Mixed Lineage Kinase 3 (MLK3) is a stress-activated MAP Kinase Kinase Kinase member, whose biological function is still elusive. While dissecting the signaling pathway mediated via MLK3, we observed a strong interaction between mammalian homolog of yeast Ste20 member, p21 activated kinase (Pak1) and MLK3. Initially, we speculated that Pak1 might regulate MLK3 kinase activities similar to its yeast counterparts. However, the observed results were counterintuitive to our speculation, and instead MLK3 directly phosphorylated and activated Pak1, which subsequently lead to NF-?B activation. These results were quite intriguing because Pak1 activation has been implicated in mammary gland hyperplasia, and recently it has been identified as a major oncogene in breast cancer. Our preliminary investigation revealed that Pak1 activity was directly regulated in a MLK3-dependent manner in breast cancer cell lines. We also observed that in primary human breast tumors, the MLK3 activities directly correlated with Pak1 and NF-?B activations, exclusively in ER and PR negative breast tumors and Pak1 was overexpressed in ER- PR- breast tumors. If these results are true then the available MLKs inhibitor, CEP-1347/CEP-11004 should induce cell death in ER- breast cancer cells? Indeed, treatment of ER-, but not ER+ breast cancer cell lines with CEP-1347 caused cell death. Based on our observations, we hypothesize that in ER- breast cancer cells, MLK3 activates Pak1 and its downstream NF-?B, leading to ER- tumorigenesis. Therefore targeting MLK3 or other MLKs could abrogate ER- breast tumors. To achieve our goals, we will determine that: (1) activation of Pak1 by MLK3 induces ER- breast cell tumorigenesis, (2) disruption of MLK3-Pak1 signaling cascade attenuates ER- breast cell tumorigenesis; and (3) the mechanism of MLK3, Pak1 and NF-?B activation in ER- breast cell tumorigenesis. It is expected that by defining the role of MLK3 in Pak1 and NF-?B activation and tumorigenesis, we might control/treat ER- breast cancers by using available inhibitors of this pathway. Interestingly, the specific inhibitor of MLK3 family, CEP-1347 has been used in clinical trials for treating neurodegenerative diseases. It has also been suggested that MLKs inhibitors might serve as a treatment for specific type of cancer, underscoring our unexpected novel results related to MLK3 role in ER- breast cancer. Taken together, the present study will lead to the identification of new prognostic factors and specific targeted therapies for difficult to treat ER- breast cancer.