Post-mortem Pituitaries Research Articles

8574 Single cell multiomics suggests tumor cell energy metabolism heterogeneity in non-functioning gonadotroph pituitary adenomas

Abstract Disclosure: F.M. Ruf-Zamojski: None. Z. Zhang: None. W. Cheng: None. G.R. Smith: None. M. Zamojski: None. N. Mendelev: None. Y. Ge: None. D. Marrero-Rodríguez: None. K. Taniguchi-Ponciano: None. C.L. Andoniadou: None. E. Zaslavsky: None. X. Chen: None. O.G. Troyanskaya: None. M. Mercado: None. S.C. Sealfon: None. Pituitary adenomas cause hormonal dysregulation and severe morbidity. The absence of clear biomarkers for prognosis/treatment and the high risk of recurrence make management challenging. Non-functioning gonadotroph pituitary adenomas (NFPAs) represent proliferation of gonadotroph lineage cells without an increase in secretion of gonadotropins. Detection is often delayed until mass effects cause visual defects. Higher resolution molecular study may improve classification, diagnosis, treatment, and provide insight into adenoma biology. We analyzed four NFPAs using unbiased genome-wide same-cell single nucleus 10X Genomics multiome assay (transcriptome and chromatin accessibility) to define individual cell identity/states in comparison with normal gonadotrope cell references from twelve age/sex-matched human post-mortem pituitaries. In total, we generated high quality multiomics single-cell data for 33,832 adenoma cells and 5,451 normal gonadotropes. We identified tumor, immune, vascular, and proliferative cells confirming cellular heterogeneity. Multidimensional scaling analysis using Manhattan distances between cell types showed that NFPA gonadotropes were closer in gene expression to healthy gonadotropes among all pituitary cell types. In addition, we observed differences in the macrophage and endothelial cell populations between tumors and healthy pituitaries. Next, we studied gene regulatory circuitry (regulated gene, modulating transcription factor and its chromatin interaction site) using the new Control of Regulation Extracted from Multiomics Assays (CREMA, [1]) multiome analysis method which showed high variation between normal and tumor gonadotropes. Within each adenoma, the NFPA tumor cells encompassed two main subgroups of cells, one group (GT+ marker tumor cells) expressing established gonadotrope markers and another group not (GT- marker tumor cells). GT+ and GT- tumor cells formed a cluster distinct from healthy gonadotropes. GT+ and GT- tumor cells differed by their level of expression of mitochondrial genes, suggesting decreased aerobic capacity associated with the GT- tumor subgroup. Interestingly, differential analyses between the healthy and the NFPA gonadotropes from same-cell multiome data highlighted dissimilar gene expression and chromatin accessibility patterns.Our newly collected single cell data characterize the difference between NFPA tumor cells and normal gonadotrope cells. Our results suggest a Warburg-effect like difference in aerobic respiratory capacity among tumor cells within the same pituitary adenoma. Overall, this study brings new insights into the molecular characteristics of NFPAs, including tumor cell heterogeneity, and represents a new resource for the field. [1] PMID: 38084892. Presentation: 6/1/2024

Single nucleus transcriptome and chromatin accessibility of postmortem human pituitaries reveal diverse stem cell regulatory mechanisms.

SUMMARYDespite their importance in tissue homeostasis and renewal, human pituitary stem cells (PSCs) are incompletely characterized. We describe a human single nucleus RNA-seq and ATAC-seq resource from pediatric, adult, and aged postmortem pituitaries (snpituitaryatlas.princeton.edu) and characterize cell-type-specific gene expression and chromatin accessibility programs for all major pituitary cell lineages. We identify uncommitted PSCs, committing progenitor cells, and sex differences. Pseudotime trajectory analysis indicates that early-life PSCs are distinct from the other age groups. Linear modeling of same-cell multiome data identifies regulatory domain accessibility sites and transcription factors that are significantly associated with gene expression in PSCs compared with other cell types and within PSCs. We identify distinct deterministic mechanisms that contribute to heterogeneous marker expression within PSCs. These findings characterize human stem cell lineages and reveal diverse mechanisms regulating key PSC genes and cell type identity.

Comprehensive Map and Functional Annotation of Human Pituitary and Thyroid Proteome.

Knowledge about human tissue proteome will provide insights into health organ physiology. To construct a comprehensive data set of human pituitary and thyroid proteins, post-mortem pituitaries and thyroids from 10 normal individuals were used. The pooled samples were prepared using two methods. One part of the sample was processed using 14 high-abundance proteins immunoaffinity column. The other part was directly subjected to digestion. Finally, a total of 7596 proteins in pituitary and 5602 proteins in thyroid with high confidence were identified, with 6623 and 4368 quantified, respectively. A total of 5781 of pituitary and 3178 of thyroid proteins have not been previously reported in the normal pituitary and thyroid proteome. Comparison of pituitary and thyroid proteome indicated that thyroid prefers to be involved in nerve system regeneration and metabolic regulation, while pituitary mainly performs functions of signal transduction and cancer modulation. Our results, for the first time, comprehensively profiled and functionally annotated the largest high-confidence data set of proteome of two important endocrine glands, pituitary and thyroid, which is important for further studies on biomarker identification and molecular mechanisms of pituitary and thyroid disorders. The mapping results can be freely downloaded at http://www.urimarker.com/pituitary/ and http://www.urimarker.com/thyroid/ . The raw data are available via ProteomeXchange with identifier PXD006471.

Histopathological features of post-mortem pituitaries: A retrospective analysis.

As a result of the use of neuroimaging techniques, silent pituitary lesions are diagnosed more and more frequently; however, there are few published post-mortem studies about this gland. Incidence data of pituitary lesions are rare and in Portugal they are outdated or even non-existent. The aim of this study is to determine the prevalence of normal patterns and incidental post-mortem pituitary pathology at Centro Hospitalar Lisboa Norte, analyzing the associations with clinical data and assessing the clinical relevance of the findings. We reviewed retrospectively and histologically 167 pituitaries of a consecutive series of autopsies from the Department of Pathology of this centre. They were done between 2012 and 2014, and in all cases medical records were reviewed. The morphological patterns observed, were classified into three major groups: 1) Normal histological patterns and variants; 2) Infectious-inflammatory pathology, metabolic and vascular disorders; 3) Incidental primary proliferation and secondary to systemic diseases. The subjects included in this study were of all age groups (from 1 day to 91 years old), 71 were female and 96 male. Fifty-seven of these glands didn't show any alteration; 51 showed colloid cysts arising from Rathke cleft; 44 presented hyperplasia in adenohypophysis and we identified 20 adenomas in 19 glands (immunohistochemically, eight PRL-producing and five ACTH-producing tumors), ten of which associated with obesity, 11 to hypertension and six to diabetes mellitus. There were two cases with metastasis. Subclinical pathology in our country is similar to that seen in other parts of the world, but at older ages.

Distinct proteomic profiles in post-mortem pituitary glands from bipolar disorder and major depressive disorder patients

Disturbances of the hypothalamic–pituitary–adrenal axis have been implicated in the pathophysiology of bipolar disorder (BD) and major depressive disorder (MDD). To examine this further, we carried out proteomic profiling of post-mortem pituitaries from 13 BD and 14 MDD patients, in comparison to 15 controls. Liquid chromatography–mass spectrometry (LC–MSE) analysis showed that BD patients had significantly increased levels of the major pituitary hormones pro-opiomelanocortin (POMC) and galanin. BD patients also showed changes in proteins associated with gene transcription, stress response, lipid metabolism and growth signalling. In contrast, LC–MSE profiling revealed that MDD patients had significantly decreased levels of the prohormone-converting enzyme carboxypeptidease E and follow-up enzymatic analysis showed decreased activity of prolyl-oligopeptidase convertase. This suggested that altered prohormone processing may occur in pituitaries of MDD patients. In addition, MDD patients had significant changes in proteins involved in intracellular transport and cytoskeletal signalling. Finally, we carried out selective reaction monitoring (SRM) mass spectrometry profiling for validation of protein changes in key biological pathways. This confirmed increased POMC levels in BD patients with no change in the levels of this prohormone in MDD. This study demonstrates that proteomic profiling analysis of the pituitary can lead to new insights into the pathophysiology of BD and MDD. Also, given that the pituitary directly releases a variety of bioactive molecules into the bloodstream, many of the proteins identified here could serve as focal points in the search for peripheral biomarkers in clinical or drug treatment studies of BD and MDD patients.

Quantitative, genome-wide analysis of the DNA methylome in sporadic pituitary adenomas

DNA methylation is one of the several epigenetic modifications that together with genetic aberrations are hallmarks of tumorigenesis including those emanating from the pituitary gland. In this study, we examined DNA methylation across 27 578 CpG sites spanning more than 14 000 genes in the major pituitary adenoma subtypes. Genome-wide changes were first determined in a discovery cohort comprising non-functioning (NF), growth hormone (GH), prolactin (PRL)-secreting and corticotroph (CT) adenoma relative to post-mortem pituitaries. Using stringent cut-off criteria, we validated increased methylation by pyrosequencing in 12 of 16 (75%) genes. Overall, these criteria identified 40 genes in NF, 21 in GH, six in PRL and two in CT that were differentially methylated relative to controls. In a larger independent cohort of adenomas, for genes in which hypermethylation had been validated, different frequencies of hypermethylation were apparent, where the KIAA1822 (HHIPL1) and TFAP2E genes were hypermethylated in 12 of 13 NF adenomas whereas the COL1A2 gene showed an increase in two of 13 adenomas. For genes showing differential methylation across and between adenoma subtypes, pyrosequencing confirmed these findings. In three of 12 genes investigated, an inverse relationship between methylation and transcript expression was observed where increased methylation of EML2, RHOD and HOXB1 is associated with significantly reduced transcript expression. This study provides the first genome-wide survey of adenoma, subtype-specific epigenomic changes and will prove useful for identification of biomarkers that perhaps predict or characterise growth patterns. The functional characterisation of identified genes will also provide insight of tumour aetiology and identification of new therapeutic targets.

Galanin and its three receptors in human pituitary adenoma

Galanin, a 29-aminoacid peptide (30 in humans), is widely distributed in the nervous and endocrine systems and exerts its actions via three G-protein-coupled receptors, GalR1-3. The galanin system has, among others, been associated with tumorigenesis. Our objective was to assess the expression of galanin and its receptors in pituitary tumors. Transcript levels of galanin and galanin receptors 1-3 (GalR1-3) were measured using quantitative real-time PCR (q-PCR) in pituitary tumors, surgically removed from thirteen patients, and twelve post mortem pituitaries. Galanin, GalR1 and GalR2 mRNA, but not GalR3 mRNA, were found in the twelve human post-mortem pituitaries. Expression of GalR1 was relatively increased in most, whereas GalR2 was decreased in some tumors. High levels of GalR3 were only found in tumors of five patients, who all relapsed shortly after surgical intervention. The results suggest that GalR3, a receptor for the neuroendocrine peptide galanin, is a potential marker for relapsing pituitary tumors. Thus, galanin receptors may play an important role in pituitary tumors, also for surgical outcome and prognosis, and may serve as a diagnostic tool. The association of GalR3 with tumor relapse suggests that antagonists to this receptor represent a potential therapeutic approach to treatment of pituitary tumors.

Endothelial Expression of Endocan Is Strongly Associated with Tumor Progression in Pituitary Adenoma

Although benign, pituitary adenomas frequently invade adjacent sinuses or recur after first surgery. To date, there is no histological marker predictive of recurrence. Angiogenic factors are candidate markers. Endocan is a proteoglycan secreted by endothelial cells, associated with an aggressive behavior in several tumor types. Endocan expression was investigated by immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR) in 18 normal post-mortem pituitaries and in 107 patients operated for a pituitary adenoma (with a follow-up of at least 8 years after surgery). In normal pituitaries, endocan was never observed in vessels but was detected in isolated endocrine cells. In adenoma tissue, we found a strong association between endocan immunoreactivity in endothelial cells and progression (P = 0.0009), as well as tumor size (P = 0.0012), raised mitotic count (P = 0.02) and p53 expression (P = 0.032). Morphometric analysis of the microvessels showed that the mean vessel area was significantly higher in the subgroup of tumors with an endothelial expression of endocan (P = 0.028), coherent with the neoangiogenesis process occurring in the pituitary. The immunolabeling of endocan in endothelial cells may therefore appear to be a relevant marker of aggressive behavior in pituitary tumors.

Metabolic, hormonal and stress-related molecular changes in post-mortem pituitary glands from schizophrenia subjects

Objectives. To identify a molecular profile for schizophrenia using post-mortem pituitaries from schizophrenia and control subjects. Methods. Molecular profiling analysis of pituitaries from schizophrenia (n = 14) and control (n = 15) subjects was carried out using a combination of liquid chromatography tandem mass spectrometry (LC-MSE), multiplex analyte profiling (MAP), two-dimensional difference gel electrophoresis (2D-DIGE) and Western blot analysis. Results. This led to identification of differentially expressed molecules in schizophrenia patients including hypothalamic–pituitary–adrenal axis-associated constituents such as cortisol, pro-adrenocorticotropic hormone, arginine vasopressin precursor, agouti-related protein, growth hormone, prolactin and secretagogin, as well as molecules associated with lipid transport and metabolism such as apolipoproteins A1, A2, C3 and H. Altered levels of secretagogin in serum from a cohort of living first onset schizophrenia patients were also detected, suggesting disease association and illustrating the potential for translating some components of this molecular profile to serum-based assays. Conclusions. Future studies on the molecules identified here may lead to new insights into schizophrenia pathophysiology and pave the way for translation of novel diagnostics for use in a clinical setting.

Altered levels of circulating insulin and other neuroendocrine hormones associated with the onset of schizophrenia

Recently, we showed that the circulating levels of insulin-related peptides and the secretory granule protein chromogranin A were increased in small cohorts of first onset schizophrenia patients. Assuming that this effect was associated with impaired insulin signalling, we investigated the possibility that secretion of other hormones is also affected in schizophrenia. Multiplex immunoassay analysis of 21 hormones and hormone-related molecules was carried out using sera from 236 first and recent onset schizophrenia patients and 230 matched controls. Serum concentrations of insulin and chromogranin A were increased in schizophrenia subjects, consistent with our previous study. In addition, we found elevated concentrations of pancreatic polypeptide, prolactin, progesterone and cortisol, and decreased levels of growth hormone. We also found that growth hormone levels were decreased in post-mortem pituitaries obtained from chronic schizophrenia patients. It will be important to determine whether any of these molecules are involved in the pathosphysiology of schizophrenia or if they reflect the associated insulin resistance. We conclude that function of multiple components of the hypothalamic-pituitary-adrenal-gonadal axis may be affected in schizophrenia. This could have important implications for future biomarker discovery efforts and personalized medicine strategies based on patient stratification for the treatment of this debilitating disorder.

Shotgun proteomic analysis of microdissected postmortem human pituitary using complementary two-dimensional liquid chromatography coupled with tandem mass spectrometer

The pituitary is responsible for multiple homeostatic functions including metabolism, growth and reproduction. Proteome analysis offers an efficient approach for a comprehensive analysis of pituitary protein expression. The pituitary is usually acquired from postmortem specimens, which may potentially affect the proteome profile by proteolysis. The aim of this study was to determine whether the postmortem pituitary could be used in proteomic analysis combining with Laser capture microdissection (LCM). Digested peptides from LCM captured prolactin (PRL) cells were separated by two dimensional-nanoscale liquid chromatography (2D-nanoLC/MS) and characterized by tandem mass spectrometry (MS). All MS/MS spectrums were searched by SEQUEST and a proteome of 1660 proteins was identified. Category analysis of the proteome revealed an extensive unbiased access to cell component proteins with diverse functional characteristics. The results demonstrated the ability of using 2D-nanoLC/MS to perform sensitive proteomic analysis on limited protein quantities through microdissection. Detailed comparisons between the proteome in question and the one derived from the prolactinoma controls at peptide and protein levels indicated that the two proteomes had similar characters. Overall, our results revealed for the first time the possibility of use of postmortem human pituitary for proteomic research which is important for further studies on disease biomarker identification and molecular mechanisms of prolactinoma tumorigenesis.

Analysis of the human pituitary proteome by data independent label‐free liquid chromatography tandem mass spectrometry

Studies of pituitary-related disorders would be facilitated by enhanced knowledge of the pituitary proteome. To construct a data set of human pituitary proteins, separate protein extracts were prepared from 15 post-mortem pituitaries and analyzed by data independent label-free nanoflow liquid chromatography mass spectrometry (nLC-MS(E) ). The detected mass/time features were aligned and quantified using the Rosetta Elucidator(®) system and annotated using results from ProteinLynx Global Server. The resulting data set comprised 1007 unique proteins, with stringent identification by a minimum of two distinct peptides. These proteins consisted predominantly of enzymes, transporters, transcription/translation factors, cell structure and secreted proteins.

Subclinical adenomas in postmortem pituitaries: classification and correlations to clinical data

The aim of this study was to examine pituitary adenomas in a series of postmortem pituitaries by use of modern technologies of immunostaining, to classify the adenomas according to the current WHO classification and to analyse the possible associations to the available clinical data. In this study, pituitaries of 3048 autopsy cases obtained from autopsy series of the years 1991-2004 were examined. A total of 334 pituitary adenomas were found in 316 pituitaries. One hundred and thirty-two sparsely granulated prolactin cell adenomas (39.5%), 75 null cell adenomas (22.5%) and 31 oncocytomas were diagnosed. Forty-six ACTH cell adenomas (13.8%, 27 densely granulated, 19 sparsely granulated) and one adenoma composed of Crooke's cells were detected. Twenty-two gonadotroph cell adenomas (6.6%), seven GH cell adenomas (four sparsely granulated, three densely granulated), one mixed GH cell-PRL cell adenoma, two TSH cell adenomas, five plurihormonal adenoma type I, four plurihormonal adenoma type II and two alpha-subunit-only adenomas were seen. Six adenomas remained unclassified because the tissue was not contained in all sections for immunohistochemistry. Seventeen pituitaries included multiple tumours. The overall tumour size ranged from 0.1 to 20 mm in diameter. Among 76 adenomas (22.7%), which had a tumour size of > or = 3 mm, only three tumours were macroadenomas corresponding to a tumour size of more than 10 mm. The evaluation of the available clinical data showed 99 cases of hypertension, 65 cases of diabetes mellitus, six patients with hyperthyroidism and four with hypothyroidism. No symptoms of adenohypophyseal hormone hypersecretion were reported. The statistical correlations to clinical data were discussed. Adenomas in postmortem pituitaries differ from those in surgical series in proportion of adenoma types and biological behaviour.

Subclinical adenomas in postmortem pituitaries: classification and correlations to clinical data

Objective: The aim of this study was to examine pituitaryadenomas in a series of postmortem pituitaries by use of modern technologies of immunostaining, to classify the adenomas according to the current WHO classification and to analyse the possible associations to the available clinical data. Methods: In this study, pituitaries of 3048 autopsy cases obtained from autopsy series of the years 1991‐2004 were examined. Results: A total of 334 pituitary adenomas were found in 316 pituitaries. One hundred and thirty-two sparsely granulated prolactin cell adenomas (39.5%), 75 null cell adenomas (22.5%) and 31 oncocytomas were diagnosed. Forty-six ACTH cell adenomas (13.8%, 27 densely granulated, 19 sparsely granulated) and one adenoma composed of Crooke’s cells were detected. Twenty-two gonadotroph cell adenomas (6.6%), seven GH cell adenomas (four sparsely granulated, three densely granulated), one mixed GH cell‐PRL cell adenoma, two TSH cell adenomas, five plurihormonal adenoma type I, four plurihormonal adenoma type II and two a-subunit-only adenomas were seen. Six adenomas remained unclassified because the tissue was not contained in all sections for immunohistochemistry. Seventeen pituitaries included multiple tumours. The overall tumour size ranged from 0.1 to 20 mm in diameter. Among 76 adenomas (22.7%), which had a tumour size of R3 mm, only three tumours were macroadenomas corresponding to a tumour size of more than 10 mm. The evaluation of the available clinical data showed 99 cases of hypertension, 65 cases of diabetes mellitus, six patients with hyper-thyroidism and four with hypothyroidism. No symptoms of adenohypophyseal hormone hypersecretion were reported. The statistical correlations to clinical data were discussed. Conclusions: Adenomas in postmortem pituitaries differ from those in surgical series in proportion of adenoma types and biological behaviour.

Preferential loss of Death Associated Protein kinase expression in invasive pituitary tumours is associated with either CpG island methylation or homozygous deletion.

Death Associated Protein kinase (DAP kinase) a novel calmodulin-dependent serine/threonine kinase was first identified as a positive mediator of programmed cell death. Loss of DAP kinase expression was first demonstrated in highly metastatic cells, whilst re-expression of the protein resulted in delayed local tumour growth and a decreased incidence of metastasis. Although loss of DAP kinase expression has been reported in several cell lines derived from human malignancies the mechanisms responsible have not been defined. In this study we have examined 32 sporadic pituitary tumours for expression of the DAP kinase protein and transcript. In addition, we examined the methylation and deletion status of the DAP kinase CpG island as possible mechanisms for the inactivation of the DAP kinase gene. Eleven of 32 (34%) tumours had undetectable DAP kinase expression, by Western blot and/or RT-PCR analysis. Loss of DAP kinase expression was significantly (P=0.004) associated with invasive tumours (10 of 17; 59%) compared to their non-invasive (1 of 15; 7%) counterparts. Of 11 tumours that failed to express DAP kinase, five (45%) showed de novo methylation of the CpG island contained within the promoter region, while four (36%) had evidence of homozygous deletion of this region. Statistical analysis showed that loss of DAP kinase expression was significantly (P=<0.001) associated with methylation or deletion of the DAP kinase CpG island. With two exceptions, none of the remaining tumours or five histologically normal post-mortem pituitaries examined had evidence of methylation or deletion within this region. To our knowledge this is the first report that describes two mutually exclusive mechanisms associated with loss of DAP kinase gene expression. In addition, we also show that loss of the DAP kinase protein and associated genetic aberrations preferentially segregates with tumours that show an invasive phenotype.

Hypermethylation of the p16CDKN2AMTS1 gene and loss of protein expression is associated with nonfunctional pituitary adenomas but not somatotrophinomas

The cyclin-dependent kinase inhibitor 2A/multiple tumor suppressor gene 1 (CDKN2A/MTS1/p16) plays an important role in the control of progression from G1 to S-phase of the cell cycle through the inhibition of CDK4-mediated RB1 phosphorylation. In this study we investigated 46 nonfunctional pituitary tumors and 21 somatotrophinomas for aberrant methylation of the CpG island contained within the CDKN2A gene as an alternative mechanism of gene silencing. We demonstrate methylation in 32/46 (70%) of nonfunctioning tumors, in contrast to 2/21 (9.5%) somatotrophinomas and 0/15 histologically normal postmortem pituitaries. Methylation in noninvasive and invasive nonfunctional tumors was approximately equal at 15/20 (75%) and 17/26 (65%), respectively. Immunohistochemical analysis showed an absence of CDKN2A protein in 25/32 (78%) methylated nonfunctioning tumors, demonstrating a highly significant overall correlation (P = 0.00007) between hypermethylation of the gene and absence of the p16 protein. The association between hypermethylation and absence of CDKN2A protein remained when the cohort of nonfunctional tumors was further subdivided into noninvasive 12/15 (80%; P = 0.004) and invasive 13/17 (76%; P = 0.01), suggesting this to be an early event in pituitary tumorigenesis. In contrast, a single invasive methylated somatotrophinoma failed to express the CDKN2A protein. These data show that hypermethylation of the CpG island within exon 1, but not exon 2, of the CDKN2A gene is frequently associated with loss of protein expression in nonfunctional pituitary tumors, but not somatotrophinomas, suggesting different tumorigenic pathways. Genes Chromosomes Cancer 24:328–336, 1999. © 1999 Wiley-Liss, Inc.

Hypermethylation of thep16/CDKN2A/MTS1 gene and loss of protein expression is associated with nonfunctional pituitary adenomas but not somatotrophinomas

The cyclin-dependent kinase inhibitor 2A/multiple tumor suppressor gene 1 (CDKN2A/MTS//p16) plays an important role in the control of progression from G to S-phase of the cell cycle through the inhibition of CDK4-mediated RBI phosphorylation. In this study we investigated 46 nonfunctional pituitary tumors and 21 somatotrophinomas for aberrant methylation of the CpG island contained within the CDKN2A gene as an alternative mechanism of gene silencing. We demonstrate methylation in 32/46 (70%) of nonfunctioning tumors, in contrast to 2/21 (9.5%) somatotrophinomas and 0/15 histologically normal postmortem pituitaries. Methylation in noninvasive and invasive nonfunctional tumors was approximately equal at 15/20 (75%) and 17/26 (65%), respectively. Immunohistochemical analysis showed an absence of CDKN2A protein in 25/32 (78%) methylated nonfunctioning tumors, demonstrating a highly significant overall correlation (P = 0.00007) between hypermethylation of the gene and absence of the p 16 protein. The association between hypermethylation and absence of CDKN2A protein remained when the cohort of nonfunctional tumors was further subdivided into noninvasive 12/15 (80%; P = 0.004) and invasive 13/17 (76%; P = 0.01), suggesting this to be an early event in pituitary tumorigenesis. In contrast, a single invasive methylated somatotrophinoma failed to express the CDKN2A protein. These data show that hypermethylation of the CpG island within exon 1, but not exon 2, of the CDKN2A gene is frequently associated with loss of protein expression in nonfunctional pituitary tumors, but not somatotrophinomas, suggesting different tumorigenic pathways.

Elevated proenkephalin-derived peptide levels in ACTH-producing adenomas: nucleus and cytoplasm localization.

The biosynthesis of met-enkephalin in human pituitary and human pituitary adenomas is still not well known. In this work, we studied the processing of proenkephalin-derived peptides in postmortem human pituitary (PMHP), ACTH-producing adenomas (ACTH-PA), nonfunctioning adenomas (NFA), and GH-producing adenomas (GH-PA). ACTH-PA contained at least 10 times more proenkephalin-derived peptides than PMHP, NFA,and GH-PA. Proenkephalin processing was different in the four tested tissues. In ACTH-PA, proenkephalin was processed to high-, intermediate-, and low-mol-wt products. The highest met-enkephalin-containing peptides levels corresponded to intermediate and low-mol-wt materials, although met-enkephalinArg-Phe and synenkephalin immunoreactivity appeared only in high-mol-wt peptides. In PMHP and NFA, met-enkephalin-Arg-Phe immunoreactivity was detected in intermediate- and low-mol-wt materials, and it was absent in GH-PA. Immunoblotting of ACTH-PA showed that met-enkephalin-Arg-Phe immunoreactivity corresponded to peptides of 44, 32-30, 27, and 17 kDa. The 32-30 and 17-kDa molecules were localized in the nuclear fraction where they were extracted after enzymatic digestion with DNase I. Plasmatic met-enkephalin levels did not increase in patients with Cushing's disease, suggesting that the pentapeptide stored in ACTH-PA was not released to the general circulation. In conclusion, we demonstrated that only ACTH-PA contained high levels of proenkephalin peptides, which were stored in cytoplasm organelles and in the nucleus, probably bound to chromatin. These results suggest an adenoma-specific physiological role of proenkephalin products.

Abundance and state of phosphorylation of the retinoblastoma gene product in human pituitary tumors

Targeted disruptions of the retinoblastoma (Rb) gene result in a high frequency of pituitary tumors in heterozygous mice. While our group and others have reported that loss of heterozygosity (LOH) at the Rb locus in human pituitary tumors is rare, these studies have not excluded small inactivating Rb-gene abnormalities more frequently found in human tumors and undetectable by LOH-PCR assays. As a more sensitive means of detecting evidence of these lesions, we have performed Western-blot analysis of several human pituitary tumors to identify Rb loss at the protein level as well as truncated forms of the Rb protein frequently associated with Rb-gene mutations. In 24 tumors, Rb protein was detected at levels 1.4- to 3.9-fold those detected in normal postmortem pituitary. There was no evidence of truncated forms of the Rb protein and only the hypophosphorylated (active) form of the protein was detected in normal and in pituitary tumor specimens. To investigate the possibility of loss of function mutations in certain tumors resulting in the expression of stable, mutant, hypophosphorylated Rb protein, we further performed SSCP analysis of exons 20 through 24 corresponding to the pocket domain of the Rb protein. Of 20 pituitary tumors examined, no mobility shifts could be demonstrated in this analysis. Our findings provide further evidence that primary Rb inactivation is not common in human pituitary tumors. Our detection of only the hypophosphorylated form of the Rb protein probably reflects the low proliferative state of these tumors.

C-myc, c-fos, and c-myb gene expression in human pituitary adenomas.

The possible roles of certain oncogenes in the development of pituitary tumors has not been investigated. We have examined the expression of c-myc, c-fos, and c-myb in a number of human pituitary tumors by ribonuclease protection assays, as these oncogenes have been implicated to have roles in the pathogenesis of other human tumors (12, 13, 15, 16). In several tumors examined (9 of 30) c-myc was expressed at levels 4-9 times greater than the level detected in normal postmortem pituitary. Although a larger percentage of negative immunohistochemical-staining tumors overexpressed c-myc, c-myc over-expression was not limited to this group of tumors. c-Fos was overexpressed in 1 of 30 tumors examined at a level 5.8-fold higher than that detected in normal postmortem pituitary. This tumor stained positive for ACTH by immunohistochemistry and was considered highly aggressive, demonstrating invasion beyond the sella turcica; however, when other ACTH-staining and invasive pituitary tumors were examined, no abnormality in the expression of c-fos was detected. In 30 tumors, c-myb was expressed at approximately the same level as that detected in normal postmortem pituitary. We conclude that c-myc is overexpressed in a subgroup of pituitary tumors and that this overexpression occurs broadly among the different groups of immunohistochemical-staining tumors. c-Fos overexpression appears to be much less common in pituitary tumors and does not necessarily correlate with the ability of the tumor to become invasive. c-Myb does not appear to have a role in the pathogenesis of pituitary tumors.