ABSTRACT: Investigating Novel Roles of Notch-1 Signaling in Estrogen Receptor-α Positive Breast Cancer
Breast cancer is a highly prevalent and life-threatening malignancy. Though therapies exist to treat breast cancer, advances in the field are necessary in order to overcome clinical hurdles such drug toxicity and cancer recurrence. Several signaling pathways are known to be dysregulated in breast cancer, and this thesis aims to describe novel crosstalk mechanisms between three distinct pathways: Estrogen Receptor-α (ERα), Notch, and Extracellular Regulated Kinase 1 and 2 (ERK1/2). ERα is overexpressed in up to 75% of all breast cancer cases. Anti-estrogen therapies are part of the standard of care for ERα positive breast cancer. We model anti-estrogen therapy by depriving cell cultures of estrogens in order to investigate the effect of these estrogens on Notch and ERK1/2 signaling. The Notch signaling pathway is commonly dysregulated in breast cancer. Drugs that target the Notch signaling pathway exist making it an attractive mode for investigation in breast cancer. MAP kinase signaling pathway is composed of four distinct signaling cascades. This thesis will focus on the Mitogen Activated Protein (MAP) kinase cascade which activates the ERK1/2 kinase, a known promoter of cellular proliferation and survival.
Classical Notch signaling is carried out by a series of proteolytic cleavages. Notably, the γ-secretase complex cleaves the membranous Notch receptor yielding a diffusible Notch intracellular domain (NICD). After cleavage, NICD translocates to the nucleus where it modulates the expression of specific target genes. Inhibitors of the γ-secretase complex can effectively attenuate this canonical Notch signaling paradigm. However, mounting evidence suggests Notch signaling functions that occur independently of this canonical pathway. Little is known about Notch signaling mechanisms that may cause Notch to behave independently of its ability to act as a transcription factor. Others have demonstrated crosstalk between the Notch, ERα and ERK1/2 signaling pathways. However, some of the observations made remain unexplained by the traditional Notch signaling paradigm. This work seeks to identify and characterize signaling crosstalk mechanisms that occur independently of the ability of Notch to act as a transcription factor. In order to distinguish between canonical and non-canonical signaling, a scheme was devised to specifically inhibit either the canonical mechanism or the non-canonical mechanism. Cellular proliferation was used to compare the effects of γ-secretase inhibition (targeting the canonical pathway) to siRNA ablation of Notch1 (targeting both canonical and non-canonical pathways) in the presence or absence of estrogen. The results from these preliminary experiments as well as the conclusions drawn from previous work suggest that a non-canonical function of Notch1 is required for cellular proliferation in an ERK1/2 and ERα dependent manner. The hypothesis drawn to describe these findings is that: the membrane-bound Notch1 receptor is required for growth and survival of MCF-7 cells in an estrogen dependent manner by activating the ERK1/2 signaling pathway. Two aims will be employed in order to test these hypotheses.
Specific Aim 1: Determine whether the membrane-bound Notch1 receptor is necessary and sufficient for ERK1/2 activation. Our preliminary data suggest that in the presence of estrogen, non-canonical Notch1 signaling is required for proliferation of MCF-7 cells, but in the absence of estrogen, the canonical function of Notch1 is required for cell proliferation. Previous published work by Rizzo, et al. demonstrated that in the presence of estrogen, Notch1 is sequestered at the cell membrane, but in the absence of estrogen, Notch1 is cleaved and activated by the γ-secretase complex. Taken together, these findings suggest a signaling role for the membrane-bound Notch1 receptor. Work performed previously in our lab by Allison Rogowski shows that Notch1 is required for ERK1/2 activation. This aim seeks to bridge these correlations and demonstrate that the membrane-bound Notch1 receptor is required for the activation of ERK1/2.
Specific Aim 2: Determine whether the Notch1-dependent effects on MCF-7 cell proliferation and survival are due to ERK1/2 activation.
The data put forth suggesting a link between Notch1 and ERK1/2 and proliferation suggests a correlation between these signaling pathways and a biological output. If a constitutively active ERK1 or ERK2 can rescue the proliferation phenotype under Notch1 knockdown conditions, then this would provide strong evidence that a crosstalk mechanism between Notch1 and ERK1/2 controls proliferation in these cells.