Discipline: Biological Sciences
Subcategory: Cancer Research
Joselyn Miller - Alabama State University
Co-Author(s): Shivani Soni, Gulnaz Javan, Aboubacar Kaba, and Jala Redmon, Alabama State University, Montgomery, AL
In the United States, colorectal cancer (CRC) is the third leading cause of cancer related morbidity and mortality. CRC is a heterogeneous disease involving various signaling pathways, and its aggressiveness and response to therapy depend on differential activation of molecular phenotypes. Therefore, identification of molecules that are involved in development and progression of CRCs will give better insight into disease progression and also help in identifying therapeutic targets. Erythroblast Macrophage Protein (Emp) is an essential protein for erythroblast and macrophage development. Our initial studies implicate abnormal expression of Emp in pathways associated with cell proliferation, growth, invasion, migration and metastasis, which are hallmarks of cancer, and provide a basis to explore its function in CRCs. Western Blot analysis was used to process Transfectants in order to determine the efficiency of down-regulation of Emp gene expression. To perform immunofluorescence studies, HCT 116/wt cells were fixed in 4% paraformaldehyde. Cells were immunolabeled with antibodies directed against beta 1 integrin, cytoskeletal proteins: talin, actinin, and vinculin, and co-immunostained with Emp. We will also extend our studies to co-localization of paxillin, a docking protein for cytoskeleton proteins and components of signaling and complexes of adhesion plaques, such as integrin kinases and the activated form of FAK, i.e., FAK-Y397Pi (phosphorylated at tyrosine 397). Primary antibodies were detected with Alexa Fluor conjugated secondary antibodies. Slides were observed under immunofluorescence microscope. Our Western blot analysis of CRC cell lines with differing DNA mismatch repair and p53 status showed that, the expression of Emp is high in primary tumors but lower in metastatic lesions. Additionally we also found that Emp interacts with focal adhesion molecules and cell motility proteins in CRC. These results confirm our hypothesis that Emp, as a component of protein complexes and a structural or signal transductional link, is involved in CRC progression. These findings have clinical implications in establishing the prognostic and predictive value of Emp in CRCs.
Funder Acknowledgement(s): National Cancer Institute P20 Partnership grant (P20 CA192973 and P20 CA192976)
Faculty Advisor: Gulnaz Javan, email@example.com
Role: The experiments that were conducted such as immunofluorescence and microscopy were done in their entirety by me within the lab. Furthermore, everyday I cultured the cells and checked their confluency daily while nourishing them in media. Furthermore, I performed the Transfection studies with the help of my mentor using Western Blot analysis. As a whole, within this project, with little to no supervision from my mentor, I was able to conduct the experimentation.