Mechanisms of B-Myb oncogenicity in ovarian cancer

B-Myb 在卵巢癌中的致癌机制

• 批准号: 9540986
• 负责人: Audra Iness
• 金额: $ 3.49万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-04-01 至 2022-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9540986
• 关键词: B-MYB Protein; Behavior; Binding; Binding Sites; Bioinformatics; Biological Assay; Biological Markers; Cell Cycle; Cell Cycle Arrest; Cell Cycle Deregulation; Cell Cycle Progression; Cell Proliferation; Cells; Clinical; Complex; Cues; Cyclin-Dependent Kinases; Data; Disease; Dreams; Epithelial Cells; Equilibrium; Evaluation; Flow Cytometry; Future; Gene Dosage; Gene Expression; Gene Expression Regulation; Gene Targeting; Genes; Genetic; Goals; Growth; Health; Human; Immunohistochemistry; In Vitro; Institutional Review Boards; Link; MYBL2 gene; Malignant Neoplasms; Malignant neoplasm of ovary; Mammalian Cell; Mammalian Oviducts; Measures; Mediating; Mitosis; Modeling; Mutation; Oncogenic; Oncoproteins; Outcome; Ovarian Serous Adenocarcinoma; Patient-Focused Outcomes; Patients; Phenotype; Play; Positioning Attribute; Prognostic Marker; Proliferating; Protein Kinase; Proteins; Protocols documentation; Recurrence; Regulator Genes; Repression; Role; S Phase; SKOV3 cells; Serine; Signal Transduction; System; Testing; Tetanus Helper Peptide; The Cancer Genome Atlas; Tissue Sample; Treatment outcome; Tumor Tissue; Work; cancer cell; cdc Genes; clinical biomarkers; gain of function; interest; knock-down; loss of function; mouse model; mutant; novel; outcome forecast; overexpression; patient response; predictive marker; protein complex; protein function; small hairpin RNA; therapeutic target; therapy resistant; transcription factor; treatment response; tumor

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.

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核心的五个 MMB（Myb-MuvB）复合物的制备方法包括将MMB（Myb-MuvB）蛋白（LIN 9、LIN 37、LIN 52、LIN 53/RBBP 4和LIN 54）与MMB（Myb-MuvB）复合以形成MMB（Myb-MuvB）复合物。MMB 复合物，反过来，促进晚期细胞周期基因的表达，通过有丝分裂的进展。交互 与另一组结合伴侣（E2 F4-DP 1和p130/p107）一起，MuvB核心可以成为 DREAM复合物（DP、RB样、E2 F和MuvB）通过抑制细胞周期基因来对抗MMB， 使电池保持静止状态。MYBL 2扩增（编码B-Myb）和MYBL 2的过表达都可以抑制B-Myb的表达。 MMB靶基因与许多癌症中的细胞增殖和不良预后相关。此外，委员会认为， 来自癌症基因组图谱的数据支持B-Myb的高表达作为高风险患者生存率差的预测因子。 级浆液性卵巢癌（HGSOC）。然而，B-Myb在HGSOC中的作用在很大程度上未被研究， B-Myb过表达改变细胞行为的机制尚不清楚。有趣的是，两者 DREAM复合物的破坏和B-Myb过表达导致类似的增殖表型。 此外，当B-Myb过度表达时，DREAM形成减少，MMB水平相当 与循环细胞相比，尽管有环境因素的提示。因此，为了阐明B- Myb的致癌性，重要的是建立B-Myb对DREAM功能的影响。我们假设 B-Myb表达增加通过结合LIN 52隔离MuvB驱动细胞增殖， 破坏DREAM介导的细胞周期基因抑制。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（稳定表达B-Myb的FTE-hTERT）用于功能获得研究。 我们还将在SKOV 3细胞（具有MYBL 2的卵巢癌细胞）中进行功能丧失和挽救研究。 扩增），同时消耗内源性B-Myb和 表达我们的异位蛋白。RT-qPCR、流式细胞术和IP/WB将用于测量靶细胞的变化。 基因表达、细胞增殖和细胞周期谱，以及MMB和DREAM复合物形成， 分别此外，我们还将评估MMB形成在介导B-Myb致癌作用中的重要性。 通过表达MuvB结合缺陷型B-Myb突变体来产生作用。为了确定我们的 研究结果对人类健康的影响，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。总之，我们试图了解B-Myb改变细胞形成的机制， 周期基因调控复合物（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确定新的预测标志物和治疗方法。 帮助治疗这种毁灭性疾病的目标。