Autophagy is a self-degradation system that is critical for maintaining cellular homeostasis during stress conditions. The 2016 Nobel Prize in Physiology or Medicine was awarded for research in autophagy. This recognition emphasizes the impact of this cell survival mechanism. During summer 2017, I worked in the Amaravadi Laboratory. The lab has a translational approach in demonstrating that autophagy inhibition increases the efficacy of anti-cancer therapy.
With the Holtz Fund, I examined the role of glucose regulating protein 78 (GRP78) in melanoma cancer cells. GRP78 is an ER chaperone protein which helps to combat ER stress that occurs when ER homeostasis is disrupted. Dr. Amaravadi has data on a series of biopsies from a patient undergoing drug treatment for melanoma. Interestingly, the expression of GRP78 increased in samples obtained at progression compared to baseline. Thus, I explored the role of GRP78 in melanoma cancer cells. When I inhibited GRP78 using siRNA, cell death increased, indicating GRP78 plays a role in cell viability.
Dr. Amaravadi has also reported that autophagy is a major mechanism of resistance during drug treatment. Therefore, I examined the effect of GRP78 on autophagy. When I inhibited GRP78 using siRNA, I found that autophagy, as measured by LC3 and p62 western blotting, decreased. This indicated that GRP78 regulates autophagy for cell survival in melanoma. Next, I examined the status of GRP78 during treatment of BRAF mutant melanoma cells with BRAF and MEK inhibitors. Surprisingly, I found that GRP78 levels increased at an earlier timepoint but decreased at a later timepoint. This raised the possibility that GRP78 was degraded during drug treatment. Recently, Arginyltransferase I (ATE1) has been shown to post-translationally modify GRP78 via arginylation (R-GRP78). This post translation modification leads to GRP78 degradation for the maintenance of cellular homeostasis under pathological conditions (Cha-Molstad, Nature Cell Biology, 2015). Therefore, I examined the status of ATE1 in melanoma cells during drug treatment. Notably, ATE1 inhibition using siRNA increased the cytotoxic effects of BRAF and MEK inhibitors in melanoma cells. Next, I wanted to see whether ATE1 mediates GRP78 degradation via arginylation during drug treatment. When ATE1 was inhibited by siRNA, GRP78 expression was consistently present; however, R-GRP78 expression declined. These studies suggest that ATE1 inhibition stabilizes R-GRP78 expression and rescues GRP78 degradation under drug treatment. Future work will require an in depth exploration of how ATE1 is regulating cell survival in melanoma cells.
The summer project was critical because it provided crucial information about melanoma cell biology. I had to become proficient in Western blot, colony formation, transformation, plasmid isolation, transfection, RNA isolation, cDNA synthesis, qPCR, and cell culture techniques. I was also expected to study the principles behind laboratory techniques and an in-depth understanding of the literature on autophagy, GRP78 and ATE1. The work that I did this summer will continue into my fall independent study. My time at the lab has morphed into an interest in achieving and MD/PhD. I am eager to continue applying classroom knowledge to the lab workbench.