured mouse pituitary cells were treated with MCH. Treatment of cultured GH adenoma cells with MCH (100 nM) induced extracellular signal-regulated kinases 1 and 2 phosphorylation, suggesting activation of MCH-R1. In aggregate, these data suggest that MCH may regulate pituitary GH secretion and imply a potential cross-talk mechanism between appetite-regulating neuropeptides and pituitary hormones.

Nitric oxide stimulates growth hormone secretion from human fetal pituitaries and cultured pituitary adenomas.

Nitric oxide (NO), a highly reactive free radical, has been identified as a neurotransmitter in the central and peripheral nervous system. NO synthase (NOS) is the enzyme responsible for NO production from L-arginine and plays an important role in regulating the release of several hypothalamic peptides. In the pituitary, NO was found to increase growth hormone (GH) secretion in several in vitro and in vivomodels. However, its role in human GH regulation is unknown. The aim of this study was to investigate the regulatory effects of NO on human GH and prolactin secretion using primary cell cultures of human fetal pituitaries and cultured hormone-secreting adenomas. Incubation of the human fetal pituitaries (21-24 wk gestation) in the presence of sodium nitroprusside (SNP; 1 mM), a NO donor, for 4 h resulted in a 50-75% increase in GH secretion, similar to the stimulatory effect evoked by growth hormone-releasing hormone (GHRH) (10 nM). However, fetal PRL secretion was not affected by SNP. GH release was also stimulated (40-70% increase) by SNP in 60% of the cultured GH-secreting adenomas studied. SNP-induced GH release was inhibited in both fetal and adenomatous cells by PTI0, a NO scavenger. The addition of cGMP (0.1-1 mM), the second messenger of multiple NO actions, enhanced fetal and adenomatous GH secretion by 55-95%. Neuronal NOS (nNOS) was expressed in normal (fetal and adult) human pituitary tissues and in GH-secreting adenomas. Examination of its functional expression using L-arginine (1 microM) yielded a 35% increase in GH release from cultured GH-secreting adenoma. This response was blocked by a NOS inhibitor with high selectivity for the neuronal enzyme and by a guanylyl cyclase inhibitor. In conclusion, NO stimulates human GH in cultured fetal pituitaries and GH-secreting adenomas. Cyclic GMP is probably involved in this hormonal regulation.