Mixed-lineage kinase 3 (MLK3), a mitogen-activated kinase kinase kinase (MAP3K), contributes to metastasis of triple-negative breast cancer (TNBC), in part through its regulation of paxillin phosphorylation and focal adhesion turnover. However the signaling mechanisms through which MLK3 promotes invasion and metastasis are not fully understood. We found that in non-metastatic, estrogen receptor positive (ER+) breast cancer cells, induced MLK3 expression robustly upregulates the oncogenic transcription factor, FOS-related antigen-1 (FRA-1), as well as matrix metalloproteinases (MMPs), MMP-1 and MMP-9. MLK3-induced MMP-1 induction is abrogated by FRA-1 silencing, demonstrating that MLK3 signals through FRA-1 to control MMP-1. In complementary experiments performed in metastatic TNBC models, high levels of FRA-1 are significantly reduced upon gene silencing of MLK3 or by MLK3 disruption using the CRISPR/Cas9n approach. Furthermore, both MMP-1 and MMP-9 are decreased upon ablation of MLK3 or MLK inhibitor treatment. Reduced endothelial permeability and transendothelial migration is observed in experiments with MLK3-depleted TNBC cells compared with parental counterparts. In addition, MLK inhibitor treatment or MLK3 depletion renders TNBC cells defective in Matrigel invasion. Furthermore, circulating tumor cells (CTCs) derived from a TNBC-bearing mouse which show an intermediate epithelial-mesenchymal phenotype display increased levels of MLK3, FRA-1, and MMP-1 compared with parental cells, supporting a role for the MLK3-FRA-1-MMP-1 signaling axis in tumor cell invasion and vascular intravasation. Consistent with the role of MMP-1 in multiple steps of metastasis, high levels of MMP-1 in breast cancer patient tumor samples are associated with increased distant metastases and poorer overall survival, particularly in TNBC. Our results demonstrating the requirement for MLK3 in controlling the FRA-1/MMP-1 axis suggest that MLK3 is a potentially effective therapeutic target for TNBC.

Citation Format: Chotirat Rattanasinchai, Brandon Llewellyn, Susan E. Conrad, Kathleen A. Gallo. Mixed-Lineage Kinase 3 signals through the FRA1-MMP1 axis to drive triple-negative breast cancer invasion and transendothelial migration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2374. doi:10.1158/1538-7445.AM2017-2374