Interplay Between Tissue Architecture and Nuclear Organization in the DNA Damage

7.项目总结 双链DNA断裂(DSB)对基因组完整性构成持续威胁。在缺乏准确信息的情况下 在修复过程中，它们会导致突变和染色体易位，从而促进癌症的进展。此外, 抗癌治疗在很大程度上依赖于在癌细胞中产生DSB的基因毒性治疗。正常组织是如何 因此，癌细胞应对双链断裂对癌症的预防和控制具有重要意义。细胞有 进化出复杂的DNA损伤反应(DDR)机制来感知DSB，激活修复途径，以及 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并定义组织架构的影响(即 多细胞结构的有组织组装)。基尖极是一个重要方面 在癌症发展过程中丢失的上皮结构。我们的初步数据显示，复员方案是 在极化组织中，通过半桥粒整合素的基底膜(BM)信号增强。这 在3D培养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观察不到， 表明对组织形态发生的依赖。核组织与组织相互联系 形态发生和癌变。在极化细胞中，核有丝分裂装置(NUMA)蛋白 DSB诱导后重新分布于胞核。NUMA在DNA损伤后迅速磷酸化，是 对维持H_2AX磷酸化(DSB的染色质标记)是必需的，并与 在DDR中起作用的染色质重塑复合体。 这些观察结果导致我们提出了一个模型，在该模型中，组织极性和核结构蛋白NUMA 合作维护基因组的完整性。这项拟议的研究将从两个角度检验这一模型：细胞 细胞核和细胞-骨髓间期。目标1将说明国家工作队在复员方案中的作用。Numa可能会 作为分子支架，促进修复因子和染色质的靶向和锚定 DNA损伤处的重构体和/或防止断裂的DNA在细胞核内自由扩散。NUMA ATM的磷酸化可能会在支架内赋予空间和时间分辨率。在接受指导的过程中 K99期，涉及NUMA和DDR因子的蛋白质相互作用将被分析。在独立期间 R00期，NUMA干扰对基因组易位频率和DSB迁移率的影响 决心测试Numa锚定DNA断裂的假设。NUMA的磷酸化功能(P- 将通过确定P-NUMA互动合作伙伴、本地化和中断P- 努玛。目标2将确定组织结构控制DSB修复的机制。 通过半桥粒整合素的机械转导或生化信号可将细胞外信号传递给 细胞核，导致影响DDR的核组织的变化。K99阶段的实验 将研究乳房组织样本中基本极性对DSB修复和核组织的影响。在……里面 R00阶段，机械转导假说将用定义的工程水凝胶进行测试 刚性，对细胞骨架动力学的干扰，以及从细胞骨架解偶联整合素。这个 生化BM信号介导基础极性对DSB修复和NUMA影响的可能性 将通过抑制整合素信号级联来解决分布问题。一种导管的三维培养模型 原位癌将用于测试改变机械或生化BM信号是否会导致减少 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指导的DNA修复项目，该项目于三年前启动，作为 学术首席研究员，并开发创新战略，与癌症负担作斗争。在此之前 投身于癌症研究，我在分子和细胞生物学以及 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机构 将为我提供一个独特的职业发展机会，让我可以开始翻译 DNA修复研究(与密歇根州立大学医学院的S.Badve和K.Hodges博士合作)。它 还将为我提供蛋白质组学和微观力学方面的培训，我可以直接将它们应用到我的项目中。 重要的是，我将征求我的导师(S·莱利耶夫尔博士)和共同导师(T·米斯特利博士和J·米斯特利博士)的建议。 Irudayaraj)，其综合专长包括乳腺癌生物学、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的进步 在研究和陪伴我过渡到独立。普渡大学非常重视癌症 研究并通过研讨会提供乳腺癌检测、治疗和预防方面的出色培训， 在NCI指定的普渡癌症研究中心内组织的课程、杂志俱乐部和务虚会。 普渡大学是生物学家、工程师和临床医生开展多学科合作的独特环境。 这一环境和我正在发展的科学网络将推动技术进步并促进 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.