Background: There is no effective adjuvant therapy for patients with advanced ACT. YAP1, a HIPPO pathway effector, interacts with Wtn\beta-catenin pathway and plays a crucial role in the maintenance of postnatal adrenal cortex and regulates cell proliferation and apoptosis in several tissues. We recently showed that overexpression of YAP1 associates with worse prognosis in our cohort of pediatric ACT (pACT).
Aim: To analyze the association of YAP1 expression in ACT with prognosis in additional cohorts of ACT and the effects of YAP1 inhibition on adrenocortical tumorigenesis.
Methods: YAP1 mRNA expression in ACT was analyzed in silico using public available data both in pACT (GSE76019; n=34) and adult ACT (TCGA; ACC=76). In vitro, H295 ACT cells were treated with Verteporfin (VP; 10uM), a FDA-approved drug that inhibits YAP1/TEAD complex. Transient YAP1 knockdown was also performed. The mRNA expression of the HIPPO pathway genes (LATS2, STK3, STK4), its target (CTGF), CTNNB1 (beta-catenin) and cell cycle progression markers (CCND1, CKD2 and CCNE1) was evaluated by qPCR. The protein expression of YAP1, phospho(p) YAP1 (Ser127), Beta-catenin, Cyclin D1 and mesenchymal markers (Vimentin, N-cadherin and Snail) was analyzed by Western blot. Cell proliferation (MTS), cell invasion (Matrigel coated transwell), cell cycle (flow cytometry) and tumorigenic potential (anchorage-independent growth/soft agar colony formation) were also assessed.
Results: Bioinformatic analysis showed that higher YAP1 mRNA expression was associated with lower disease free survival (DFS) in pediatric patients (P=0.001) and also with lower DFS (P=0.0004) and total survival in adults (P=0.009). In vitro, VP reduced cell proliferation (48h=-53% and 72h=-67%; P<0.05), invasion (-85%;P<0.05) and abolished anchorage-independent growth. VP reduced mRNA expression of CTNNB1 (P<0.05), cell cycle genes (CCND1, CDK2 and CCNE1;P<0.005) and CTGF (P<0.05) while increased mRNA expression of the HIPPO pathway genes (LATS2, STK3 and STK4; P<0.05). VP reduced protein levels of YAP1 and pYAP1 (-88% and -94%;P<0.0005), B-catenin (-50%,P<0.001) and the mesenchymal markers Vimentin (-36%,P<0.01), N-cadherin (-66%,P<0.001) and Snail (-53%,P<0.05). YAP1 knockdown reduced cell tumorigenic potential, the expression of mesenchymal markers (Vimentin:-42%,P<0.05; N-cad: -68%,P<0.001) and Cyclin D1 (-43%,P<0.005), while induced G0/G1 cell cycle arrest.
Conclusions: YAP1 overexpression is a marker of unfavorable prognosis in pediatric and adult ACT patients. YAP1 signaling inhibition has antitumor effects in ACT cells, as shown by decreased cell proliferation, Beta-catenin downregulation, reduction of cell invasion, loss of anchorage-independent growth and reversion of epithelial-mesenchymal transition. YAP1 inhibition by Verteporfin may emerge as a potential adjuvant therapy for patients with ACT.
19 Sep 2019 - 21 Sep 2019