ACTH-Receptor-initiated Signaling in Adrenal Tumorigenesis

The ACTH-receptor is a seven-transmembrane G-protein-coupled receptor. Its signaling is mainly transduced via the cAMP pathway, but activation of phopholipase C and the MAPK pathway as well as opening the calcium channels have been described. Although ACTH-R activation induces potential mitogens such as jun, fos, and myc in addition to activating the MAPK pathway, there is strong evidence for a growth-inhibiting effect in vitro. In undifferentiated adrenocortical carcinomas, expression of the ACTH-R is frequently lost, which is paralleled by extensive tumor growth. This has led to the hypothesis that ACTH acts as a differentiation factor and inhibits tumor growth. Unfortunately, the effects of ACTH on adrenal proliferation in vivo cannot be differentiated from effects of other POMC derived peptides such as N-terminal POMC. Although several lines of evidence suggest that the net effect of ACTH-R mediated signal transduction is more anti-proliferative, this concept awaits confirmation. In order to address this question, we established an in vivo tumor model using Y6 cells – mutants of the mouse adrenocortical cell line Y1 that do not express the ACTH-R. Transfection of these cells with a human ACTH-R cDNA (Y6-ACTHR) led to stable expression of functioning ACTH-R. Control cells were transfected with the empty vector pcDNA3.1. In vitro studies using conventional cell counts and thymidine incorporation assays confirmed the growth-inhibiting effect of ACTH that was most pronounced with a dose of 10-8 M ACTH 1-24. Higher doses diminished this effect. Injection of both cell lines in the left and right neck region of mice resulted in an ACTH-R-positive and -negative tumor within the same mouse. A slow release formula of ACTH 1-24 was intraperitoneally administered in three different doses corresponding to a low and high physiological and a pharmacological dose. The biological effects of ACTH were monitored by measuring endogenous ACTH in plasma and determining thymus and adrenal weight. ACTH 1-24 significantly reduced tumor weight in ACTH-R-positive tumors in a U-shape manner with the maximum effect at a high physiological dose. No significant difference in tumor mass was observed in ACTH-R-negative tumors. Expression of the proliferation marker PCNA, determined by Western Blot and immunohistochemistry, was lower in ACTH-R-positive tumors after treatment at the high physiological dose. In conclusion, the ACTH-R mediated an anti-proliferative effect in this model when stimulated with physiological doses of ACTH. Pharmacological doses might partially reverse this effect, showing to a dual role of ACTH in adrenal tumor growth.