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.
Alexander Gorshtein, Hadara Rubinfeld, Efrat Kendler, Marily Theodoropoulou, Vesna Cerovac, Günter K Stalla, Zvi R Cohen, Moshe Hadani, and Ilan Shimon
Yong Lin, Xiaofei Jiang, Ye Shen, Min Li, Huili Ma, Mingzhao Xing, and Yuan Lu
Genetic alterations in the PIK3CA gene of the phosphoinositide 3-kinase (PI3K)/AKT pathway have been found in many human tumors, but they have not been explored in pituitary tumors. We undertook the present study to explore mutations and amplifications of the PIK3CA gene in pituitary tumors. DNA sequencing and real-time quantitative PCR were used to examine mutations and amplifications respectively, on genomic DNA samples isolated from 353 cases of pituitary tumors, and immunohistostaining was used to assess PIK3CA expression. About 8 out of 91 (9%) invasive pituitary tumors versus 0 out of 262 (0%) noninvasive tumors were found to harbor somatic mutations in exons 9 and 20 of the PIK3CA gene (P<0.001), and the mutation was associated with increased disease recurrence. Genomic PIK3CA amplifications (defined as ≥4 copies) were observed in both invasive and noninvasive tumors, with a prevalence of around 20–40% in various types of pituitary tumors. PIK3CA protein overexpression was observed in cases with high PIK3CA copy number. RAS mutations were also examined and found in 6 out of the 91 (7%) invasive tumors. PIK3CA amplifications were mutually exclusive with PIK3CA or RAS mutations (P<0.001). This study demonstrated for the first time relatively common PIK3CA mutations and amplifications as well as RAS mutations and their tendency of mutual exclusivity in pituitary tumors. The data provide strong genetic evidence supporting a role of the PI3K/AKT signaling pathway in the tumorigenesis of pituitary tumors, particularly the invasive types.
G Mantovani, D Treppiedi, E Giardino, R Catalano, F Mangili, P Vercesi, M Arosio, A Spada, and E Peverelli
Although generally benign, pituitary tumors are frequently locally invasive, with reduced success of neurosurgery and unresponsive to pharmacological treatment with somatostatin or dopamine analogues. The molecular basis of the different biological behavior of pituitary tumors are still poorly identified, but a body of work now suggests that the activity of specific cytoskeleton proteins is a key factor regulating both the invasiveness and drug resistance of these tumors. This review recapitulates the experimental evidence supporting a role for the actin-binding protein filamin A (FLNA) in the regulation of somatostatin and dopamine receptors expression and signaling in pituitary tumors, thus in determining the responsiveness to currently used drugs, somatostatin analogues and dopamine receptor type 2 agonists. Regarding the regulation of invasive behavior of pituitary tumoral cells, we bring evidence to the role of the actin-severing protein cofilin, whose activation status may be modulated by dopaminergic and somatostatinergic drugs, through FLNA involvement. Molecular mechanisms involved in the regulation of FLNA expression and function in pituitary tumors will also be discussed.
A L Ogilvy-Stuart and S M Shalet
Eric Monsalves, Kyle Juraschka, Toru Tateno, Sameer Agnihotri, Sylvia L Asa, Shereen Ezzat, and Gelareh Zadeh
Pituitary adenomas are common intracranial neoplasms. Patients with these tumors exhibit a wide range of clinically challenging problems, stemming either from results of sellar mass effect in pituitary macroadenoma or the diverse effects of aberrant hormone production by adenoma cells. While some patients are cured/controlled by surgical resection and/or medical therapy, a proportion of patients exhibit tumors that are refractory to current modalities. New therapeutic approaches are needed for these patients. Activation of the AKT/phophotidylinositide-3-kinase pathway, including mTOR activation, is common in human neoplasia, and a number of therapeutic approaches are being employed to neutralize activation of this pathway in human cancer. This review examines the role of this pathway in pituitary tumors with respect to tumor biology and its potential role as a therapeutic target.
Katharina Lampichler, Patricio Ferrer, Greisa Vila, Mirjam I Lutz, Florian Wolf, Engelbert Knosp, Ludwig Wagner, Anton Luger, and Sabina Baumgartner-Parzer
The Hedgehog (Hh) pathway is an important regulator of early tissue patterning and stem cell propagation. It was found to be aberrantly activated in numerous types of human cancer and might be relevant in cancer stem cells. The identification of adult stem cells in the pituitary raised the question if tumor-initiating cells and Hh signaling are involved in pituitary adenoma formation. The present study aimed at the evaluation of Hh signaling in relation to stem cell and cell cycle markers in 30 human pituitary adenomas and in cultured murine adenoma cells. Therefore, expression levels of components of the Hh pathway, stem cell marker SOX2, cell cycle regulator tumor-protein 53 (TP53), proliferation marker Ki67 (MKI67) and superoxide dismutase 1 (SOD1) were evaluated in 30 human pituitary adenomas in comparison to control tissue. Modulation of cell function and target gene expression by the inhibition and activation of the Hh pathway were studied in murine adenoma cells. We show that transcription factor glioma-associated oncogene 1 (GLI1) is overexpressed in 87% of all pituitary adenomas. The expression of GLI1 significantly correlated with that of SOX2, TP53, MKI67 and SOD1. Inhibition of GLI1 resulted in the downregulation of the above genes and severe cell death in mouse adenoma cells. On the other hand, activation of the Hh pathway increased cell viability and target gene expression. In conclusion, our findings point toward an alternative, ligand-independent Hh pathway activation with GLI1 playing a major role in the cell survival of pituitary adenoma cells.
W E Farrell and R N Clayton
Throughout the genome CpG dinucleotides are found at one-fifth of their expected frequency and their rarity is further marked by the fact that 70% are methylated. In contrast, CpG islands (CGI), found associated with the promoters of many genes, have maintained their expected frequency of this dinucleotide, and remain unmethylated. Inappropriate methylation of CGIs is associated with histone deacetylation and gene silencing, while methylation of CpGs outside of CGIs is associated with significantly higher mutation rates. Methylation of CGIs is a frequent event in numerous tumour types including those that arise within the pituitary gland. Several studies now show highly frequent methylation of the p16 gene that is significantly associated with loss of cognate protein and that appears to be an early change in pituitary tumorigenesis. Collectively, studies show that somatotrophinomas are an infrequent target for p16 CGI methylation. However, in this pituitary tumour subtype, loss of pRb is associated with either CGI methylation or micro-deletion within the protein-pocket binding domain. As in other tumour types loss of p16 or RB1 appear to be mutually exclusive events in non-functional adenomas and somatotrophinomas respectively. Investigation of the Death Associated Protein Kinase gene shows that loss of its protein (DAPK), a pro-apoptotic molecule, in pituitary tumours is also associated with either methylation or deletion within its associated CGI. In the case of DAPK, however, these changes segregate with invasive pituitary tumours irrespective of tumour subtype. Methylation represents a positive signal that can be detected with exquisite sensitivity; in addition, this change targets multiple genes that show tumour type specificity. Taken together, the detection of DNA methylation changes, using either a panel of predefined marker-islands, or CGI arrays, provides the opportunity to generate "methylation profiles". This new knowledge will increase our understanding of tumour biology and could ultimately aid medical management in these different tumour types, including those of pituitary origin.
D Dworakowska, E Wlodek, C A Leontiou, S Igreja, M Cakir, M Teng, N Prodromou, M I Góth, S Grozinsky-Glasberg, M Gueorguiev, B Kola, M Korbonits, and A B Grossman
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.
Tomoko Sekiya, Marcello D Bronstein, Katiuscia Benfini, Viviane C Longuini, Raquel S Jallad, Marcio C Machado, Tatiana D Goncalves, Luciana H Osaki, Leonardo Higashi, Jose Viana-Jr, Claudio Kater, Misu Lee, Sara Molatore, Guilherme Francisco, Roger Chammas, Michel S Naslavsky, David Schlesinger, Patricia Gama, Yeda A O Duarte, Maria Lucia Lebrão, Mayana Zatz, Osorio Meirelles, Bernardo Liberman, Maria Candida B V Fragoso, Sergio P A Toledo, Natalia S Pellegata, and Rodrigo A Toledo
Germline mutations in p27 kip1 are associated with increased susceptibility to multiple endocrine neoplasias (MEN) both in rats and humans; however, the potential role of common polymorphisms of this gene in endocrine tumor susceptibility and tumorigenesis remains mostly unrecognized. To assess the risk associated with polymorphism rs2066827 (p27-V109G), we genotyped a large cohort of Brazilian patients with sporadic endocrine tumors (pituitary adenomas, n=252; pheochromocytomas, n=125; medullary thyroid carcinoma, n=51; and parathyroid adenomas, n=19) and 885 population-matched healthy controls and determined the odds ratios and 95% CIs. Significant associations were found for the group of patients with pituitary adenomas (P=0.01), particularly for those with ACTH-secreting pituitary adenomas (P=0.005). In contrast, no association was found with GH-secreting pituitary tumors alone or with the sporadic counterpart of MEN2-component neoplasias. Our in vitro analyses revealed increased colony formation and cell growth rate for an AtT20 corticotropin mouse cell line overexpressing the p27-V109G variant compared with cells transfected with the WT p27. However, the genotypic effects in genetic and in vitro approaches were divergent. In accordance with our genetic data showing specificity for ACTH-secreting pituitary tissues, the overexpression of p27-V109G in a GH3 somatotropin rat cell line resulted in no difference compared with the WT. Pituitary tumors are one of the major clinical components of syndromes associated with the p27 pathogenic mutations MENX and MEN4. Our genetic and in vitro data indicate that the common polymorphism rs2066827 may play a role in corticotropinoma susceptibility and tumorigenesis through a molecular mechanism not fully understood thus far.
Giampaolo Trivellin, Ricardo R Correa, Maria Batsis, Fabio R Faucz, Prashant Chittiboina, Ivana Bjelobaba, Darwin O Larco, Martha Quezado, Adrian F Daly, Stanko S Stojilkovic, T John Wu, Albert Beckers, Maya B Lodish, and Constantine A Stratakis
Cushing’s disease (CD) in children is caused by adrenocorticotropic hormone (ACTH)-secreting pituitary adenomas. Germline or somatic mutations in genes such as MEN1, CDKIs, AIP, and USP8 have been identified in pediatric CD, but the genetic defects in a significant percentage of cases are still unknown. In this study, we investigated the orphan G-protein-coupled receptor GPR101, a gene known to be involved in somatotropinomas, for its possible involvement in corticotropinomas. We performed GPR101 sequencing, expression analyses by RT-qPCR and immunostaining, and functional studies (cell proliferation, pituitary hormone secretion, and cAMP measurement) in a series of patients with sporadic CD secondary to ACTH-secreting adenomas in whom we extracted DNA from peripheral blood and pituitary tumor samples (n=36). No increased GPR101 expression was observed in tumors compared with normal pituitary (NP) tissues, nor did we find a correlation between GPR101 and ACTH expression levels. Sequence analysis revealed a very rare germline heterozygous GPR101 variant (p.G31S) in one patient with CD. Overexpression of the p.G31S variant did not lead to increased growth and proliferation, although modest effects on cAMP signaling were observed. GPR101 is not overexpressed in ACTH-secreting tumors compared with NPs. In conclusion, rare germline GPR101 variant was found in one patient with CD, but in vitro studies did not support a consistent pathogenic effect. GPR101 is unlikely to be involved in the pathogenesis of CD.