Role of beta arrestins and G proteins in mediating DRD2 signaling in pituitary tumors

Emanuela Esposito; Federica Mangili; Muro Genesio Di; Barbieri Anna Maria; Donatella Treppiedi; federico arlati; Rosa Catalano; Giusy Marra; emma nozza; Maura Arosio; Giovanna Mantovani; Erika Peverelli

doi:10.1530/endoabs.81.P179

Dopamine receptor type 2 (DRD2) agonists (DA) are the first-choice treatment for prolactin (PRL)-secreting pituitary tumors, but poorly effective in non-functioning (NF)-PitNETs. Along with G protein-dependent signaling, DRD2 also mediates non-canonical beta-arrestin-dependent pathways, where reduction of AKT phosphorylation plays a leading role for the antiproliferative effect of DRD2 in pituitary tumors. Through UNC9994 and MLS1547, a beta-arrestin 2-biased and a G protein-biased agonist, respectively, the present study aimed to clarify the role of G proteins and beta-arrestin 2 in mediating DRD2 signaling in rat tumoral lactotroph cells MMQ and in human primary cultured NF-PitNET cells. In MMQ cells, treatment with UNC9994 reduced cell proliferation (-41.4±20% at 100 nM, P<0.01 vs bas) with a greater efficacy compared to cabergoline (-22.2 at 100 nM±10.9%, P<0.01 vs bas), while MLS1547 treatment resulted into a slight lowering of cell proliferation (-10.8±7.4% at 100 nM, P<0.05 vs bas). Accordingly, UNC9994 was more efficient in reducing AKT phosphorylation (-45.5±16%, P<0.01 vs bas) than cabergoline, whereas an increased AKT phosphorylation was detected after MLS1547 treatment. Consistently, cabergoline and UNC9994 treatments determined a significant reduction of cyclin D3 (-14.7±5.8%, P<0.01 vs bas and -18.8±9%, P<0.05 vs bas, respectively), together with an upregulation of p27/Kip1 (+35±20%, P<0.05 vs bas and +41.6±20.5%, P<0.05 vs bas, respectively). Beta-arrestin 2 silencing reverted either UNC9994 and cabergoline anti-proliferative effects, as well as their effects on AKT phosphorylation. Pretreatment with pertussis toxin (PTX) maintained the antiproliferative effects of cabergoline (-16.6±3.6%, P<0.001 vs bas) and UNC9994 (-31±1.9%, P<0.001 vs bas), while it abolished the ability of MLS1547 in reducing cell proliferation. After 6 h treatment with MLS1547, cell migration showed a considerable reduction (-44±10% at 1μM, P<0.001 vs bas), to a greater extent than cells treated with UNC9994 (-31±19% at 1μM, P<0.01 vs bas). 5 out of 8 human primary cultured NF-PitNET cells in vitro responsive to cabergoline antiproliferative effects (-31±9.4%, P<0.001 vs bas) showed a significant reduction of cell proliferation after UNC9994 and MLS1457 treatments (-27.4±7.5%, P<0.001 vs bas, and -21.7±9.3%, P<0.01 vs bas, respectively). On the other hand, 3 out of 8 NF-PitNETs that did not respond to cabergoline appeared to be unresponsive also to UNC9994 and MLS1547. In conclusion, our data demonstrated a relevant role for the beta-arrestin 2-dependent pathway in regulating DRD2 inhibitory effects on tumoral growth, whereas the canonical G protein-mediated signaling seemed to be key in controlling cell migration.

Endocrine Abstracts

P179