Untargeted Lipidomics Reveals Specific Lipid Abnormalities in Nonfunctioning Human Pituitary Adenomas.

The pituitary gland is a small but important organ located in the base of the brain. Although mostly noncancerous, pituitary adenomas (PAs) can cause serious health problems such as headaches, visual field defects, double vision, and hypopituitarism by invasion of regional structures. Nonfunctioning PAs (NFPAs) approximately account for one-third of PAs manifested by no circulating hormone hypersecretion. Lipid reprogramming has been recognized as a hallmark of tumor cells and proven to play a crucial role in tumorigenesis. However, the lipid molecular pathogenesis of NFPAs has remained obscure to date. To uncover lipid alterations that may contribute to the development of NFPAs and define their molecular characteristics, we investigated tissue lipids of patients with NFPAs including eight null cell adenomas (NCAs) and eight oncocytomas (OCMs) and of five normal pituitary glands as the control (Ctrl) using nontargeted lipidomics based on ultrahigh-performance liquid chromatography-Orbitrap Q-Exactive HF mass spectrometry. The lipidomic results were further validated in another set of subjects consisting of 8 NCAs, 10 OCMs, and 6 Ctrls to define crucial lipids discriminating NFPAs from the normal pituitary tumors. Lipidomic analyses revealed that OCM showed more pronounced changes in lipid compositions than NCA and Ctrl. As expected, mitochondria abundant cardiolipins were remarkably increased in OCM, which was accordant with the biochemical evidence of mitochondria hyperplasia in OCM. Significantly increased levels of phospholipids (PLs), especially arachidonic acid (AA)-enriched PLs, were unique characteristics of lipid profiling in OCM vs Ctrl. Our results indicate that AA-PLs may have diagnostic potential for OCM.

Metabolic profiling reveals distinct metabolic alterations in different subtypes of pituitary adenomas and confers therapeutic targets.

BACKGROUND: Pituitary adenomas are common brain tumors. Although transsphenoidal surgery are able to achieve extensive tumor removal, the rate of recurrence ranges from 5 to 20% depending on the different subtype. Further understanding of these tumors is needed to develop novel strategies to improve the prognosis of patients. But their metabolic characteristics are largely unknown. METHODS: We used metabolomic, transcriptomic, and proteomic approaches to systematically investigate eight subtypes of pituitary adenomas and normal pituitary glands. By blocking IDH2, we investigate IDH2 play an inhibitory role in GH tumor cell growth and tumor secretion. RESULTS: We found that all of the pituitary adenomas displayed downregulated glucose metabolism and glycolysis compared to normal tissues. Together with the differences in amino acids and fatty acids, we categorized these tumors into three clusters. We then re-established the reprogrammed metabolic flux in pituitary adenomas based on multiomic analyses. Take growth hormone-secreting pituitary adenomas as an example, we revealed that IDH2 is a key player in the reprogrammed metabolism of such tumors. By blocking IDH2, we confirmed that IDH2 is a potential target for the inhibition of tumor cell growth and tumor secretion. CONCLUSIONS: Our study first uncovered the metabolic landscape of pituitary adenomas and demonstrated a possible way to inhibit tumor growth by regulating aberrant metabolism.

Enhancement of mitochondrial biogenesis and paradoxical inhibition of lactate dehydrogenase mediated by 14-3-3η in oncocytomas.

Oncocytomas represent a subset of benign pituitary adenomas that are characterized by significant mitochondrial hyperplasia. Mitochondria are key organelles for energy generation and metabolic intermediate production for biosynthesis in tumour cells, so understanding the mechanism underlying mitochondrial biogenesis and its impact on cellular metabolism in oncocytoma is vital. Here, we studied surgically resected pituitary oncocytomas by using multi-omic analyses. Whole-exome sequencing did not reveal any nuclear mutations, but identified several somatic mutations of mitochondrial DNA, and dysfunctional respiratory complex I. Metabolomic analysis suggested that oxidative phosphorylation was reduced within individual mitochondria, and that there was no reciprocal increase in glycolytic activity. Interestingly, we found a reduction in the cellular lactate level and reduced expression of lactate dehydrogenase A (LDHA), which contributed to mitochondrial biogenesis in an in vitro cell model. It is of note that the hypoxia-response signalling pathway was not upregulated in pituitary oncocytomas, thereby failing to enhance glycolysis. Proteomic analysis showed that 14-3-3η was exclusively overexpressed in oncocytomas, and that 14-3-3η was capable of inhibiting glycolysis, leading to mitochondrial biogenesis in the presence of rotenone. In particular, 14-3-3η inhibited LDHA by direct interaction in the setting of complex I dysfunction, highlighting the role of 14-3-3η overexpression and inefficient oxidative phosphorylation in oncocytoma mitochondrial biogenesis. These findings deepen our understanding of the metabolic changes that occur within oncocytomas, and shine a light on the mechanism of mitochondrial biogenesis, providing a novel perspective on metabolic adaptation in tumour cells. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Integrative proteomics and transcriptomics identify novel invasive-related biomarkers of non-functioning pituitary adenomas.

Non-functioning pituitary adenomas (NFPAs) are usually macroadenomas and display invasion into surrounding tissues. The treatment for invasive NFPAs is still challenging. This study describes the differential patterns of gene expression between invasive and non-invasive NFPAs and identifies novel biomarkers involved in invasion of NFPAs for diagnosis and treatment. Using gene microarray technology, we examined the gene expression profile and found 1160 differentially expressed messenger RNA (mRNA) between invasive and non-invasive NFPAs. Then, we examined the protein profile by liquid chromatography tandem mass spectrometry (LC-MS/MS) and found 433 differentially expressed proteins between invasive and non-invasive NFPAs. Subsequently, we integrated the proteomics and transcriptomics datasets and identified 29 common changed molecules. Through bioinformatics analysis using Ingenuity Pathway Analysis (IPA) software, we showed that the 29 molecules were enriched in 25 canonical signaling pathways, 25 molecular and cellular functions, and 2 networks. Eight genes were identified involved in the invasion function by the molecular and cellular functions analysis, including CAT, CLU, CHGA, EZR, KRT8, LIMA1, SH3GLB2 and SLC2A1. Furthermore, we validated the decreased CHGA expression and increased CLU expression in invasive NFPAs by qRT-PCR and Western blot. Our study demonstrated that integration of proteomics and transcriptomics could prove advantageous for accelerating tumor biomarker discovery and CHGA and CLU might be important novel biomarkers and therapeutic targets for invasion of NFPAs.

Lower PRDM2 expression is associated with dopamine-agonist resistance and tumor recurrence in prolactinomas.

BACKGROUND: Dopamine agonists (DAs) are the first-line treatment for prolactinomas, which account for 25-30% of functioning pituitary adenomas, and bromocriptine (BRC) is the only commercially available DAs in China. However, tumors are resistant to therapy in 5-18% of patients. METHODS: The exomes of six responsive prolactinomas and six resistant prolactinomas were analyzed by whole-exome sequencing. RESULTS: Using stringent variant calling and filtering parameters, ten somatic variants that were mainly associated with DNA repair or protein metabolic processes were identified. New resistant variants were identified in multiple genes including PRDM2, PRG4, MUC4, DSPP, DPCR1, RP1L1, MX2, POTEF, C1orf170, and KRTAP10-3. The expression of these genes was then quantified by real-time reverse-transcription PCR (RT-qPCR) in 12 prolactinomas and 3 normal pituitary glands. The mRNA levels of PRDM2 were approximately five-fold lower in resistant prolactinomas than in responsive tumors (p < 0.05). PRDM2 protein levels were lower in resistant prolactinomas than in responsive tumors, as determined by Western blotting and immunohistochemical analysis (p < 0.05). Overexpression of PRDM2 upregulated dopamine receptor D2 (D2DR) and inhibited the phosphorylation of ERK1/2 in MMQ cells. PRDM2 showed a synergistic effect with BRC on the inhibition of prolactin (PRL) secretion and MMQ cell viability, and low PRDM2 expression was associated with tumor recurrence. CONCLUSIONS: PRDM2 downregulation may play a role in dopamine-agonist resistance and tumor recurrence in prolactinomas.

Identification of a subtype-specific ENC1 gene related to invasiveness in human pituitary null cell adenoma and oncocytomas.

Non-functioning pituitary adenomas (NFPAs) may be locally invasive. Surgery is a treatment option, but unlike the case for functional pituitary adenomas, there are almost no drug treatments available for NFPAs. Markers of invasiveness are needed to guide therapeutic decision-making and identify potential adjuvant drugs. Owing to the highly heterogeneous nature of NFPAs, little is known regarding the subtype-specific gene expression profiles associated with invasiveness. To identify important biomarkers of invasiveness, we selected 23 null cell adenomas and 20 oncocytomas. These tumors were classified as invasive or non-invasive adenomas based on magnetic resonance imaging, pathology slides and surgical findings. Firstly, we observed that there were significant differences in expression between invasive (n = 3) and non-invasive (n = 4) adenomas by gene expression microarray. A total of 1,188 genes were differentially expressed in the invasive and non-invasive adenomas. Among these 1,188 genes, 578 were upregulated and 610 were downregulated in invasive adenomas. Secondly, the expression of ENC1, which displayed the significant alterations, was further confirmed by qRT-PCR and Western blot analysis in all 43 tumor samples and three normal pituitary glands. Low levels of ENC1 were found in tumor samples, while high levels were detected in normal pituitary glands. Interestingly, the ENC1 expression level was low in invasive null cell adenomas compared with non-invasive adenomas, but this relationship was not observed in invasive oncocytomas. Immunohistochemistry also demonstrated that the staining of ENC1 was different between invasive and non-invasive null cell adenomas. In addition, bioinformatics studies, including gene ontology and protein interaction analyses, were also performed to better understand the critical role of ENC1 in the development and progression of null cell adenomas and oncocytomas. Consequently, ENC1 may be an important biomarker for null cell adenomas and oncocytomas, and it is specific to invasive null cell adenomas.

Low levels of PRB3 mRNA are associated with dopamine-agonist resistance and tumor recurrence in prolactinomas.

Prolactinomas, or prolactin-secreting adenomas, constitute the most common type of hyperfunctioning pituitary adenoma. Dopamine agonists are used as first-line medication for prolactinomas, but the tumors are resistant to the therapy in 5-18 % of patients. To explore potential mechanisms of resistance to bromocriptine (a dopamine agonist), we analyzed six responsive prolactinomas and six resistant prolactinomas by whole-exome sequencing. We identified ten genes with sequence variants that were differentially found in the two groups of tumors. The expression of these genes was then quantified by real-time reverse-transcription PCR (RT-qPCR) in the 12 prolactinomas and in six normal pituitary glands. The mRNA levels of one of the genes, PRB3, were about fourfold lower in resistant prolactinomas than in the responsive tumors (p = 0.02). Furthermore, low PRB3 expression was also associated with tumor recurrence. Our results suggest that low levels of PRB3 mRNA may have a role in dopamine-agonist resistance and tumor recurrence of prolactinomas.

Integration of Proteomics and Metabolomics Revealed Metabolite-Protein Networks in ACTH-Secreting Pituitary Adenoma.

An effective treatment for the management of adrenocorticotropic hormone-secreting pituitary adenomas (ACTH-PA) is currently lacking, although surgery is a treatment option. We have integrated information obtained at the metabolomic and proteomic levels to identify critical networks and signaling pathways that may play important roles in the metabolic regulation of ACTH-PA and therefore hopefully represent potential therapeutic targets. Six ACTH-PAs and seven normal pituitary glands were investigated via gas chromatography-mass spectrometry (GC-MS) analysis for metabolomics. Five ACTH-PAs and five normal pituitary glands were subjected to proteomics analysis via nano liquid chromatography tandem-mass spectrometry (nanoLC-MS/MS). The joint pathway analysis and network analysis was performed using MetaboAnalyst 3.0. software. There were significant differences of metabolites and protein expression levels between the ACTH-PAs and normal pituitary glands. A proteomic analysis identified 417 differentially expressed proteins that were significantly enriched in the Myc signaling pathway. The protein-metabolite joint pathway analysis showed that differentially expressed proteins and metabolites were significantly enriched in glycolysis/gluconeogenesis, pyruvate metabolism, citrate cycle (TCA cycle), and the fatty acid metabolism pathway in ACTH-PA. The protein-metabolite molecular interaction network identified from the metabolomics and proteomics investigation resulted in four subnetworks. Ten nodes in subnetwork 1 were the most significantly enriched in cell amino acid metabolism and pyrimidine nucleotide metabolism. Additionally, the metabolite-gene-disease interaction network established nine subnetworks. Ninety-two nodes in subnetwork 1 were the most significantly enriched in carboxylic acid metabolism and organic acid metabolism. The present study clarified the pathway networks that function in ACTH-PA. Our results demonstrated the presence of downregulated glycolysis and fatty acid synthesis in this tumor type. We also revealed that the Myc signaling pathway significantly participated in the metabolic changes and tumorigenesis of ACTH-PA. This data may provide biomarkers for ACTH-PA diagnosis and monitoring, and could also lead to the development of novel strategies for treating pituitary adenomas.

Solitary subdural osteoma: A case report and literature review.

Osteomas attached to the meninges unrelated to bone are extremely rare and their etiology has not been discussed previously in the English literature. Here, we report the case of a 54-year-old male patient with a right frontal subdural osteoma. The patient presented with a ~5-month history of intermittent dizziness. Non-contrasted computerized tomography demonstrated a densely calcified mass attached to the inner surface of the right frontal skull. Magnetic resonance imaging (MRI) revealed this mass could be enhanced by contrast MRI. Intraoperatively, the hard mass was noted to be attached to the inner layer of the dura mater, and not associated with the bone. Histopathological examination revealed lamellated bony trabeculae lined by osteoblasts and the intertrabecular marrow spaces occupied by adipose tissue, which contributed to the MRI enhancement. It was speculated that subdural osteomas arose from ectopic osteoblasts derived from the embryological neural crest cells. The context of intertrabecular bone marrow contributed to the enhancement on MRI. Simple excision is the treatment of choice for symptomatic lesions.

Overexpression of the cell adhesion molecule claudin-9 is associated with invasion in pituitary oncocytomas.

Pituitary oncocytoma is a subtype of nonfunctioning pituitary adenomas with the potential to be locally invasive. Currently, surgery is the most effective treatment, whereas functional pituitary adenomas can be treated by drugs. We analyzed the invasiveness of pituitary oncocytomas to identify biomarkers that may be useful for guiding future therapeutic decision making. To identify important biomarkers of pituitary oncocytomas, 20 oncocytomas that were negative for hormone-specific immunostaining were confirmed by anti-mitochondria antibody immunostaining and electron microscopy. Our clinical phenotype data showed a prominent male predilection (85%). These tumors were classified as invasive or noninvasive based on preoperative magnetic resonance imaging, intraoperative records, and pathology slide. We observed significantly different expression profiles between pituitary oncocytomas (n = 3) and normal pituitary glands (n = 3). A total of 1937 genes were differentially expressed between the pituitary oncocytomas and normal pituitary glands. Among these 1937 genes, 954 were up-regulated and 983 were down-regulated in pituitary oncocytomas. The most significantly altered gene, claudin-9 (CLDN9), was further confirmed by quantitative real-time polymerase chain reaction, Western blot, and immunohistochemical staining in 10 invasive pituitary oncocytoma samples and 10 noninvasive pituitary oncocytoma samples. High levels of CLDN9 were found in the pituitary oncocytomas, whereas low levels were detected in normal pituitary glands. In addition, the CLDN9 expression level was higher in invasive oncocytomas compared with noninvasive oncocytomas. Bioinformatics Gene Ontology analysis was performed to better understand the critical role of CLDN9 in the development and progression of oncocytomas. Consequently, CLDN9 may be an important biomarker for invasive pituitary oncocytomas.