ResearchPad - pituitary-tumors-i Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[SAT-313 Synaptojanin 2 Binding Protein Is Required for Efficient Somatostatin Receptor 2A Phosphorylation by G Protein Coupled Receptor Kinases and Signaling to the ERK/MAPK Pathway]]> Somatostatin receptor 2A (Sst2A) agonists are the primary pharmacological treatment for growth hormone (GH) secreting pituitary tumors to inhibit GH secretion and limit the destructive effects of these tumors. Sst2A agonists are also widely used as imaging agents for neuroendocrine tumors, and are under investigation for treatment of these cancers. However, despite the clinical importance of Sst2A agonists, regulatory mechanisms controlling Sst2A internalization and signaling are not fully understood. Sst2A contains a C-terminal PDZ binding site that serves as a docking platform for PDZ domain containing proteins. In an unbiased screen for PDZ domain proteins that can interact with Sst2A, we identified Synaptojanin 2 binding protein (SYNJ2BP) / Outer mitochondrial protein 25 (OMP25). SYNJ2BP is an outer mitochondrial protein that has been shown previously to promote the internalization of the Activin Type II receptor. We find that SYNJ2BP interacts with Sst2A in a ligand-dependent manner. Importantly, we show that SYNJ2BP promotes interaction of Sst2A with the G protein couple receptor kinase 2 (GRK2), and that siRNA-mediated knockdown of SYNJ2BP dramatically inhibits Sst2A phosphorylation by endogenous GRKs. Moreover, overexpression of SYNJ2BP strongly potentiates Sst2A phosphorylation on the GRK phosphorylation sites. Modulation of SYNJ2BP expression also regulates somatostatin-stimulated β-arrestin recruitment to the plasma membrane. Interestingly, in contrast to the large effects on Sst2A phosphorylation and β-arrestin recruitment, modulation of SYNJ2BP expression only caused a small change in ligand-stimulated receptor internalization. When we assessed downstream signaling, we found that SYNJ2BP overexpression potently stimulated receptor-dependent activation of ERK1/2, but had little effect on the ability of Sst2A to repress forskolin-stimulated cAMP production. Taken together, these data demonstrate for the first time that interaction of Sst2A with a PDZ domain protein affects receptor regulation and signaling. Because multiple PDZ domain containing proteins have been shown to interact with Sst2A, these data also suggest that interaction of Sst2A with this class of proteins may be an important regulatory mechanism to bias its function within the cell.

<![CDATA[SAT-309 Integrated Analysis of Pituitary Adenoma Using Novel Approach of Non-Target Proteomics Along with RNA-Sequencing Analyses]]> Objective: To clarify the relationship between proteomic expression and clinical feature of pituitary adenoma. Methods: We have previously developed non-target proteomics analysis, which enables to detect and quantify approximately 7,000 to 9,000 kinds of protein weave, in parallel with RNA-seq analysis, and then subjected to 14 cases of pituitary adenoma surgically removed at Chiba University Hospital. Bioinformatic evaluation including DEGs, heatmap and PCA analyses was performed to reveal underlying their molecular pathogenesis. Results: We successfully identified 789 differentially expressed proteins and 593 DEGs in non-target proteomics and RNA-seq, respectively. Intriguingly, PCA analysis demonstrated that tumors were clearly divided into 3 groups based on protein expression profile; functional pituitary adenomas consisting of two subtypes depending on Pit1 and T-pit linage, and non-functional tumors consisting of two distinct subtypes, with properties close to functional tumors and unique characteristics of hard tumor difficult to remove by endoscopic surgery. To address the underlying molecular biological functions in each group clustering analysis and heat-map were performed and we found that 3 groups were separated clearly with their own both gene and protein expression profile. Indeed, for instance, GO term of plasma membrane part was significantly enriched in hard tumor group, pathways of GH receptor signaling, GH hormone synthesis as in GH-positive group. Conclusions: We herein demonstrate that pituitary adenoma can be uniquely separated into certain categories through our novel non-target proteomics with coupling to RNA-seq, particularly providing novel group of hard tumor characteristics with enriched expression of both protein and mRNA in plasma membrane part. Thus our method would be beneficial and useful to elucidate underlying molecular pathogenesis for pituitary tumors, while further analysis is required.

<![CDATA[SAT-304 Pituitary Stem Cells May Drive Adenomas Causing Cushing’s Disease]]> Introduction:Cell rests of self-renewing Sox2+ progenitor cells have been identified in the normal pituitary glands1, however their role in human pituitary tumorigenesis is not understood. Adrenocorticotropic hormone (ACTH) producing microadenomas that cause Cushing’s disease frequently (~70%) lack pathogenic genetic mutations.2 In mice, targeted expression of oncogenic β-catenin in Sox2+ cells generate microadenomas. Interestingly, the Sox2+ cells reside within the adjacent normal gland and drive adenomas in a paracrine fashion.3 We hypothesized that Sox2+ progenitors in human pituitary gland may drive the formation of microadenomas that cause Cushing’s disease (CD).

Methods:Four ACTH producing adenomas and two non-functional adenomas (NFPA) with separately annotated adjacent normal tissue (henceforward called ‘microenvironment’) were procured for this study (NCT00060541). We performed RNA deep sequencing (RNAseq) and compared expression of lineage-specific markers and progenitor markers using two-sample T-tests after testing for variance equality and using Welch’s approximation for degrees of freedom.

Results:We found expected overexpression of ACTH preprohormone POMC in CD adenomas compared to adjacent microenvironment (?-fold) and NFPA (?-fold). The microenvironment in Cushing’s disease showed increased expression of progenitor markers including SOX2, SOX9, CDH1, GRFA2, and KLF4 compared with microenviroment in NFPA. Likewise, the Cushing’s disease microenvironment showed increased expression ofPOMC (26.98 - fold, P = 0.004) as well as PRLR (FC 17.39, P = 0.006) and GH1 (FC 29.91, P = 0.003) implying that increased Sox2+ progenitors contribute to terminally differentiated corticotrope, lactotroph and somatotroph lineages in-vivo.

Conclusions:We report increased expression of several progenitor markers and concomitant elevation in tissues-specific markers in the microenvironment of Cushing’s disease patients. Our results indicate that increased pituitary progenitors in the microenvironment of human corticotropinomas may signal in paracrine fashion and may contribute to the pathogenesis of Cushing’s disease.

References:1. Cox, B. et al. J. Endocrinol.234, R135-R158 (2017).2. Bi, W. L. et al. Clin. Cancer Res.23, 1841-1851 (2017).3. Andoniadou, C. L. et al. Cell Stem Cell13, 433-445 (2013).

<![CDATA[SAT-300 Evading Death: Noxa in Cushing’s Disease Pituitary Adenomas]]> Introduction: Recurrence of Cushing’s disease (CD) caused by benign pituitary microadenomas are challenging clinical problems. Mechanisms underlying adenoma formation and recurrence remain unknown. PMAIP1 gene codes for Noxa, a Bcl-2 homology 3 (BH3) pro-apoptotic protein frequently downregulated in malignant human tumors.1-6 The role of dysregulated apoptosis remains largely unknown in benign tumors and in CD. We hypothesized that altered expression of Noxa protein is a pro-survival adaptation employed by CD adenomas.

Methods: Syngeneic human pituitary adenoma and adjacent normal gland pairs (n=2), and an additional CD adenoma were analyzed with RNAseq. 10 CD, 1 growth hormone (GH) and 1 non-functioning adenoma (NFPA) underwent immunohistochemical (IHC) analysis for Noxa expression, which was graded by a neuropathologist as 0=none, 1=light, 2=medium, 3=strong. Staining grade represents relative protein expression.

Results: Compared to adjacent normal pituitary tissue, we found that adenomas (n = 3) had a 3.76 fold increase in PMAIP1 mRNA. However, there was attenuated Noxa IHC staining in adenomas compared to normal pituitary in 8 of 10 CD patients (2:3, respectively), but similar staining in 2 of 10 CD patients (2:2 and 2-3:2-3). In GH and NFPA, we found similar patterns of Noxa suppression in the adenomas compared to the normal gland.

Conclusion: Despite elevated PMAIP1 (Noxa) gene expression in adenomas compared to adjacent normal gland in CD, protein expression was reduced in adenomas. This downregulation of Noxa protein expression may contribute to reduced apoptosis of tumor cells. These findings suggest that CD adenomas gain pro-survival advantage by downregulating Noxa protein at post-transcriptional or post-translational level.

References 1. Escobar, D. et al. Cell Death Dis.6, 1-14 (2015).2. Brinkmann, K. et al. Cell Rep.3, 881-891 (2013).3. Liu, Y. L. et al. Oncotarget5, 11237-11251 (2014).4. Dengler, M. A. et al. Cell Death Dis.5, 1-10 (2014).5. Liang, L. et al. J. Oral Pathol. Med.48, 52-59 (2019).6. Tahir, S. K. et al. Cancer Res.67, 1176-1183 (2007).

<![CDATA[SAT-301 Relationship Between Clinicopathological Aspects and MSH6/MSH2 and PD-L1 Expressions in Clinically Nonfunctioning Pituitary Adenomas]]> Introduction: Mismatch repair (MMR) genes are associated with the MMR mechanism that corrects DNA polymerase misincorporation errors. We analyzed the aggressive pituitary adenomas (PAs) associated with Lynch syndrome due to germline mutation in the MMR gene. Reduced expression of MMR genes mutS homologs 6/2 (MSH6/2) directly promotes PA growth (1, 2). MMR gene expression and programmed cell death 1 ligand 1 (PD-L1) expression are involved in tumor immunity with immune checkpoint inhibitors, but the direct association in PAs is not fully understood. Hypothesis and Objectives: MSH6/2 and PD-L1 expression could affect PA proliferation and invasion by pathological classification of nonfunctioning (NF) PAs because the proliferation and invasiveness differ depending on the PA histological subtype. In this study, we therefore analyzed the correlation between MSH6/2 and PD-L1 mRNA expression levels and clinicopathological factors related to tumor proliferation using human NFPAs. Experimental Design: We performed immunohistochemistry to classify the NFPAs into gonadotroph adenomas (GAs), silent corticotroph adenomas (SCAs), null cell adenomas (NCAs) and pituitary transcription factor 1 (PIT1) lineage PAs according to 2017 WHO classifications. Quantitative analyses were by real-time PCR to detect MSH6/2 and PD-L1 mRNA expressions in NFPAs (n = 89). We also performed statistical analyses of the expressions and clinicopathological factors such as Knosp Grade and histological subtypes. We investigated the effect of MSH6 knockout on cell proliferation and PD-L1 expression in AtT-20ins cells. Major Results: MSH6/2 expression was positively associated with PD-L1 expression. MSH6/2 and PD-L1 expressions are significantly lower in invasive NFPAs with Knosp Grade 3–4 or recurrence than in non-invasive NFPAs with Knosp Grade 1–2. Their expression is significantly lower in SCAs and NCAs than in GAs. Although MSH6/2 expression also tends to be lower, the PD-L1 expression tends to be higher in PIT1 lineage PAs, which is unlike SCAs and NCAs. MSH6 knockout in AtT-20ins significantly decreased PD-L1 expression with cell proliferation promotion. Interpretation of results and Conclusion: MSH6/2 and PD-L1 expressions of SCAs, NCAs, and PIT1 lineage PAs compared to GAs were thought to contribute to their clinically aggressive behaviors. The molecular mechanism of the difference in clinical features of NFPAs was partially elucidated. In particular, reduced expressions of MSH6/2 were thought to be useful for predicting the proliferation and invasiveness of NFPAs.

References: (1) Uraki S et al., Endocr J. 2017;64(9):895–906 (2) Uraki S et al., J Clin Endocrinol Metab. 2018;103(3):1171–1179. Declarations of conflicts of Interest: No authors declare any conflicts of interest.

<![CDATA[SAT-306 Muscarinic and Adrenergic Receptor Cooperativity in a Human Adrenocortical Carcinoma Cell Line]]> The role of autonomic receptors in the regulation of the adrenal cortex is poorly understood. We recently showed that activation of M3 muscarinic receptors stimulates intracellular calcium oscillations, aldosterone production, and expression of CYP11B2 (1). The present study explores the relationship between muscarinic and adrenergic receptors in corticosteroid production. Using live-cell fluorescence imaging of HAC15 adrenocortical cells with the calcium-sensitive probe Fluo-4, we have shown that stimulation of adrenergic receptors with the endogenous, non-selective adrenergic agonist norepinephrine (10μM) enhances intracellular Ca2+ oscillations caused by the cholinergic agonist carbachol (1μM). However, Ca2+ is not affected by norepinephrine alone. Adrenergic enhancement of carbachol-induced Ca2+ oscillations is blocked by the ⍺ adrenergic receptor antagonist phentolamine, but not by the β adrenergic receptor antagonist propanolol. Specifically, ⍺2 and β2 antagonists (such as yohimbine and butoxamine, respectively) significantly suppressed the norepinephrine effect, but ⍺1 and β1 antagonists (such as tamsulosin and metoprolol, respectively) had no effect. RT qPCR identified ⍺2A receptors as the most abundant adrenergic receptor in HAC15 cells. Saturation experiments using 3H-NMS and 3H-Rauwolscine confirmed the presence of muscarinic M2 and M3 receptors as well as ⍺2A receptors. Using competition radiolabeled binding assays we explored the cooperation between M2/M3 and ⍺2A adrenergic receptors. Our results suggest that autonomic regulation of intracellular Ca2+ depends on an interplay of M3 and ⍺2A receptors. Additional experiments will use ELISA methods to determine the functional impact of autonomic receptor cooperativity on steroid synthesis and secretion.

References: (1) Malaiyandi et al., Mol Cell Endocrinol. 2018 478: 1-9.

<![CDATA[SAT-311 Role of SREBP in Regulation of Corticotroph Tumor ACTH Secretion and Cell Proliferation]]> Cushing Disease (CD) is a life-threatening condition with suboptimal medical treatment. To identify drugs that not only inhibit ACTH secretion to attain eucortisolemia but also inhibit tumor growth, we conducted a high throughput screen employing a novel “gain of signal” ACTH AlphaLISA assay. From a kinase inhibitor library containing 430 compounds, we identified the dual PI3K/HDAC inhibitor, CUDC-907, as a potent inhibitor of both in vitro and in vivo corticotroph tumor ACTH secretion and growth. By stepwise comparison of CUDC-907 with mono-functional PI3K and HDAC inhibitors, we demonstrated that CUDC-907 exerts its inhibitory effect on ACTH secretion primarily through its inhibition of HDAC activity at the POMC transcriptional level; while PI3K-mediated inhibition of corticotroph cell viability further contributes to reduced ACTH secretion. We also used RNA-seq to characterize the global transcriptiome changes associated with CUDC-907 treatment. Hierarchical clustering showed that 1432 differentially expressed genes (DEGs, p≤0.05 and fold-change≥1.5) were altered by CUDC-907 treatment in comparison to the vehicle-treated control cells. Gene ontology (GO) analysis of 456 downregulated and 976 upregulated DEGs revealed that the most enriched biological processes were cholesterol biosynthesis (GO:0006695, p=1.977e-17) and the type I interferon signaling pathway (GO: 0060337, p=4.928e-7) respectively. Further analysis demonstrated downregulation of the membrane-bound transcription factor sterol regulatory element binding proteins (SREBPs). Downregulation of SREBP-2 by CUDC-907 as well as the several other target enzymes in the cholesterol biosynthesis and uptake pathway including IDI2, NSDHL, MVD, and HMGCR, was confirmed by real-time PCR. To further characterize a role for SREBP-2 in regulation of corticotroph tumor ACTH secretion and proliferation, we employed siRNA targeting endogenous SREBP-2 (SREBP-2 mRNA, Control vs. siRNA 1±0.03 vs. 0.6±0.08, p<0.05), and demonstrated that knockdown of SREBP-2 not only inhibited POMC mRNA expression (POMC mRNA, 1±0.03 vs. 0.7±0.01, p<0.01), and ACTH secretion (ACTH (ng/mL) 29±0.4 vs. 23±0.3, p<0.005), but also suppressed cell proliferation (Relative Proliferation Rate, 1±0.01 vs. 0.7±0.01, p<0.005). This was further confirmed by overexpression of cleaved mature SREBP-2, which led to increased POMC expression and cell proliferation. We demonstrate for the first time the role of the SREBP-mediated cholesterol biosynthesis pathway in regulation of corticotroph tumor POMC regulation and growth. Our studies identify SREBP and cholesterol biosynthesis as a therapeutic target in CD.

<![CDATA[SAT-314 Pituitary and Neuroendocrine Tumors Exhibit Distinct Transcriptomes on Single Cell RNA Sequencing]]> <![CDATA[SAT-315 CircVPS13c Promotes Tumor Growth and Invasiveness in Pituitary Adenoma by Downregulating IFITM1]]> <![CDATA[SAT-LB61 METTL3 Promotes Sparsely Granulated GH-Secreting Pituitary Adenomas to Behavior as Densely Granulated Adenomas]]> <![CDATA[SAT-312 Beta-Arrestin 2 Is Required for Dopamine Receptor Type 2 Inhibitory Effects on Akt Phosphorylation and Cell Proliferation in Pituitary Tumors]]> <![CDATA[SAT-310 Analysis of the Influence of Glucocorticoid Receptor Gene Polymorphism BCL-1 on Clinical and Psychiatric Manifestations in Brazilian Patients with Cushing’s Disease]]> <![CDATA[SAT-303 3D Mapping of the Human Growth Hormone Locus Identifies Putative Regulatory Hubs for Genes Involved in Cellular Signalling and Cancer-Related Pathways]]> <![CDATA[SAT-305 Veldoreotide (COR005) Mechanisms of Action Studies: Comparison to Octreotide and Pasireotide]]> <![CDATA[SAT-LB60 Discordant Biological Parameters of Remission in Acromegaly Do Not Increase the Risk of Hypertension or Diabetes: A Study With the Liege Acromegaly Survey Database]]> <![CDATA[SAT-308 Effect of Silibinin on ACTH Synthesis and Secretion in Human Adenomatous Corticotropes in Vitro]]>