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OR02-4 YAP1 Overexpression Associates With Worse Prognosis In Patients With Adrenal Tumors And YAP1 Inhibition By Verteporfin Reduces Tumorigenicity And Reverses Epithelial-mesenchymal Transition In Adrenocortical Tumor Cells

Abstract Background: There is no effective adjuvant therapy for patients with advanced adrenocortical tumors (ACT). Abnormal YAP1, a HIPPO pathway effector, is involved in some types of cancers. Recently, we showed association of the overexpression of YAP1 in pediatric ACT (pACT) with worse prognosis, but its association with tumorigenicity and metastasis in ACT or its involvement in adult ACT (aACT) is unknown. Aims:To analyze the association of YAP1 expression in ACT with prognosis and the effect of YAP1 inhibition on tumor growth and epithelial-mesenchymal transition (EMT) in adrenocortical tumor cells.Methods: The YAP1 mRNA expression in pACT and aACT was analyzed using data available in the Gene Expression Omnibus database (GSE76019, ACT=34) and The Cancer Genome Atlas database (ACC, n=76), respectively. In vitro, we treated NCI-H295 adrenocortical tumor cells with Verteporfin (10uM), a FDA-approved drug that inhibits YAP1/TEAD complex. We evaluated mRNA expression of the HIPPO pathway genes (LATS2, STK3, STK4), its targets (CTGF), TEAD4, CTNNB1 and cycle cell progression markers (CCND1, CKD2 and CCNE1) by qPCR. In addition, we analyzed YAP1, phosphoYAP1 (Ser127), Beta-catenin and mesenchymal markers (Vimentin, N-cadherin and the transcription factor, Snail1) protein expression by Western blot (WB). Cell proliferation (MTS), cell invasion (transwell system), and tumorigenesis (soft agar colony formation assay) were assessed after VP treatment. Results:Higher YAP1 mRNA expression was associated with lower disease free survival (DFS) and lower total survival in adults with and with lower DFS in pediatric patients. Verteporfin considerably reduced cell proliferation (-53.2% after 48h and -67.7% after 72h), cell invasion (-85.6%) and the tumorigenicity, shown by reduction of anchorage-independent growth. Verteporfin antitumor effect on ACT cells was shown by reduced mRNA expression of the Beta-catenin encoding gene (CTNNB1; p<0.05), cell cycle progression genes (CCND1, CDK2 and CCNE1; p<0.005) and YAP1 target gene (CTGF) and TEAD4 (p<0.0001), besides increased mRNA expression of core kinase genes of the HIPPO pathway (LATS2, STK3 and STK4; p<0.05). Verteporfin reduced protein levels of YAP1 (-88.5%), phosphoYAP1 (-94.6%) (p<0.0005), Beta-catenin (-50.4%, p<0.001) and the mesenchymal markers Vimentin (-36%, p<0.01), N-cadherin (-66%, p<0.001) and Snail1 (-53%, p<0.05).Conclusions: YAP1 overexpression is a marker in worse prognosis in ACT. YAP1 inhibition by Verteporfin reduced tumor growth through activation of HIPPO pathway, inhibition of cell cycle progression, reduction of Beta-catenin expression, loss of capacity of anchorage-independent growth, reversed EMT by reduction of mesenchymal markers and reduced cell viability in adrenal cells. These results indicate to YAP1 inhibition by Verteporfin as a novel potential adjuvant therapy for patients with ACT.