Interplay Between Tissue Architecture and Nuclear Organization in the DNA Damage

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。因此，正常组织和癌细胞如何应对双链断裂对癌症的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，并确定组织结构(即多细胞结构的有组织组装)对DDR的影响。基尖极性是上皮结构的一个重要方面，在癌症发展过程中丢失了。我们的初步数据表明，基底膜(BM)信号通过半桥粒整合素在极化组织中增强了DDR。在3D培养中，这种效应在非肿瘤细胞和恶性细胞中都能观察到，但在扁平细胞单层中观察不到，这表明对组织形态发生的依赖。核组织与组织形态发生和癌变密切相关。在极化细胞中，在DSB诱导后，核有丝分裂装置(NUMA)蛋白重新分布在细胞核中。NUMA在DNA损伤后迅速磷酸化，是维持H_2AX磷酸化所必需的(DSB的染色质标记)，并与在DDR中发挥作用的染色质重塑复合体相互作用。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合作来维持基因组的完整性。这项拟议的研究将从两个角度测试这一模型：细胞核和细胞-骨髓界面。目标1将说明国家工作队在复员方案中的作用。NUMA可以作为一种分子支架，促进修复因子和染色质重构体在DNA损伤处的靶向和锚定，和/或防止DNA断裂在细胞核内的自由扩散。ATM对NUMA的磷酸化可能会在支架内赋予空间和时间分辨率。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。在独立的R00阶段，NUMA中断对基因组易位频率和DSB迁移率的影响将被确定，以检验NUMA锚定DNA断裂的假设。将通过确定P-NUMA相互作用伙伴、定位和干扰P-NUMA来解决NUMA在DDR中的作用。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能将细胞外信号传递到细胞核，导致影响DDR的核组织变化。K99阶段的实验将检验基本极性对乳房组织样本中DSB修复和核组织的影响。在R00阶段，机械转导假说将通过定义刚性的工程水凝胶、干扰细胞骨架动力学以及从细胞骨架解偶联整合素来检验。生化BM信号介导基础极性对DSB修复和NUMA分布的影响的可能性将通过抑制整合素信号级联来解决。导管原位癌的3D培养模型将被用来测试改变机械或生化的BM信号是否会导致癌细胞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。作为一名学术首席研究员，我的职业目标是扩大我目前在DNA修复方面的指导项目，该项目于三年前启动，并开发出与癌症负担作斗争的创新策略。在投身癌症研究之前，我已经在分子和细胞生物学方面建立了坚实的背景，并在荧光技术方面获得了广泛的专业知识，这些技术将应用于这个项目。K99机制将为我提供一个独特的职业发展机会，使我能够启动DNA修复研究的翻译方面(与密歇根大学医学院的S.Badve博士和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.Lelievre博士)和共同导师(T.Misteli博士和J.Irudayaraj博士)的建议，他们的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，并指导我向独立的过渡。普渡大学非常专注于癌症研究，并通过研讨会、课程、期刊俱乐部和在NCI指定的普渡癌症研究中心内组织的务虚会，在乳腺癌检测、治疗和预防方面提供出色的培训。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。这种环境和我正在发展的科学网络将推动技术进步并促进概念发展。 公共卫生相关性： 虽然健康细胞中的DNA损伤促进了癌症的进展，但化疗和放射治疗造成的DNA损伤在杀死癌细胞方面起到了重要作用。这项拟议的研究旨在了解组织内细胞的有组织组装如何影响DNA修复。获得这些知识对于提高抗癌治疗的疗效和制定癌症预防策略非常重要。

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. PUBLIC HEALTH RELEVANCE: While DNA lesions in healthy cells promote cancer progression, DNA damage by chemo- and radiotherapies is instrumental in killing cancer cells. The proposed research aims at understanding how the organized assembly of cells within tissues influences DNA repair. Gaining this knowledge is important to enhance the efficacy of anticancer treatments, and to develop cancer prevention strategies.