Research Project
10112196
Project Summary-Abstract B-Myb is an oncoprotein involved in cell cycle gene regulation. B-Myb contacts the MuvB core of five proteins (LIN9, LIN37, LIN52, LIN53/RBBP4, and LIN54) to form the MMB (Myb-MuvB) complex. The MMB complex, in turn, promotes expression of late cell cycle genes for progression through mitosis. By interacting with an alternative set of binding partners (E2F4-DP1 and p130/p107), the MuvB core can become part of the DREAM complex (DP, RB-like, E2F, and MuvB), which opposes MMB by repressing cell cycle genes, maintaining the cell in a quiescent state. Both MYBL2 amplification (encoding B-Myb) and over-expression of MMB target genes are associated with cell proliferation and poor prognosis in many cancers. Furthermore, data from The Cancer Genome Atlas supports high expression of B-Myb as a predictor of poor survival in high grade serous ovarian carcinoma (HGSOC). However, the role of B-Myb in HGSOC is largely unstudied and the mechanism by which B-Myb overexpression alters cellular behavior is not well understood. Interestingly, both disruption of the DREAM complex and B-Myb overexpression result in a similar proliferative phenotype. Additionally, when B-Myb is over-expressed, DREAM formation is diminished and MMB levels are comparable to those of cycling cells, despite environmental cues for arrest. Therefore, to elucidate the mechanism of B- Myb's oncogenicity, it is important to establish the effect of B-Myb on DREAM function. We hypothesize that increased expression of B-Myb drives cell proliferation by sequestering MuvB, via binding LIN52, and disrupting DREAM-mediated repression of cell cycle genes. To test our hypothesis, we will employ human immortalized fallopian tube epithelial cells (FTE-hTERT stably expressing B-Myb) for gain of function studies. We will also perform loss of function and rescue studies in SKOV3 cells (ovarian cancer cells with MYBL2 amplification) using a tet-inducible dual expression system to simultaneously deplete endogenous B-Myb and express our ectopic protein. RT-qPCR, flow cytometry, and IP/WB will be used to measure changes in target gene expression, cell proliferation and cell cycle profile, as well as MMB and DREAM complex formation, respectively. Additionally, we will assess the importance of MMB formation in mediating B-Myb's oncogenic effects by expressing a MuvB-binding deficient B-Myb mutant. To establish the relevance of our findings to human health, we will determine the effect of B-Myb levels on DREAM target gene expression in HGSOC tissue samples. We will relate our findings to the treatment responses and outcomes of these patients. Overall, we seek to understand the mechanisms by which B-Myb alters the formation of cell cycle gene regulatory complexes (DREAM and MMB), cell cycle gene expression, and promotes proliferative cellular phenotypes in ovarian cancer. Our ultimate goal is to identify novel predictive markers and therapeutic targets to aid in the treatment of this devastating disease.
