The malignant phenotype in breast cancer is driven by aberrant signal transduction pathways. Mixed-lineage kinase-3 (MLK3) is a mammalian mitogen-activated protein kinase kinase kinase (MAP3K) that activates multiple MAPK pathways. Depending on the cellular context, MLK3 has been implicated in apoptosis, proliferation, migration and differentiation. Here we investigated the effect of MLK3 and its signaling to MAPKs in the acquisition of malignancy in breast cancer. We show that MLK3 is highly expressed in breast cancer cells. We provide evidence that MLK3's catalytic activity and signaling to c-jun N-terminal kinase (JNK) is required for migration of highly invasive breast cancer cells and for MLK3-induced migration of mammary epithelial cells. Expression of active MLK3 is sufficient to induce the invasion of mammary epithelial cells, which requires AP-1 activity and is accompanied by the expression of several proteins corresponding to AP-1-regulated invasion genes. To assess MLK3's contribution to the breast cancer malignant phenotype in a more physiological setting, we implemented a strategy to inducibly express active MLK3 in the preformed acini of MCF10A cells grown in 3D Matrigel. Induction of MLK3 expression dramatically increases acinar size and modestly perturbs apicobasal polarity. Remarkably, MLK3 expression induces luminal repopulation and suppresses the expression of the pro-apoptotic protein BimEL, as has been observed in Her2/Neu-expressing acini. Taken together, our data show that MLK3-JNK-AP-1 signaling is critical for breast cancer cell migration and invasion. Our current study uncovers both a proliferative and novel antiapoptotic role for MLK3 in the acquisition of a malignant phenotype in mammary epithelial cells. Thus, MLK3 may be an important therapeutic target for the treatment of invasive breast cancer.

MLK3 (mixed lineage kinase 3) is a MAP3K [MAPK (mitogen-activated protein kinase) kinase kinase] that activates multiple MAPK pathways, including the JNK (c-Jun N-terminal kinase) pathway. Immunoblotting of lysates from cells ectopically expressing active MLK3 revealed an additional immunoreactive band corresponding to a CTF (C-terminal fragment) of MLK3. In the present paper we provide evidence that MLK3 undergoes proteolysis to generate a stable CTF in response to different stimuli, including PMA and TNFalpha (tumour necrosis factor alpha). The cleavage site was deduced by Edman sequencing as between Gln251 and Pro252, which is within the kinase domain of MLK3. Based on our homology model of the kinase domain of MLK3, the region containing the cleavage site is predicted to reside on a flexible solvent-accessible loop. Site-directed mutagenesis studies revealed that Leu250 and Gln251 are required for recognition by the 'MLK3 protease', reminiscent of the substrate specificity of the coronavirus 3C and 3CL proteases. Whereas numerous mammalian protease inhibitors have no effect on MLK3 proteolysis, blockade of the proteasome through epoxomicin or MG132 abolishes PMA-induced production of the CTF of MLK3. This CTF is able to heterodimerize with full-length MLK3, and interact with the active form of the small GTPase Cdc42, resulting in diminished activation loop phosphorylation of MLK3 and reduced signalling to JNK. Thus this novel proteolytic processing of MLK3 may negatively control MLK3 signalling to JNK.

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