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 testp= 0.01) and Overall Survival (OS) (log-rank testp= 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 testp= 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.
背景：肺腺癌（LUAD）作为肺癌亚型中最常见且恶性程度最高的类型，亟需寻找新的治疗靶点与预后标志物。抗氧化蛋白1（ATOX1）铜伴侣蛋白与RhoA/Rho激酶1（ROCK1）是近年来在多种癌症中发现的新型抗肿瘤靶点，但二者在LUAD中的预后价值及协同抑制作用尚不明确。 方法：本研究重新分析了2019年发表的LUAD开放获取蛋白质组学数据，探究ATOX1/ROCK1表达模式的预后价值，并采用本院35例TNM III/IV期LUAD患者组织样本进行免疫组化验证。通过双荧光染色确认ATOX1/ROCK1在Lewis肺癌（LLC）细胞中的共表达与定位；采用CCK-8实验与Transwell实验评估ATOX1/ROCK1 siRNA或抑制剂处理后LLC细胞增殖与迁移能力变化；Western blot验证siRNA转染后蛋白表达水平。进一步在LLC同种异体移植模型及MNU诱导肿瘤模型中评估ATOX1/ROCK1靶向药物的联合治疗效果。 结果：蛋白质组学数据分析显示，ATOX1/ROCK1双低表达患者具有更长的无病生存期（DFS）（时序检验p=0.01）和总生存期（OS）（时序检验p=8.2×10−3），且该表达模式与MCM家族蛋白低表达相关。临床队列验证证实，免疫组化定义的ATOX1/ROCK1低表达亚型患者OS最佳（时序检验p=2.4×10−3）。体外实验显示ATOX1与ROCK1在LLC细胞中高共表达，通过siRNA或抑制剂联合抑制二者可显著降低肿瘤增殖能力。值得注意的是，ATOX1转录抑制会上调ROCK1表达，而ROCK1抑制会促进ATOX1表达。迁移能力分析同样观察到ATOX1/ROCK1共抑制的协同效应。动物实验进一步证明，联合靶向ATOX1与ROCK1在LLC移植模型及MNU诱导模型中均能显著提升治疗效果。 结论：ATOX1/ROCK1低表达模式可作为LUAD良好预后的预测指标，联合抑制这两个靶点比单一靶向治疗能产生更显著的抗肿瘤效果。
肿瘤(癌症)患者之家