Non-cell autonomous consequences of cytoplasmic DNA

项目摘要/摘要 基因组不稳定是癌症的一个标志，并可能导致染色体分离错误的高比率 有丝分裂细胞分裂，可产生称为微核的异常结构，使错误的分离陷入困境 染色体。微核容易受到大规模DNA损伤，引发灾难性的粉碎化 被困住的染色体变成小的DNA片段，这一过程被称为染色蓟马。除了……之外 来自微核的DNA片段产生基因组重排，从而推动癌症的发展，也可以 在细胞质中错误积累并持续存在。这些细胞质DNA是由胞质DNA传感器检测到的 CGAS，导致细胞自主激活刺痛途径，以触发先天免疫反应。 尽管cGAS-STING信号机制已经很好地定义，但细胞质DNA的命运如下 CGAS的认可仍然是一个严重的知识鸿沟。在这里，我建议研究非细胞自主角色 细胞质DNA，这对基因不稳定的癌细胞如何诱导相互作用具有关键意义 细胞对肿瘤微环境的反应。我推测细胞质DNA来源于 微核中的碎裂染色体被输出，供邻近细胞摄取。为了测试这一点 假设，我将利用一个实验系统，使染色体特异性诱导微核和 然后追踪含有可选择标记的特定细胞质DNA片段。 在目标1中，我将确定细胞质DNA是否以及如何释放到细胞外环境中 促进邻近细胞中cGAS-STING通路的非细胞自主激活。我会进一步确定 来自宿主细胞胞质的胞外DNA能否被受体细胞摄取，这将 通过使用基于dCas9的细胞质DNA报告器的活细胞成像进行监测。在《目标2》中，我将追踪 将细胞质DNA掺入受体细胞核，并确定这些片段是否能整合 进入宿主基因组。这将结合细胞遗传学和全基因组测序进行研究。 目的：探讨人细胞间DNA横向转移的可能性。尽管经常发生在 细菌，细胞间DNA转移一直是在人类癌症背景下测试的一个长期挑战。 这些研究有可能通过促进我们对癌细胞相互作用的理解而产生广泛的影响， 包括将癌基因和/或突变从染色体不稳定的癌细胞转移到非癌症细胞 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.