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

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. 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. 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 = 0.01) and Overall Survival (OS) (log-rank test = 8.2 × 10), 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 = 2.4 × 10). 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. 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.