Role of miR-4732-3p in breast cancer brain metastasis and brain metastatic tumor microenvironment

In this study, we aimed to identify microRNAs (miRNAs) that may play important roles in breast cancer brain metastasis (BCBM). To this end, we conducted miRNA-sequencing of extracellular vesicles isolated from the serum samples of 6 BCBM patients and 8 Stage I/II/III breast cancer patients, and identified 49 circulating miRNAs that were upregulated in BCBM patients compared to Stage I/II/III breast cancer. Upon further analysis, we identified miR-4732-3p to be upregulated in brain-tropic triple-negative breast cancer (TNBC) cell lines, compared to parental lines. MiR-4732-3p overexpression increased metastatic properties of TNBC cells, including proliferation, migration, invasion, and maintenance of a mesenchymal state. Additionally, we observed that extracellular vesicles secreted from miR-4732-3p-overexpressing TNBC cells significantly activated astrocytes. To determine how miR-4732-3p-activated astrocytes may activate further astrocytes and also BCBM cells, we conducted cytokine arrays to identify significantly dysregulated cytokines, and found that multiple cytokines including FLT3LG can be upregulated in miR-4732-3p-overexpressing astrocytes. We further showed that FLT3LG significantly activates astrocytes and promotes astrocyte proliferation. In agreement with the cytokine array results, FLT3LG mRNA is upregulated in astrocytes overexpressing miR-4732-3p. Finally, using TargetScan and GeneCards, we identified multiple potential miR-4732-3p target genes that are transcriptional repressors of FLT3LG, suggesting their potential roles in activating FLT3LG expression. These results collectively demonstrate that breast cancer-derived miR-4732-3p may play a novel important role in BCBM by promoting metastatic properties of BCBM cells, and activating astrocytes in the brain-metastatic tumor microenvironment through FLT3LG secretion.