Impact of mtDNA Mutations and Transfer on Tumor Growth Dynamics

mtDNA 突变和转移对肿瘤生长动态的影响

• 批准号: 10439441
• 负责人: Amy Katherine Yu
• 金额: $ 5.18万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10439441
• 关键词: 4T1; Address; Aerobic; Affect; Cancer Biology; Cell Line; Cell Respiration; Cell Survival; Cell physiology; Cells; Clinical; DNA; DNA Sequence; Data; Dependence; Disease; Disease Progression; Engineering; Event; Evolution; Experimental Designs; Generations; Genes; Genome; Glycolysis; Growth; Haplotypes; Hybrids; Impairment; In Vitro; Isoleucine-Specific tRNA; Kinetics; Light; Link; Malignant Neoplasms; Mammary Neoplasms; Measurement; Metabolic; Metabolism; Methods; Mitochondria; Mitochondrial DNA; Mus; Mutation; Nature; Neoplasm Metastasis; Non-Malignant; Normal Cell; Nuclear; Nucleotide Biosynthesis; Organelles; Outcome; PTGS1 gene; Patient-Focused Outcomes; Phenotype; Physicians; Primary Neoplasm; Process; Reactive Oxygen Species; Research; Respiration; Scientist; Signal Transduction; Stress; Stromal Cells; System; Testing; Time; Tissue Sample; Transplantation; Tumor Cell Line; Tumorigenicity; Variant; behavior in vitro; cancer cell; cell behavior; cell growth; cell motility; epigenome; epithelial to mesenchymal transition; experimental study; in vivo; insight; malignant breast neoplasm; mammary epithelium; melanoma; member; metabolic profile; mitochondrial DNA mutation; mitochondrial dysfunction; mitochondrial genome; mouse model; mutant; neoplastic cell; pressure; respiratory; rho; subcutaneous; tool; tumor; tumor growth; tumor heterogeneity; tumor metabolism; tumor microenvironment; tumor progression; uptake

PROJECT SUMMARY/ABSTRACT Beyond generating ATP for cellular processes, mitochondria also provide metabolites and signaling factors that orchestrate cell survival, proliferation, and metabolism. It has been known since the 1930s that most cancer cells rely mainly on glycolysis even in aerobic conditions. Mitochondria nonetheless remain essential for metabolic plasticity, the generation of reactive oxygen species (ROS), and nucleotide biosynthesis, all factors that contribute to metastasis and poor outcomes in cancer. The direct impact of mitochondrial dysfunction, such as mutations in the mitochondrial genome (mtDNA), has been difficult to study without experimental tools for editing mtDNA into desired sequences. It remains unknown whether differences in the mtDNA sequence are sufficient to alter the growth kinetics of a tumor, which is a fundamental lack of understanding with potential clinical implications. Furthermore, it is unclear whether tumor cells with deleterious mtDNA mutations will mitigate an impairment in respiration over the natural course of tumor growth, during an epithelial-to- mesenchymal transition, and with metastasis. Cancer cells lacking endogenous mtDNA (ρ0) have previously been shown to restore oxidative metabolism by acquiring mitochondria by organelle transfer from non- malignant cells in the tumor microenvironment. However, this highly artificial system relies upon extreme selective pressure in ρ0 cancer cells that do not exist in nature. Whether the native mtDNA status in mtDNA- resident (ρ+) tumor cells changes with tumor progression has not been determined. To address this key question, it is necessary to generate tumor cells with different known mtDNA mutations in an isogenic nuclear background. Members of my thesis lab developed an in vitro method for permanently transferring isolated mitochondria into ρ0 recipient cells of choice, enabling generation of cancer clones with specific mtDNA mutations and identical nuclear genomes. In Aim 1 studies, I will use B16 mouse melanoma and 4T1 mammary cancer cells as the nuclear backgrounds and generate cancer cells with a panel of wild- type and mutant mtDNA sequences. I will quantify how these mutations alter their metabolism, ROS levels, and invasive capacities in vitro. In Aim 2, I will inject these cells, along with ρ+ and ρ0 controls, into mice subcutaneously and quantify how mtDNA mutations affect kinetics of tumor growth and metastasis. In Aim 3, I will purify tumor cells from local and disseminated disease and analyze changes in mtDNA sequences and tumor cell function following metastasis. Due to differences in respiratory capacities and metabolic profiles between cells containing different mtDNA, I anticipate growth rates and rates of metastasis will vary between lines even before potential mitochondrial acquisition from the normal microenvironment. Understanding the dependence of cancer cells on their mtDNA and whether mtDNA is exchanged in a ρ+ tumor cell setting will provide fresh insight into tumor heterogeneity and evolution in cancer metabolism as a whole.

项目概要/摘要 除了为细胞过程产生 ATP 之外，线粒体还提供代谢物和信号因子， 协调细胞的存活、增殖和新陈代谢。自 20 世纪 30 年代以来，人们就知道大多数癌症 即使在有氧条件下，细胞也主要依赖糖酵解。尽管如此，线粒体仍然至关重要 代谢可塑性、活性氧 (ROS) 的产生和核苷酸生物合成，所有因素 导致癌症转移和不良结果。线粒体功能障碍的直接影响， 例如线粒体基因组 (mtDNA) 的突变，如果没有实验工具就很难研究 用于将 mtDNA 编辑成所需的序列。目前尚不清楚线粒体DNA序列是否存在差异 足以改变肿瘤的生长动力学，这是从根本上缺乏对潜在的了解 临床意义。此外，尚不清楚具有有害线粒体DNA突变的肿瘤细胞是否会 减轻肿瘤自然生长过程中的呼吸损伤，在上皮到- 间质转化，并伴有转移。缺乏内源性 mtDNA (ρ0) 的癌细胞先前已 已被证明可以通过从非细胞器转移获得线粒体来恢复氧化代谢 肿瘤微环境中的恶性细胞。然而，这个高度人工的系统依赖于极端的 自然界中不存在的ρ0癌细胞的选择压力。 mtDNA 中的天然 mtDNA 状态是否为 驻留（ρ+）肿瘤细胞随肿瘤进展的变化尚未确定。 为了解决这个关键问题，有必要产生具有不同已知 mtDNA 突变的肿瘤细胞 等基因核背景。我的论文实验室的成员开发了一种永久的体外方法 将分离的线粒体转移到所选的 ρ0 受体细胞中，从而能够生成癌症克隆 特定的线粒体DNA突变和相同的核基因组。在目标 1 研究中，我将使用 B16 小鼠黑色素瘤 和 4T1 乳腺癌细胞作为核背景，并产生具有一组野生型的癌细胞 类型和突变型 mtDNA 序列。我将量化这些突变如何改变它们的新陈代谢、ROS水平、 和体外侵袭能力。在目标 2 中，我将这些细胞以及 ρ+ 和 ρ0 对照注射到小鼠体内 皮下注射并量化 mtDNA 突变如何影响肿瘤生长和转移的动力学。在目标 3 中，我 将从局部和播散性疾病中纯化肿瘤细胞，并分析 mtDNA 序列的变化和 转移后的肿瘤细胞功能。由于呼吸能力和代谢特征的差异 在含有不同 mtDNA 的细胞之间，我预计生长率和转移率会有所不同 甚至在从正常微环境中潜在获取线粒体之前就已经出现了这种情况。了解 癌细胞对其 mtDNA 的依赖性以及 mtDNA 是否在 ρ+ 肿瘤细胞环境中交换将 为整个癌症代谢的肿瘤异质性和进化提供新的见解。