Chrysanthia Leontiou

The Role of the Aryl Hydrocarbon Receptor-Interacting Protein Gene in Familial and Sporadic Pituitary Adenomas

CONTEXT Mutations have been identified in the aryl hydrocarbon receptor-interacting protein (AIP) gene in familial isolated pituitary adenomas (FIPA). It is not clear, however, how this molecular chaperone is involved in tumorigenesis. OBJECTIVE AIP sequence changes and expression were studied in FIPA and sporadic adenomas. The function of normal and mutated AIP molecules was studied on cell proliferation and protein-protein interaction. Cellular and ultrastructural AIP localization was determined in pituitary cells. PATIENTS Twenty-six FIPA kindreds and 85 sporadic pituitary adenoma patients were included in the study. RESULTS Nine families harbored AIP mutations. Overexpression of wild-type AIP in TIG3 and HEK293 human fibroblast and GH3 pituitary cell lines dramatically reduced cell proliferation, whereas mutant AIP lost this ability. All the mutations led to a disruption of the protein-protein interaction between AIP and phosphodiesterase-4A5. In normal pituitary, AIP colocalizes exclusively with GH and prolactin, and it is found in association with the secretory vesicle, as shown by double-immunofluorescence and electron microscopy staining. In sporadic pituitary adenomas, however, AIP is expressed in all tumor types. In addition, whereas AIP is expressed in the secretory vesicle in GH-secreting tumors, similar to normal GH-secreting cells, in lactotroph, corticotroph, and nonfunctioning adenomas, it is localized to the cytoplasm and not in the secretory vesicles. CONCLUSIONS Our functional evaluation of AIP mutations is consistent with a tumor-suppressor role for AIP and its involvement in familial acromegaly. The abnormal expression and subcellular localization of AIP in sporadic pituitary adenomas indicate deranged regulation of this protein during tumorigenesis.

Octreotide and the mTOR Inhibitor RAD001 (Everolimus) Block Proliferation and Interact with the Akt-mTOR-p70S6K Pathway in a Neuro-Endocrine Tumour Cell Line

Background/Aim: The mode of action of the somatostatin analog octreotide on neuro-endocrine tumour proliferation is largely unknown. Overexpression of the proto-oncogene Akt/PKB (protein kinase B) has been demonstrated in certain neuro-endocrine tumours: Akt activates downstream proteins including mTOR and p70S6K, which play an important role in cell proliferation. RAD001 (everolimus) is a novel agent that is being trialled in the treatment of neuro-endocrine tumours, and is known to interact with mTOR. We explored the mechanism of action of octreotide, RAD001, and their combination on cell proliferation and kinase activation in a neuro-endocrine tumour cell line (rat insulinoma cell line, INS1). Methods: Proliferation assays were used to determine the effects of octreotide, RAD001, and their combination on cell proliferation. Western blotting was used to characterize the expression of phosphorylated Akt, phosphorylated TSC2, phosphorylated mTOR, and phosphorylated 70S6K. Results: Treatment with octreotide and RAD001 inhibited proliferation and attenuated phosphorylation of all downstream targets of Akt: TSC2, mTOR, and p70S6K. Conclusions: In this cell model, octreotide and RAD001 appear to act through a similar pathway and inhibit the Akt-mTOR-p70S6 kinase pathway downstream of Akt. There may be some overlapping effects of the two inhibitors on the mTOR pathway, although it is likely that other additional effects may differentiate the two agents.

Activation of RAF/MEK/ERK and PI3K/AKT/mTOR pathways in pituitary adenomas and their effects on downstream effectors

Raf/MEK/ERK and phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) cascades are key signalling pathways interacting with each other to regulate cell growth and tumourigenesis. We have previously shown B-Raf and Akt overexpression and/or overactivation in pituitary adenomas. The aim of this study is to assess the expression of their downstream components (MEK1/2, ERK1/2, mTOR, TSC2, p70S6K) and effectors (c-MYC and CYCLIN D1). We studied tissue from 16 non-functioning pituitary adenomas (NFPAs), six GH-omas, six prolactinomas and six ACTH-omas, all collected at transsphenoidal surgery; 16 normal autopsy pituitaries were used as controls. The expression of phospho and total protein was assessed with western immunoblotting, and the mRNA expression with quantitative RT-PCR. The expression of pSer217/221 MEK1/2 and pThr183 ERK1/2 (but not total MEK1/2 or ERK1/2) was significantly higher in all tumour subtypes in comparison to normal pituitaries. There was no difference in the expression of phosphorylated/total mTOR, TSC2 or p70S6K between pituitary adenomas and controls. Neither c-MYC phosphorylation at Ser 62 nor total c-MYC was changed in the tumours. However, c-MYC phosphorylation at Thr58/Ser62 (a response target for Akt) was decreased in all tumour types. CYCLIN D1 expression was higher only in NFPAs. The mRNA expression of MEK1, MEK2, ERK1, ERK2, c-MYC and CCND1 was similar in all groups. Our data indicate that in pituitary adenomas both the Raf/MEK/ERK and PI3K/Akt/mTOR pathways are upregulated in their initial cascade, implicating a pro-proliferative signal derangement upstream to their point of convergence. However, we speculate that other processes, such as senescence, attenuate the changes downstream in these benign tumours.

Somatostatin analogs modulate AIP in somatotroph adenomas: the role of the ZAC1 pathway.

CONTEXT Somatotroph adenomas harboring aryl hydrocarbon receptor interacting protein (AIP) mutations respond less well to somatostatin analogs, suggesting that the effects of somatostatin analogs may be mediated by AIP. OBJECTIVE The objective of the investigation was to study the involvement of AIP in the mechanism of effect of somatostatin analogs. DESIGN In the human study, a 16-wk somatostatin analog pretreatment compared with no pretreatment. In the in vitro cell line study, the effect of somatostatin analog treatment or small interfering RNA (siRNA)/plasmid transfection were studied. SETTING The study was conducted at a university hospital. PATIENTS Thirty-nine sporadic and 10 familial acromegaly patients participated in the study. INTERVENTION Interventions included preoperative lanreotide treatment and pituitary surgery. OUTCOME For the human study, GH and IGF-I levels, AIP, and somatostatin receptor staining were measured. For the cell line, AIP and ZAC1 (zinc finger regulator of apoptosis and cell cycle arrest) expression, metabolic activity, and clone formation were measured. RESULTS Lanreotide pretreatment reduced GH and IGF-I levels and tumor volume (all P < 0.0001). AIP immunostaining was stronger in the lanreotide-pretreated group vs. the surgery-only group (P < 0.001). After lanreotide pretreatment, the AIP score correlated to IGF-I changes in females (R = 0.68, P < 0.05). Somatostatin receptor staining was not reduced in samples with AIP mutations. In GH3 cells, 1 nm octreotide increased AIP mRNA and protein (both P < 0.01) and ZAC1 mRNA expression (P < 0.05). Overexpression of wild-type (but not mutant) AIP increased ZAC1 mRNA expression, whereas AIP siRNA knockdown reduced ZAC1 mRNA (both P < 0.05). The siRNA-mediated knockdown of AIP led to an increased metabolic activity and clonogenic ability of GH3 cells compared with cells transfected with a nontargeting control (both P < 0.001). CONCLUSION These results suggest that AIP may play a role in the mechanism of action of somatostatin analogs via ZAC1 in sporadic somatotroph tumors and may explain their lack of effectiveness in patients with AIP mutations.

PPAR-gamma expression in pituitary tumours and the functional activity of the glitazones: evidence that any anti-proliferative effect of the glitazones is independent of the PPAR-gamma receptor

Objective   It has been reported that both normal pituitary and pituitary tumours express PPAR‐γ, a nuclear hormone receptor, the expression being more abundant in pituitary tumours, and that this is the basis for the reported antiproliferative effects of the thiazolidinedione, rosiglitazone, in animal models. However, the mechanisms for the responsivity to rosiglitazone have remained unclear.

Ghrelin in neuroendocrine organs and tumours

Ghrelin is a 28 amino-acid hormone with multiple functions. It is predominantly produced by the stomach but has also been detected in other organs, including the small intestine, pancreas, hypothalamus and pituitary, as well as in the immune system and almost every other normal human tissue examined. It is also present in neuroendocrine tumours, pituitary adenomas, endocrine tumours of the pancreas, breast tumours, and thyroid and medullary thyroid carcinomas. Ghrelin is a brain-gut peptide with growth hormone-releasing and appetite-inducing activities, and is the endogenous ligand of the G protein-coupled growth hormone secretagogue receptor (GHS-R). In this review we comprehensively summarize the available data regarding (a) the expression of ghrelin and the GHS-R in normal endocrine tissues and in pituitary adenomas and neuroendocrine tumours, (b) the levels of circulating ghrelin in patients with pituitary adenomas and neuroendocrine tumours and (c) the effects of ghrelin administration in these patients on the levels of other hormones and on the rate of proliferation of the tumour. It is clear that ghrelin has many more functions and is involved in many more processes than was initially postulated, and its endocrine, paracrine and autocrine effects play a role in its physiological and pathophysiological functions.

The CB1 receptor mediates the peripheral effects of ghrelin on AMPK activity but not on growth hormone release

This study aimed to investigate whether the growth hormone release and metabolic effects of ghrelin on AMPK activity of peripheral tissues are mediated by cannabinoid receptor type 1 (CB1) and the central nervous system. CB1‐knockout (KO) and/or wild‐type mice were injected peripherally or intracerebroventricularly with ghrelin and CB1 antagonist rimonabant to study tissue AMPK activity and gene expression (transcription factors SREBP1c, transmembrane protein FAS, enzyme PEPCK, and protein HSL). Growth hormone levels were studied both in vivo and in vitro. Peripherally administered ghrelin in liver, heart, and adipose tissue AMPK activity cannot be observed in CB1‐KO or CB1 antagonist‐treated mice. Intracerebroventricular ghrelin treatment can influence peripheral AMPK activity. This effect is abolished in CB1‐KO mice and by intracerebroventricular rimonabant treatment, suggesting that central CB1 receptors also participate in the signaling pathway that mediates the effects of ghrelin on peripheral tissues. Interestingly, in vivo or in vitro growth hormone release is intact in response to ghrelin in CB1‐KO animals. Our data suggest that the metabolic effects of ghrelin on AMPK in peripheral tissues are abolished by the lack of functional CB1 receptor via direct peripheral effect and partially through the central nervous system, thus supporting the existence of a possible ghrelin‐cannabinoid–CB1–AMPK pathway.—Kola, B., Wittman, G., Bodnár, I., Amin, F., Lim, C. T., Oláh, M., Christ‐Crain, M., Lolli, F., van Thuijl, H., Leontiou, C. A., Füzesi, T., Dalino, P., Isidori, A. M., Harvey‐White, J., Kunos, G., Nagy, G. M., Grossman, A. B., Fekete, C., Korbonits, M., The CB1 receptor mediates the peripheral effects of ghrelin on AMPK activity but not on growth hormone release. FASEB J. 27, 5112–5121 (2013). www.fasebj.org

AIP, a Protein Mutated in Familial Acromegaly, Plays a Role in the Regulation of Cell Proliferation and Shows Cell-Type Specific Subcellular Localisation

a Endocrinology, b Histopathology, and c Cardiac, Vascular & Inflammation Research, Barts and the London Medical School, d Division of Molecular and Cellular Neuroscience, Institute of Ophthalmology, University College London, London, e Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford , UK, and f Section of Endocrinology, University of Illinois at Chicago, Chicago, Ill. , USA