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MLK3 Meets Pak1 in Breast Cancer: A Prognostic Factor and a Therapeutics Target
Mixed Lineage Kinase 3 (MLK3) is a stress-activated kinase, whose biological function is still not well understood. While dissecting the signaling pathway mediated through 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-kB activation. This result was quite intriguing because Pak1 activation has been implicated in mammary gland hyperplasia. 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 breast tumors, but not in normal mammary tissues that were derived from same patients, the MLK3 activities directly correlated with Pak1 and NF-kB activations, exclusively in ER and PR negative breast tumors.
Based on our novel observations, we hypothesize that in ER- breast cancer cells, MLK3 might activate NF-kB and thus tumorogenesis, via Pak1 activation. We also hypothesize that estrogen might negatively regulate MLK3 kinase activities and pro-apoptotic functions in ER+ breast cancers. To achieve our goal, three aims are proposed: (1) Determine whether activation of Pak1 by MLK3 induces ER- breast cell tumorogenesis, (2) Determine whether disruption of MLK3-Pak1 signaling cascade attenuates ER- breast cell tumorogenesis; and (3) Determine the mechanism of MLK3 mediated Pak1 activation in ER- breast cell tumorogenesis. Various methods, including generation of stable cell lines either with knockdown or overexpressed MLK3 and/or Pak1, SCID mice xenografts, kinase assay, immunoprecipitation, and Western blotting would be utilized.
It is expected that by defining the role of MLK3 in Pak1 and NF-kB activation and tumorogenesis, we might control/treat ER- breast cancers by using available inhibitors of this pathway. Interestingly, the specific inhibitor of MLK3 family, CEP-1347 is under clinical trials for treating neurodegenerative diseases. It has been suggested that MLKs inhibitors might serve as a treatment for specific type of cancer, underscoring our unexpected novel function of MLK3 in ER- breast cancer treatment. Taken together, the present study will lead to the identification of new prognostic factors and specific targeted therapies for breast cancer.
Gonadal hormone estrogen and its receptor (i.e. ER) play a pivotal role in the pathogenesis of breast cancer. ER+ breast cancers generally have a better prognosis and are often responsive to anti-estrogen therapy. Unfortunately ER- breast cancers are more aggressive and un-responsive to anti-estrogens. Furthermore, 20-30% of ER+ tumors are resistant to anti-estrogen therapy and 20-40% of primary breast cancers are inherently resistant to the hormonal therapy. Identification of non-hormonal targets is thus critical in developing effective therapies to all forms of breast cancer. Members of protein kinase family have often been targeted for therapeutic purposes in different types of cancer, including breast cancer. Several of the inhibitors of protein kinases are under clinical trials that promise cure of specific types of cancer. These protein kinase inhibitors were designed and synthesized based on the biological functions, and the roles of these kinases in signal transduction pathways. Recently, we have observed a novel protein kinase-signaling pathway in ER- breast tumors and cell lines. Our preliminary results suggest that this pathway might successfully be targeted to inhibit tumor growth, especially in ER- breast cancers. We observed that a protein kinase called Mixed Lineage Kinase 3 (MLK3) might cause tumorogenesis in ER- breast cancers by phosphorylating and activating another protein kinase called p21-activated protein kinase-1 (Pak1).
Since the small molecule inhibitors of MLK3 and peptide inhibitors of Pak1 are already available, we propose to examine our hypothesis; whether this pathway can be targeted to control unregulated cell growth (i.e. cancer) in ER- breast cancers. To achieve our goal, we propose three aims: (1) Determine whether activation of Pak1 by MLK3 induces ER- breast cell tumorogenesis, (2) Determine whether disruption of MLK3-Pak1 signaling cascade attenuates ER- breast cell tumorogenesis; and (3) Determine the mechanism of MLK3 mediated Pak1 activation in ER- breast cell tumorogenesis. The goal of this proposal will be achieved by using cell culture system, xenografts SCID mouse model, and genetical modulation, either by knockdown or overexpression of MLK3 and/or Pak1 in breast cell lines.
It is expected that by defining the MLK3-Pak1 signaling in breast cancers, we may use the inhibitors of either MLK3 or Pak1 for ER- breast cancer treatment in future. Overall, the present study will provide insights for the development of novel prognostic factors and specific therapeutic targets in breast cancer.