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Interplay Between Tissue Architecture and Nuclear Organization in the DNA Damage

DNA 损伤中组织结构和核组织之间的相互作用

基本信息

批准号：
8534728
负责人：
Pierre-Alexandre Vidi
金额：
$ 8.99万
依托单位：
PURDUE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-08-21 至 2014-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8534728
关键词：
Acinus organ component Address Affect Architecture Basement membrane Biochemical Breast Breast Cancer Detection Cancer Biology Cancer Burden Cancer Control Cell Culture Techniques Cell Cycle Progression Cell Nucleus Cell Survival Cells Cellular biology Chromatin Chromatin Remodeling Factor Chromosomal translocation Collaborations Communication Cues Cytoskeleton DNA DNA Damage DNA Double Strand Break DNA Repair DNA lesion Data Defect Dependency Development Diffusion Engineering Environment Epithelial Epithelium Excision Extracellular Matrix Fluorescence Fluorescent in Situ Hybridization Fostering Frequencies Genome Genomic Instability Genomics Goals Histologic Hydrogels Integrins Interphase Journals Knowledge Laboratories Lead Location Maintenance Malignant Neoplasms Mammary Gland Parenchyma Measures Mechanics Mediating Mentors Mitotic Spindle Apparatus Modeling Molecular Molecular Chaperones Molecular and Cellular Biology Monitor Morphogenesis Mutation NCI Center for Cancer Research Neoadjuvant Therapy Nodule Non-Malignant Noninfiltrating Intraductal Carcinoma Normal tissue morphology Nuclear Nuclear Protein Pathway interactions Patients Phase Phosphorylation Prevention Prevention strategy Principal Investigator Proteins Proteomics Radiation therapy Recruitment Activity Research Resolution Role Sampling Shapes Signal Transduction Site Solid Specimen Sterile coverings Structural Protein Structure Techniques Testing Tetanus Helper Peptide Texture Tissue Banking Tissue Banks Tissue Model Tissue Sample Tissues Training Universities Work anticancer research anticancer treatment cancer cell cancer initiation cancer prevention cancer therapy carcinogenesis career career development cell assembly chemotherapy coping extracellular fascinate fighting innovation insight killings malignant breast neoplasm medical schools monolayer multidisciplinary new technology polarized cell prevent public health relevance repaired research study response scaffold three-dimensional modeling tumor tumor progression tumorigenesis

项目摘要

DESCRIPTION (provided by applicant): Double-strand DNA breaks (DSBs) constitute a constant threat for genome integrity. In absence of accurate repair, they lead to mutations and chromosomal translocations promoting cancer progression. In addition, anticancer therapies largely rely on genotoxic treatments generating DSBs in cancer cells. How normal tissues and cancer cells cope with DSBs has therefore major implications for cancer prevention and control. Cells have evolved elaborate DNA damage response (DDR) mechanisms to sense DSBs, activate repair pathways, and control cell cycle progression to prevent the propagation of genomic instability. A great challenge is to understand the DDR in the context of tissues and to define the influence of the tissue architecture (i.e., the organized assembly of multicellular structures) on the DDR. Basoapical polarity is an essential aspect of epithelial architecture that is lost during cancer development. Our preliminary data indicate that the DDR is enhanced in polarized tissues by basement membrane (BM) signaling through hemidesmosomal integrins. This effect is observed both for non-neoplastic and malignant cells in 3D culture, but not in flat cell monolayers, indicating dependency on tissue morphogenesis. Nuclear organization is interconnected with tissue morphogenesis and carcinogenesis. In polarized cells, the nuclear mitotic apparatus (NuMA) protein redistributes in the nucleus after DSB induction. NuMA is rapidly phosphorylated upon DNA damage, is necessary for the maintenance of H2AX phosphorylation (a chromatin mark at DSBs), and interacts with the WICH chromatin remodeling complex that functions in the DDR. These observations led us to propose a model in which tissue polarity and the nuclear structural protein NuMA cooperate to maintain genome integrity. The proposed research will test this model from two angles: the cell nucleus and the cell-BM interphase. Aim 1 will be to characterize the role of NuMA in the DDR. NuMA may serve as a molecular scaffold facilitating the targeting and anchorage of repair factors and chromatin remodelers at DNA lesions and/or preventing free diffusion of broken DNA in the nucleus. NuMA phosphorylation by ATM may confer spatial and temporal resolution within the scaffold. During the mentored K99 phase, protein interactions involving NuMA and DDR factors will be analyzed. During the independent R00 phase, the effect of NuMA disruption on genomic translocation frequencies and DSB mobility will be determined to test the hypothesis that NuMA anchors DNA breaks. The function of NuMA phosphorylation (P- NuMA) in the DDR will be addressed by identifying P-NuMA interaction partners, localizing, and disrupting P- NuMA. Aim 2 will be to define the mechanism by which tissue architecture controls DSB repair. Mechanotransduction or biochemical signaling via hemidesmosomal integrins may convey extracellular cues to the cell nucleus, leading to changes in nuclear organization affecting the DDR. Experiments in the K99 phase will examine the influence of basal polarity on DSB repair and nuclear organization in breast tissue samples. In the R00 phase, the mechanotransduction hypothesis will be tested with engineered hydrogels of defined stiffness, interference with the cytoskeleton dynamics, and uncoupling integrins from the cytoskeleton. The possibility that biochemical BM signals mediate the effect of basal polarity on DSB repair and NuMA distribution will be addressed by inhibiting integrin signaling cascades. A 3D culture model of ductal carcinoma in situ will be used to test if altering mechanical or biochemical BM signaling leads to decreased DSB repair in cancer cell. I am fascinated by the organization of the cell nucleus and by the mechanisms that maintain genome integrity. My career goal is to expand my current mentored project on DNA repair, initiated three years ago, as an academic principal investigator and to develop innovative strategies to fight the cancer burden. Before embracing a career in cancer research, I have built a solid background in molecular and cellular biology and acquired extensive expertise in fluorescence techniques that will be applied to this project. The K99 mechanism would offer me a unique opportunity of career development by allowing me to initiate a translational aspect of research on DNA repair (collaboration with Drs. S. Badve and K. Hodges at the IU School of Medicine). It would also provide me training in proteomics and micromechanics that I could directly apply to my project. Importantly, I will seek advice from my Mentor (Dr. S. Lelievre) and co-mentors (Drs. T. Misteli and J. Irudayaraj) whose combined expertise include breast cancer biology, 3D tissue models, nuclear organization, DNA repair, and the application of new technology to cell biology. This mentoring team will assess my progress in research and chaperone my transition to independence. Purdue University has a very strong focus on cancer research and offers excellent training in breast cancer detection, treatment, and prevention with seminars, courses, journal club, and retreats organized within the NCI-designated Purdue Center for Cancer Research. Purdue is a unique environment for multidisciplinary endeavors between biologists, engineers, and clinicians. This milieu and my developing scientific network will drive technical advances and foster conceptual development.

描述（由申请人提供）：双链DNA断裂（DSB）对基因组完整性构成持续威胁。在缺乏准确修复的情况下，它们导致突变和染色体易位，促进癌症进展。此外，抗癌疗法很大程度上依赖于在癌细胞中产生DSB的遗传毒性治疗。因此，正常组织和癌细胞如何科普DSB对癌症预防和控制具有重要意义。细胞已经进化出复杂的DNA损伤反应（DDR）机制来感知DSB，激活修复途径，并控制细胞周期进程，以防止基因组不稳定性的传播。一个巨大的挑战是在组织的背景下理解DDR并定义组织结构的影响（即，多细胞结构的有组织组装）。基底顶端极性是上皮结构的一个重要方面，在癌症发展过程中丢失。我们的初步数据表明，DDR增强极化组织的基底膜（BM）信号通过半桥粒整合素。在3D培养物中非肿瘤细胞和恶性细胞都观察到这种效应，但在扁平细胞单层中没有观察到，表明依赖于组织形态发生。核组织与组织形态发生和癌变相互关联。在极化细胞中，核有丝分裂器（NuMA）蛋白在DSB诱导后重新分布在细胞核中。NuMA在DNA损伤后迅速磷酸化，是维持H2 AX磷酸化（DSB处的染色质标记）所必需的，并与在DDR中起作用的WICH染色质重塑复合物相互作用。这些观察使我们提出了一个模型，其中组织极性和核结构蛋白NuMA合作，以保持基因组的完整性。本研究将从细胞核和细胞-骨髓间期两个角度对该模型进行验证。目标1将是说明NuMA在复员方案中的作用。NuMA可作为分子支架，促进修复因子和染色质重塑物在DNA损伤处的靶向和锚定和/或防止断裂DNA在细胞核中的自由扩散。通过ATM的NuMA磷酸化可以赋予支架内的空间和时间分辨率。在指导的K99阶段，将分析涉及NuMA和DDR因子的蛋白质相互作用。在独立R 00阶段，将确定NuMA破坏对基因组易位频率和DSB迁移率的影响，以检验NuMA锚定DNA断裂的假设。DDR中NuMA磷酸化（P-NuMA）的功能将通过鉴定P-NuMA相互作用伴侣、定位和破坏P-NuMA来解决。目的2将是确定的机制，组织结构控制DSB修复。通过半桥粒整合素的机械转导或生化信号传导可以将细胞外信号传递到细胞核，导致影响DDR的核组织的变化。K99阶段的实验将检查基底极性对乳腺组织样本中DSB修复和核组织的影响。在R 00阶段，机械转导假说将测试工程水凝胶定义的刚度，干扰细胞骨架动力学，并从细胞骨架解偶联整合素。生物化学BM信号介导基底极性对DSB修复和NuMA分布的影响的可能性将通过抑制整联蛋白信号级联来解决。原位导管癌的3D培养模型将用于测试改变机械或生物化学BM信号传导是否导致癌细胞中DSB修复减少。我对细胞核的组织和维持基因组完整性的机制着迷。我的职业目标是扩大我目前的指导项目DNA修复，三年前开始，作为一个学术首席研究员，并制定创新的战略，以打击癌症负担。在从事癌症研究之前，我已经在分子和细胞生物学方面建立了坚实的背景，并获得了将应用于该项目的荧光技术方面的广泛专业知识。K99机制将为我的职业发展提供一个独特的机会，让我能够开始DNA修复研究的转化方面（与S。Badve和K. Hodges at the IU School of Medicine）。它还将为我提供蛋白质组学和微观力学方面的培训，我可以直接应用于我的项目。重要的是，我会向我的导师（S博士）寻求建议。Lelievre）和共同导师（T. Misteli和J. Irudayaraj），他们的综合专业知识包括乳腺癌生物学，3D组织模型，核组织，DNA修复以及新技术在细胞生物学中的应用。这个指导团队将评估我在研究方面的进展，并监督我向独立的过渡。普渡大学非常重视癌症研究，并在NCI指定的普渡癌症研究中心内组织研讨会，课程，期刊俱乐部和务虚会，提供乳腺癌检测，治疗和预防方面的优秀培训。普渡大学是生物学家，工程师和临床医生之间的多学科努力的独特环境。这种环境和我不断发展的科学网络将推动技术进步，促进概念发展。