Drug resistance mechanisms in dopamine agonist-resistant prolactin pituitary neuroendocrine tumors and exploration for new drugs.

BACKGROUND: The treatment of dopamine agonists (DA) resistant prolactinomas remains a formidable challenge, as the mechanism of resistance is still unclear, and there are currently no viable alternative drug therapies available. This study seeks to investigate the mechanism of DA resistance in prolactinomas and identify new potentially effective drugs. METHODS: To explore the mechanism of DA resistance in prolactinomas, this study conducted transcriptome sequencing analysis on 27 cases of DA-resistant prolactinomas and 10 cases of sensitive prolactinomas. In addition, single-cell sequencing analysis was performed on 3 cases of DA-resistant prolactinomas and 3 cases of sensitive prolactinomas. Furthermore, to screen for potential therapeutic drugs, the study successfully established an organoids model for DA-resistant prolactinomas and screened 180 small molecule compounds using 8 organoids. The efficacy of the identified drugs was verified through various assays, including CCK-8, colony formation, CTG, and flow cytometry, and their mechanisms of action were confirmed through WB and IHC. The effectiveness of the identified drugs was evaluated both in vitro and in vivo. RESULTS: The results of transcriptome sequencing and single-cell sequencing analyses showed that DA resistance in prolactinomas is associated with the upregulation of the Focal Adhesion (FA) signaling pathway. Additionally, immunohistochemical validation revealed that FAK and Paxillin were significantly upregulated in DA-resistant prolactinomas. Screening of 180 small molecule compounds using 8 organoids identified Genistein as a potentially effective drug for DA-resistant prolactinomas. Experimental validation demonstrated that Genistein inhibited the proliferation of pituitary tumor cell lines and organoids and promoted apoptosis in pituitary tumor cells. Moreover, both the cell sequencing results and WB validation results of the drug-treated cells indicated that Genistein exerts its anti-tumor effect by inhibiting the FA pathway. In vivo, experiments also showed that Genistein can inhibit subcutaneous tumor formation. CONCLUSION: DA resistance in prolactinomas is associated with upregulation of the Focal Adhesion (FA) signaling pathway, and Genistein can exert its anti-tumor effect by inhibiting the expression of the FA pathway.

PAM variants in patients with thyrotrophinomas, cyclical Cushing's disease and prolactinomas.

Introduction: Germline loss-of-function variants in PAM, encoding peptidylglycine α-amidating monooxygenase (PAM), were recently discovered to be enriched in conditions of pathological pituitary hypersecretion, specifically: somatotrophinoma, corticotrophinoma, and prolactinoma. PAM is the sole enzyme responsible for C-terminal amidation of peptides, and plays a role in the biosynthesis and regulation of multiple hormones, including proopiomelanocortin (POMC). Methods: We performed exome sequencing of germline and tumour DNA from 29 individuals with functioning pituitary adenomas (12 prolactinomas, 10 thyrotrophinomas, 7 cyclical Cushing's disease). An unfiltered analysis was undertaken of all PAM variants with population prevalence <5%. Results: We identified five coding, non-synonymous PAM variants of interest amongst seven individuals (six germline, one somatic). The five variants comprised four missense variants and one truncating variant, all heterozygous. Each variant had some evidence of pathogenicity based on population prevalence, conservation scores, in silico predictions and/or prior functional studies. The yield of predicted deleterious PAM variants was thus 7/29 (24%). The variants predominated in individuals with thyrotrophinomas (4/10, 40%) and cyclical Cushing's disease (2/7, 29%), compared to prolactinomas (1/12, 8%). Conclusion: This is the second study to demonstrate a high yield of suspected loss-of-function, predominantly germline, PAM variants in individuals with pathological pituitary hypersecretion. We have extended the association with corticotrophinoma to include the specific clinical entity of cyclical Cushing's disease and demonstrated a novel association between PAM variants and thyrotrophinoma. PAM variants might act as risk alleles for pituitary adenoma formation, with a possible genotype-phenotype relationship between truncating variants and altered temporal secretion of cortisol.

An aggressive cabergoline-resistant, temozolomide-responsive macroprolactinoma due to a germline SDHB pathogenic variant in the absence of paraganglioma or pheochromocytoma.

Context: Germline succinate dehydrogenase subunit B (SDHB) pathogenic variants are characteristic of familial paraganglioma (PGL) syndrome type 4. This syndrome frequently presents with abdominal PGL and has high tendency for locally aggressive behavior and distant metastasis. The vast majority of pituitary adenomas (PAs) are sporadic. However, PAs can be part of a number of familial tumor syndromes such as multiple endocrine neoplasia type 1 (MEN 1) or more rarely in association with pheochromocytoma and PGL (referred to as 3P syndrome). Only a limited number of PAs in association with SDHB-related PGL has been reported and the vast majority occurred subsequently or simultaneously with pheochromocytoma/PGL (collectively abbreviated as PPGL). In this report, we describe a young patient who had a giant pituitary macroprolactinoma resistant to large doses of cabergoline (CBG) and external beam radiotherapy (XRT). The patient did not have personal history of PPGL but was found to carry a germline SDHB pathogenic variant. Case report: A 38-year-old woman presented with headache, visual disturbances and galactorrhea and was found to have a 34-mm macroprolactinoma. She was treated with CBG 3-4 mg per week but PA continued to grow and caused significant cranial pressure symptoms. She underwent two transsphenoidal surgeries with rapid tumor recurrence after each one. She received XRT but PA continued to grow. She was finally treated with temozolomide with excellent response. Whole exome and subsequent Sanger sequencing confirmed that she has a pathogenic monoallelic SDHB mutation (NM_003000:c.C343T, p.R115*). PA tissue showed loss of heterozygosity for the same mutation and absent SDHB immunostaining confirming the pathogenic role of this SDHB mutation. Conclusion: Germline SDHB mutations can rarely cause PA in the absence of PPGL. They should be considered as a possible cause of aggressiveness and resistance to dopamine agonists in similar cases.

Genetic testing in prolactinomas: a cohort study.

BACKGROUND: Prolactinomas represent 46%-66% of pituitary adenomas, but the prevalence of germline mutations is largely unknown. We present here the first study focusing on hereditary predisposition to prolactinoma. OBJECTIVE: We studied the prevalence of germline mutations in a large cohort of patients with isolated prolactinomas. MATERIALS AND METHODS: A retrospective study was performed combining genetic and clinical data from patients referred for genetic testing of MEN1, AIP, and CDKN1B between 2003 and 2020. SF3B1 was Sanger sequenced in genetically negative patients. RESULTS: About 506 patients with a prolactinoma were included: 80 with microprolactinoma (15.9%), 378 with macroprolactinoma (74.7%), 48 unknown; 49/506 in a familial context (9.7%). Among these, 14 (2.8%) had a (likely) pathogenic variant (LPV) in MEN1 or AIP, and none in CDKN1B. All positive patients had developed a macroprolactinoma before age 30. The prevalence of germline mutations in patients with isolated macroprolactinoma under 30 was 4% (11/258) in a sporadic context and 15% (3/20) in a familial context. Prevalence in sporadic cases younger than 18 was 15% in men (5/33) and 7% in women (4/57). No R625H SF3B1 germline mutation was identified in 264 patients with macroprolactinomas. CONCLUSIONS: We did not identify any LPVs in patients over 30 years of age, either in a familial or in a sporadic context, and in a sporadic context in our series or the literature. Special attention should be paid to young patients and to familial context.

The clinical and therapeutic profiles of prolactinomas associated with germline pathogenic variants in the aryl hydrocarbon receptor interacting protein (AIP) gene.

Introduction: Prolactinomas are the most frequent type of pituitary adenoma encountered in clinical practice. Dopamine agonists (DA) like cabergoline typically provide sign/ symptom control, normalize prolactin levels and decrease tumor size in most patients. DA-resistant prolactinomas are infrequent and can occur in association with some genetic causes like MEN1 and pathogenic germline variants in the AIP gene (AIPvar). Methods: We compared the clinical, radiological, and therapeutic characteristics of AIPvar-related prolactinomas (n=13) with unselected hospital-treated prolactinomas ("unselected", n=41) and genetically-negative, DA-resistant prolactinomas (DA-resistant, n=39). Results: AIPvar-related prolactinomas occurred at a significantly younger age than the unselected or DA-resistant prolactinomas (p<0.01). Males were more common in the AIPvar (75.0%) and DA- resistant (49.7%) versus unselected prolactinomas (9.8%; p<0.001). AIPvar prolactinomas exhibited significantly more frequent invasion than the other groups (p<0.001) and exhibited a trend to larger tumor diameter. The DA-resistant group had significantly higher prolactin levels at diagnosis than the AIPvar group (p<0.001). Maximum DA doses were significantly higher in the AIPvar and DA-resistant groups versus unselected. DA-induced macroadenoma shrinkage (>50%) occurred in 58.3% in the AIPvar group versus 4.2% in the DA-resistant group (p<0.01). Surgery was more frequent in the AIPvar and DA- resistant groups (43.8% and 61.5%, respectively) versus unselected (19.5%: p<0.01). Radiotherapy was used only in AIPvar (18.8%) and DA-resistant (25.6%) groups. Discussion: AIPvar confer an aggressive phenotype in prolactinomas, with invasive tumors occurring at a younger age. These characteristics can help differentiate rare AIPvar related prolactinomas from DA-resistant, genetically-negative tumors.

Therapeutic response to pazopanib: case report and literature review on molecular abnormalities of aggressive prolactinomas.

Introduction: Aggressive prolactinomas (APRLs) pose a significant clinical challenge due to their high rate of regrowth and potentially life-threatening complications. In this study, we present a case of a patient with an APRL who had a trial of multiple therapeutic modalities with the aim to provide a review of molecular abnormalities and management of APRLs by corroborating our experience with previous literature. Methods: A total of 268 articles were reviewed and 46 were included. Case reports and series, and studies that investigated the molecular and/or genetic analysis of APRLs were included. Special care was taken to include studies describing prolactinomas that would fall under the APRL subtype according to the European Society of Endocrinology guidelines; however, the author did not label the tumor as "aggressive" or "atypical". Addiontionally, we present a case report of a 56-year-old man presented with an invasive APRL that was resistant to multiple treatment modalities. Results: Literature review revealed multiple molecular abnormalities of APRLs including mutations in and/or deregulation of ADAMTS6, MMP-9, PITX1, VEGF, POU6F2, CDKN2A, and Rb genes. Mismatch repair genes, downregulation of microRNAs, and hypermethylation of specific genes including RASSF1A, p27, and MGMT were found to be directly associated with the aggressiveness of prolactinomas. APRL receptor analysis showed that low levels of estrogen receptor (ER) and an increase in somatostatin receptors (SSTR5) and epidermal growth factor receptors (EGFR) were associated with increased invasiveness and higher proliferation activity. Our patient had positive immunohistochemistry staining for PD-L1, MSH2, and MSH6, while microarray analysis revealed mutations in the CDKN2A and POU6F2 genes. Despite undergoing two surgical resections, radiotherapy, and taking dopamine agonists, the tumor continued to progress. The patient was administered pazopanib, which resulted in a positive response and the patient remained progression-free for six months. However, subsequent observations revealed tumor progression. The patient was started on PD-L1 inhibitor pembrolizumab, yet the tumor continued to progress. Conclusion: APRLs are complex tumors that require a multidisciplinary management approach. Knowledge of the molecular underpinnings of these tumors is critical for understanding their pathogenesis and identifying potential targets for precision medical therapy.

[Modern concepts of genetic and immunohistochemical features of prolactin-secreting pituitary adenomas].

Prolactinomas are the most common secreting adenomas of the pituitary. In 20% of cases resistance to dopamine-agonists treatment is observed. Medical therapy resistance causes progression of pathological symptoms of hyperprolactinemia and negative topographic and anatomical changes of prolactinoma. The causes of ineffectiveness of dopamine agonists therapy are not fully understood as well as approaches to managing patients require clarification. Current concepts of resistance are based on the data obtained as a result of surgery or after a period of long-term ineffective therapy. Thus, it is very important to find methods of assessing the sensitivity of prolactin-secreting adenomas to drug therapy before surgical treatment. Genetic and immunohistochemical studies find special place among these methods, making it possible to predict adenoma's response to drug therapy at early diagnostic stage. Obtained results will allow us to form personalized algorithm for managing patients.

CircOMA1 modulates cabergoline resistance by downregulating ferroptosis in prolactinoma.

PURPOSE: Prolactinomas are one of the most common pituitary neuroendocrine tumors (PitNETs), accounting for approximately 50% of all pituitary tumors. Dopamine agonists are the main treatment for prolactinoma, but a small number of patients are still resistant to pharmacotherapy. Recent discoveries have revealed that ferroptosis is involved in regulating tumor drug resistance. However, the role of ferroptosis in prolactinoma has not been reported. In this study, we aimed to explore the mechanism of a circRNA in ferroptosis in prolactinoma. METHODS: The expression of circOMA1 in prolactinoma tissues was examined by quantitative reverse transcription PCR (qRT-PCR). The biological function of circOMA1 was evaluated in vitro and in vivo. To explore the role of ferroptosis in prolactinoma, we used qRT-PCR and western blotting. Glutamate-cysteine ligase, modifier subunit (GCLM) was predicted to be a direct target gene of miR-145-5p by bioinformatics analysis, which was confirmed by luciferase reporter assays. RESULTS: circOMA1 was overexpressed in drug-resistant prolactinoma tissues compared with sensitive prolactinoma samples. We further found that circOMA1 promoted MMQ cells growth in vivo and in vitro. In addition, GCLM was directly targeted by miR-145-5p and indirectly regulated by circOMA1. Importantly, circOMA1 induced ferroptosis resistance through the increased expression of Nrf2, GPX4, and xCT, and circOMA1 attenuated CAB-induced ferroptosis in MMQ cells in vivo and in vitro. CONCLUSION: The present study demonstrates that circOMA1 attenuates CAB efficacy through ferroptosis resistance and may be a new therapeutic target for the individualized treatment of DA-resistant prolactinoma patients.

PRL-R Variants Are Not Only Associated With Prolactinomas But Also With Dopamine Agonist Resistance.

CONTEXT: Knockout prolactin receptor gene (PRL-R) mice are animal models for prolactinomas and PRL acts via autocrine/paracrine inhibiting lactotroph proliferation. Recently, variants of the PRL-R were identified in prolactinoma patients and their frequency was higher compared to individuals from the genomic database. OBJECTIVE: We analyzed PRL-R variants frequency in an extensive cohort of prolactinoma patients and evaluated their association with clinical, laboratorial, and imaging characteristics and hormonal response to cabergoline. DESIGN: Observational, retrospective, and cross-sectional study. SETTING: This study took place at the Neuroendocrinology Unit of Clinics Hospital, Medical School of University of São Paulo, Brazil, a tertiary referral center. PATIENTS AND METHODS: Study participants included adults with sporadic prolactinomas treated with cabergoline, where response to therapy was defined by prolactin normalization with up to 3 mg/week doses. DNA was extracted from blood samples and the PRL-R was analyzed by polymerase chain reaction techniques and automatic sequencing. The association of PRL-R variants with serum prolactin levels, maximal tumor diameter, tumor parasellar invasiveness, and response to cabergoline was analyzed. RESULTS: We found 6 PRL-R variants: p.Ile100(76)Val, p.Ile170(146)Leu, p.Glu400(376)Gln/p.Asn516(492)Ile, p.Glu470Asp e p.Ala591Pro; the last 2 are newly described in prolactinomas' patients. The variants p.Glu400(376)Gln/p.Asn516(492)Ile and p.Ala591Pro were more frequent amongst patients compared to genomic databases, and the p.Asn516(492)Ile showed pathogenic potential using in silico analysis as previously described. PRL-R variants were associated with male sex (P = 0.015), higher serum PRL levels (P = 0.007), larger tumors (P = 0.001), and cabergoline resistance (P < 0.001). CONCLUSIONS: The prolactin/prolactin receptor system seems to be related to prolactinoma tumorigenesis and cabergoline resistance. Additional studies are needed to better understand the PRL-R variants' role and their potential as therapeutic targets.

MicroRNA-137 inhibits pituitary prolactinoma proliferation by targeting AKT2.

PURPOSE: Prolactinoma is the most common type of pituitary adenoma. Most prolactinoma need medical treatment, but some of them are aggressive and require surgery. In previous decades, some miRNAs have been manifested as oncogenes or tumor suppressors. Consequently, miRNAs' abnormal expression involves tumorigenesis, invasion, and metastasis of different types of tumors, including pituitary tumors. The current study aim to explore the aggressiveness-associated miRNAs in prolactinoma and underlying molecular mechanisms based on the bioinformatic analysis and fundamental experiment studies. METHODS: GSE46294 miRNA expression profile from the Gene Expression Omnibus (GEO) database was downloaded. Differentially expressed miRNAs (DEMs) were filtered from this data. Subsequently, the target genes of downregulated miRNAs were analyzed by Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment. RT-qPCR, western blot, and CCK-8 assays were used to validate the effect of miR-137 on the proliferation of MMQ cells through AKT2. Finally, the binding site of rat miR-137 to AKT2 were predicted by Targetscan and Bibiserv database, and verified by double luciferase reporter assay. RESULTS: Twenty-four changed DEMs (fourteen upregulated and ten downregulated) were identified. Target genes of downregulated DEMs were classified into three groups by GO terms. KEGG pathway enrichment analysis revealed these target genes enriched in the PI3K-Akt pathway. We also confirmed that miR-137 can target AKT2 and inhibit the proliferation of MMQ cells induced by AKT2. CONCLUSION: MiR-137 suppressed prolactinomas' aggressive behavior by targeting AKT2.

A Genetic Assessment of Dopamine Agonist-Induced Impulse Control Disorder in Patients With Prolactinoma.

CONTEXT: Dopamine agonist (DA)-induced impulse control disorder (ICD) represents a group of behavioral disorders that are increasingly recognized in patients with prolactinoma. OBJECTIVE: We aimed to examine the genetic component of the underlying mechanism of DA-induced ICD. METHODS: Patients with prolactinoma receiving dopamine agonist (cabergoline) treatment were included in the study. These patients were divided into 2 groups: patients who developed ICD due to DA and patients who did not. Patients were evaluated for polymorphisms of the DRD1, DRD3, COMT, DDC, GRIN2B, TPH2, OPRK1, OPRM1, SLC6A4, SLC6A3, HTR2A genes. RESULTS: Of the 72 patients with prolactinoma using cabergoline, 20 were diagnosed with ICD. When patients with and without ICD were compared according to genotype frequencies, OPRK1/rs702764, DRD3/rs6280, HTR2A/rs6313, SLC6A4/rs7224199, GRIN2B/rs7301328, TPH2/rs7305115, COMT/rs4680, DRD1/rs4532 polymorphisms significantly increased in patients with DA-induced ICD. CONCLUSION: Our results show that multiple neurotransmission systems affect DA-induced ICD in patients with prolactinoma.

Giant prolactinoma in children and adolescents: a single-center experience and systematic review.

PURPOSE: Giant prolactinoma (GP) in childhood and adolescence is a rare entity with scarce literature. We aimed to describe clinical features, biochemistry, radiology, genetics, management, and outcome in pediatric (≤ 20 years) GP. METHODS: Retrospective record review of 18 pediatric GP patients from our center and systematic review including these and 77 from the literature (total cohort: 95). RESULTS: GP constituted 20% of our pediatric prolactinoma cohort. In the total cohort (age: 15.4 ± 3.5 years), the majority (77, 82.8%) were males. Mass effect symptoms (88.6%), and pubertal delay/arrest in males (82.1%) were frequent. Median basal prolactin was 8649 (3246-17,532) ng/ml and the maximum tumor dimension was 5.5 ± 1.5 cm. MEN1 and AIP mutations were noted in 7 (21.9%) and 6 (18.8%) patients, respectively. Males with central hypogonadism had baseline bi-testicular volume of 20.2 ± 8.4 cc, lower LH than FSH (-2.04 ± 0.9 vs. -0.7 ± 1.6 SDS, p = 0.0075), and mostly, normal inhibin B. Majority (49/76, 64.5%) received dopamine agonist (DA) as first-line treatment with additional therapy in 35% (17/49). DA monotherapy arm had less frequent central hypothyroidism (42.9% vs 87.1%, p = 0.002) and central adrenal insufficiency (7.1% vs 66.7%, p = 0.0003) than multimodal therapy. A smaller tumor dimension (4.7 vs. 5.7 cm, p = 0.04) was associated with normoprolactinemia on DA monotherapy and AIP mutations (33.3% vs. nil, p = 0.02) with multimodal therapy. CONCLUSION: GP is characterized by male predominance with frequent delay/arrest of puberty (82%), but relative sparing of the FSH-inhibin B axis in boys. DA monotherapy may be preferred as the first-line therapy in pediatric GP.

Sex-specific Regulation of Prolactin Secretion by Pituitary Bradykinin Receptors.

Sex differences in the control of prolactin secretion are well documented. Sex-related differences in intrapituitary factors regulating lactotroph function have recently attracted attention. Sex differences in prolactinoma development are well documented in clinic, prolactinomas being more frequent in women but more aggressive in men, for poorly understood reasons. Kallikrein, the enzyme releasing kinins has been found in the pituitary, but there is no information on pituitary kinin receptors and their function. In the present work, we characterized pituitary bradykinin receptors (BRs) at the messenger RNA and protein levels in 2 mouse models of prolactinoma, Drd2 receptor gene inactivation and hCGß gene overexpression, in both males and females, wild type or genomically altered. BR B2 (B2R) accounted for 97% or more of total pituitary BRs in both models, regardless of genotype, and was present in lactotrophs, somatotrophs, and gonadotrophs. Male pituitaries displayed higher level of B2R than females, regardless of genotype. Pituitary B2R gene expression was downregulated by estrogen in both males and females but only in females by dopamine. Activation of B1R or B2R by selective pharmacological agonists induced prolactin release in male pituitaries but inhibited prolactin secretion in female pituitaries. Increased B2R content was observed in pituitaries of mutated animals developing prolactinomas, compared to their respective wild-type controls. The present study documents a novel sex-related difference in the control of prolactin secretion and suggests that kinins are involved, through B2R activation, in lactotroph function and prolactinoma development.

The SF3B1R625H mutation promotes prolactinoma tumor progression through aberrant splicing of DLG1.

BACKGROUND: Recently, a hotspot mutation in prolactinoma was observed in splicing factor 3b subunit 1 (SF3B1R625H), but its functional effects and underlying molecular mechanisms remain largely unexplored. METHODS: Using the CRISPR/Cas9 genome editing system and rat pituitary GH3 cells, we generated heterozygous Sf3b1R625H mutant cells. Sanger and whole-genome sequencing were conducted to verify the introduction of this mutation. Transcriptome analysis was performed in SF3B1-wild-type versus mutant human prolactinoma samples and GH3 cells. RT-PCR and minigene reporter assays were conducted to verify aberrant splicing. The functional consequences of SF3B1R625H were evaluated in vitro and in vivo. Critical makers of epithelial-mesenchymal transition and key components were detected using western blot, immunohistochemistry, and immunofluorescence. Suppressing proteins was achieved using siRNA. RESULTS: Transcriptomic analysis of prolactinomas and heterozygous mutant cells revealed that the SF3B1R625H allele led to different alterations in splicing properties, affecting different genes in different species. SF3B1R625H promoted aberrant splicing and DLG1 suppression in both rat cells and human tumors. In addition, SF3B1R625H and knocking down DLG1 promoted cell migration, invasion, and epithelial-mesenchymal transition through PI3K/Akt pathway. CONCLUSIONS: Our findings elucidate a mechanism through which mutant SF3B1 promotes tumor progression and may provide a potent molecular therapeutic target for prolactinomas with the SF3B1R625H mutation.

MicroRNAs in Dopamine Agonist-Resistant Prolactinoma.

Dopamine agonists (DAs) are preferred for the treatment of prolactinomas and are usually very effective. Nonetheless, 20-30% of bromocriptine- and approximately 10% of cabergoline-treated individuals exhibit resistance to DAs. In addition, the mechanism underlying this phenomenon remains elusive. In this study, we summarize the major findings regarding the role of microRNAs (miRNAs) in the pathogenesis of DA-resistant prolactinoma (DARP). Currently available evidence suggests that miRNAs are usually dysregulated in DARP and that, although controversial, the dysregulated miRNAs target the transforming growth factor (TGF)-ß, dopamine 2 receptor (D2R), or estradiol (E2)/estrogen receptor (ER) signaling pathways to mediate the therapeutic effect of DAs. These findings provide new incentives for research on innovative strategies for predicting patients' responsiveness to dopamine therapies and for developing treatment approaches. Unfortunately, recent studies tended to focus exclusively on the differential miRNA expression profiles between DARP and dopamine-sensitive prolactinoma, and no definitive consensus has been reached regarding the role of these miRNAs in the modulation mechanism. Therefore, current and future efforts should be directed toward the exploration of the mechanism underlying the dysregulation of miRNAs as well as of the target proteins that are affected by the dysregulated miRNAs. Furthermore, the modulation of the expression of dysregulated miRNAs, which target the D2R, TGF-ß, or E2/ER signaling pathways, might be a promising alternative to treat patients with DARP and improve their prognosis.

IL-18 expression in clinical human pituitary adenoma.

BACKGROUND: IL-18 is known as an interferon-inducing factor that belongs to the IL-1 family, and is synthesized as an inactive precursor protein. OBJECTIVE: The present study aims to investigate the expression of IL-18, IL-18R, R and IL-18 binding protein (BP) mRNA in various types of human pituitary tumors, such as adrenocorticotropic hormone (ACTH), growth hormone (GH), prolactin (PRL), thyroid stimulating hormone (TSH)-producing adenomas and non-function adenomas. METHODS: Pituitary adenoma tissues were obtained during the surgery of 41 patients: nine patients had ACTH-producing pituitary adenomas, nine patients had GH-producing pituitary adenomas, five patients had TSH-producing pituitary adenomas, seven patients had PRL-producing pituitary adenomas, and 11 patients had non-functioning adenomas. The mRNA expression levels of IL-18, IL-18BP, IL-18R and IL-18R were quantified using real-time quantitative PCR. RESULTS: The mRNA expression of IL-18 was significantly higher in ACTH-, GH- and PRL-producing adenomas, when compared to non-function tumors. Similarly, a significantly higher mRNA expression of IL-18BP and IL-18R was observed in ACTH-, GH- and PRL-producing adenomas, when compared with non-functional adenomas. In contrast, no upregulation of IL-18R mRNA was observed in any of the pituitary adenomas. CONCLUSIONS: The mRNA levels of IL-18, IL-18BP and IL-18R are significantly elevated in clinical pituitary tumors, such as ACTH-, GH- and PRL-producing adenomas, when compared to non-functional adenomas. These present results suggest the possibility that IL-18 may be involved in the pathogenesis of pituitary adenoma.

Tumor stem-like cells isolated from MMQ cells resist to dopamine agonist treatment.

Although tumor stem-like cells (TSLCs) have been studied in a range of malignant tumors, evidence for the presence of these cells in pituitary adenomas needs further exploration. Here, we identified a small subset of sphere-forming cells possess tumor stem-like cell properties in rat prolactinoma MMQ cells, which resist to dopamine agonist treatment. Comparing to MMQ cells, sphere-forming cells showed higher cell viability after dopamine agonist (DA) treatment. Furthermore, the cells showed lower expression of prolactin (PRL) and dopamine 2 receptor (D2R). On the contrary, the daughter tumor cells differentiated from these cells restored the sensitivity to DA and showed high expression of PRL and D2R. The lower D2R expression and DA resistance might be due to DNA hypermethylation of D2R promoter. Our study demonstrates that the sphere-forming cells isolated from MMQ cells possess the trait of TSLCs and resist to DA treatment, which offers the opportunity to further investigate the mechanisms underlying tumor recurrence based on TSLCs.

A germline c.1546dupC MEN1 mutation in an MEN1 family: A case report.

RATIONALE: Multiple endocrine neoplasia type 1 (MEN1) is a rare tumor syndrome with an autosomal dominant inheritance, and genetic testing for MEN1 gene is important for both affected individuals and their relatives. We present a 2-person family affected by a germline c.1546dupC MEN1 mutation, and one of them had a full-spectrum of MEN-related endocrine tumors. PATIENT CONCERNS: A female patient aged 32 years presented with jejunal ulcer perforation due to gastrinoma. DIAGNOSES: We conducted genetic analysis and extensive biochemical/radiological evaluation for detecting other endocrine tumors. Multiple pancreatic neuroendocrine tumors (NETs), prolactinoma and primary hyperparathyroidism were diagnosed, and a frame-shift mutation, NM_130799.1:c.1546dupC (p.Arg516Profs∗15), was detected. One daughter of the proband, aged 12 years, had the same mutation for MEN1. INTERVENTION: She underwent pancreatic surgery for pancreatic NETs and total parathyroidectomy for primary hyperparathyroidism. OUTCOMES: After pancreatic surgery, long-term symptoms of epigastric soreness, acid belching, sweating, and palpitation in fasting were improved. Hypercalcemia was improved after parathyroidectomy and she was supplemented with oral calcium and vitamin D. Her daughter showed normal biochemical surveillance until 15 years of age. LESSONS: We report 2 people in a family affected by MEN1 with the heterozygous germline c.1546dupC mutation, a variant that should be surveilled for early development of full-blown MEN1-associated endocrine tumors.

MicroRNA­29­3p regulates the ß­catenin pathway by targeting IGF1 to inhibit the proliferation of prolactinoma cells.

The present study aimed to analyze the effects and underlying mechanisms of microRNA (miR)­29­3p on the proliferation and secretory abilities of prolactinoma cells by targeting insulin­like growth factor (IGF)­1/ß­catenin. The relationship between miR­29a­3p and the survival of prolactinoma cells was analyzed with the Kaplan­Meier method in reference to The Cancer Genome Atlas. The expression levels of miR­29a­3p and IGF­1 in MMQ and GH3 cells were detected. A dual­luciferase reporter gene assay was performed to verify the combination of miR­29a­3p and IGF­1. Cells were transfected with a miR­29a­3p mimic and/or IGF­1 pcDNA3.1 to analyze the effects on the proliferation, apoptosis and secretion of prolactin (PRL) and growth hormone (GH) of prolactinoma cells. The effects on ß­catenin in the cytoplasm and nucleus were investigated by western blot analysis. The results showed that miR­29a­3p expression was low in MMQ and GH3 cells. Overexpression miR­29a­3p inhibited IGF­1 mRNA and protein expression. miR­29a­3p inhibited cell proliferation and PRL and GH expression, and promoted apoptosis by inhibiting IGF­1. Increasing the expression of miR­29a­3p increased ß­catenin levels in the cytoplasm, whereas IGF­1 promoted ß­catenin activation and entry into the nucleus, and reversed the inhibitory effects of miR­29a­3p on ß­catenin. To conclude, miR­29a­3p inhibited the proliferation and secretory abilities of prolactinoma cells by inhibiting nuclear translocation of ß­catenin via a molecular mechanism that is inseparable from IGF­1.

A mutation of the ß-domain in POU1F1 causes pituitary deficiency due to dominant PIT-1ß expression.

BACKGROUND: POU1F1 encodes both PIT-1α, which plays pivotal roles in pituitary development and GH, PRL and TSHB expression, and the alternatively spliced isoform PIT-1ß, which contains an insertion of 26-amino acids (ß-domain) in the transactivation domain of PIT-1α due to the use of an alternative splice acceptor at the end of the first intron. PIT-1ß is expressed at much lower levels than PIT-1α and represses endogenous PIT-1α transcriptional activity. Although POU1F1 mutations lead to combined pituitary hormone deficiency (CPHD), no patients with ß-domain mutations have been reported. RESULTS: Here, we report that a three-generation family exhibited different degrees of CPHD, including growth hormone deficiency with intrafamilial variability of prolactin/TSH insufficiency and unexpected prolactinoma occurrence. The CPHD was due to a novel POU1F1 heterozygous variant (c.143-69T>G) in intron 1 of PIT-1α (RefSeq number NM_000306) or as c.152T>G (p.Ile51Ser) in exon 2 of PIT-1ß (NM_001122757). Gene splicing experiments showed that this mutation yielded the PIT-1ß transcript without other transcripts. The lymphocyte PIT-1ß mRNA expression was significantly higher in the patients with the heterozygous mutation than a control. A luciferase reporter assay revealed that the PIT-1ß-Ile51Ser mutant repressed PIT-1α and abolished transactivation capacity for the rat prolactin promoter in GH3 pituitary cells. CONCLUSIONS: We describe, for the first time, that the PIT-1ß mutation can cause CPHD through a novel genetic mechanism, such as PIT-1ß overexpression, and that POU1F1 mutation might be associated with a prolactinoma. Analysis of new patients and long-term follow-up are needed to clarify the characteristics of PIT-1ß mutations.