Despite improvements in early detection and treatment, breast cancer metastasis to distant sites remains a clinical problem and is a major cause of death. Thus, discovery of key molecular players that drive cancer cell local invasion and metastasis could reveal novel therapeutic targets for combating such diseases. Mitogen-activated protein kinases (MAPKs) mainly comprise c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), p38, ERK5/BMK and control a variety of intercellular events. Mixed-lineage kinase 3 (MLK3), an MAP3K, can activate all the MAPK signaling pathways. We find that MLK3 is highly expressed in breast cancer cells compared with mammary epithelial cells. Active MLK and signaling to JNK are required for migration of breast cancer cells. Conversely, induced overexpression of MLK3 promotes migration and invasion of mammary epithelial cells. Consistent with this, MLK3 activates AP-1 transcription factor through JNK, which induces the expression of invasion genes. In contrast, Inhibition of AP-1 blocks MLK3-induced invasion. In a 3D extracellular matrix culture model, MLK3 increases acinar size, perturbs apico-basal polarity and induces luminal repopulation through inhibition of pro-apoptotic protein BimEL. Taken together, we demonstrate that MLK3 is critical for breast cancer cell migration, invasion and acquisition of a malignant phenotype of mammary epithelial cells. In addition to induction of invasion gene expression, we also hypothesize MLK3 signaling can directly impact the cell migration machinery in breast cancer cells. We show that the prometastatic factors, CXCL12 and HGF, signal through MLK3-JNK to promote phosphorylation of focal adhesion scaffold protein, paxillin on Ser 178 (by JNK), which is required for migration of breast cancer cells. Furthermore, we show that MLK3 promotes phosphorylation of paxillin at Tyr 118 (by FAK/Src), possibly through facilitating the recruitment of FAK to S178-phosphorylated paxillin. Supporting this model, expression of nonphosphorylatable paxillin mutant S178A blocks Y118 phosphorylation of paxillin. Y118 phosphorylation of paxillin is a critical event for focal adhesion turnover by limiting activation of small GTPase Rho. We show silencing or inhibition of Mlk3, inhibition of JNK, or expression of paxillin S178A all increase Rho activity, suggesting that the MLK3-JNK-paxillin axis limits Rho activity in breast cancer cells. Consistently, MLK3 knockdown in breast cancer cells increases focal adhesion numbers, suggesting that loss of MLK3 causes a defect in focal adhesion turnover. Using mouse xenograft models, we provide evidence that MLK3 is necessary for metastases of human breast cancer cells to lung. Furthmore, paxillin phosphorylation on S178 is elevated in high metastatic cells. We conclude that Inhibition of MLK3-paxillin axis may be a novel therapeutic strategy to prevent breast cancer metastasis.
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