ECE2023 Oral Communications Oral Communications 9: Adrenal and Cardiovascular Endocrinology 2 (6 abstracts)
FLNA-binding partner Wee1 as a new potential pharmacological target in adrenocortical carcinomas
Emanuela Esposito 1,2 , Rosa Catalano 1 , Emma Nozza 1,2 , Anna Maria Barbieri 1 , Sara Maioli 1 , Jens Albrecht Ernst Geginat 1 , Giusy Marra 1 , Donatella Treppiedi 3 , Genesio Di Muro 1,4 , Federica Mangili 3 , Costanze Hantel 5,6 , Serena Palmieri 3 , Sofia Frigerio 3 , Emanuele Ferrante 3 , Maura Arosio 1,3 , Giovanna Mantovani 1,3 & Erika Peverelli 1,3
1University of Milan, Department of Clinical Sciences and Community Health, Milan, Italy; 2University of Milan, PhD Program in Experimental Medicine, Milan, Italy; 3Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Endocrinology Unit, Milan, Italy; 4University Sapienza of Rome, PhD Program in Endocrinological Sciences, Rome, Italy; 5University Hospital Zurich (USZ) and University of Zurich (UZH), Department of Endocrinology, Diabetology and Clinical Nutrition, Zurich, Switzerland; 6University Hospital Carl Gustav Carus Dresden, Medizinische Klinik und Poliklinik III, Dresden, Germany
The cytoskeletal actin-binding protein filamin A (FLNA) is poorly expressed in adrenocortical carcinomas (ACC) compared to adenomas (ACA), and this might contribute to sustain the increased cell proliferation by downregulating IGF1R expression and its downstream signaling. In mouse neural progenitor cells, increased protein expression levels of the CDK1 kinase Wee1 have been found after loss of FLNA. This protein has a leading role in regulating the G2-M checkpoint and functions as a mitotic inhibitor. Wee1 is overexpressed in several cancer types and its pharmacological inhibitor Adavosertib (AZD1775) is currently undergoing clinical trials. Aims of the present project are to investigate the role of FLNA in regulating Wee1, the effects of Wee1 inhibition on cell proliferation and apoptosis, as well as the effects of FLNA levels on AZD efficacy in human H295R and MUC-1 cell lines. The analysis of protein expression levels of FLNA and Wee1 in 6 ACC and 8 normal adrenal tissues revealed that ACC express lower levels of FLNA (0.4±0.7 and 2.9±0.9, respectively, P<0.05), while significantly increased Wee1 (0.26±0.1 and 0.01±0.06, respectively, P<0.001). In MUC-1 cells a correlation between Wee1 and FLNA expression was shown. Indeed, FLNA silencing induced an increased expression of Wee1, phosphorylated CDK1 and cyclin B1 (+1.6±0.2, +1.7±0.4 and +1.4±0.2 fold, P<0.001 vs negative control, respectively). On the contrary, FLNA overexpression resulted into a reduced expression of Wee1, phosphorylated CDK1 and cyclin B1 (-51±5%, P<0.001, −60±5%, P<0.001, −67±20%, P<0.01 vs mock). Treatment with AZD1775 induced a dose-dependent reduction of cell proliferation (-32±4.6%, P<0.001; −78±4.8%, P<0.001 vs bas at 250 nM) and an increase of apoptosis (+6±1.5-fold, P<0.001; +4±0.5-fold, P<0.001 vs bas at 1 μM) in H295R and MUC-1 cell lines, respectively. Flow cytometric analysis of apoptotic subpopulations showed that, in MUC-1, Wee1 inhibition specifically stimulated an increase of the early apoptotic cells (+5-fold, P<0.001 vs bas at 1 μM). Moreover, AZD1775 induced a time-dependent reduction of CDK1 phosphorylation, with a maximum after 2 hours (-67±9.5%, P<0.01; −90.5±1.7%, P<0.001 vs bas at 250 nM in H295R and MUC-1, respectively). Interestingly, FLNA knockdown potentiated AZD1775 effects on cell proliferation (-51±6.4%, P<0.01 vs negative control at 250 nM) in MUC-1 cells. In conclusion, this work demonstrates that low FLNA levels in ACC correlate to a high Wee1 expression, contributing to an increased cell growth. Moreover, it proposes Wee1 inhibition as a new potential therapeutic approach for ACC, particularly for those lacking FLNA.
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Endocrine Abstracts
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