Date on Master's Thesis/Doctoral Dissertation

8-2015

Document Type

Master's Thesis

Degree Name

M.S.

Department

Pharmacology and Toxicology

Degree Program

Pharmacology and Toxicology, MS

Committee Chair

Ceresa, Brian

Committee Co-Chair (if applicable)

Bates, Paula

Committee Member

Bates, Paula

Committee Member

Beverly, Levi

Committee Member

Clark, Geoffrey

Committee Member

Siskind, Leah

Subject

Ligands; Apoptosis; Epidermal growth factor--Receptors

Abstract

Background: The Epidermal Growth Factor Receptor (EGFR) is a 170- kilodalton transmembrane protein that belongs to the ErbB family of receptor tyrosine kinases. Upon ligand-mediated activation, the EGFR responsible for cell growth, proliferation, and tissue homeostasis in epithelial cells; however, the EGFR is overexpressed in many human malignancies including MDA-MB-468 cells, a metastatic breast epithelial cell line. Previous studies have indicated that within the MDA-MB-468 cell line, receptors at the cell surface promote cell growth when activated with Epidermal Growth Factor (EGF) ligand. Activated receptors that are internalized to the endosomes however induce apoptosis. This contrasting response at different cellular locations is defined as spatial regulation. The overarching research goal of this thesis is to better understand the spatial regulation within these cells, and identify the effector proteins responsible for the difference in signals emanated at the cell membrane versus intracellularly. The goals of this thesis were to first determine whether other EGFR ligands, Betacellulin (BTC) and Transforming Growth Factor-alpha (TGFA), are able to induce apoptosis in the MDA-MB-468 cell line. The second goal was to identify effectors downstream of EGFR activation that could have potential implications in EGFR-mediated apoptosis. Methods: An MTT assay was conducted in order to evaluate the viability of the MDA-MB-468 cells after treatment and activation with 3 EGFR ligands: EGF, BTC, or TGFA. EGF activity was measured as a function of receptor phosphorylation. Western blot analysis was conducted after MDAMB- 468 cell exposure to increasing concentrations of the three ligands. This was done in order to identify any variances or similarities in phosphorylation patterns amongst the three ligands. Lastly, cell morphology was observed after the cells were exposed to 16 nM concentrations of each ligand for 24 hours. A different approach to assessing EGFR spatial regulation was then employed. MDA-MB- 468 cells were subjected to time course and dose response experiments, prior to the ultimate assessment of various different effectors. Results: All ligands induced dose dependent decreases in cell viability. All three ligands signaled through the EGFR, as measured by receptor phosphorylation, with similar phosphorylation patterns. Western blot analyses indicate a dose dependent increase in EGFR phosphorylation in response to all ligands. MDA-MB-468 cells were round in morphology at 24-hour time points with exposure to high concentrations of all ligands. An effector screening was conducted, which resulted in the discovery of upregulated STAT3 activity, preferentially with high EGF concentrations. This suggests the potential for STAT3 to mediate apoptosis, and be spatially regulated in MDA-MB0468 cells. After confirming the upregulation of STAT3 in an EGFR dependent manner, commercially available inhibitors of STAT3 were employed; however the inhibitors exhibited non-specific effects, in vitro. Conclusions: These data suggest that EGFR signaling in MDAMB- 468 cells is not ligand specific, and that activation and internalization of the receptor in this cell line with any endogenous ligand will result in cell death. The results indicate a potential role of STAT3 in EGFR-induced apoptosis. Future experiments will entail employing siRNA targeting STAT3 in order to determine the role of STAT3 in EGFR-induced apoptosis.

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