Abstract Adrenocortical carcinomas (ACC) are rare tumors with no effective therapy for patients with advanced or metastatic disease. Therefore, efforts have been made to develop new therapeutic approaches for effective ACC treatment. Studies suggest that the dysregulation of Aurora kinases might be strongly associated with tumorigenesis and that Aurora kinases are involved in the promotion of the epithelial-mesenchymal transition (EMT) and invasion in different cancer cells. Molecular markers for EMT include decreased expression of E-cadherin, nuclear localization of β-catenin, and upregulation of genes related to the Wnt/β-catenin pathway, such as Cyclin D1 and c-Myc. Thus, the aim of this study was to investigate whether Aurora kinases are involved in the promotion of EMT in ACC cells. NCI-H295 cells were treated with 50 nM of the pan-Aurora kinase inhibitor, AMG 900, for 6, 24, and 48 hours. Multiple Reaction Monitoring proteomic approach was performed to screen proteins that are known to be involved in EMT. Total and enriched membrane, nucleus, and cytosol proteins were obtained by different centrifugation grades and detergents. Western blotting was performed for protein expression and subcellular localization. Cell cycle, proliferation, and migration assays were performed to study the effect of AMG 900 in NCI-H295 cells. MRM studies showed increased expression of E-cadherin 24 and 48 hours after AMG 900 treatment and decreased expression of N-cadherin after 48 hours of treatment. Conversely, our data showed increased of β-catenin expression and Vimentin expression after 48 hours of treatment. Moreover, our results showed higher expression of β-catenin in the nucleus when compared to control. Western blotting analysis confirmed these findings and gene expression analysis showed increased of c-Myc, one of the main products of β-catenin nuclear translocation, after 24 hours of treatment. We performed functional assays in order to check whether the studied pathways would affect NCI-H295 cell proliferation and migration, and our results showed that inhibition of Aurora kinases leads to cell cycle arrest in mitosis and decreases cell proliferation and migration. Further studies need to be performed combining Aurora kinases and β-catenin inhibitors; however, our results suggest that Aurora kinases might regulate proteins involved in EMT process during ACC progression. Citation Format: Andrea G. Maria, Carolina H. Thomé, Kleiton S. Borges, Mariana L. Grassi, Augusto F. Andrade, Regia C.P. Lira, Fabio R. Faucz, Constantine A. Stratakis, Luiz G. Tone, Vitor M. Faca. Uncovering unknown Aurora kinase signaling in adrenal tumor cell line, NCI-H295 [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A070.