Dual-Targeting of ATOX1 and ROCK1: A Potent Strategy to Potentiate the Inhibition of Lung Adenocarcinoma...

Background: Lung adenocarcinoma (LUAD), the most prevalent and malignant form of lung cancer subtypes, is in urgent need of additional therapeutic targets and prognostic indicators. Antioxidant 1 (ATOX1) copper chaperone and RhoA/Rho kinase 1 (ROCK1) are novel anti-tumour targets in cancers. However, their prognostic value and synergistic inhibitory effect remain unclear in LUAD. Methods: We re-analyzed the open-access proteomic landscape study of LUAD in 2019 and investigated the prognostic value of ATOX1/ROCK1 expression patterns. Then we verified it immunohistochemically using an independent cohort from our hospital enrolling 35 patients with TNM stage III/IV LUAD. In vitro, double fluorescence was used to confirm the co-expression and location of ATOX1/ROCK1. The CCK-8 assay and Transwell assay were carried out to assess the changes in proliferation and migration of Lewis lung carcinoma (LLC) cells following treatment with ATOX1/ROCK1 si-RNA or inhibitory drugs. Western blot was used to confirm protein expression after si-RNA transfection. Moreover, ATOX1/ROCK1-targeted drugs' therapeutic effects were further investigated in the LLC allogeneic transplantation model and MNU-induced tumour model. Results: Firstly, according to the ATOX1/ROCK1 expression pattern derived from proteomic data, double-low expression of ATOX1/ROCK1 indicated a better Disease Free Survival (DFS) (log-rank test p = 0.01) and Overall Survival (OS) (log-rank test p = 8.2 × 10^-3), whose expression was also correlated with the lower expression of MCM family proteins. Further, we verified this prognostic correlation in our cohort. The IHC-defined ATOX1/ROCK1 low subtype also had the best OS (log-rank test p = 2.4 × 10^-3). In vitro, double fluorescence confirmed that ATOX1/ROCK1 was highly expressed together in Lewis cells. Co-inhibition of ATOX1 and ROCK1 either by siRNA transfection or inhibitory drugs could lead to a significant decrease in tumour proliferation. Interestingly, transcriptional inhibition of ATOX1 can lead to the up-regulation of ROCK1, while inhibition of ROCK1 resulted in the promotion of ATOX1. Moreover, in the analysis of migration ability, a similar synergistic effect from the co-inhibition of ATOX1/ROCK1 was also observed. Finally, the Lewis and Mnu-induced allogeneic transplantation model also demonstrated a greatly improved therapeutic effect by combining targeting ATOX1 and ROCK1. Conclusions: Collectively, our results suggest that a low expression pattern of ATOX1/ROCK1 can predict better clinical outcomes in LUAD. Combining the inhibition of these two targets can reach a significantly better therapeutic effect than targeting either alone.