Responses to Corticotrophin-Releasing Hormone and Dexamethasone in a Large Series of Human Adrenocorticotrophic Hormone-Secreting Pituitary Adenomas In Vitro Reveal Manifold Corticotroph Tumoural Phenotypes

Abstract

Patients with Cushing's disease are known to present a variable secretory response to stimulatory and inhibitory challenges. Evaluation of the secretory behaviour of pituitary adrenocorticotrophic hormone (ACTH)-secreting adenomas in vitro aids in the comprehension of its behaviour in vivo; however, given the small size of these tumours and the consequent paucity of material available to in vitro studies, a comprehensive study on the secretory behaviour of human corticotroph tumours has not yet been performed. The present study aimed to assess the spectrum of responses to the two main corticotroph modulators, corticotrophin-releasing hormone (CRH) and dexamethasone, in a large series of human ACTH-secreting pituitary tumours. Seventy-two ACTH-secreting pituitary tumours were collected during surgery and established in culture. Specimens were incubated with 10 nm CRH and/or 10 nm dexamethasone for 4 h and 24 h. Secretion in unstimulated, control wells was set at 100% and changes in ACTH concentrations by at least 20% were considered as responses. Parallel experiments in 12 rat anterior pituitary primary cultures were evaluated. A marked ACTH increase was observed during incubation with CRH in 70% of tumoural specimens at 4 h (range 124-3500% of control wells) and in 57% at 24 h (range 122-3323%). Dexamethasone reduced ACTH secretion in almost 50% of tumours (range 78-2% of control at 4 h; 76-3% at 24 h), whereas it did not affect ACTH medium levels in 30% of specimens and induced a paradoxical ACTH increase in 20% of tumours (range 130-327% of control at 4 h; 156-348% at 24 h). By comparison, CRH uniformly increased ACTH levels in rat anterior pituitary primary cultures (mean 745 ± 84% at 4 h; 347 ± 25% at 24 h), whereas dexamethasone decreased ACTH levels by 40-50% in all experiments. In conclusion, the present study of a large series of human ACTH-secreting pituitary tumours in vitro revealed a considerable variability in the responses to CRH and dexamethasone. This finding indicates the existence of multiple corticotroph tumoural phenotypes and may account for the different responses to physiological and pharmacological modulators in vivo.

Sexual Dimorphism in Cellular and Molecular Features in Human ACTH-Secreting Pituitary Adenomas

“…MIB-1 staining was <2% in all but one specimen; this adenoma presented MIB-1 index 9%, mitosis count 3 per 10 high power field and had been collected from a female patient in whom surgery proved successful. As per our previous publications [53,96,97], the presence of corticotrope cells in fresh adenoma specimens was assured by abundant ACTH secretion in culture medium [98]; as regards formalin-fixed specimens, abundant POMC and absent GH, PRL, PIT1, LHB, FSHB expression was documented by microarray analysis [13].…”

“…Specimens were established in culture according to our standard protocol [98,99]; primary cultures were incubated in serum-free DMEM + 0.1% bovine serum albumin (BSA) containing 10 nM CRH or 10 nM dexamethasone. Wells treated with DMEM + BSA only represented control secretion.…”

“…Intra-assay coefficient of variation is 7.9% and assay sensitivity 1.2 pg/mL. Given the considerable variability in ACTH adenoma concentrations [98], ACTH concentrations were normalized to number of cells per well and responses to CRH and dexamethasone expressed relative percent of secretion in wells incubated with DMEM + BSA only (control = 100%) for statistical analyses. Plated cells counts were comparable between specimens from female and male patients (138,305 ± 18,460 vs. 110,449 ± 23,372 cells per well, respectively, N.S.…”

Gene expression profiling in human corticotroph tumours reveals distinct, neuroendocrine profiles

“…Of note, an increasing body of evidence indicates that ACTH‐secreting adenomas differ markedly among themselves regarding their responses to the main modulators of corticotroph secretion, such as corticotrophin‐releasing factor and steroids, as well as ancillary modulators, such as vasopressin analogues, gonadotrophin‐releasing hormone . Differences in in vitro ACTH secretory patterns have also been observed in response to dopamine or somatostatin receptor agonists, which obviously translates into different responsiveness to these drugs.…”

Proopiomelanocortin, glucocorticoid, and CRH receptor expression in human ACTH-secreting pituitary adenomas

“…The aim of the present study was to expand upon our recent findings on a large series of ACTH-secreting pituitary adenomas [ 9 ]. In this study, we reported on the marked variability in ACTH secretion by human corticotrope tumors in vitro.…”