Access Type

Open Access Dissertation

Date of Award


Degree Type


Degree Name



Cancer Biology

First Advisor

Stephen P. Ethier


We have previously shown that SUM-149 human breast cancer cells require an AREG/EGFR autocrine loop for cell proliferation. We also demonstrated that AREG can increase EGFR stability and promote EGFR localization to the plasma membrane. In the presented dissertation we successfully knocked-down AREG expression in SUM-149 cells by lenti-viral infection of AREG shRNA. In the absence of AREG expression, SUM-149 cell growth was slowed, but not completely inhibited. Furthermore, cells infected with AREG shRNA constructs showed an increase in EGFR protein expression by western blot. Immunofluorescence and confocal microscopy showed that following AREG knock-down, EGFR continued to localize to the cell surface. Soft agar assays demonstrated that AREG knock-down cells retain anchorage-independent growth capacity. Additionally mammosphere forming assays and Adefluor staining analysis showed that knock-down of AREG expression did not affect the expression of stem cell 158 phenotypes. However, following AREG knock-down, SUM-149 cells demonstrated a dramatic decrease in their ability to invade a Matrigel matrix. Consistent with this observation, microarray analysis comparing cells infected with a non-silencing vector to the AREG knock-down cells, identified genes associated with the invasive phenotype such as RHOB and DKK1, and networks associated with cell motility such as integrinlinked kinase signaling, and focal adhesion kinase signaling. AREG was also found to modulate WNT and Notch signaling in SUM-149 cells. In an additional microarray study, changes in gene expression were analyzed from cDNA transcribed from RNA isolated from MCF10A cells growing in the presence of AREG or EGF and after 24 hours withdrawl of the respective ligand. Genes regulating WNT signaling, but not NOTCH signaling, were altered in the MCF10A cells. Thus, the pathway that AREG/EGFR signaling effects is contextually dependent on the cell type that it is functioning in. We conclude that AREG functions in regulating the invasive phenotype, and we propose that this regulation may be through altered signaling that occurs when AREG activates plasma membrane localized EGFR.