Tumours of the anterior pituitary can manifest from all endocrine cell types but the mechanisms for determining their specification are not known. The Hippo kinase cascade is a crucial signalling pathway regulating growth and cell fate in numerous organs. There is mounting evidence implicating this in tumour formation, where it is emerging as an anti-cancer target. We previously demonstrated activity of the Hippo kinase cascade in the mouse pituitary and nuclear association of its effectors YAP/TAZ with SOX2-expressing pituitary stem cells. Here, we sought to investigate whether these components are expressed in the human pituitary and if they are deregulated in human pituitary tumours. Analysis of pathway components by immunofluorescence reveals pathway activity during normal human pituitary development and in the adult gland. Poorly differentiated pituitary tumours (null-cell adenomas, adamantinomatous craniopharyngiomas (ACPs) and papillary craniopharyngiomas (PCPs)), displayed enhanced expression of pathway effectors YAP/TAZ. In contrast, differentiated adenomas displayed lower or absent levels. Knockdown of the kinase-encoding Lats1 in GH3 rat mammosomatotropinoma cells suppressed Prl and Gh promoter activity following an increase in YAP/TAZ levels. In conclusion, we have demonstrated activity of the Hippo kinase cascade in the human pituitary and association of high YAP/TAZ with repression of the differentiated state both in vitro and in vivo. Characterisation of this pathway in pituitary tumours is of potential prognostic value, opening up putative avenues for treatments.
Paraskevi Xekouki, Emily J Lodge, Jakob Matschke, Alice Santambrogio, John R Apps, Ariane Sharif, Thomas S Jacques, Simon Aylwin, Vincent Prevot, Ran Li, Jörg Flitsch, Stefan R Bornstein, Marily Theodoropoulou, and Cynthia L Andoniadou
Freya Mertens, Lies Gremeaux, Jianghai Chen, Qiuli Fu, Christophe Willems, Heleen Roose, Olivier Govaere, Tania Roskams, Carolina Cristina, Damasia Becú-Villalobos, Mark Jorissen, Vincent Vander Poorten, Marie Bex, Johannes van Loon, and Hugo Vankelecom
Pituitary adenomas cause significant endocrine and mass-related morbidity. Little is known about the mechanisms that underlie pituitary tumor pathogenesis. In the present study, we searched for a side population (SP) in pituitary tumors representing cells with high efflux capacity and potentially enriched for tumor stem cells (TSCs). Human pituitary adenomas contain a SP irrespective of hormonal phenotype. This adenoma SP, as well as the purified SP (pSP) that is depleted from endothelial and immune cells, is enriched for cells that express ‘tumor stemness’ markers and signaling pathways, including epithelial–mesenchymal transition (EMT)-linked factors. Pituitary adenomas were found to contain self-renewing sphere-forming cells, considered to be a property of TSCs. These sphere-initiating cells were recovered in the pSP. Because benign pituitary adenomas do not grow in vitro and have failed to expand in immunodeficient mice, the pituitary tumor cell line AtT20 was further used. We identified a SP in this cell line and found it to be more tumorigenic than the non-SP ‘main population’. Of the two EMT regulatory pathways tested, the inhibition of chemokine (C-X-C motif) receptor 4 (CXCR4) signaling reduced EMT-associated cell motility in vitro as well as xenograft tumor growth, whereas the activation of TGFβ had no effect. The human adenoma pSP also showed upregulated expression of the pituitary stem cell marker SOX2. Pituitaries from dopamine receptor D2 knockout (Drd2 −/−) mice that bear prolactinomas contain more pSP, Sox2+, and colony-forming cells than WT glands. In conclusion, we detected a SP in pituitary tumors and identified TSC-associated characteristics. The present study adds new elements to the unraveling of pituitary tumor pathogenesis and may lead to the identification of new therapeutic targets.
Chiara Villa, Maria Stefania Lagonigro, Flavia Magri, Maria Koziak, Marie-Lise Jaffrain-Rea, Raja Brauner, Jerome Bouligand, Marie Pierre Junier, Federico Di Rocco, Christian Sainte-Rose, Albert Beckers, François Xavier Roux, Adrian F Daly, and Luca Chiovato
Mutations of the aryl hydrocarbon receptor interacting protein (AIP) gene are associated with pituitary adenomas that usually occur as familial isolated pituitary adenomas (FIPA). Detailed pathological and tumor genetic data on AIP mutation-related pituitary adenomas are not sufficient. Non-identical twin females presented as adolescents to the emergency department with severe progressive headache caused by large pituitary macroadenomas require emergency neurosurgery; one patient had incipient pituitary apoplexy. Post-surgically, the patients were found to have silent somatotrope adenomas on pathological examination. Furthermore, the light microscopic, immunohistochemical, and electron microscopic studies demonstrated tumors of virtually identical characteristics. The adenomas were accompanied by multiple areas of pituitary hyperplasia, which stained positively for GH, indicating somatotrope hyperplasia. Genetic analyses of the FIPA kindred revealed a novel E216X mutation of the AIP gene, which was present in both the affected patients and the unaffected father. Molecular analysis of surgical specimens revealed loss of heterozygosity (LOH) in the adenoma but showed that LOH was not present in the hyperplastic pituitary tissue from either patient. AIP immunostaining confirmed normal staining in the hyperplastic tissue and decreased staining in the adenoma in the tumors from both patients. These results demonstrate that patients with AIP germline mutation can present with silent somatotrope pituitary adenomas. The finding of somatotrope hyperplasia unaccompanied by AIP LOH suggests that LOH at the AIP locus might be a late event in a potential progression from hyperplastic to adenomatous tissue.
M Theodoropoulou, I Cavallari, L Barzon, D M D'Agostino, T Ferro, T Arzberger, Y Gr√ºbler, L Schaaf, M Losa, F Fallo, V Ciminale, G K Stalla, and U Pagotto
Pituitary adenomas represent one of the key features of multiple endocrine neoplasia type 1. The gene involved in this syndrome (MEN1) is a putative tumor suppressor, that codes for a 610-amino acid nuclear protein termed 'menin'. Analyses of sporadic pituitary adenomas have so far failed to reveal MEN1 mutations or defects in MEN1 transcription in these tumors. In the present study we detected menin protein expression in a panel of normal and tumoral pituitary tissues, using a monoclonal antibody against the carboxy-terminus of menin. In the normal human pituitary gland, strong nuclear staining for menin was detectable in the majority of the endocrine cells of the anterior lobe, without a clear association with a particular hormone-producing type. In sporadic pituitary adenomas, menin expression was variable, with a high percentage of cases demonstrating a significant decrease in menin immunoreactivity when compared with the normal pituitary. Interestingly, metastatic tissues derived from one pituitary carcinoma had no detectable menin levels. Altogether, our data provide the first information regarding the status of menin expression in human normal and neoplastic pituitary as determined by immunohistochemistry (IHC).
Vladimir Vasilev, Adrian F Daly, Giampaolo Trivellin, Constantine A Stratakis, Sabina Zacharieva, and Albert Beckers
Familial isolated pituitary adenoma (FIPA) is one of the most frequent conditions associated with an inherited presentation of pituitary tumors. FIPA can present with pituitary adenomas of any secretory/non-secretory type. Mutations in the gene for the aryl-hydrocarbon receptor interacting protein (AIP) have been identified in approximately 20% of FIPA families and are the most frequent cause (29%) of pituitary gigantism. Pituitary tumors in FIPA are larger, occur at a younger age and display more aggressive characteristics and evolution than sporadic adenomas. This aggressiveness is especially marked in FIPA kindreds with AIP mutations. Special attention should be paid to young patients with pituitary gigantism and/or macroadenomas, as AIP mutations are prevalent in these groups. Duplications on chromosome Xq26.3 involving the gene GPR101 lead to X-linked acrogigantism (X-LAG), a syndrome of pituitary gigantism beginning in early childhood; three kindreds with X-LAG have presented in the setting of FIPA. Management of pituitary adenomas in the setting of FIPA, AIP mutations and GPR101 duplications is often more complex than in sporadic disease due to early onset disease, aggressive tumor growth and resistance to medical therapy.
Odelia Cooper, George Vlotides, Hidenori Fukuoka, Mark I Greene, and Shlomo Melmed
The role of ErbB family in discreet pituitary functions is reviewed. Several ErbB receptor ligands, EGF, TGFα, and heregulin are differentially expressed in normal gonadotroph and lacto-somatotroph lineages, and other elements of the anterior pituitary. ErbB receptors, i.e. EGFR and ErbB2, are also localized to the anterior pituitary with preferential EGFR lactosomatotroph expression. EGF regulates CRH and ACTH secretion and corticotroph proliferation as well as exhibiting autocrine and paracrine effects on gonadotrophs and on lactosomatotroph proliferation, gene and protein expression, and hormonal secretion. EGF and EGFR are expressed in both functioning and non-functioning pituitary adenomas, with higher expression in more aggressive tumor subtypes. ErbB2 receptor is detected in all tumor subtypes, particularly in invasive tumors. ErbB tyrosine kinase inhibitors regulate hormonal secretion, cell morphology, and proliferation in lacto-somatotroph tumors, reflecting the emerging application of targeted pituitary therapeutics.
M Muşat, M Korbonits, B Kola, N Borboli, M R Hanson, A M Nanzer, J Grigson, S Jordan, D G Morris, M Gueorguiev, M Coculescu, S Basuand, and A B Grossman
Pituitary tumours have previously been shown to harbour several abnormalities that cause deregulation of the cell cycle, particularly down-regulation of expression of the cyclin-dependent kinase inhibitor p27. However, it has been unclear whether these are the primary initiating events, or are secondary to other more proximate alterations in signalling pathways. In other cellular systems the Akt signalling pathway has been associated with downstream modulation of cell-cycle control. The aim of the present study was to test the hypothesis that Akt signalling is enhanced in pituitary tumours, and to see if changes in Akt expression are related to previous findings on low expression levels of the nuclear cell-cycle inhibitor p27 in pituitary tumours. We examined normal and adenomatous human pituitary tissue for mRNA and protein expression of Akt1, Akt2 and p27, and the activation of Akt, as well the phosphatase involved in the inactivation of Akt, phosphatase and tensin homologue deleted on chromosome 10 (PTEN). In pituitary adenomas Akt1 and Akt2 mRNA were found to be over-expressed compared with normal pituitary, while PTEN transcripts showed similar levels between the two tissue types. Immunohistochemical expression of phospho-Akt was found to be higher in the tumours than normal pituitaries, while the protein expression of nuclear p27 and PTEN was lower in the adenomas. However, the expression of p27 and Akt were not directly correlated. PTEN sequencing revealed no mutation in the coding region of the gene in pituitary adenomas, and thus we did not locate a cause for the increased phosphorylation of Akt. In summary, we have shown over-expression and activation of the Akt pathway in pituitary tumours, and we speculate that cell-cycle changes observed in such tumours are secondary to these more proximate alterations. Since Akt is a major downstream signalling molecule of growth factor-liganded tyrosine kinase receptors, our data are most compatible with an abnormality at this level as the primary driver of pituitary tumorigenesis.
K Revill, K J Dudley, R N Clayton, A M McNicol, and W E Farrell
The imprinted gene, neuronatin (NNAT), is one of the most abundant transcripts in the pituitary and is thought to be involved in the development and maturation of this gland. In a recent whole-genome approach, exploiting a pituitary tumour cell line, we identified hypermethylation associated loss of NNAT. In this report, we determined the expression pattern of NNAT in individual cell types of the normal gland and within each of the different pituitary adenoma subtypes. In addition, we determined associations between expression and CpG island methylation and used colony forming efficiency assays (CFE) to gain further insight into the tumour-suppressor function of this gene. Immunohistochemical (IHC) co-localization studies of normal pituitaries showed that each of the hormone secreting cells (GH, PRL, ACTH, FSH and TSH) expressed NNAT. However, 33 out of 47 adenomas comprising, 11 somatotrophinomas, 10 prolactinomas, 12 corticotrophinomas and 14 non-functioning tumours, irrespective of subtype failed to express either NNAT transcript or protein as determined by quantitative real-time RT-PCR and IHC respectively. In normal pituitaries and adenomas that expressed NNAT the promoter-associated CpG island showed characteristics of an imprinted gene where ∼50% of molecules were densely methylated. However, in the majority of adenomas that showed loss or significantly reduced expression of NNAT, relative to normal pituitaries, the gene-associated CpG island showed significantly increased methylation. Induced expression of NNAT in transfected AtT-20 cells significantly reduced CFE. Collectively, these findings point to an important role for NNAT in the pituitary and perhaps tumour development in this gland.
M Fuertes, M Sapochnik, L Tedesco, S Senin, A Attorresi, P Ajler, G Carrizo, A Cervio, G Sevlever, J J Bonfiglio, G K Stalla, and E Arzt
Increased levels of the proto-oncogene pituitary tumor-transforming gene 1 (PTTG) have been repeatedly reported in several human solid tumors, especially in endocrine-related tumors such as pituitary adenomas. Securin PTTG has a critical role in pituitary tumorigenesis. However, the cause of upregulation has not been found yet, despite analyses made at the gene, promoter and mRNA level that show that no mutations, epigenetic modifications or other mechanisms that deregulate its expression may explain its overexpression and action as an oncogene. We describe that high PTTG protein levels are induced by the RWD-containing sumoylation enhancer (RWDD3 or RSUME), a protein originally identified in the same pituitary tumor cell line in which PTTG was also cloned. We demonstrate that PTTG and RSUME have a positive expression correlation in human pituitary adenomas. RSUME increases PTTG protein in pituitary tumor cell lines, prolongs the half-life of PTTG protein and regulates the PTTG induction by estradiol. As a consequence, RSUME enhances PTTG transcription factor and securin activities. PTTG hyperactivity on the cell cycle resulted in recurrent and unequal divisions without cytokinesis, and the consequential appearance of aneuploidies and multinucleated cells in the tumor. RSUME knockdown diminishes securin PTTG and reduces its tumorigenic potential in a xenograft mouse model. Taken together, our findings show that PTTG high protein steady state levels account for PTTG tumor abundance and demonstrate a critical role of RSUME in this process in pituitary tumor cells.
V V Vax, M Gueorguiev, I I Dedov, A B Grossman, and M Korbonits
The oncogenes and/or tumour suppressor genes which may be involved in the transformation process for the vast majority of pituitary tumours remain unknown. There is substantial evidence for derangement of cell cycle control in such tumours, but cell cycle protein mutations identified in other human malignancies are restricted to only a very small subset of sporadic pituitary neoplasms. Kr√ºppel-like factors are DNA-binding transcriptional regulators with diverse effects including the upregulation of the cell cycle protein p21(WAF1/CIP1). It has been reported that the Kr√ºppel-like transcription factor 6 (KLF6) gene is mutated in a proportion (15-55%) of human prostate cancers, and more recent data are emerging regarding mutated KLF6 in nasopharyngeal carcinomas, astrocytoid gliomas and colorectal cancer. We therefore speculated that other tumours such as pituitary adenomas might also harbour such mutations that may be involved in the control of cell proliferation in the pituitary. The aim of the current study was thus to analyse the KLF6 gene for mutations in sporadic pituitary tumours. We analysed 60 pituitary adenomas (15 GH-, four ACTH-, two PRL-secreting and 39 non-functioning) with direct sequence analysis of exons 2 and 3 of the KLF6 gene, the region where most of the previously described mutations are located. Three non-functioning pituitary adenomas of the 60 pituitary tumours (5%) had two identical sequence changes in exon 2 (missense mutation Val165Met, 523G-->A and a silent substitution in Ser77Ser codon 261C-->T). Analysis of genomic DNA extracted from peripheral lymphocytes in one patient confirmed these changes to be present in the germline and they therefore probably represent polymorphisms, although we cannot exclude the possibility that these are predisposing germline mutations. We conclude that mutations of the KLF6 gene are unlikely to play an important role in sporadic pituitary tumorigenesis.