Mechanisms of B-Myb oncogenicity in ovarian cancer

项目摘要-摘要 B-Myb是一种参与细胞周期基因调控的癌蛋白。B-Myb联系MuvB核心五人组 蛋白质(LIN9、LIN37、LIN52、LIN53/RBBP4和LIN54)形成MMB(Myb-MuvB)复合体。MMB 复合体反过来又通过有丝分裂促进细胞周期晚期基因的表达。通过互动 通过一组可选的结合伙伴(E2F4-DP1和p130/p107)，MuvB核心可以成为 梦想复合体(DP、RB样蛋白、E2F和MuvB)，通过抑制细胞周期基因来对抗MMB， 将电池保持在静止状态。MYBL2扩增(编码B-Myb)和过表达 在许多癌症中，MMB靶基因与细胞增殖和预后不良有关。此外， 来自癌症基因组图谱的数据支持B-Myb的高表达是高危患者生存不良的预测因素 恶性浆液性卵巢癌(HGSOC)。然而，B-Myb在HGSOC中的作用在很大程度上还没有研究，而且 B-Myb过表达改变细胞行为的机制尚不清楚。有趣的是，两者 梦想复合体的破坏和B-Myb的过度表达导致了类似的增殖表型。 此外，当B-Myb过度表达时，梦的形成减少，MMB水平相当 尽管有逮捕的环境线索，但还是有循环细胞的迹象。因此，要阐明B-的作用机制。 MYB的致瘤性，确定B-Myb对梦功能的影响是很重要的。我们假设 B-Myb表达增加通过结合LIN52隔离MuvB，从而推动细胞增殖 打乱梦对细胞周期基因的抑制。为了验证我们的假设，我们将使用人类 永生化输卵管上皮细胞(FTE-hTERT稳定表达B-Myb)用于功能研究。 我们还将在SKOV3细胞(带有MYBL2的卵巢癌细胞)中进行功能丧失和挽救研究 扩增)使用tet可诱导的双表达系统同时耗尽内源B-Myb和 表达我们的异位蛋白。将使用RT-qPCR、流式细胞术和IP/WB来测量靶细胞的变化 基因表达、细胞增殖和细胞周期分布，以及MMB和DREAM复合体的形成， 分别进行了分析。此外，我们将评估MMB的形成在介导B-Myb致癌过程中的重要性 表达MuvB结合缺陷B-Myb突变体的效果。为了确立我们的 对人类健康的发现，我们将确定B-Myb水平对DREAM靶基因表达的影响 在HGSOC组织样本中。我们将把我们的发现与治疗反应和结果联系起来。 这些病人中。总体而言，我们试图了解B-Myb改变细胞形成的机制 周期基因调控复合体(DREAM和MMB)，细胞周期基因表达，并促进增殖 卵巢癌的细胞表型。我们的最终目标是识别新的预测标记物和治疗 帮助治疗这种毁灭性疾病的目标。

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.