SCHWEINFURTHIN-MEDIATED EFFECTS ON EGFR AND AKT IN CANCER CELLS
Open Access
- Author:
- Fernando, Shanika
- Graduate Program:
- Biomedical Sciences
- Degree:
- Master of Science
- Document Type:
- Master Thesis
- Date of Defense:
- December 31, 2019
- Committee Members:
- Raymond J Hohl, Thesis Advisor/Co-Advisor
Jeffrey D Neighbors, Committee Member
Nadine Hempel, Committee Member
Hong-Gang Wang, Committee Member
Ralph Lauren Keil, Program Head/Chair - Keywords:
- EGFR
Glioblastoma
Non-small cell lung cancer
AKT
MAPK
Schweinfurthin
Glycosylation - Abstract:
- Schweinfurthins are a class of plant-derived compounds with potential anticancer effects against a select subset of cancer cells within the NCI-60 panel. Synthetic analogs of schweinfurthins alter oncogenic signaling and homeostasis of multiple processes such as cytoskeleton and the mevalonate pathway, especially within difficult-to-treat cancers such as glioblastomas. The full schweinfurthin mechanism of action is unclear, but a combinatorial set of genetic mutations may influence sensitivity to schweinfurthins. Recently published work extends our knowledge into schweinfurthins with reports that these analogs bind to the cholesterol binding site of oxysterol binding proteins to impair intracellular cholesterol transport. This result is associated with decreased glycosylation of EGFR and decreased PI3K-AKT-mTOR signaling. Problematically, these changes were reported to occur in treated schweinfurthin-sensitive and –resistant cells, which fail to account for differential toxicity of schweinfurthins. The current need for alternative, novel therapeutics to circumvent non-specific and, sometimes, toxic effects of compounds, such as lovastatin, is desirable. Schweinfurthins are an intriguing alternative, although questions remain as to whether schweinfurthin-mediated effects are due to mevalonate-derived metabolites like cholesterol and dolichol. We chose to investigate effects of two schweinfurthin analogs on cancer cells that are either schweinfurthin-sensitive (SF-295 cells) or -resistant (A549 cells). MTT assays of vehicle- vs. analog-treated cells reveals that only SF-295 cells were sensitive to these compounds. I hypothesized that schweinfurthins dysregulate EGFR and AKT signaling within SF-295 cells, not A549 cells, because A549 cells have compensatory pathways to circumvent the effects of schweinfurthins. This pathway includes oncogenic activity of K-Ras, a constitutively active protein from a class of Ras isoforms that can withstand fluctuations in membrane cholesterol levels. Analyses of protein expression and phosphorylation of EGFR reveals that SF-295 cells, but not A549 cells, experience a decrease in apparent molecular weight of EGFR and an increase in phosphorylation of EGFR at tyrosine residues 1068 (which activates AKT) and 1173 (which activates MAPK). It is important to note that much of these phosphorylations are found on lower apparent molecular weight forms of EGFR, which we suspect are immature, unmodified forms of the receptors. These suspicions are bolstered by our experiments that show tunicamycin, which inhibits glycosylation, mediates similar shifts of EGFR to lower apparent molecular weight forms in SF-295 cells. Furthermore, schweinfurthin analog TTI-3066 reduces EGF-induced levels of phospho-EGFR(Y1068) and phospho-AKT(S473). It is important to acknowledge that schweinfurthin effects on EGFR phosphorylation appeared as trends, but were not statistically significant. Nevertheless, these trends support de-coupling of phospho-EGFR from its effectors. While investigations into EGFR-MAPK signaling revealed a lack of an effect by schweinfurthins, we found that treatments with schweinfurthins on their own induce significant decreases in phospho-AKT levels in SF-295 cells, not A549 cells. Taken together, our results suggest that schweinfurthin may exert its antitumor activity by de-coupling the EGFR-AKT signaling through impairment of EGFR glycosylation.