Corticotroph Tumor Cells Research Articles

PCSK1N as a tumor size marker and an ER stress response protein in corticotroph pituitary adenomas.

Silent corticotroph adenoma (SCA) exhibits more tumor aggressiveness features than functioning adenomas (FCA). We aimed to investigate PCSK1N expression in CA and examine if ER stress-induced responses affect cell survival in a corticotroph tumor cell model. Clinical and imaging characteristics were recorded in 33 patients with FCA (20 women, 11 macroadenomas) and 18 SCA (8 women, all macroadenomas). Gene expression of proopiomelanocortin (POMC), T-box transcription factor 19(TBX19)/TPIT, proprotein convertase subtilisin/kexin type 1(PCSK1)/PC1/3, and its inhibitor PCSK1N, was measured by RT-qPCR in adenoma tissue.Mouse pituitary corticotroph tumor (AtT-20) cells were treated with tanespimycin (17-AAG), a HSP90 chaperone inhibitor, to induce ER stress, followed by gene and protein analyses. POMC, TPIT, and PCSK1 expression were higher, whereas PCSK1N was lower in FCA compared to SCA. PCSK1N correlated with POMC (rs= -0.514, p <0.001), TPIT (rs= -0.386, p = 0.005), PCSK1 (rs= -0.3691, p = 0.008), and tumor largest diameter (rs= 0.645, p <0.001), in all CA. Induction of ER stress by 17-AAG in AtT-20 cells led to a decrease of POMC and an increase of PCSK1N gene expression at 24h. Moreover, a downregulation of cell cycle, apoptosis, and senescence pathways, and alterations in cell adhesion and cytoskeleton were observed at the protein level. PCSK1N is higher in SCA compared with FCA, and associated with corticotroph cell markers and tumor size. PCSK1N is likely to be part of the adaptive response to ER stress, potentially conferring a survival advantage to the corticotroph tumor cell in conjunction with other proteins.

ER stress causes variable ACTH production and secretion in corticotroph tumour cells

ER stress causes variable ACTH production and secretion in corticotroph tumour cells

Circulating levels of mir-375 in cushing's disease patients and evaluation of its role in the regulation of sstr2 expression in murine pituitary corticotroph tumor cell model

Circulating levels of mir-375 in cushing's disease patients and evaluation of its role in the regulation of sstr2 expression in murine pituitary corticotroph tumor cell model

SAT618 Uncovering The Role Of O-glcnacylation In Pituitary Adenoma Pathogenesis: Insights From Corticotroph Tumors

Abstract Disclosure: L.J. Massman: None. R. Gonzalez: None. B. Laing: None. D. Coss: None. A.G. Ioachimescu: None. N. Zwagerman: None. S. Olivier-Van Stichelen: None. Functioning corticotroph pituitary adenomas are associated with high mortality, morbidity, and a high propensity of biochemical recurrence postoperatively. Silent corticotroph adenomas cause mass-effect manifestations and have an increased risk for tumor regrowth. The pathogenetic mechanisms responsible for the clinical course of corticotroph PA are poorly understood. In this study, we investigated the role of the O-GlcNAc posttranslational modification in their pathophysiology. O-GlcNAcylation is involved in protein signaling pathways and deregulated in cancers and diabetes. First, we performed RNA-sequencing in functioning and silent corticotroph and gonadotroph adenomas (n=25). We discovered that O-GlcNAc enzymes were upregulated in ACTH-secreting corticotroph tumors and correlated with larger, more invasive tumors. Also, an in vitro mechanistic analysis of corticotroph tumor cells (ATt-20) demonstrated that O-GlcNAcylation was essential for their proliferation and secretory ability. Specifically, O-GlcNAcylation affected Proopiomelanocortin mRNA splicing and stability, suggesting a new mechanism of fast hormonal response in corticotropes. Overall, our study indicates a key role for O-GlcNAcylation in corticotroph adenomas’ cellular proliferation and hypersecretion. Presentation: Saturday, June 17, 2023

Somatostatin receptor type 2 (SSTR2) expression regulation by miR-375 in a murine pituitary corticotroph tumor cell model

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

PMON149 Identification of TR4 Modulators for Treatment of Cushing Disease.

Abstract Cushing Disease (CD) is caused by an adrenocorticotropic hormone (ACTH)-secreting pituitary tumor that causes excess adrenal-derived cortisol. It is a life-threatening "orphan disease" with an annual health care cost &amp;gt;7-fold higher than average patients. Surgical removal is the current first-line therapy but the disease frequently recurs. Repeat surgery, radiation therapy and bilateral adrenalectomy are not always successful and associated with significant morbidity. Currently available drugs or those in clinical trials for CD do not target the pituitary corticotroph tumor itself and escape from control is common with long-term use. A clear unmet need for efficacious and safe therapies that offer biochemical and tumor control exists. We hypothesize that direct targeting of corticotroph tumors to modulate ACTH and in turn, glucocorticoid secretion is the optimal way to treat CD. We recently demonstrated that the orphan testicular receptor 4 (TR4, also known as NR2C2) is a potent regulator of hypothalamic-pituitary-adrenal (HPA) axis function and directly regulates pro-opiomelanocortin (POMC) gene transcription and ACTH secretion. To identify and characterize TR4 small molecule modulators, we used the Flexi Vector system to fuse the TR4 224-615aa LBD to the 3' GAL4 DBD to generate a chimeric fusion plasmid (pBIND-TR4-LBD) that constitutively expresses a Renilla luciferase serving as an internal reporter control. We then transiently transfected our chimeric pBIND-TR4-LBD construct into human embryonic kidney (HEK293) cells which express an exogenous reporter construct pGL4.35 (GloResponse, luc2P/9XGAL4UAS-HEK293 Cell Line, Promega) that is driven by multiple copies of a GAL4 responsive upstream activating sequence (UAS). Following treatment with TR4 ligands or modulators, our TR4-LBD undergoes a conformational change and recruits additional cofactors and RNA polymerase II to the complex to drive GAL4-directed firefly luciferase expression. This dual-luciferase format allows us to detect compound cytotoxicity or off-target change in expression during chemical screening, greatly improving data quality. In a pilot drug screen of a LOPAC library and using robust z-score statistics (z-score&amp;gt;3), representing a luciferase to renilla induction &amp;gt; 3 standard deviations of baseline variability, we have identified several G-protein coupled ion channel regulators as potential "hit" TR4 modulators. These identified GPCRs regulate phosphatidylinositol and cyclic AMP effector systems, and activate several signaling pathway kinases. Mode-of-action and dose-response evaluation to calculate EC50 of these potential TR4 hit compounds is under investigation and lead compounds will be further validated using a POMC transactivation assay in murine and human corticotroph tumor cells. Our studies have begun to identify and rigorously validate compounds that efficiently and specifically abrogate TR4 actions to inhibit ACTH secretion and may lead to the discovery of safe and efficacious lead TR4 inhibitory compounds that can be further advanced as potential drug therapies for CD. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Improved pasireotide response in USP8 mutant corticotroph tumours in vitro.

Cushing's disease is a rare but devastating and difficult to manage condition. The somatostatin analogue pasireotide is the only pituitary-targeting pharmaceutical approved for the treatment of Cushing's disease but is accompanied by varying efficacy and potentially severe side effects. Finding means to predict which patients are more likely to benefit from this treatment may improve their management. More than half of corticotroph tumours harbour mutations in the USP8 gene, and there is evidence of higher somatostatin receptor 5 (SSTR5) expression in the USP8-mutant tumours. Pasireotide has a high affinity for SSTR5, indicating that these tumours may be more sensitive to treatment. To test this hypothesis, we examined the inhibitory action of pasireotide on adrenocorticotrophic hormone synthesis in primary cultures of human corticotroph tumour with assessed USP8 mutational status and in immortalized murine corticotroph tumour cells overexpressing human USP8 mutants frequent in Cushing's disease. Our in vitro results demonstrate that pasireotide exerts a higher antisecretory response in USP8-mutant corticotroph tumours. Overexpressing USP8 mutants in a murine corticotroph tumour cell model increased endogenous somatostatin receptor 5 (Sstr5) transcription. The murine Sstr5 promoter has two binding sites for the activating protein 1 (AP-1) and USP8 mutants possibly to mediate their action by stimulating AP-1 transcriptional activity. Our data corroborate the USP8 mutational status as a potential marker of pasireotide response and describe a potential mechanism through which USP8 mutants may regulate SSTR5 gene expression.

P720R USP8 Mutation Is Associated with a Better Responsiveness to Pasireotide in ACTH-Secreting PitNETs.

Simple SummarySomatic mutations in USP8 gene have been identified in 11–60% of patients with ACTH-secreting pituitary tumors. This study investigated the impact of USP8 mutations on corticotroph tumor cells responsiveness to the treatment with the somatostatin analog pasireotide. Although we found that the somatostatin receptor targeted by pasireotide, SSTR5, was upregulated in cells transfected with USP8 mutants, pasireotide failed to reduce ACTH secretion in cells expressing mutated USP8, except for the mutation P720R. Overall these data demonstrate that different USP8 mutations exert opposite effects on the responsiveness of ACTH-secreting pituitary tumors to pasireotide.Somatic mutations in the ubiquitin specific peptidase 8 (USP8) gene have been associated with higher levels of somatostatin (SS) receptor subtype 5 (SSTR5) in adrenocorticotroph hormone (ACTH)-secreting pituitary neuroendocrine tumors (PitNETs). However, a correlation between the USP8 mutational status and favourable responses to pasireotide, the somatostatin multi-receptor ligand acting especially on SSTR5, has not been investigated yet. Here, we studied the impact of USP8 mutations on pasireotide responsiveness in human and murine corticotroph tumor cells. SSTR5 upregulation was observed in USP8 wild-type primary tumor cells transfected with S718del USP8 mutant. However, cell transfection with S718del USP8 and C40-USP8 mutants in in vitro sensitive cultures from USP8 wild-type tumors abolished their ability to respond to pasireotide and did not confer pasireotide responsiveness to the in vitro resistant culture. Pasireotide failed to reduce ACTH secretion in primary cells from one S718P USP8-mutated tumor but exerted a strong antisecretory effect in primary cells from one P720R USP8-mutated tumor. In agreement, AtT-20 cells transfection with USP8 mutants led to SSTR5 expression increase but pasireotide could reduce ACTH production and cyclin E expression in P720R USP8 overexpressing cells, only. In situ Proximity Ligation Assay and immunoflurescence experiments revealed that P720R USP8 mutant is still able to bind 14-3-3 proteins in AtT-20 cells, without affecting SSTR5 localization. In conclusion, P720R USP8 mutation might be considered as a molecular predictor of favourable response to pasireotide in corticotroph tumor cells.

Impact of USP8 mutations on corticotroph tumor cells responsiveness to pasireotide

Impact of USP8 mutations on corticotroph tumor cells responsiveness to pasireotide

154 Pituitary Adenomas Evade Apoptosis via Noxa Degradation in Cushing’s Disease

INTRODUCTION: Cushing’s disease (CD) arising from ACTH-secreting pituitary adenomas is associated with severe morbidity and mortality. Pathogenic mechanisms of sporadic CD are poorly understood. Using novel syngeneic, pairwise transcriptomic analysis of surgical tissues, we identified transcriptional upregulation of PMAIP1 (encoding pro-apoptotic noxa) in CD adenomas compared to adjacent normal gland. METHODS: We investigated noxa protein levels by immunohistochemistry in 10 CD adenoma/normal pairs and by Western blotting in two syngeneic pairs of primary human cell lines from CD adenomas. We validated our key findings in ATT20 murine pituitary corticotroph tumor cells and in corticotroph-enriched murine cell lines mCortEFGF. Proteasomal-mediated degradation was assessed by cycloheximide chase assay. Bortezomib was used for proteasomal inhibition. Cell viability was assessed using CellTiterGlo (Promega). RESULTS: Despite PMAIP1 mRNA overexpression in CD, we found consistently low noxa protein in CD adenomas compared to surrounding normal glands but not in growth hormone secreting adenomas or prolactinomas. A cycloheximide assay demonstrated proteasomal-mediated noxa degradation mCortEFGF cells, which was rescued by proteasomal inhibition using bortezomib. Bortezomib treatment decreased cell viability and induced apoptosis in mCortEFGF cells, and also inhibited growth with apoptosis induction in CD adenoma primary cells. CONCLUSION: We identified proteasomal-mediated noxa degradation as a mechanism of escape from apoptosis in CD adenomas despite PMAIP1 transcriptional upregulation. Proteasomal inhibition using bortezomib rescued noxa protein levels and induced apoptosis in a dose-dependent fashion. Bortezomib, an FDA approved medication for mantle cell lymphoma, may represent a novel therapeutic target for patients with CD.

PI3K inhibition by BKM120 results in anti-proliferative effects on corticotroph tumor cells.

Cushing's disease is associated with significant morbidity; thus, additional tumor-directed drugs with the potential to exert antineoplastic effects on corticotroph adenoma cells are desired. The phosphoinositide-3-kinase (PI3K)/protein kinase B (AKT) pathway, which plays regulatory role in cell survival and proliferation, is activated in pituitary adenomas. The present study evaluated the effects of BKM120 (Buparlisib), an oral PI3K inhibitor, on cell viability, apoptosis, cell cycle phase distribution, and ACTH production in mouse corticotroph tumor cells. AtT-20/D16v-F2 mouse pituitary corticotroph tumor cells were treated with increasing concentrations of BKM120 or vehicle. Cell viability was measured using an MTS-based assay. Apoptosis was evaluated by Annexin V staining. Cell cycle analysis was performed by propidium iodide DNA staining and flow cytometry. Gene expression of cell cycle regulators (Cdkn1b, Ccnd1, Ccne1, Cdk2, Cdk4, Myc, and Rb1) was assessed by qPCR. Protein expression of p27, total and phosphorylated Akt was assessed by Western blot. ACTH levels were measured in the culture supernatants by chemiluminescent immunometric assay. Treatment with BKM120 decreased AtT-20/D16v-F2 cell viability, induced a G0/G1 cell cycle arrest, reduced the phosphorylation of Akt at Serine 473, and increased p27 expression. Furthermore, BKM120 treatment diminished ACTH levels in the cell culture supernatants. In vitro inhibition of PI3K/AKT pathway by BKM120 resulted in anti-proliferative effects on corticotroph tumor cells, decreasing cell viability and ACTH production. These encouraging findings shape the path for further experiments with the inhibition of PI3K/AKT pathway in Cushing's disease.

Effects of tubastatin A on adrenocorticotropic hormone synthesis and proliferation of AtT-20 corticotroph tumor cells.

Cushing's disease is an endocrine disorder characterized by hypercortisolism, mainly caused by autonomous production of ACTH from pituitary adenomas. Autonomous ACTH secretion results in excess cortisol production from the adrenal glands, and corticotroph adenoma cells disrupt the normal cortisol feedback mechanism. Pan-histone deacetylase (HDAC) inhibitors inhibit cell proliferation and ACTH production in AtT-20 corticotroph tumor cells. A selective HDAC6 inhibitor has been known to exert antitumor effects and reduce adverse effects related to the inhibition of other HDACs. The current study demonstrated that the potent and selective HDAC6 inhibitor tubastatin A has inhibitory effects on proopiomelanocortin (Pomc) and pituitary tumor-transforming gene 1 (Pttg1) mRNA expression, involved in cell proliferation. The phosphorylated Akt/Akt protein levels were increased after treatment with tubastatin A. Therefore, the proliferation of corticotroph cells may be regulated through the Akt-Pttg1 pathway. Dexamethasone treatment also decreased the Pomc mRNA level. Combined tubastatin A and dexamethasone treatment showed additive effects on the Pomc mRNA level. Thus, tubastatin A may have applications in the treatment of Cushing's disease.

PI3K Inhibition by BKM120 Results in Antiproliferative Effects on Corticotroph Tumor Cells

Purpose: Cushing’s disease is associated with significant morbidity, thus additional tumor-directed drugs with the potential to exert antineoplastic effects on corticotroph adenoma cells are desired. The PI3K (phosphoinositide-3-kinase)/AKT (protein kinase B) pathway, which plays regulatory roles in cell survival and proliferation, is activated in pituitary adenomas. The present study evaluated the effects of BKM120 (Buparlisib), an oral PI3K inhibitor, in corticotroph tumor cells. Methods: AtT-20/D16v-F2 mouse pituitary corticotroph tumor cells were treated with increasing concentrations of BKM120 or vehicle. Cell viability was measured using MTS-based assay. Apoptosis was evaluated by Annexin V staining. ACTH levels were measured in the culture supernatants by chemiluminescent immunometric assay. Cell cycle analysis was performed by propidium iodide DNA staining and flow cytometry. Gene expression of cell cycle regulators (Cdkn1b, Rb1, Ccnd1, Cdk4, Cdk2, and Myc) was assessed by qPCR. Protein expression of p27, p70 S6 Kinase, p85 S6 Kinase, and phosphorylated AKT was assessed by Western blot. Results: Treatment with BKM120 decreased AtT-20/D16v-F2 cell proliferation and ACTH levels in the cell culture supernatants. Furthermore, BKM120 treatment diminished the phosphorylation of AKT at residue 473, increased p27 expression and induced a G0/G1 cell cycle arrest. Conclusion:In vitro inhibition of PI3K/AKT pathway by BKM120 resulted in antiproliferative effects on corticotroph tumor cells, decreasing cell viability and ACTH production. These encouraging findings shape the path for further experiments with the inhibition of PI3K/AKT pathway in Cushing’s disease.

A Prospective Trial With Ketoconazole Induction Therapy and Octreotide Maintenance Treatment of Cushing’s Disease

Abstract Context: The lack of efficacy of octreotide in the medical treatment of Cushing’s disease (CD) may result from suppressive effects of hypercortisolism on somatostatin receptor subtype 2 (SST2) expression by corticotroph adenomas. We previously demonstrated that SST2 mRNA expression levels in corticotroph tumor cells from patients preoperatively treated with cortisol-lowering therapy are significantly higher compared to those from patients with uncontrolled CD at time of surgery. It may be hypothesized that control of cortisol production induced by steroidogenesis inhibitors may reverse SST2 expression and increase efficacy of tumor-directed therapy with octreotide. Objective: To evaluate the efficacy of a sequential strategy of initiation treatment with ketoconazole (KTC) to reduce cortisol levels, followed by octreotide as maintenance therapy in patients with CD. Patients and Design: 14 adult patients with CD were prospectively enrolled. All patients started on KTC (600-800 mg/day), and once cortisol levels were normalized, octreotide 20 mg/4 weeks was initiated which could eventually be increased to 30 mg/4 weeks. After two months of combined therapy, patients were maintained on octreotide monotherapy until the end of the study period (9 months). Treatment success was assessed by the mean of 2 collections of urinary free cortisol (UFC) levels. Results: The mean age of our study population (14 patients) was 48.6 years, 64% (n=8) were female, 85% (n=12) were newly diagnosed and naïve in treatment. Ketoconazole was able to normalize UFC level in 11 (79%) patients. Subsequently, octreotide effectively sustained normal UFC levels in 3 patients (27%) (responders). Four (36%) other patients showed a partial response to octreotide. In 3 patients, normal UFC levels were sustained for one- or two-months following discontinuation of KTC and in the other partial responder, the UFC levels at follow-up decreased by at least 50% of the baseline levels. The remaining 4 (36%) patients developed hypercortisolism as soon as ketoconazole was stopped (non-responders). Responders to octreotide had lower UFC levels at baseline when compared to partial and non-responders (1.40 ± 0.06 vs. 2.05 ± 0.20 ULN, p=0.08). Two of three responders showed improvement in weight, waist circumference, and systolic and diastolic blood pressure during the treatment period. In terms of side effects, one patient discontinued KTC because of gastrointestinal intolerance and 5 patients had a transient increase in liver enzymes. Conclusions: This proof-of-concept study shows that the sequential treatment with ketoconazole to lower cortisol levels followed by octreotide to maintain normal cortisol production seems effective in a subset of patients with mild CD. Ongoing studies aim to evaluate whether this is the result from increased SST2 expression in corticotroph adenomas.

New Transcriptional Insights into Silent and Active Corticotroph Pituitary Tumors at Single Cell Resolution

Silent pituitary corticotroph tumors derive from the Tpit (aka TBX19) pituitary lineage. Accounting for ~ 30% of corticotroph tumors, they are not infrequently clinically aggressive and invade locally into adjacent sellar structures, making complete surgical resection challenging and contributing to their higher recurrence rates. How silent and active corticotroph tumor subtypes differ is not clear although some studies reported that silent corticotroph tumors exhibit reduced PC1 expression causing impaired POMC processing. We used single cell RNAseq to compare the transcriptome between silent (n = 2) and active (n = 4) corticotroph tumors at the single cell level. We obtained an average of 265 million reads, and 24,682 genes per patient with an average of 1,240 genes expressed and 3,5442 unique molecular identifiers (UMIs) detected per cell. We further defined 5 distinct cell populations from a total of 23,269 cells, namely tumor cells (62%), stromal cells (25%), immune cells (7%), progenitor cells (5%), and a minor population of erythrocytes (1%). Tumor cells clustered in an origin-dependent manner and all expressed POMC and TBX19. However, the gene signatures of silent and active corticotroph tumors differed in 3 major aspects. Firstly, and supporting prior studies, a series of hormone processing peptidase genes, including PC1 (aka PCSK1), PDIA3, SEC11C, SPCS1 and CTSB, were reduced in silent corticotroph tumors (p=5.54e-5) compared to active corticotroph tumors. Secondly, genes involved in organization of secretory vesicles such as SCG5, TIMP1, VGF, SYT17, LGALS3 and CALY were also reduced in silent corticotroph tumors, which could further compound their inability to secrete ACTH. Thirdly, the silent corticotroph tumors exhibited several features of endothelial-to-mesenchymal transition (EMT), including increased expression of the mesenchymal genes CDH2 (aka NCAD), COL1A1, PCDH9, FGF5, ID2 (p=8.4e-3), and loss of EPCAM, which regulate cell migration and movement. Upstream analysis suggested that aberrant STAT3 activation may be related to these changes. Consequently, we noted that the stromal content was higher in silent corticotroph tumors (47.5% vs. 18.13%), concordant with the observed EMT de-differentiation of tumor cells. In summary, our scRNAseq analysis provides an unprecedented precise investigation of the transcriptomic features of thousands of heterogenous corticotroph tumor cells simultaneously. We demonstrate that although silent corticotroph tumor cells still express the pituitary lineage markers PITX1 and TBX19, they exhibit EMT, potentially affording increased migratory capacity at the cost of reduced neuroendocrine function with inability to produce and secrete mature ACTH. Our findings provide novel insights into the pathogenesis of silent versus active corticotroph tumor, but may reveal novel molecular targets for treatment of these challenging tumors.

A human ACTH-secreting corticotroph tumoroid model: Novel Human ACTH-Secreting Tumor Cell in vitro Model

BackgroundCushing disease (CD), although rare, is a life-threatening disorder caused by an adrenocorticotropic hormone (ACTH)-secreting pituitary adenoma, which leads to excess adrenal-derived cortisol. Efficacious and safe medical therapies that control both hormonal hypersecretion and pituitary corticotroph tumor growth remain an unmet need in the management of CD. Translational research in pituitary tumors has been significantly hampered by limited quantities of surgically resected tissue for ex vivo studies, and unavailability of human pituitary tumor cell models. MethodsTo characterize human corticotroph tumors at the cellular level, we employed single cell RNA-sequencing (scRNA-seq) to study 4 surgically resected tumors. We also used microarrays to compare individualized paired consecutive culture passages to understand transcriptional shifts as in vitro cultures lost ACTH secretion. Based on these findings, we then modified our in vitro culture methods to develop sustained ACTH-secreting human corticotroph tumoroid cultures. FindingsscRNA-seq identified 4 major cell populations, namely corticotroph tumor (73.6%), stromal (11.2%), progenitor (8.3%), and immune cells (6.8%). Microarray analysis revealed striking changes in extracellular matrix, cell adhesion and motility-related genes concordant with loss of ACTH secretion during conventional 2D culture. Based on these findings, we subsequently defined a series of crucial culture nutrients and scaffold modifications that provided a more favorable trophic and structural environment that could maintain ACTH secretion in in vitro human corticotroph tumor cultures for up to 4 months. InterpretationOur human corticotroph tumoroid model is a significant advance in the field of pituitary tumors and will further enable translational research studies to identify critically needed therapies for CD. FundingThis work was partly funded by NCI P50-CA211015 and the Warley Trust Foundation.

Filamin A is required for somatostatin receptor type 5 expression and pasireotide-mediated signaling in pituitary corticotroph tumor cells

Somatostatin receptor type 5 (SST5) represents the main pharmacological target in the treatment of adrenocorticotroph hormone (ACTH)-secreting tumors. However, molecular predictors of responsiveness to pasireotide require further investigation. The cytoskeleton protein filamin A (FLNA) modulates the responsiveness to somatostatin analogs (SSA) treatment in other types of pituitary tumors by regulating somatostatin receptor type 2 (SST2)/dopamine receptor type 2 (DRD2) expression and activity. Here, we aimed to test the involvement of FLNA in the modulation of SST5 response to SSA in human and murine tumor corticotrophs. Western blot analysis of human corticotropinomas showed that FLNA and SST5 correlate. Both in human primary cultures and AtT-20 cells, FLNA genetic silencing caused a decrease of receptor expression level. Moreover, pasireotide-mediated SST5 downregulation observed in AtT-20 control cells was no further detected in FLNA silenced cells. In AtT-20 cells, in situ PLA experiments revealed an increased number of SST5-FLNA complexes following pasireotide incubation. Finally, FLNA knock down abolished pasireotide-induced SST5 actions on hormone secretion, cell proliferation and apoptosis. In conclusion, FLNA is implicated in SST5 expression modulation and signaling.

Involvement of histone deacetylase 1/2 in adrenocorticotropic hormone synthesis and proliferation of corticotroph tumor AtT-20 cells

Cushing’s disease is mainly caused by autonomous production of adrenocorticotropic hormone (ACTH) from pituitary adenomas. In our previous study, a histone deacetylase (HDAC) inhibitor, trichostatin A, inhibited cell proliferation and ACTH production via decreased pituitary tumor-transforming gene 1 (PTTG1) in AtT-20 mouse corticotroph tumor cells. In the present study, we examined the effects of romidepsin, a potent and selective HDAC1/2 inhibitor, on cell proliferation and ACTH synthesis. To elucidate further potential mechanisms of romidepsin, we examined the effects of HDAC1/2 on proopiomelanocortin (Pomc) and Pttg1 mRNA levels and cell proliferation. Small interfering RNA-mediated knockdown was used to decrease HDAC1 or 2. Romidepsin treatment decreased Pomc and Pttg1 mRNA levels, and cell proliferation. The drug also increased Hdac1 and decreased Hdac2 mRNA levels. Hdac1 knockdown decreased basal Pttg1 mRNA levels and cell proliferation, but not Pomc mRNA levels. Romidepsin treatment decreases ACTH synthesis in corticotroph tumor cells. Romidepsin suppresses cell proliferation via PTTG1. HDAC1 is also involved in the proliferation of corticotroph cells via PTTG1.

Differential effects of β-arrestin1 and β-arrestin2 on somatostatin receptors in murine AtT-20 corticotroph tumor cells.

Autonomous production of adrenocorticotropic hormone (ACTH) from pituitary corticotroph adenomas is the primary cause of Cushing's disease. Somatostatin receptor, a G protein-coupled receptor (GPCR), types 2 (SSTR2) and 5 (SSTR5) mRNA expression is greater than that of other SSTR subtypes in human corticotroph adenomas. Further, the multiligand SOM230 shows potent effects in decreasing ACTH plasma levels and urinary free cortisol levels in patients with Cushing's disease. We previously showed that both Sstr2 and Sstr5 mRNA levels were unaffected by SOM230 treatment, suggesting that both receptors might not be downregulated by the agonist. Intracellular molecules, such as β-arrestins, modulate ligand activated-receptor responses. In the present study, we determined regulation of β-arrestin1 and β-arrestin2 by SOM230 and dexamethasone in murine AtT-20 corticotroph tumor cells. In addition, we examined the effects of β-arrestin1 and β-arrestin2 on Sstr mRNA and their protein levels. SOM230 treatment increased β-arrestin1 mRNA levels and did not alter β-arrestin2 mRNA levels. SOM230 treatment could induce β-arrestin1 production in corticotroph tumor cells. Dexamethasone treatment decreased β-arrestin2 mRNA levels. β-arrestin2 knockdown increased proopiomelanocortin, and both Sstr2 and Sstr5 mRNA and their protein levels. The β-arrestin2 knockdown-increased proopiomelanocortin mRNA levels were canceled by SOM230 treatment.

OR06-03 Serotonin Regulates Corticotroph Tumor Cell Proliferation and ACTH Secretion

Serotonin (5-HT) is an important hormonal modulator and neurotransmitter, and 5-HT has been demonstrated in pituitary tissue from several species. Previous responses to 5-HT antagonists have been reported in some patients with Cushing disease although this effect was unsustained, and ACTH and cortisol levels actually increased in some patients. To better understand the role of serotonin in the regulation of corticotroph tumor growth and ACTH secretion, we first measured serotonin levels in supernatants (SNs) derived from murine corticotroph tumor AtT20 cells. We demonstrated that AtT20 cells secrete serotonin (2±0.05ng/105cells/24h) which was ~50% of the levels secreted by the serotonin secreting mid-gut carcinoid BON-1 cell-line (4.2±0.05ng/105cells/24h). In contrast, serotonin secretion was not detected in rat pituitary tumor lactotroph (GH3) or human embryonic kidney (HEK293) cell SNs, or in our Ham’s F12 medium control. Immunocytochemical staining using serotonin specific antibodies demonstrated that serotonin was diffusively present in AtT20 cell cytoplasm, suggesting that serotonin was endogenously generated in the AtT20 cells. Using real-time PCR, we demonstrated that both serotonin synthesis enzymes, tyrosine hydroxylase-1 (THP1) and -2 (THP2) are expressed in AtT20 cells with higher relative THP1 expression, confirming endogenous corticotroph pituitary tumor serotonin production. We further demonstrated that the synthetic glucocorticoid Dexamethasone (100nM x 24h) suppressed TPH1 expression and inhibited corticotroph tumor serotonin secretion. We next evaluated the actions of serotonin and various serotonin antagonists on corticotroph tumor cell proliferation and ACTH secretion. Ritanserin (10-5M) inhibited murine corticotroph tumor proliferation by 5% and inhibited ACTH secretion by ~25%. In contrast, metergoline (10-5M) and the orally administered TPH inhibitor, telotristat etiprate (10-5M for 1-3 days), inhibited ACTH secretion by 50% and 30% respectively but did not reduce cell proliferation, suggesting that Metergoline and telotristat may regulate ACTH secretion independently of their anti-proliferative effect. Addition of serotonin (10-4~10-5M) to corticotroph tumor cells cultured in reduced serum (OptiMEM) or no serum conditions, resulted in an 80% increase in cell proliferation but had little effect on ACTH secretion with a 1-4% increase. In summary, we have demonstrated that serotonin is synthesized in and secreted from corticotroph tumor cells and that serotonin plays a role in regulation of corticotroph tumor proliferation and ACTH secretion in vitro. Our findings using inhibitors of serotonin action indicate potential for targeting this pathway as a novel treatment for patients with CD.