Liang-dong Li, He-fen Sun, Shui-ping Gao, Hong-lin Jiang, Xin Hu, Wei Jin

Breast cancer, Fudan University Shanghai Cancer Center

Objective:Despite advances in basic and clinical research, metastasis remains the leading cause of death in breast cancer patients. Genetic abnormalities in mitochondria, including mutations affecting complex I and oxidative phosphorylation, are found in breast cancers and might facilitate metastasis. The purpose of this study is to explore the way by which the NDUFB9, an accessory subunit of the mitochondrial membrane respiratory chain NADH dehydrogenase (complex I), affect MDA-MB-231 breast cancer cell proliferation and metastasis. Method: In this study, firstly we used quantitative proteomic analyses (iTRAQ-nano-HPLC-

MS/MS) to identify that NDUFB9, was down-regulated in highly metastatic MDA-MB-231HM cell compared to its parental MDA-MB-231cell. Furthermore, we constructed stable MDA-MB-231 cells, in which NDUFB9 was knockdown, to detect the changes in cell proliferation, migration, and invasion. To explore the mechanism involved in this process, we detected the production of mitochondrial ROS and cell NAD+/NADH, determined mtDNA content, and measured glucose uptake, lactate production that were associated with mitochondrial metabolism in cells loss of NDUFB9 and the control. Then the EMT makers as well as the Akt/mTOR/p70S6K signaling pathway were detected. Western blot analysis, Quantitative real-time PCR, Lentivirus packaging and infection, Cell proliferation assays, Cell cycle analysis via flow cytometry, Transwell assays, Kinetic Wound-Healing Assay, Mitochondrial ROS Determination, NAD+/NADH analysis in cell, Mt-DNA content detection, Glucose uptake assay, Lactate production assay etc., were performed in this study. Result: The quantitative proteomic analyses, comparing a highly metastatic cancer cell line and a parental breast cancer cell line revealed that NDUFB9, was down-regulated in highly metastatic breast cancer cells. Using online "Kaplan Meier plotter” (KM plotter) database, in which updated gene expression data and survival information are supported for a total of 4142 breast cancer patients, we found that the majority of other subunits (NDUFB1-8/11) of the NADH dehydrogenase family had significant prognostic value (RFS/OS/DMFS/PPS) for breast cancer patients. Furthermore, we demonstrated that loss of NDUFB9 promotes MDA-MB-231 cells proliferation, migration, and invasion because of elevated levels of mtROS, disturbance of the NAD+/NADH balance, and depletion of mtDNA. Finally, we also showed that, the EMT and activation of Akt/mTOR/p70S6K signaling pathway might be involved in this mechanism. Conclusion: Genes encoding complex I components have significant breast cancer prognostic value. Our findings showed that complex I deficiency, including subunits dysfunction, unbalanced NAD+/NADH, and reduction of mtDNA content could profoundly enhance the aggressiveness of human breast cancer cells, and might serve as a potential and important biomarker to guide further research or clinical treatments with specific targeted therapies aimed at ROS and the Akt/mTOR pathway.

Key Words: NDUFB9  complex I deficiency  metastatic breast cancer