Non-cell autonomous consequences of cytoplasmic DNA

项目总结/摘要 基因组不稳定性是癌症的标志，并且在癌症发生过程中可能导致染色体分离错误的高发生率。 有丝分裂细胞分裂，这可以产生异常的结构，称为微核， 染色体细胞核容易受到大规模DNA损伤，引发灾难性的粉碎， 被包裹的染色体变成小的DNA片段，这一过程被称为染色体碎裂。除了 产生基因组重排，驱动癌症的发展，来自微核的DNA片段也可以 错误积累并持续存在于细胞质中。这些细胞质DNA被细胞质DNA传感器检测到 cGAS，导致STING途径的细胞自主激活以触发先天免疫应答。 尽管cGAS-STING信号传导机制已经被很好地定义，但细胞质DNA在 cGAS的认可仍然是一个关键的知识差距。在这里，我建议研究非细胞自主角色 细胞质DNA，这对基因组不稳定的癌细胞如何引起细胞间的相互作用具有重要意义。 肿瘤微环境的细胞反应。我假设细胞质DNA来源于 微核中的碎片染色体被输出以供邻近细胞吸收。为了验证这一 假设，我将利用一个实验系统，使染色体特异性诱导微核， 细胞质DNA，随后追踪具有选择标记的特异性细胞质DNA片段。 在目标1中，我将确定细胞质DNA是否以及如何被释放到细胞外环境中， 促进相邻细胞中cGAS-STING途径的非细胞自主激活。我将进一步确定 来源于宿主细胞细胞质的细胞外DNA是否可以被受体细胞摄取，这将 通过使用基于dCas 9的细胞质DNA报告基因的活细胞成像来监测。在目标2中，我将跟踪 将细胞质DNA掺入受体细胞核中，并确定这些片段是否可以整合 植入宿主基因组这将使用细胞遗传学和全基因组测序相结合的方法进行研究 研究人类细胞间横向DNA转移的可能性。尽管经常发生在 细菌，细胞间DNA转移一直是人类癌症背景下测试的长期挑战。 这些研究通过推进我们对癌细胞相互作用的理解， 包括癌基因和/或突变从染色体不稳定的癌细胞转移到非癌细胞 肿瘤微环境中的细胞。

PROJECT SUMMARY/ABSTRACT Genomic instability is a hallmark of cancer and can drive high rates of chromosome segregation errors during mitotic cell division, which can generate abnormal structures called micronuclei that entrap mis-segregated chromosomes. Micronuclei are susceptible to massive DNA damage, triggering the catastrophic pulverization of the entrapped chromosome into small DNA fragments, a process termed chromothripsis. In addition to generating genomic rearrangements that drive cancer development, DNA fragments from micronuclei can also mis-accumulate and persist in the cytoplasm. These cytoplasmic DNAs are detected by the cytosolic DNA sensor cGAS, resulting in the cell-autonomous activation of the STING pathway to trigger an innate immune response. Although the cGAS-STING signaling mechanisms are well defined, the fate of cytoplasmic DNAs following recognition by cGAS remains a critical knowledge gap. Here I propose to investigate non-cell autonomous roles of cytoplasmic DNAs, which hold critical implications in how genomically unstable cancer cells can elicit inter- cellular responses with the tumor microenvironment. I hypothesize that cytoplasmic DNAs derived from fragmented chromosomes in micronuclei become exported for uptake by neighboring cells. To test this hypothesis, I will leverage an experimental system enabling chromosome-specific induction of micronuclei and cytoplasmic DNA followed by tracking of specific cytoplasmic DNA fragments that harbor a selectable marker. In Aim 1, I will determine whether and how cytoplasmic DNAs are released into the extracellular environment to facilitate non-cell autonomous activation of the cGAS-STING pathway in adjacent cells. I will further determine whether extracellular DNA derived from the cytoplasm of host cells can be taken up by recipient cells, which will be monitored by live-cell imaging using a dCas9-based cytoplasmic DNA reporter. In Aim 2, I will track the incorporation of cytoplasmic DNAs into recipient cell nuclei and determine whether these fragments can integrate into the host genome. This will be studied using a combination of cytogenetics and whole-genome sequencing to investigate the possibility of lateral DNA transfer between human cells. Despite occurring frequently in bacteria, inter-cellular DNA transfer has been a longstanding challenge to test in the context of human cancer. These studies have potential for broad impact by advancing our understanding of cancer cell interactions, including the transfer of oncogenes and/or mutations from chromosomally unstable cancer cells to non-cancer cells in the tumor microenvironment.