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.

项目总结/摘要 除了为细胞过程产生ATP外，线粒体还提供代谢物和信号因子， 协调细胞存活、增殖和代谢。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。然而，线粒体仍然是至关重要的， 代谢可塑性，活性氧（ROS）的产生，核苷酸的生物合成，所有因素 导致癌症转移和预后不良。线粒体功能障碍的直接影响， 例如线粒体基因组（线粒体DNA）的突变，如果没有实验工具就很难研究 用于将mtDNA编辑成所需序列。目前尚不清楚线粒体DNA序列的差异是否 足以改变肿瘤的生长动力学，这是一个根本缺乏了解的潜力， 临床意义此外，目前还不清楚带有有害mtDNA突变的肿瘤细胞是否会 在肿瘤生长的自然过程中，在上皮细胞到 间质转化和转移。缺乏内源性mtDNA（ρ0）的癌细胞以前 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，这种高度人工化的系统依赖于极端的 在自然界中不存在的ρ0癌细胞中的选择压力。是否天然mtDNA在mtDNA中的状态- 尚未确定驻留（ρ+）肿瘤细胞随肿瘤进展的变化。 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体外方法， 将分离的线粒体转移到选择的ρ0受体细胞中，使得能够产生具有 特定的mtDNA突变和相同的核基因组。在Aim 1研究中，我将使用B16小鼠黑色素瘤 和4 T1乳腺癌细胞作为核背景，并产生具有一组野生型- 型和突变型mtDNA序列。我将量化这些突变如何改变他们的新陈代谢，活性氧水平， 和体外侵袭能力。在目标2中，我将这些细胞与ρ+和ρ0对照一起沿着注射到小鼠体内 皮下注射和量化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， 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将在 甚至在从正常微环境中获得潜在线粒体之前。了解 癌细胞对其mtDNA的依赖性以及mtDNA是否在ρ+肿瘤细胞环境中发生交换， 为肿瘤异质性和癌症代谢的整体演变提供了新的见解。