Hadara Rubinfeld

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.

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.

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 vivo models. 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 PT10, 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 µM) 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.

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.

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.

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.

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.

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

Summary 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.