Access Type

Open Access Dissertation

Date of Award

January 2014

Degree Type


Degree Name



Cancer Biology

First Advisor

Julie L. Boerner

Second Advisor

Raymond Mattingly


Triple negative breast cancer (TNBC) patients suffer from a highly malignant and aggressive cancer that lacks an effective targeted therapeutic. Although many TNBCs, both in vitro and in vivo, have increased expression of epidermal growth factor receptor (EGFR), EGFR targeted inhibitors, such as gefitinib (GEF), have yet to demonstrate efficacy. Using mass spectrometry to identify pathways that remain activated in the presence of GEF, we found that components of the mTOR signaling pathway remain phosphorylated. While inhibiting mTOR with temsirolimus (TEM) decreased mTOR signaling, EGFR signaling pathways remained activated and the TNBC cell lines continued to proliferate. However, dual treatment with TEM and GEF synergistically decreased cell viability in TNBC cells. Interestingly, abrogation of both EGFR and mTOR signaling did not alter the phosphorylation of key growth signaling molecules including MAPK and AKT. Instead, our data have identified the translational control pathway, specifically, eIF4B as a potentially key regulatory point in EGFR and mTOR inhibitor synergy. Further, we have also identified the transcription factor, STAT3 as another regulatory point in the EGFR and mTOR inhibitor synergy. Therefore, in this study we hypothesized that mTOR inhibition can sensitize TNBC cells to EGFR TKIs through the inhibition of eIF4B and STAT3 phosphorylation.

eIF4B enhances the helicase activity of eIF4A during translation initiation. As expected, knockdown of eIF4B expression decreased cell viability comparable to the decrease observed with the combination treatment. Importantly, we have identified p70S6K and p90RSK as kinases directly responsible for eIF4B phosphorylation, such that both molecules need to be inactivated in order for eIF4B phosphorylation to be abrogated. This inactivation correlated with a loss of cell growth and viability and a decrease in clonogenic cell survival, potentially through alterations in the cell cycle. Furthermore, cap-dependent translation was inhibited to a greater extent in the combination treatment than GEF or TEM alone. Taken together these data suggest that EGFR and mTOR inhibitor combination abrogates cell growth, viability, and survival via disruption of translational control mechanisms through eIF4B.

STAT3 is a widely considered oncogenic transcription factor that has been implicated in a variety of cancer types. We found a decrease in phospho-STAT3 with the GEF+TEM combination. Further DNA binding ELISAs found STAT3 activity was also significantly decreased with the combination. Overexpression of a constitutively active STAT3 plasmid found that STAT3 activation negates the GEF+TEM synergetic effect on cell viability. Together, these studies suggest a role for STAT3 in EGFR and mTOR inhibitor synergy.

Taken together these data suggest that in the presence of activated MAPK and AKT, EGFR and mTOR inhibitors abrogate growth, viability, and survival via disruption of eIF4B and STAT3 phosphorylation leading to decreased translation and transcription factor DNA binding, respectively, in TNBC cell lines. The effect translation has on cancer cells in regard to the mTOR and EGFR pathways is largely unexplored in TNBC and further implicates eIF4B as a protein of interest in understanding the gefitinib and temsirolimus synergy. TNBC patients currently have limited treatment options and our data suggest that including an mTOR inhibitor along with an EGFR inhibitor in TNBC with increased EGFR expression should be further explored.