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Abstract

The main function of glucocorticoids in corticotroph cells is to suppress proopiomelanocortin, the precursor of the stress hormone adrenocorticotropin (ACTH). Cushing?s disease is a rare but severe neuroendocrine condition caused by partially glucocorticoid resistant corticotroph adenomas, which consequently secrete excessive amounts of ACTH in an uncontrolled fashion. The patients suffer from chronic hypercortisolism due to excessive stimulation of the adrenal glands by ACTH to produce glucocorticoids. Impairing mutations of the glucocorticoid receptor (GR) only sporadically explain the reduced glucocorticoid sensitivity in the adenomas ? the molecular mechanism behind the partial resistance is poorly understood.
The function of GR depends on direct interactions with the molecular chaperone Hsp90. Both the reduction and overexpression of Hsp90 impedes GR activity in different experimental settings. Therefore, the expression of the inducible Hsp90\ensuremath{α} isoform was determined in biopsy specimens of corticotroph pituitary adenomas from patients with Cushing?s disease. Its strong overexpression compared to normal human pituitary cells paved the way to study its role in the function of corticotroph adenomas using small molecules which target Hsp90.
The three distinct Hsp90 inhibitors 17?AAG, Novobiocin and Silibinin showed antiproliferative effects in AtT?20 cells through the degradation of the oncogenic client kinase Cdc2, a hallmark of pharmacologic inhibition of Hsp90. Surprisingly, only the N?terminal Hsp90 inhibitor 17?AAG caused the degradation of GR, as was reported also for other Geldanamycin?based Hsp90 inhibitors. Neither Silibinin nor the C?terminal Hsp90 inhibitor Novobiocin affected GR protein levels. These converging effects led to the assumption that both compounds bind to the same domain in Hsp90. It was shown here that Novobiocin displaces Silibinin from the C?terminal domain of Hsp90, and that these compounds dissociate mature GR from Hsp90 at the biochemical level. As a result, increased levels of mature receptor were present in the cell able to bind glucocorticoids with high affinity. This novel molecular mechanism proved to potentiate GR transcriptional activity in AtT?20 cells. The potentiation in GR activity also led to enhanced suppression of ACTH elicited by low concentrations of Dexamethasone in AtT?20 cells and in primary cultures of human corticotroph adenomas from patients with Cushing?s disease. In contrast, Silibinin did not show effects on rat normal pituitary cells. Finally, Silibinin reduced tumor growth, partially reverted hormonal alterations, and alleviated symptoms in a mouse allograft model for Cushing?s disease.
These results suggest that the regulation of GR sensitivity by overexpressed Hsp90 may represent a pharmacologically reversible mechanism in the pathogenesis of this disease. Together, a proof of principle is provided that the clinically safe Hsp90 inhibitor Silibinin potentially restores glucocorticoid sensitivity in corticotroph adenomas in vitro and in vivo, and that it might be used to treat Cushing?s patients in the future.