Neuromedin B Receptor Antagonist Suppresses Pomc Expression in AtT-20 Cells and Human Corticotroph Adenoma Cells

Objective: We previously reported that Neuromedin B (NMB) is expressed in murine pituitary corticotrophs under adrenal insufficiency (1). Because NMB is also expressed in several cancer cells and stimulates ACTH secretion, we hypothesized that NMB is related to corticotroph adenoma cell proliferation and hormone secretion. To examine this hypothesis, we investigated the expression of NMB and its receptor NMBR in human corticotroph adenoma and the effects of a NMBR antagonist on AtT-20 cells, a mouse corticotroph adenoma cell line, and patient-derived corticotroph adenoma cells. Methods: 1. NMB and NMBR expression in human pituitary adenoma: We performed real-time qPCR and immunostaining on human pathological specimens of corticotrophs, somatotrophs, and non-functioning pituitary adenoma to investigate NMB and NMBR expression. 2. Experiments in AtT-20 cells: We extracted mRNAs and proteins from AtT-20 cells after incubation with 100nM NMBR antagonist PD168368, and performed real-time qPCR and western blotting analyses to investigate Pomc expression. 3. Experiments in patient-derived corticotroph adenoma cells: We isolated surgically resected human corticotroph adenoma cells from patients who underwent trans-sphenoidal surgery and investigated POMC mRNA expression by real-time qPCR after incubation with PD168368. Statistical analysis: One-way ANOVA was employed to compare values among multiple groups. If the ANOVA revealed significant differences, the Tukey-Kramer post-hoc test was employed to compare values between two specific groups. Dunnett’s post-hoc test was employed to compare values with the control group. Statistical significance was defined as p < 0.05. Results: 1. NMB and NMBR expression levels were significantly higher in human corticotroph adenoma (13 and 33 times higher than non-functioning adenoma, respectively) than in somatotroph adenoma (2 and 3 times higher than non-functioning adenoma, respectively) and non-functioning adenoma in the qPCR analyses. Immunostaining confirmed higher expression of NMB and NMBR in corticotroph adenoma than in somatotroph and non-functioning adenoma. 2. Treatment with 100 nM PD168368 significantly suppressed Pomc mRNA and protein expression in AtT-20 cells by 22%±3% and 25%±10%, respectively. 3. Treatment with 1 µM PD168368 significantly suppressed POMC mRNA expression in human corticotroph adenoma cells by 18%±1%. Conclusions: NMB and NMBR were both expressed in human corticotroph adenoma, suggesting that NMB may stimulate adenoma cell proliferation and hormone secretion in autocrine or paracrine manners. Because the NMBR antagonist suppressed Pomc expression in both AtT-20 cells and human corticotroph adenoma cells, it may represent a potential treatment for Cushing disease. Reference: (1) Kameda H et al., Endocrinology 2014;155(7):2492-9.

Pituitary Somatotroph Adenoma Cell-Derived Exosomes: Characterization of Novel Non-Hormonal Functions

Exosomes, small extracellular vesicles carrying lipids, proteins, DNA and RNA, enable intercellular communication. Pituitary-derived exosomes have not been well validated, and as no human pituitary cell lines are available, we characterized exosomes derived from rat somatotroph tumor cells (GH1 and GH3). Rat FR and H9C2 cells were used as non-pituitary controls. Exosomes were isolated from serum-free culture supernatants by combining ultrafiltration and ultracentrifugation to eliminate hormone contamination. Derived exosomes were analyzed by NanoSight to visualize, size, and count particles. Exosomal proteins were extracted and exosome markers including TSG101, ALIX, CD63, HSP70, HSP90 detected by Western Blot. The exosome inhibitor GW4869 (10 µM, 30 h) reduced exosome release (up to 81%), whereas treating cells with hydrocortisone (0.1 µM, 72 h) increased exosome production (up to 42%) in GH1 and GH3 cells. Exosomal shuttle RNA characterized by RNA-Seq showed distinct pituitary vs non-pituitary exosome RNA profiles. Selected miRNAs assessed in exosomes and corresponding cells by qRT-PCR validated exosomal RNA-seq and suggested that miRNA signatures in exosomes and in respective cells of origin were concordant. Next, we explored downstream signaling of GH1-derived exosomes (GH1-exo) in vitro and in vivo and studied biological actions in normal hepatocytes and in malignant cells. As evidenced by mRNA-seq, GH1-exo distinctly altered signaling pathways in rat primary hepatocytes, vs pathways elicited by GH or PRL (0.5 µg/mL, 24 h). GH1-exo, FR-exo or vehicle were intravenously injected to 4-week-old female Wistar rats twice weekly for 4 weeks (5*109 exo/200 g, n=3), and livers dissected for mRNA-seq. Among GH1-exo specifically regulated genes, EIF2AK/ATF4, involved in cAMP responses and amino acid biosynthesis, were attenuated. In hepatocytes, GH1-exo suppressed up to 65% of nascent protein synthesis and reduced forskolin (10 µM)-stimulated cAMP activity by 19%, while GH (0.01-1 µg/mL) did not affect this pathway. Notably, GH1-exo also attenuated malignant cell motility. Both GH1-exo incubation or GH1 cell co-culture (48 h) suppressed migration, invasion and wound healing of HCT116 cancer cells by up to 70%. In contrast, treatment with rGH (0.5 µg/mL) increased HCT116 motility. Intravenous administration of GH1-exo (1010 exo/mouse, twice a week for 5 weeks) decreased metastatic tumor volume by 40% in nude mice harboring splenic HCT116 implants (5*105 cells/mouse, n=10), and especially abrogated hepatic metastases. mRNA-seq of GH1-exo treated HCT116 cells vs controls indicated dysregulated p53 and MAPK pathways, which may partially explain mechanisms underlying motility attenuation. The results elucidate novel biological actions of somatotroph adenoma cell-derived exosomes and suggest exosomes as non-hormonal messengers produced by pituitary tumors.