Notch-1 is a cell fate regulatory protein and a potent breast oncogene. Notch-1 and its ligand Jagged-1 are over-expressed in human breast cancers that are associated with poor overall survival (Reedijk, Odorcic et al. 2005). Deregulated Notch signaling may contribute to tumorigenesis by increasing proliferation, inhibiting differentiation, and preventing apoptosis (Miele, Golde et al. 2006). The mitogen-activated protein kinase (MAPK) pathway is a critical cell signaling pathway that has been implicated in the development and progression of cancer (Hanahan and Weinberg 2000). Four major MAPK pathways are involved in both cell growth and apoptosis. The regulation of these pathways is critical for cell fate decisions (Boutros, Chevet et al. 2008). One of the major MAPK subfamilies is the Extracellular Signal Regulated Kinases 1/2 (ERK ½, or p44/p42) signaling cascade. Hyper-activated ERK1/2 has been implicated in a large subset of mammary tumors and low ERK1/2 activity in primary breast tumors correlates with longer relapse-free survival (Mueller, Flury et al. 2000). Activation of ERK1/2 results in signals that stimulate proliferation (Pearson, Robinson et al. 2001), differentiation (Pearson, Robinson et al. 2001), survival (Pearson, Robinson et al. 2001), angiogenesis (Pages, Milanini et al. 2000), motility (Joslin, Opresko et al. 2007), and invasion (Price, Avraham et al. 2002); all of which contribute to breast cancer progression.
The overall objective of this thesis is to identify a novel crosstalk between Notch-1 and ERK1/2 signaling and to understand the mechanism of this crosstalk in breast cancer. The hypothesis of this project is: Notch-1 specifically activates ERK1/2 in multiple breast cancer subtypes through Aim 1A. Inhibition of MKP-1, a negative regulator of ERK1/2; Aim 1B. Activation of a receptor tyrosine kinase; Aim 2. Upregulation of MEK1/2, the activating kinase of ERK.
The first aim defines the crosstalk between Notch-1 and MKP-1. We demonstrate that Notch-1 downregulates transcription of MKP-1, but upregulates MKP-1 protein levels, suggesting that Notch-1 regulation of ERK may be downstream of MKP-1. Additionally, the first aim defines Notch crosstalk with receptor tyrosine kinases (RTKs) in MCF-7 and BT474 breast cancer cells. In a cell line with high ERK1/2 activity (MCF-7), the vascular endothelial growth factor receptor 3 (VEGFR3) was highly phosphorylated. Activity of VEGFR3 was abrogated upon Notch-1 inhibition, which correlated with a decrease in ERK activity. BT474 sensitive cells, which have low ERK activity, also had low VEGFR3 phosphorylation, which was not affected by Notch-1. The differences in VEGFR3 activity may account for the varying levels of ERK phosphorylation among cell lines, but Notch-1 activation of ERK is most probably not through activation of an RTK. In the second aim, overexpression of Notch-1 induced ERK phosphorylation and this effect was abrogated by inhibition of MEK. Inhibiting the proteasome caused a significant increase in ERK phosphorylation and Notch-1 protein, indicating that Notch-1 stabilization may be critical for ERK activation. Taken together, these results suggest that Notch-1 is activating ERK1/2 in multiple subsets of breast cancer. This novel crosstalk may be regulated by stabilization of Notch-1 protein, which appears to involve MEK1/2 or ERK1/2 activity. Future investigations will aim to determine the exact mechanism of Notch-1 activation of ERK and the role of MEK or ERK in Notch-1 stabilization.