RGCC-mediated PLK1 activity drives breast cancer lung metastasis by phosphorylating AMPKa2 to activate oxidative phosphorylation and fatty acid oxidation
Background: More than 90% mortality of triple negative breast cancer (TNBC) patients are attributed to cancer metastasis with organotropism. Lung is the frequent site of TNBC metastasis. However, the precise mechanism of lung-specific metastasis of TNBC, particularly the driving factors, is not well understood. Methods: RNA sequencing was performed to identify patterns of gene expression associated with lung metastatic behavior using 4T1-LM3, 231-LM3 and their parental cells (4T1-P, 231-P). Expressions of RGCC, encoding the completion 32 protein response protein, were detected in TNBC cells and tissues by qRT-PCR, western blotting and IHC. Kinase activity assay were performed to evaluated PLK1 kinase activity and the amount of phosphorylated AMP-activated protein kinase a2 (AMPKa2). RGCC-mediated metabolism was determined by UHPLC system. Oxidative phosphorylation was evaluated by JC-1 staining and oxygen consumption rate (OCR) assay. Fatty acid oxidation assay, NADPH and ROS was applied to measure the status of RGCC-mediated fatty acid oxidation. The chemical sensitivity of cells was evaluated by CCK8 assay. Results: RGCC is aberrantly upregulated in pulmonary metastatic cells. High level of RGCC is significantly related with lung metastasis rather than other organ metastases. RGCC can effectively promote the kinase activity of PLK1, and activated PLK1 phosphorylates AMPKa2 to facilitate TNBC lung metastasis. Mechanistically, Survival and colonization of TNBC cells in the lungs by upregulating oxidative phosphorylation of mitochondria to obtain more energy and promoting fatty acid oxidation to produce abundant NADPH and sustains redox homeostasis, thus prevents excessive accumulation of potentially detrimental ROS in metastatic tumor cells, ultimately establishing metastases. Importantly, targeting RGCC in combination with paclitaxel/carboplatin effectively suppresses pulmonary metastases of TNBC in a mouse model. Conclusions: The enhanced RGCC is closely associated with lung-specific metastasis of TNBC. RGCC leads to activation of AMPKa2 and downstream signaling through RGCC-driven PLK1 activity to facilitate TNBC lung metastasis, which may determine a potential value of RGCC-driven OXPHOS and fatty acid oxidation as an important therapeutic target. Overall design: Methods: The total RNA was extracted from 4T1-LM3, MDA-MB-231/LM3 and their corresponding parental cells using TRIzol reagent (Invitrogen). Subsequently, RNA was purified through rRNA depletion and then subjected to cDNA synthesis and RNA amplification. Next, RNA sequencing was carried out by Shengyin Biotech (Shanghai, China), and differential gene expression (DEG) analysis was performed using the DESeq2 package.