Notch-1 Specifically Activates Erk1/2 in Multiple Breast Cancer Subtypes

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 (ERK1/2, 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

NOTCH-1 and NOTCH-4 are novel gene targets of PEA3 in breast cancer: novel therapeutic implications

INTRODUCTION: Women with triple-negative breast cancer have the worst prognosis, frequently present with metastatic tumors and have few targeted therapy options. Notch-1 and Notch-4 are potent breast oncogenes that are overexpressed in triple-negative and other subtypes of breast cancer. PEA3, an ETS transcription factor, is also overexpressed in triple-negative and other breast cancer subtypes. We investigated whether PEA3 could be the critical transcriptional activator of Notch receptors in MDA-MB-231 and other breast cancer cells. METHODS: Real-time PCR and Western blot analysis were performed to detect Notch-1, Notch-2, Notch-3 and Notch-4 receptor expression in breast cancer cells when PEA3 was knocked down by siRNA. Chromatin immunoprecipitation was performed to identify promoter regions for Notch genes that recruited PEA3. TAM-67 and c-Jun siRNA were used to identify that c-Jun was necessary for PEA3 enrichment on the Notch-4 promoter. A Notch-4 luciferase reporter was used to confirm that endogenous PEA3 or AP-1 activated the Notch-4 promoter region. Cell cycle analysis, trypan blue exclusion, annexin V flow cytometry, colony formation assay and an in vivo xenograft study were performed to determine the biological significance of targeting PEA3 via siRNA, Notch signaling via a γ-secretase inhibitor, or both. RESULTS: Herein we provide new evidence for transcriptional regulation of Notch by PEA3 in breast cancer. PEA3 activates Notch-1 transcription in MCF-7, MDA-MB-231 and SKBr3 breast cancer cells. PEA3 activates Notch-4 transcription in MDA-MB-231 cells where PEA3 levels are endogenously high. In SKBr3 and BT474 breast cancer cells where PEA3 levels are low, overexpression of PEA3 increases Notch-4 transcripts. Chromatin immunoprecipitation confirmed the enrichment of PEA3 on Notch-1 and Notch-4 promoters in MDA-MB-231 cells. PEA3 recruitment to Notch-1 was AP-1-independent, whereas PEA3 recruitment to Notch-4 was c-JUN-dependent. Importantly, the combined inhibition of Notch signaling via a γ-secretase inhibitor (MRK-003 GSI) and knockdown of PEA3 arrested growth in the G(1 )phase, decreased both anchorage-dependent and anchorage-independent growth and significantly increased apoptotic cells in vitro. Moreover, either PEA3 knockdown or MRK-003 GSI treatment significantly reduced tumor growth of MDA-MB-231 xenografts in vivo. CONCLUSIONS: Taken together, the results from this study demonstrate for the first time that Notch-1 and Notch-4 are novel transcriptional targets of PEA3 in breast cancer cells. Targeting of PEA3 and/or Notch pathways might provide a new therapeutic strategy for triple-negative and possibly other breast cancer subtypes

Estimating national-level measles case–fatality ratios in low-income and middle-income countries: an updated systematic review and modelling study

To understand current and prevent future measles mortality burden, it is critical to have robust estimates of measles case fatality. Dynamic estimates of measles case fatality ratios (CFR) that are age-, location-, and time-specific are essential to capture variation in underlying population-level factors that contribute systematically to increases or decreases in case fatality, such as vaccination coverage and measles incidence. In this study, we updated estimates of measles CFR by expanding upon previous systematic reviews and implementing a meta-regression model