Jacob Hoj and Dr. Marc Hansen, Department of Physiology and Developmental Biology
The Rho Kinase Pathway is a cellular pathway that is thought to be involved in cancer metastasis, as previous research has indicated1. As indicated by the name, the Rho group of proteins are central to this pathway. Over the past year, we set out to analyze this pathway and to further our understanding of its role in epithelial-mesenchymal transition (EMT), a biological process common in many metastatic cancers. Through experimental analysis, we were able to look at this pathway in ways that no one else has been able to. While our results were very intriguing, obtaining the data was an arduous process. Multiple experiments failed along the way, and multiple set-backs forced us to restart on many of the experiments and data analysis we had already performed. These set-backs were frustrating at times, but helped us achieve a more accurate portrayal of the data we wanted to publish. In the coming months, the data we have gathered will be presented at the Tumor Invasion and Metastasis Conference held by the American Association for Cancer Research in San Diego this January. We also hope to publish our data in a scientific journal once all the final details have been made, and will be submitting our research for publication during the 2013 winter semester.
When I started on this project, I had very little knowledge of the Rho Kinase pathway. This entire research process has been a remarkable experience for me. I have not only gained a better understanding of the Rho Kinase pathway, but have learned invaluable problem-solving and leadership skills. As mentioned in the paragraph above, we experienced multiple set-backs. For example, the techniques used to generate all data in this project require a perfectly sterile environment to avoid the risk of cell contamination. When contaminations do occur, there is often no way to identify how exactly it happened. These contaminations are extremely detrimental to the progress of the research because it puts a halt on everything we do as we are forced to start all over again from the beginning. Over the summer we experienced two or three of these contaminations, but we came back each time with a greater determination to finish this project and get the results we were hoping for.
A couple months into the project, my mentor, Dr. Marc Hansen, left on sabbatical for a year. Since we were only able to communicate via Skype and email, it left me with greater responsibility and accountability in the oversight of this project. This helped me learn many important leadership lessons, as I was placed in a situation where I had to delegate some of the experiments to other members of the lab. Although it was very convenient having my mentor around to guide me through some of the steps, I learned a great deal about myself as I was forced to solve the problems we faced on my own.
As far as the results of our research go, we discovered many interesting characteristics about the Rho Kinase pathway. My favorite experiment throughout the whole process was the “Blebbistatin Washout Experiment”. We looked at different steps along the Rho pathway, and decided to target Myosin II, a protein involved in the process of EMT that is indirectly turned on or off with activation/inactivation of RhoA. We treated RhoA cells with blebbistatin, a Myosin II inhibitor, for 2.5 hours, and then washed the blebbistatin out to see of the cells recovered their Myosin II contractility. Using a live-cell imaging microscope to document the process, we discovered that the cells expressed similar characteristics to metastatic cancer cells undergoing the process of EMT. In other words, we discovered a new way to stimulate the EMT process, thereby shedding new light on the dynamics of the Rho Kinase pathway.
Another interesting experiment we did was to analyze how cells constantly expressing RhoA would affect the EMT process; in other words, we wanted to determine a role for RhoA in cancer metastasis. We treated these RhoA mutant cells with a chemical known as hepatocyte growth factor (HGF), a chemical that stimulates cells to become metastatic. We tested two cell lines expressing different variants of RhoA, and found that in both cell lines, EMT was inhibited when the RhoA mutant cells were treated with HGF. This provides potential opportunity for anti-cancer drug development, as we discovered that RhoA inhibits EMT.
I would say that our hypotheses were relatively accurate; we had hypothesized that the Rho Kinase pathway played an important role in cancer metastasis. We were most surprised, however, by our “blebbistatin washout experiment”, because we didn’t expect to see results as dramatic as we did. All in all, we have been very pleased with the data we have generated, and we hope that it sparks new discoveries in the future relating to this topic.
As for the future of this specific research project, we are in the process of preparing our data for the AACR Tumor Invasion and Metastasis Conference in San Diego this upcoming January 20-23. Our abstract proposal for the conference has already been accepted, and we are making the final adjustments to our data to make sure it is ready to be presented by this date. We will also be submitting the findings of this data for publication. We hope that our findings will lead to the development of new anti-cancer drugs because of the increased understanding of the Rho Kinase pathway our research has uncovered.
References
- Yanagisawa, M., et al., A p120 Catenin Isoform Switch Affects Rho Activity, Induces Tumor Cell Invasion, and Predicts Metastatic Disease. The Journal of Biological Chemistry, 2008. 283(26): p. 18344-18354.