Pituitary Corticotroph Research Articles

Transcription factor Creb3l1 regulates the synthesis of prohormone convertase enzyme PC1/3 in endocrine cells.

Transcription factor cAMP responsive element‐binding protein 3 like 1 (Creb3l1) is a non‐classical endoplasmic reticulum stress molecule that is emerging as an important component for cellular homeostasis, particularly within cell types with high peptide secretory capabilities. We have previously shown that Creb3l1 serves an important role in body fluid homeostasis through its transcriptional control of the gene coding for antidiuretic hormone arginine vasopressin in the neuropeptide‐rich magnocellular neurones of the supraoptic nucleus. In response to osmotic stimuli such as dehydration, vasopressin magnocellular neurones undergo remarkable transcriptome changes, including increased Creb3l1 expression, to ensure that the supply of vasopressin meets demand. To determine where else Creb3l1 fits into the secretory cell supply chain, we performed RNA‐sequencing of Creb3l1 knockdown anterior pituitary mouse corticotroph cell line AtT20. The target chosen for further investigation was Pcsk1, which encodes proprotein convertase enzyme 1 (PC1/3). PC1/3 is crucial for processing of neuropeptides and peptide hormones such as pro‐opiomelanocortin (POMC), proinsulin, proglucagon, vasopressin and oxytocin. Viral manipulations in supraoptic nuclei by over‐expression of Creb3l1 increased Pcsk1, whereas Creb3l1 knockdown decreased Pcsk1 expression. In vitro promoter activity and binding studies showed that Creb3l1 was a transcription factor of the Pcsk1 gene binding directly to a G‐box motif in the promoter. In the dehydrated rat anterior pituitary, Creb3l1 and Pcsk1 expression decreased in parallel compared to control, supporting our findings from manipulations in AtT20 cells and the supraoptic nucleus. No relationship was observed between Creb3l1 and Pcsk1 expression in the neurointermediate lobe of the pituitary, indicating a different mechanism of PC1/3 synthesis by these POMC‐synthesising cells. Therefore, Creb3l1, by regulating the expression of Pcsk1, does not control the processing of POMC peptides in the intermediate lobe.

Paediatric Cushing's disease - a literature review of epidemiology, pathogenesis, clinical symptoms, and diagnostics.

Cushing's disease (CD) is characterised by excess production of adrenocorticotropic hormone (ACTH) by a pituitary corticotroph adenoma, which results in hypercortisolaemia. CD is extremely rare in the paediatric population, and few paediatric endocrinology centres have experience in diagnosing and treating this disease. The clinical presentation of hypercortisolaemia is variable, so proper and rapid diagnosis of CD is often challenging. The molecular pathogenesis of CD was largely unknown until recently. The latest research has revealed somatic mutations in the USP8 gene as the most common pathogenic molecular variants of this disease. Herein, we describe the current state of knowledge of paediatric CD epidemiology, molecular pathogenesis, clinical symptoms, and diagnostics.

MGMT expression in pituitary corticotroph adenomas and its relationship to clinical, pathological, and ultrastructural parameters in patients with Cushing's disease.

Transsphenoidal surgery is the treatment of choice in Cushing's disease (CD), although even late recurrences occur in some patients. Low expression of O-6-methylguanine-DNA methyltransferase (MGMT) has been linked to ahigh risk of relapse in pituitary tumours, but the evidence for corticotroph adenomas is limited. Therefore, we investigated whether MGMT expression was associated with CD remission or clinicopathological markers of tumour aggressiveness among patients with corticotroph adenomas. We included 72 consecutive patients (83% female, mean age ±SD: 44.15 ±15.15 years) with CD, who underwent transsphenoidal adenomectomy between 2012 and 2018. The invasiveness of corticotroph tumours was assessed based on the Knosp scale. Immunohistochemistry was used to analyse MGMT expression as well as the proliferation markers (Ki-67, p53, mitotic index). Electron microscopy was used to categorise tumours into densely or sparsely granulated. Early biochemical remission was evaluated in all patients 6 months after pituitary surgery. Early remission was observed in 47 (65%) patients 6 months after surgery. MGMT expression was > 75% in half of all tumours, < 25% in 14 tumours, and 25-50% or 50-75% in 11 tumours. Lower MGMT expression was associated with alarger tumour diameter (p = 0.001), higher adrenocorticotropic hormone (ACTH) concentration (p = 0.002), higher p53 expression (p = 0.026), and higher frequency of sparsely granulated corticotroph adenomas (p = 0.009). Low MGMT expression was significantly related to lower frequency of early clinical remission (p = 0.005). MGMT predicted the outcomes of transsphenoidal surgery for CD. Pituitary corticotroph adenomas with low MGMT expression may be associated with increased invasiveness and poorer prognosis.

Expression of EGFR in Pituitary Corticotroph Adenomas and Its Relationship With Tumor Behavior.

Objective: Epidermal growth factor receptor (EGFR) has been found to localize in several human neoplasms and has been shown to have a significant correlation with adenomaigenesis and patient prognosis. EGFR is also overexpressed in pituitary corticotroph adenomas. However, its clinical significance and relationship with tumor behavior, especially tumor recurrence status, remain obscure. The purpose of the present study was to identify the expression patterns of EGFR and its downstream signaling pathway molecules in pituitary corticotroph adenomas and to investigate the association of EGFR with clinicopathological characteristics and tumor recurrence.Methods: Fifty-two sporadic pituitary adenoma specimens and six normal pituitary glands were collected. The expression levels of EGFR and its downstream signaling molecules in each sample were evaluated and quantified using immunohistochemistry and Western blot. The relationships of EGFR expression with clinicopathological characteristics and tumor recurrence status were analyzed.Results: EGFR was overexpressed in 55.8% of the pituitary corticotroph adenomas and in 1 of 6 of the normal adenohypophysial tissues. The expression degree was significantly higher in pituitary corticotroph adenomas than in normal adenohypophysial tissues. In EGFR-overexpressing adenomas, the downstream pathway phosphorylated Erk (p-Erk) was also significantly activated. Moreover, the expression levels of EGFR were positively correlated with the adrenocorticotropic hormone (ACTH) and cortisol levels but were not correlated with age, sex or symptom duration. The expression levels of EGFR, phosphorylated EGFR (p-EGFR) and p-Erk were significantly up-regulated in the recurrent adenoma group compared with those in the non-recurrent adenoma group (all p < 0.05). The expression levels of EGFR were strongly correlated with the recurrence-free interval (p = 0.005, CC = −0.31).Conclusion: The expression levels of EGFR and its downstream pathway components were significantly increased in pituitary corticotroph adenomas compared to the levels in normal adenohypophysial tissues. EGFR expression levels were positively associated with the ACTH and cortisol levels and with tumor recurrence status. Pituitary corticotroph adenomas with high EGFR expression levels were correlated with an increased recurrence rate and a decreased recurrence-free interval. EGFR could be used as a promising biomarker for predicting pituitary corticotroph tumor recurrence.

Crosstalk of BMP-4 and RA signaling pathways on Pomc gene regulation in corticotrophs.

Retinoic acid (RA), an active metabolite of Vitamin A, and bone morphogenetic protein 4 (BMP-4) pathways control the transcription of pro-opiomelanocortin (Pomc), the precursor of ACTH. We describe a novel mechanism by which RA and BMP-4 act together in the context of pituitary corticotroph tumoral cells to regulate Pomc transcription. BMP-4 and RA exert a potentiated inhibition on Pomc gene expression. This potentiation of the inhibitory action on Pomc transcription was blocked by the inhibitory SMADs of the BMP-4 pathway (SMAD6 and SMAD7), a negative regulator of BMP-4 signaling (TOB1) and a blocker of RA pathway (COUP-TFI). AtT-20 corticotrophinoma cells express RA receptors (RARB, RXRA and RXRG) which associate with factors of BMP-4 (SMAD4 and SMAD1) signaling cascade in transcriptional complexes that block Pomc transcription. COUP-TFI and TOB1 disrupt these complexes. Deletions and mutations of the Pomc promoter and a specific DNA-binding assay show that the complexes bind to the RARE site in the Pomc promoter. The enhanced inhibitory interaction between RA and BMP-4 pathways occurs also in another relevant corticotroph gene promoter, the corticotropin-releasing hormone receptor 1 (Crh-r1). The understanding of the molecules that participate in the control of corticotroph gene expression contribute to define more precise targets for the treatment of corticotrophinomas.

O-linked β-N-acetylglucosamine transferase is involved in pro-opiomelanocortin gene expression in mouse pituitary corticotroph AtT-20 cells

Glucocorticoids and glucocorticoid receptors (GRs) suppress pituitary pro-opiomelanocortin (Pomc) gene expression. O-linked β-N-acetylglucosamine (O-GlcNAc) modification, mediated by O-GlcNAc transferase (OGT), plays an important role during gene transcription. However, whether OGT is involved in the GR-mediated transrepression that occurs in pituitary corticotroph cells is currently unknown. Here, we report that OGT regulates Pomc expression in the mouse corticotroph cell line AtT-20. The overexpression of OGT has an additive effect on the GR-mediated negative transcription pathway. Both the knockdown of OGT by RNA interference and the use of a chemical OGT inhibitor abolished the repressive effects of Pomc expression induced by GRs. OGT inhibition leads to both the decreased recruitment of GRs and the increased recruitment of RNA polymerase II to the Pomc locus. O-GlcNAc modification is involved in the negative regulation of Pomc transcription in corticotroph cells. OGT may be a promising therapeutic target for the treatment of Cushing’s disease.

Canine Ependymoma: Diagnostic Criteria and Common Pitfalls.

Reports of canine ependymoma are generally restricted to single case reports with tumor incidence estimated at 2% to 3% of primary central nervous system (CNS) tumors. While most commonly reported in the lateral ventricle, tumors can occur anywhere in the ventricular system and in extraventricular locations. Rosettes and pseudorosettes are a common histologic feature; however, these features can be mimicked by other CNS neoplasms. Thirty-seven potential ependymoma cases were identified in a retrospective database search of 8 institutions, and a histologic review of all cases was conducted. Of 37 cases, 22 candidate cases were further subjected to a consensus histologic and immunohistochemical review, and only 5 of 37 (13.5%) were conclusively identified as ependymoma. The neuroanatomic locations were the lateral ventricle (3/5), third ventricle (1/5), and mesencephalic aqueduct (1/5). Subtypes were papillary (4/5) and tanycytic (1/5). Histologic features included rosettes (5/5), pseudorosettes (5/5), ependymal canals (2/5), tanycytic differentiation (1/5), blepharoplasts (1/5), ciliated cells (1/5), and high nuclear to cytoplasmic ratio (5/5). Immunolabeling for GFAP (4/4) and CKAE1/3 (3/4) was found in pseudorosettes, rosettes, and scattered individual neoplastic cells. Diffuse but variably intense cytoplasmic S100 immunolabeling was detected in 3 of 4 cases. Olig2 intranuclear immunolabeling was observed in less than 1% of the neoplastic cells (3/3). Tumors that had pseudorosettes and mimicked ependymoma included oligodendroglioma, choroid plexus tumor, pituitary corticotroph adenoma, papillary meningioma, and suprasellar germ cell tumor. These findings indicate that canine ependymoma is an extremely rare neoplasm with histomorphologic features that overlap with other primary CNS neoplasms.

TSP-1 is downregulated and inversely correlates with miR-449c expression in Cushing's disease.

The pathogenesis of Cushing's disease, which is caused by pituitary corticotroph adenoma, remains to be studied. Secreted angioinhibitory factor thrombospondin‐1 (TSP‐1) is an adhesive glycoprotein that mediates cell‐to‐cell and cell‐to‐matrix interactions and is associated with platelet aggregation, angiogenesis and tumorigenesis. We have found that the expression of TSP‐1 is significantly lower in human pituitary corticotroph tumours compared with normal adenohypophysis. This study aims to elucidate the role of TSP‐1 in regulating the tumour function of pituitary adenomas. Forced overexpression of TSP‐1 in a murine AtT20 pituitary corticotroph tumour cell line decreased corticotroph precursor hormone proopiomelanocortin (POMC) transcription and adrenocorticotropic hormone (ACTH) secretion. Functional studies showed that TSP‐1 overexpression in pituitary adenoma cells suppressed proliferation, migration and invasion. We have demonstrated that TSP‐1 is a direct target of miR‐449c. Further study showed that miR‐449c activity enhanced tumorigenesis by directly inhibiting TSP‐1 expression. Low expression of lncTHBS1, along with low expression of TSP‐1, was associated with the high expression of miR‐449c in Cushing's disease patients. Furthermore, RNA‐immunoprecipitation associates miR‐449c with lncTHBS1 suggesting that lncTHBS1 might be a negative regulator of miR‐449c. Taken together, this study has demonstrated that lncTHBS1 might function as competing endogenous RNA for miR‐449c, which could suppress the development of Cushing's disease.

MON-444 Defining Remission In Cushing Syndrome After Tumor Resection In Cases Of Non-suppressed Postoperative Serum Cortisol Levels

Background: Remission of Cushings syndrome (CS) is generally defined as morning serum cortisol (F) values <5mcg/dL or Urine Free Cortisol < 28 - 56 nmol/d (<10-20/ μg/d) after tumor resection, as normal pituitary corticotropes are suppressed by sustained hypercortisolism (1). In cases where eucortisolemia is achieved by pharmacologic therapy preoperatively, the corticotrope cells can recover their function leading to detectable postoperative cortisol levels not always indicative of unsuccessful tumor resection. Here we describe the use of the 1mg dexamethasone suppression test (DST) and night time serum cortisol as tools to establish criteria for remission in patients where normal pituitary corticotrophs may not have been suppressed. Methods: We retrospectively reviewed 19 patients (pts) with biochemical evidence of ACTH dependent CS who underwent tumor resection. All had measurable serum F measurements within post op day 7 and underwent a 1mg DST to determine if surgery was successful. Remission was defined as serum F value of <1.8 mcg/dl after DST. Recognizing that DST results can be affected by various factors such as concurrent medications or dexamethasone metabolism, in cases where post DST serum F was > 1.8mcg/dl, late night serum F levels were collected at 2330 and 2359. Definition of remission was established as late night F level < 7.5mcg/dl.Results: Of 19 pts, 11 had Ectopic ACTH Syndrome (EAS) and 8 had Cushing’s disease (CD). The median age was 54, 12/19 were female. 15 were deemed to be in surgical remission, 10 having passed the DST with F value <1.8 (range <1 - 5.4mcg/dl). The remaining 5 had night serum F values <7.5mcg/dl (range <1-5.9). 3/10 pts had DST results >2mcg/dl; one had a 2cm adrenal nodule secondary to CD, one had bilateral adrenal hyperplasia, and in the 3rd case a dexamethasone level was not measured. These 3 cases underwent 24 hour urine cortisol measurements, all found within the lower end of the reference range; 5mcg/24hr, 8mcg/24h and 5.2ug/24hr respectively. 12/15 had documented or reported history of cyclic CS and/or were treated with steroidogenesis inhibitors prior to surgery. 21% (4/19) pts were concluded to have persistent hypercortisolemia based on abnormal DST results (F range 4.7-10.8mcg/dl) with 2 also having abnormal night serum F (>20mcg/d). All had CD, and of these 2 had no tumor identified on pathology, one had persistent disease requiring a repeat pituitary surgery with subsequent cure and positive adenoma on pathology, and the 4th had residual disease. Conclusion: In cases of cyclic CS, or where medical management was used prior to surgical resection, normal corticotrophs may not be suppressed, resulting in measurable or normal serum cortisol values post operatively. We suggest the use of the 1 mg DST and/or night time serum cortisol samples to differentiate between persistent hypercortisolism and remission after successful tumor resection.

MON-469 Pituitary Corticotroph Lesions: Clinical, Biochemical, and Pathological Characterization of Cushing Disease, Silent Corticotroph Adenomas, and Corticotroph Hyperplasia

Pituitary adenomas with positive ACTH staining are derived from the corticotroph lineage. Most corticotroph adenomas are small and secrete ACTH, causing a form of hypercortisolism known as Cushing disease (CD); however, a subset do not secrete ACTH and are referred to as silent corticotroph adenomas (SCA). Rarely, hypercortisolemic patients who undergo surgery are diagnosed with the ill-defined entity of corticotroph hyperplasia (CH). In order to better define CH and compare the clinical, laboratory, and imaging parameters of CD, SCA, and CH, comprehensive chart review was performed for all patients at our institution within the past ten years who underwent transsphenoidal surgery for a pituitary mass showing diffuse ACTH immunostaining in lesional tissue. All cases were re-reviewed by an experienced neuropathologist to confirm the diagnoses. Patients with clinical and biochemical evidence of ACTH-dependent hypercortisolemia and an ACTH-positive adenoma were classified as CD (n=59). Patients without hypercortisolemia but with ACTH-staining adenomas were classified as SCA (n=32). Patients clinically suspected of having Cushing disease whose surgical specimen did not show adenoma but consisted only of abnormally expanded acini with ACTH-positive cells were classified as CH (n=13). Average (± SD) pre-operative cortisol (mcg/dL)/ ACTH (pg/mL) measurements were: CD 23.7±8.1 / 66.4±40.1; SCA 12.7±6.2 / 44.4±38.0; CH 24.5±5.9 / 63.8±35.1. SCA patients had larger tumors and presented with more symptoms of compression whereas both CD and CH patients presented with characteristic features of hypercortisolism: weight gain, hypertension, hirsutism, and skin changes (p<0.05 compared to SCA). Compared to CD patients, CH patients more frequently exhibited mood changes (p=0.007) and cognitive dysfunction (p=0.002). CH patients without prior pituitary surgery on average had a greater absolute number of negative late-night salivary cortisol results than CD patients (p=0.006). Of the patients with adequate follow-up, CH patients had higher cortisol levels 6 months post-operatively (p=0.007). Most patients with CH (8/13, 61.5%) had discrete lesions identified on pre-operative MRI. CD patients were more likely to have a higher MIB-1 proliferative index than those of CH patients (CD: 2.8±3.7, CH: 0.4±0.2, p=0.029). In conclusion, SCAs are more likely to cause symptoms of compression, while CD and CH cause symptoms related to hypercortisolism. There may be subtle differences between CD and CH patients, but both groups manifest with ACTH-dependent hypercortisolism and can be nearly indistinguishable pre-operatively. The difference in MIB-1 indices suggests that CH may represent a reactive or pre-neoplastic condition as opposed to undersampled adenoma. Further studies are needed to better understand the pathophysiology of corticotroph cell hyperplasia and its relation to CD.

SAT-LB077 Remission and Mortality Rates in Patients with Cushing Disease According to Their Tumor Size

Objective It has been reported that Cushing’s disease due to macroadenoma is characterized by different biochemical features compared with microadenoma. We aimed to analyze and compare the clinical and biochemical features of patients with micro and macroadenomas causing Cushing disease. Methods We conducted a retrospective review of electronic and paper medical records that included 139 patients (108 microdenomas and 40 macroadenomas) with Cushing disease diagnosis who were studied and treated at the (undisclosed), in Mexico City from 2000 to 2017. Clinical characteristics, biochemical analysis of hypercorticolism, pituitary imaging findings, treatment modalities, remission as well as mortality rates were recorded. Results The mean age was 32.5 years (range 24-37) for microadenoma and 30.0 years (range 23-38) for macroadenoma (p=0.65). Female predominance was seen both in microadenoma (91.7%) and macroadenoma (87.1%) patients. Biochemical analysis of hypercortisolism including basal cortisol, ACTH, 24-hour urinary cortisol levels, and responsivity to low-dose dexamethasone suppression were similar between both groups. Remission and mortality rates after initial pituitary surgery for microadenoma and macroadenoma were 82.5 vs 17.5% (p=0.049) and 40.0% vs 60.0% (p=0.024), respectively. Using log-rank test we confirmed higher remission rates in microadenomas (p=0.049) and higher mortality risk with macroadenomas (p=0.024). Conclusion Our study suggest that patients with microadenomas showed better remission rates and lower mortality than those cases with macroadenomas, despite similar biochemical activity and treatment for CD. Therefore, further research is mandatory to identify those risk factors that gave different prognosis Reference Aydin, K & Dagdelen, S & Ozgen, Burce & Oruckaptan, H & Soylemezoglu, F & Erbas, T. (2014). Pituitary Corticotroph Microadenomas Versus Macroadenomas Causing Cushing’s Disease. Journal für Klinische Endokrinologie und Stoffwechsel. 7. 358-362. Keywords Cushing’s disease, corticotroph macroadenoma, corticotroph microadenoma Unless otherwise noted, all abstracts presented at ENDO are embargoed until the date and time of presentation. For oral presentations, the abstracts are embargoed until the session begins. s presented at a news conference are embargoed until the date and time of the news conference. The Endocrine Society reserves the right to lift the embargo on specific abstracts that are selected for promotion prior to or during ENDO.

MON-256 Co-Occurrence of Parathyroid and Corticotroph Adenoma in a Pediatric Patient without MEN1

Introduction: We report a pediatric case of primary hyperparathyroidism due to a parathyroid adenoma and Cushing’s disease due to a pituitary adenoma. Clinical Case: The patient presented at 13.5 years old for hypertension that was discovered during an exam for weight loss, headaches, and paresthesias. Evaluation was significant for elevated blood pressure (148/98 mmHg), obesity (BMI 96.5%), striae, and he was found to be midpubertal. Tests showed calcium levels 10.7 (RR 8.6-10.6 mg/dL), aldosterone 2.4 (RR 4.0-31.0), and 24 hour urine cortisol/creatinine 451 ug/g (prepubertal RR< 25, >18 yo <32 ug/g). Renin, urine metanephrines and normetanephrine, TSH, and free T4 were all unremarkable. MRI and CT showed absence of the septum pellucidum and mild cortical atrophy. PTH was 76.6 pg (calcium 11.4). Subsequent sestamibi scan showed a solitary parathyroid adenoma. Family history was negative for endocrinopathy. MEN1 sequencing, prolactin, gastrin, and VIP levels were normal. Minimally invasive parathyroid adenomectomy was performed 3 months post-presentation with normalization of calcium and PTH and improved blood pressures. The patient was then lost to endocrinology follow up until 18.5 years of age. In the interval, he continued to have persistent hypertension despite antihypertensive medication and normal PTH and calcium levels. Exam at this time showed a blood pressure of 137/98 mmHg, obesity with BMI of 29.8 kg/m2, and increased abdominal striae. Tests showed AM cortisol of 39.1 ug/dL, ACTH of 85 (RR 6-55 pg/mL), and 24 hour urine cortisol/creatinine of 1306 ug/g. MRI showed a 7 mm hypo-enhancing lesion in the left pituitary. Follow-up MRI of the brain with contrast revealed a 7 mm pituitary adenoma. Inferior petrosal sinus sampling showed peak ACTH levels of 10869 pg/mL (left) and 2738 pg/mL (right). RET analysis and neurosurgical pituitary adenomectomy are planned. Conclusion: Co-occurrence of a pituitary corticotroph and parathyroid adenoma without MEN1 mutation is rare and, to our knowledge has not been reported in a pediatric patient.

The role of Crooke's changes in recurrence and remission after gamma knife radiosurgery.

The objective of this study is to evaluate the role of Crooke's changes (CC) in normal the peri-tumoral anterior pituitary gland, in patients with Cushing's disease (CD) with a histopathological confirmed corticotroph adenoma, and determine if there is any difference in the recurrence and remission rates in CD patients after treatment with Gamma Knife Radiosurgery (GKRS). All patients treated with GKRS for CD from 2005 to 2016at our institution were identified. Patients had a confirmed adrenocorticotropic (ACTH)-secreting adenoma, i.e. corticotroph adenoma, and normal pituitary gland included in the surgical specimen, and specimens were stained with hematoxylin and eosin and also immunostaining for cytokeratin and ACTH. Statistical analyses were performed in a total of 61 patients who met the inclusion criteria. Additionally, we analyzed 20 patients in each group, with and without CC, after they were matched in a propensity score fashion. Endocrine remission defined as, a normal 24h urine free cortisol while off suppressive medication, occurred in 48 patients (78.7%), with 76.9% in those with CC and 81.8% in those without CC. There was no statistical significant difference between the two groups in regarding remission (p = 0.312) or recurrence (p = 0.659) in either the unmatched or matched cohorts. The presence or absence of CC in normal pituitary gland does not appear to confer a lower rate of remission or a higher rate of recurrence after GKRS. Patients with pituitary corticotroph adenomas that present with CC features may be well served by Stereotactic radiosurgery (SRS).

Proton Sensitivity of Corticotropin-Releasing Hormone Receptor 1 Signaling to Proopiomelanocortin in Male Mice.

Because an acidic cellular microenvironment is engendered by inflammation and may determine cell differentiation, we elucidated the impact of acidic conditions on induction of proopiomelanocortin (POMC) expression. Here, we demonstrate mechanisms for proton sensitivity of CRH receptor 1 (CRHR1) signaling to POMC and ACTH production. Low pH (6.8) resulted in doubling of POMC expression and ACTH production in pituitary cell line AtT-20 and in primary mouse pituitary cells. Using CRISPR knockout, we show that CRHR1 is necessary for acid-induced POMC expression, and this induction is mediated by CRHR1 histidine residues and calmodulin-dependent protein kinase II in both pituitary corticotroph cells and in nonpituitary cell lines expressing ectopic ACTH. In contrast, CRH ligand binding affinity to CRHR1 was decreased with acidic pH, implying that proton-induced POMC expression prevails in acidic conditions independently of CRH ligand binding. The results indicate that proton-induced CRHR1 signaling regulates ACTH production in response to an acidic microenvironment.

Effect of Vasopressin V1b Receptor Blockade on Activity of the Hypothalamic-Pituitary-Adrenal Axis in Old Monkeys with Depression-Like and Anxious Behavior Subjected to Stress or Injected with Vasopressin.

The effect of selective antagonist of the arginine vasopressin (AVP) V1b receptors on the secretion of ACTH and corticosteroids in response to insulin-induced hypoglycemia and injection of AVP is studied in old Macaca mulatta females with depression-like and anxious behavior. Intravenous antagonist in a dose of 1.1-1.7 μg/kg inhibits the increase of ACTH concentration, induced by hypoglycemia or injection of AVP. The degree of increase in the concentrations of hydrocortisone and dehydroepiandrosterone sulfate has not changed or increased. The effects of AVP antagonist prove that previously detected disorders in the reaction of the hypothalamic-pituitary-adrenal system in old Macaca mulatta with depression-like and anxious behavior could be caused by excessive activation of vasopressin V1b receptors on the pituitary corticotrophs, while the use of V1b receptor antagonists seems to be a promising method for prevention of these disorders.

Somatostatin receptor subtype 5 modifies hypothalamic-pituitary-adrenal axis stress function.

Pituitary corticotroph somatostatin receptor subtype 5 (SSTR5) signals to inhibit adrenocorticotrophin (ACTH) secretion. As ACTH deficiency results in attenuated adrenal cortisol production and an impaired stress response, we sought to clarify the role of SSTR5 in modifying the hypothalamic/pituitary/adrenal (HPA) axis. We generated Tg HP5 mice overexpressing SSTR5 in pituitary corticotrophs that produce the ACTH precursor proopiomelanocortin (POMC). Basal ACTH and corticosterone were similar in HP5 and WT mice, while HP5 mice showed attenuated ACTH and corticosterone responses to corticotrophin releasing hormone (CRH). HP5 mice exhibited attenuated corticosterone responses upon a restraint stress test and inflammatory stress following LPS injection, as well as increased anxiety-like and depressive-like behavior on open field and forced swim tests. Pituitary corticotroph CRH receptor subtype 1 (CRHR1) mRNA expression and ACTH responses to CRH were also attenuated in HP5 mice. In AtT20 cells stably overexpressing SSTR5, CRHR1 expression and cAMP response to CRH were reduced, whereas both were increased after SSTR5 KO. In elucidating mechanisms for these observations, we show that SSTR5-induced miR-449c suppresses both CRHR1 expression and function. We conclude that corticotroph SSTR5 attenuates HPA axis responses via CRHR1 downregulation, suggesting a role for SSTR5 in the pathogenesis of secondary adrenal insufficiency.

Metformin suppresses growth and adrenocorticotrophic hormone secretion in mouse pituitary corticotroph tumor AtT20 cells.

Pituitary corticotroph tumors lead to excess adrenocorticotrophic hormone (ACTH) secretion, resulting in Cushing's disease (CD), which is associated with significant mortality. Standard treatments include neurosurgery, radiotherapy and medical therapy. Both surgery and radiotherapy have undesirable complications and high recurrence rates. At present, there is only one medical option available that targets pituitary adenoma and ACTH secretion, the drug pasireotide. However, hyperglycemia is common during pasireotide treatment. In addition, some patients have discontinued pasireotide treatment because of hyperglycemia-related adverse events or uncontrolled diabetes. New medical treatments directly targeting the corticotroph cells and suppressing ACTH secretion are urgently required. Metformin is a commonly used antidiabetic drug that has been widely used to control the hyperglycemia that occurs in patients with CD, which is secondary to both cortisol excess and pasireotide treatment. Recent studies suggest that metformin has direct anticancer activities against many tumor cell lines. In the present study, we investigated whether metformin exerts an anti-tumor effect by directly targeting pituitary corticotroph tumors and exploring the underlying mechanisms. Using the mouse corticotroph tumor cells, AtT20 cells, we report that metformin inhibited cell proliferation, promoted cell apoptosis and decreased ACTH secretion but did not block the cell cycle in cells. The apoptosis induced by metformin was accompanied by increased caspase-3 activity. Meanwhile, metformin down-regulated the anti-apoptotic protein B cell lymphoma 2 (Bcl-2) but up-regulated the pro-apoptotic protein Bcl2-associated X (BAX), which suggests the involvement of the mitochondrial-mediated apoptosis pathway. Furthermore, metformin promoted AMP-activated protein kinase (AMPK) phosphorylation but inhibited insulin-like growth factor-1 receptor (IGF-1R) expression, protein kinase B (PKB/AKT) phosphorylation and mammalian target of rapamycin (mTOR) phosphorylation. Finally, the IGF-1R inhibitor picropodophyllin (PPP) significantly inhibited the cell proliferation of AtT20 cells. We conclude that metformin inhibits cell proliferation and induces apoptosis in AtT20 cells by activating AMPK/mTOR and inhibiting IGF-1R/AKT/mTOR signaling pathways. Metformin may have direct antitumor activity against pituitary corticotroph tumors.

Genomic insights into Cushing syndrome

In the setting of Cushing syndrome, genomic analyses can be performed either in tumors responsible for endogenous Cushing, or in patients exposed to glucocorticoid excess. Genomics of tumors identified several new genes – including ZNRF3 in adrenocortical carcinomas, PRKACA in cortisol-producing adrenal adenomas, ARMC5 in primary macronodular adrenal hyperplasia and USP8 in pituitary corticotroph adenomas. These genes shed new lights on the mechanisms responsible for these tumors. Integrated genomic studies of adrenal carcinomas identified distinct molecular classes, with remarkably different prognostic outcome. Beyond the mechanistic novelties, a new generation of prognostic markers emerges, with potentially important impact on patients care. For the future, genomic efforts should be pursued, focusing on poorly characterized tumors responsible for Cushing syndrome – including endocrine tumors secreting ACTH. In addition, epigenomics is emerging as an outstanding set of tools for characterizing tumors, unraveling unprecedented aspects of tumorigenesis. Applying these tools to endocrine tumors responsible for Cushing syndrome may also lead to important discoveries. Genomics of patients exposed to glucocorticoid excess is an emerging research field. Proof of principle studies have been performed, identifying molecular markers of glucocorticoid excess in blood. Research efforts should now concentrate on markers of mild glucocorticoid excesses – endogenous or exogenous –, owing to their high prevalence in general population. In addition, markers of individual susceptibility to each type of glucocorticoid complication are needed. It remains to be determined whether genomics can identify such markers.

Pituitary-Directed Therapies for Cushing's Disease.

Cushing’s disease (CD) is caused by a pituitary corticotroph neuroendocrine tumor inducing uncontrolled hypercortisolism. Transsphenoidal surgery is the first-line treatment in most cases. Nonetheless, some patients will not achieve cure even in expert hands, others may not be surgical candidates and a significant percentage will experience recurrence. Many patients will thus require medical therapy to achieve disease control. Pharmacologic options to treat CD have increased in recent years, with an explosion in knowledge related to pathophysiology at the molecular level. In this review, we focus on medications targeting specifically pituitary adrenocorticotropic hormone-secreting tumors. The only medication in this group approved for the treatment of CD is pasireotide, a somatostatin receptor ligand. Cabergoline and temozolomide may also be used in select cases. Previously studied and abandoned medical options are briefly discussed, and emphasis is made on upcoming medications. Mechanism of action and available data on efficacy and safety of cell cycle inhibitor roscovitine, epidermal growth factor receptor inhibitor gefitinib, retinoic acid, and silibinin, a heat shock protein 90 inhibitor are also presented.

Decreased expression of SFRP2 promotes development of the pituitary corticotroph adenoma by upregulating Wnt signaling.

Cushing's disease is primarily caused by pituitary adrenocorticotropin-secreting adenoma. However, its pathogenesis has remained obscure. In the present study, whole transcriptome analysis was performed by RNA sequencing (RNA-Seq) and expression of secreted frizzled-related protein 2 (SFRP2) was decreased in corticotroph tumors compared with normal pituitary glands. Furthermore, the RNA-Seq results were validated and the expression of SFRP2 in tumor tissues was analyzed by comparing another cohort of 23 patients with Cushing's disease and 3 normal human pituitary samples using reverse transcription-quantitative polymerase chain reaction, western blot and immunohistochemistry staining. Clinically, there was an association between lower SFRP2 expression and aggressive adenoma characteristics, including larger size and invasiveness. Conversely, SFRP2 overexpression reduced the ability of AtT20 cells to proliferate and migrate, and reduced production of the adrenocorticotrophic hormone in vitro. Mechanistically, overexpressed SFRP2 reduced the level of β-catenin in the cytoplasm and nucleus, and decreased Wnt signaling activity in AtT20 cells. Therefore, SFRP2 appears to act as a tumor suppressor in Cushing's disease by regulating the activity of the Wnt signaling pathway.