Interplay Between Tissue Architecture and Nuclear Organization in the DNA Damage

7.项目摘要 双链DNA断裂（DSB）对基因组的完整性构成了持续的威胁。在没有准确的 修复时，它们会导致突变和染色体易位，促进癌症进展。此外，本发明还提供了一种方法， 抗癌疗法主要依赖于在癌细胞中产生DSB的遗传毒性治疗。正常组织是如何 因此，癌细胞科普DSB对于癌症预防和控制具有重要意义。细胞具有 进化出复杂的DNA损伤反应（DDR）机制来感知DSB，激活修复途径， 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，的 多细胞结构的有组织组装）。基底顶端极性是 在癌症发展过程中失去的上皮结构。我们的初步数据表明，民主德国是 通过半桥粒整合素的基底膜（BM）信号传导在极化组织中增强。这 在3D培养物中观察到对非肿瘤细胞和恶性细胞的影响，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。核组织与组织相互连接 形态发生和致癌作用。在极化细胞中，核有丝分裂器（NuMA）蛋白 在DSB诱导后重新分布于核中。NuMA在DNA损伤后迅速磷酸化， 维持H2 AX磷酸化（DSB处的染色质标记）所必需，并与 在DDR中起作用的WICH染色质重塑复合物。 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 共同维护基因组的完整性拟议中的研究将从两个角度测试这一模型： 核和细胞-BM间期。目标1将是说明NuMA在复员方案中的作用。NuMA可能 作为分子支架，促进修复因子和染色质的靶向和锚定 在DNA损伤处的重塑和/或阻止细胞核中断裂DNA的自由扩散。NUMA 通过ATM的磷酸化可以赋予支架内的空间和时间分辨率。在指导期间， K99期，将分析涉及NuMA和DDR因子的蛋白质相互作用。在独立期间 在R 00期，将研究NuMA破坏对基因组易位频率和DSB迁移率的影响。 决定测试NuMA锚定DNA断裂的假设。NuMA磷酸化（P- 将通过确定P-NuMA互动伙伴、本地化和破坏P-NuMA来解决复员方案中的NuMA问题。 NuMA。目的2将是确定的机制，组织结构控制DSB修复。 通过半桥粒整联蛋白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 在R 00阶段，将用定义的工程化水凝胶测试机械转导假设。 刚度，干扰细胞骨架动力学，以及从细胞骨架解偶联整联蛋白。的 生物化学BM信号介导基底极性对DSB修复和NuMA影响的可能性 通过抑制整联蛋白信号级联来解决分布问题。乳腺导管的三维培养模型 原位癌将用于测试改变机械或生物化学BM信号传导是否导致降低的 癌细胞中的DSB修复。 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，并制定创新战略，以打击癌症负担。之前 我从事癌症研究，在分子和细胞生物学方面有着坚实的背景， 在荧光技术方面获得了广泛的专业知识，将应用于该项目。K99机制 将为我提供一个独特的职业发展机会，让我开始翻译方面的工作， DNA修复的研究（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，我可以直接应用到我的项目中。 重要的是，我会向我的导师（S博士）寻求建议。Lelièvre）和共同导师（T. Misteli和J. Irudayaraj），其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。这个指导小组将评估我的进步 帮助我研究和独立普渡大学非常关注癌症 研究并通过研讨会提供乳腺癌检测、治疗和预防方面的优秀培训， 课程，杂志俱乐部，并在NCI指定的普渡癌症研究中心组织务虚会。 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， 发展

7. Project Summary 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. Lelièvre) 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.