Many important cellular events are regulated by antagonistic mechanisms, and understanding how factors involved in the regulation of tumorigenesis execute their antagonistic functions is particularly important. Histone H2A mono-ubiquitination at K119 mediated by polycomb repressive complex 1 (PRC1) is an important epigenetic mark associated with epithelial-mesenchymal transition (EMT) and cell invasion during early metastasis. However, less is known about the function of histone H2A deubiquitination during these processes. Our long-term research goal is to elucidate the molecular mechanism underlying the regulation of histone H2A ubiquitination and deubiquitination, and determine the role of this histone modification in the regulation of cell cycle and tumorigenesis. To this end, the goal of the proposed research is to study the function of histone H2A deubiquitination mediated by ubiquitin specific peptidase 16 (USP16) in the regulation of EMT and invasion of breast cancer cells. Our preliminary data has shown that BMI1, a member of PRC1, promotes EMT and invasion of breast cancer cells. Since USP16 specifically deubiquitinates H2A at K119, the central hypothesis of this application is that the USP16 antagonizes the function of BMI1 and inhibits the EMT and invasion of breast cancer cells. Furthermore, we have previously identified USP16 as a novel substrate of polo-like kinase 1 (PLK1). Therefore, the role of PLK1 in USP16-mediated regulation of EMT and cell invasion will also be determined. The rationale for this hypothesis rests on the following: once the role of USP16-mediated H2A deubiquitination in the regulation of EMT and invasion of cancer cells is established, a novel tumorigenesis regulatory mechanism that involves both the ubiquitination and deubiquitination of H2A can be defined. The central hypothesis will be tested through the activities including: (1) Determine whether USP16-mediated deubiquitination of histone H2A inhibits EMT and invasion of breast cancer cells. Following overexpression and knockdown of USP16, the changes in the expression of EMT markers and invasiveness of breast cancer cells will be analyzed. (2) Determine the role of PLK1 in USP16-mediated regulation of EMT and invasion of breast cancer cells. As the phosphorylation sites on USP16 have been identified, we will utilize both non- phosphorylatable and phosphomimic USP16 constructs to carefully evaluate the function of USP16 phosphorylation by PLK1 in regulating EMT and cell invasion. The expected outcomes of this study include the identification of ubiquitination and deubiquitination of histone H2A as an important mechanism for the regulation of metastasis in breast cancer, and the revealing of a cell cycle regulation-independent function of PLK1 in tumorigenesis. Knowledge of these mechanisms may contribute to the development of novel approaches in the prevention and treatment of metastatic breast cancer. !
Many important cellular events are regulated by antagonistic mechanisms. This application proposes to study the function of USP16-mediated histone H2A deubiquitination in breast cancer cells by antagonizing histone H2A mono-ubiquitination. This study is expected to reveal a new mechanism of metastasis regulation and may contribute to the development of novel approaches in the prevention and treatment of metastatic breast cancer. !
