Dissecting the Role of Mixed Lineage Kinase 3 (MLK3) in Breast Cancer Invasion and Metastasis

DISSECTING THE ROLE OF MIXED LINEAGE KINASE 3 (MLK3) IN BREAST CANCER INVASION AND METASTASIS By Chotirat Rattanasinchai MLK3 is a mitogen-activated protein kinase kinase kinase (MAP3K) protein which can activate multiple MAPK pathways. MLK3 has been recently shown to be critical for breast cancer metastasis. In this thesis, the mechanisms through which MLK3 signaling controls discrete steps of cancer invasion and metastasis have been investigated. MLK3 was found to regulate the expression of FOS-related antigen-1 (FRA-1), a member of activator protein-1 (AP1) transcription factor family. Overexpression of catalytically active MLK3, but not catalytically inactive MLK3, induced expression of FRA-1, suggesting that MLK3 utilizes its kinase activity rather than scaffolding function to control FRA-1 levels. In addition, MLK3 overexpression was sufficient to drive cancer cell migration in non-invasive, estrogen receptor positive (ER+) breast cancer cells. Conversely, deletion of MLK3 in highly metastatic, triple-negative breast cancer (TNBC) cells decreased FRA-1 expression at both the transcript and protein levels. Similarly, FRA-1 level was reduced upon treatment of TNBC cells with CEP-1347 or URMC-099, two structurally distinct, adenosine triphosphate (ATP)-competitive, MLK inhibitors. These data, together with the findings in ER+ breast cancer cells, indicate that MLK3 kinase activity is required for FRA-1 expression. MLK3 is capable of activating several MAPK pathways. Experiments utilizing small molecule inhibitors targeting specific MAPK pathways identified JNK and ERK pathways as mediators for MLK3-induced FRA-1 expression. FRA-1 is an oncogenic transcription factor controlling a subset of invasion genes. The work described in this thesis demonstrates a role for MLK3 in regulating matrix metalloproteinase (MMP) levels in breast cancer cells. In ER+ breast cancer cells, MLK3 overexpression induced expression of MMP-1 and MMP-9, two well-characterized, FRA-1 target genes. Silencing of FRA-1 in MLK3-overexpressing ER+ breast cancer cells attenuated MMP-1 and MMP-9 expression indicating that FRA-1 is a required intermediary in MLK3induced MMP regulation. Furthermore, MLK3 deletion or MLK inhibitor treatment was sufficient to decrease both MMP-1 and MMP-9 levels in TNBC cells. MMPs are known to facilitate cancer invasion. Overexpression of MLK3 in ER+ breast cancer cells, which induces both MMP-1 and MMP-9, enhanced the ability of these cells to invade through a thin layer of Matrigel and this invasion was attenuated upon FRA-1 silencing. Moreover, MLK3 deletion or MLK inhibitor treatment also blocked Matrigel invasion of highly invasive TNBC cells. Consistent with a role for MMP-1 in vascular intravasation, deletion of MLK3 which downregulates MMP-1 expression rendered TNBC cells defective in both endothelial permeability and transendothelial migration. Upregulation of FRA-1 and MMP-1 was observed in circulating tumor cells (CTCs) derived from TNBC-bearing mice when compared with parental TNBC cells, further supporting the role of MLK3/ MMP-1 axis in vascular intravasation. High levels of MMP-1 in breast cancer patients are strongly associated with poor prognosis, including increased distant metastases, shortened recurrence, and poorer overall survival. This investigation has deciphered key components of the MLK3 signaling pathways that control the transcription factor FRA-1 and MMP target genes during cancer invasion and intravasation. These studies support the idea that targeting MLK3 is a promising therapeutic strategy for combating TNBC metastasis. Copyright by CHOTIRAT RATTANASINCHAI 2017