结直肠癌（CRC）可分为四种分子亚型（CMS），其中CMS1与最佳预后相关，而CMS4，即间质亚型，预后最差。虽然线粒体被认为是众多信号通路的中心枢纽，但多年来对线粒体代谢的研究一直被忽视。线粒体复合物I（CI）在能量和反应性氧化物（ROS）的产生中起着双重作用。然而，CI对癌症发生的可能贡献仍不清楚。本研究的目的是在CMS分类的视角下研究CRC中的CI在体外模型中的作用。我们使用生化测量、生物能学分析和蛋白质免疫印迹来表征属于CMS1和CMS4亚组的LoVo和MDST8细胞系中的CI表达、功能和氧化还原平衡。细胞增殖和迁移通过xCELLigence技术进行评估。通过过量产生或清除线粒体ROS（mtROS）来分析mtROS对增殖、迁移和间质标志物的影响。免疫荧光分析了聚焦粘附激酶（FAK）及其活化。我们评估了根据CMS分类在CRC队列中的两种CI评分的分布以及它们与患者生存的相关性。我们发现CI在CMS4细胞中被下调并与升高的mtROS相关。我们首次证明在这些迁移细胞中，mtROS产生不仅通过CI活性的变化，还通过乙酰化/失活超氧化物歧化酶2（SOD2），一个重要的线粒体抗氧化酶来维持在最佳水平。我们表明，促进或清除mtROS都减轻了CMS4细胞的迁移。我们的结果还指出，mtROS介导的聚焦粘附激酶（FAK）活化可能维持其迁移表型。通过使用CRC患者的队列，我们证明CI在CMS4亚组中表达下调，并且低CI表达与预后差相关。患者数据集揭示CI和上皮间质转变（EMT）通路之间存在反向相关。我们展示了CI的抑制有助于提高mtROS，这可能促进了MDST8的迁移并可能解释了CMS4肿瘤的特异性EMT特征。这些数据揭示了线粒体CI在CRC中的新角色，并且其生物学后果可以通过抗氧化剂或促氧化剂药物在临床实践中作为靶点。©2023。作者。

Colorectal cancer (CRC) can be classified into four molecular subtypes (CMS) among which CMS1 is associated with the best prognosis, while CMS4, the mesenchymal subtype, has the worst outcome. Although mitochondria are considered to be hubs of numerous signaling pathways, the study of mitochondrial metabolism has been neglected for many years. Mitochondrial Complex I (CI) plays a dual role, both in energy and reactive oxygen species (ROS) production. However, the possible contribution of CI to tumorigenesis in cancer remains unclear. The purpose of this study was to investigate the CI under the prism of the CMS classification of CRC in ex vivo models.Biochemical dosages, bioenergetics analysis and western-blot were used to characterize CI expression, function and redox balance in LoVo and MDST8 cell lines, belonging to CMS1 and CMS4 subgroups, respectively. Cell proliferation and migration were assessed by xCELLigence technology. Overproduction or scavenging of mitochondrial ROS (mtROS) were performed to analyze the effect of mtROS on proliferation, migration, and mesenchymal markers. Focal adhesion kinase (FAK) and its activation were analyzed by immunofluorescence. We assessed the distribution of two CI scores in CRC cohorts according to CMS classification and their relevance for patient survival.We found that CI is downregulated in CMS4 cells and is associated with elevated mtROS. We establish for the first time that in these migrating cells, mtROS production is maintained at optimal levels not only through changes in CI activity but also by inactivation/acetylation of superoxide dismutase 2 (SOD2), a major mitochondrial antioxidant enzyme. We show that promoting or scavenging mtROS both mitigate CMS4 cells' migration. Our results also point to a mtROS-mediated focal adhesion kinase (FAK) activation, which likely sustains their migratory phenotype. Using cohorts of CRC patients, we document that the expression of CI is downregulated in the CMS4 subgroup, and that low CI expression is associated with poor prognosis. Patients' datasets reveal an inverse correlation between CI and the epithelial-mesenchymal transition (EMT) pathway.We showed that inhibition of CI contributes to heighten mtROS, which likely foster MDST8 migration and might account for the specific EMT signature of CMS4 tumors. These data reveal a novel role of mitochondrial CI in CRC, with biological consequences that may be targeted with anti- or pro-oxidant drugs in clinical practice.© 2023. The Author(s).