Ava Samson
For many years, copper was overlooked as an important metal in biological processes. Researchers identified that too much copper was toxic, but it is only very recently that copper has been discovered to affect signaling pathways in cells. These signaling pathways are extremely important as they lead to proliferation of cells.
One of the first significant research papers to touch on copper’s role in the signaling pathway for cancer cells was written by Donita Brady. Brady focused on cancer that has a mutation in the BRAF protein, a protein that is involved in receiving growth signals and activating other proteins down the pathway. BRAF directly activates MEK1/2 by phosphorylation which is the act of adding a phosphate group to the protein. Active MEK1/2 phosphorylates and activates ERK1/2 which sends signals to the nucleus of the cell to proliferate. The mutation is called BRAFV600E which means that the amino acid, valine, at the 600th residue was changed to glutamic acid due to a genetic mutation. This mutation causes the signal pathway to be overly active, leading to high cell replication and therefore tumor growth. With that, researchers have attempted to target the MEK1/2 protein in order to stop the over proliferation of cells. Brady’s research is unique in the fact that it focuses on the copper binding area of MEK1 and how that may influence cell production. MEK1 has been found to bind copper in two locations, so the lab wanted to observe if copper binding contributed to the function of it.
In the first experiment, they used Mouse Embryonic Fibroblast cells (MEFs) and transformed them to have the BRAFV600E mutation. They then tested two groups of the BRAFV600E cells; one group was transformed so that the copper transport protein, CTR1, was not expressed in the cells while the other group maintained the copper transport protein. The absence of CTR1 means that the cells were no longer able to bring copper through the outer membrane, leading to lower copper levels within the cells. After transformation, CTR1 was added back to the cells in a rescue experiment. In one group, wild type CTR1 was added, and in the other group a mutated version of CTR1 was added.
The phosphorylated ERK1/2 was observed to see if MEK1 was still functioning as seen in figure b. Figures a and b are both immunoblots which are gels that have antibodies that are correlated to specific proteins. When the gels are run, the proteins stick to the antibodies and create a band. The thickness of the bands indicates how much of it was present in the sample; a thick band means a high amount of protein and a light band means a low amount of protein. Figure b shows a much lighter band in the cells that lack CTR1 as well as the cells that lack CTR1 and had the mutated CTR1 added.
After testing the CTR1 transformed cells, they decided to mutate the copper binding domain of MEK1 to see if that would also affect the function of MEK1. There are two copper binding domains in MEK1, one at the 187th and 188th amino acids and another at the 230th and 239th amino acids. In one group of cells they mutated the 187th and 188th amino acids and in another group they mutated the 230th and 239th amino acids. In the other group they mutated all four and named it the Copper Binding Mutation (CBM). The cells with one mutation showed a much lower amount of phosphorylated ERK while the CBM cells had virtually none. This means that mutating the copper binding domain of MEK1 causes it to lose its function in the MAPK signaling pathway.
More experiments similar to this were done and showed that mutating the copper binding site in MEK1 reduces tumor growth in BRAFV600E cells because it debilitates the function of MEK1.
In another experiment by the same lab, the effect of copper binding to ULK1/2 was tested. ULK1/2 is a part of the autophagic signaling pathway in cells. Autophagy is the process in which cells degrade damaged cellular materials.
The figure above shows cells from mice that were given the BRAFV600E mutated lung adenocarcinoma. CRISPR Cas-9 was used to knock out the gene that produces ULK1, then they added new versions of the gene back into the cells, one wild type and one with the copper binding mutation. LC3I and II are indicators of the function of ULK1 as ULK1 helps facilitate the breakdown of LC3I to LC3II. In the clone number 5, there is LC3I present which shows that LC3 was not fully synthesized from I to II, particularly in the CBM cells. This means that when copper binding is not able to occur in ULK1, the ULK1 does not function properly. Additionally, figure B shows that the phosphorylated ATG (P-ATG13) is almost non-existent in the CBM cells. This also shows a loss in function of ULK1 as it phosphorylates ATG.
References
- Brady, D., Crowe, M., Turski, M. et al. Copper is required for oncogenic BRAF signalling and tumorigenesis. Nature 509, 492–496 (2014). https://doi.org/10.1038/nature13180
- Tsang, T., Gu, X., Davis, C. I., Posimo, J. M., Miller, Z. A., & Brady, D. C. (2022). BRAFV600E-Driven Lung Adenocarcinoma Requires Copper to Sustain Autophagic Signaling and Processing. Molecular cancer research : MCR, 20(7), 1096–1107. https://doi.org/10.1158/1541-7786.MCR-21-0250