MLK3 regulates FRA-1 and MMPs to drive invasion and transendothelial migration in triple-negative breast cancer cells

Mixed-lineage kinase 3 (MLK3), a mitogen-activated protein kinase kinase kinase (MAP3K), has critical roles in metastasis of triple-negative breast cancer (TNBC), in part by regulating paxillin phosphorylation and focal adhesion turnover. However the mechanisms and the distinct step(s) of the metastatic processes through which MLK3 exerts its influence are not fully understood. Here we report that in non-metastatic, estrogen receptor-positive breast cancer (ER+ BC) cells, induced MLK3 expression robustly upregulates the oncogenic transcription factor, FOS-related antigen-1 (FRA-1), which is accompanied by elevation of matrix metalloproteinases (MMPs), MMP-1 and MMP-9. MLK3-induced ER+ BC cell invasion is abrogated by FRA-1 silencing, demonstrating that MLK3 drives invasion through FRA-1. Conversely, in metastatic TNBC models, high FRA-1 levels are significantly reduced upon depletion of MLK3 by either gene silencing or by the CRISPR/Cas9n editing approach. Furthermore, ablation of MLK3 or MLK inhibitor treatment decreases expression of both MMP-1 and MMP-9. Consistent with the role of tumor cell-derived MMP-1 in endothelial permeability and transendothelial migration, both of these are reduced in MLK3-depleted TNBC cells. In addition, MLK inhibitor treatment or MLK3 depletion, which downregulates MMP-9 expression, renders TNBC cells defective in Matrigel invasion. Furthermore, circulating tumor cells derived from TNBC-bearing mice display increased levels of FRA-1 and MMP-1 compared with parental cells, supporting a role for the MLK3–FRA-1–MMP-1 signaling axis in vascular intravasation. Our results demonstrating the requirement for MLK3 in controlling the FRA-1/MMPs axis suggest that MLK3 is a promising therapeutic target for treatment of TNBC.