Roles for Intracellular pH Dynamics in Cancer

细胞内 pH 动态在癌症中的作用

• 批准号: 10121379
• 负责人: DIANE L BARBER
• 金额: $ 32.02万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-06-01 至 2023-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10121379
• 关键词: Acids; Address; Affect; Alzheimer' s disease related dementia; Attention; Award; Behavior; Biological Assay; Biosensor; C9ORF72; CRISPR/Cas technology; Catabolism; Cell Survival; Cell model; Cells; Charge; Clinical; Data; Dementia; Disease; Disease Progression; Equilibrium; Family; Funding; Funding Opportunities; Future; Gene Mutation; Human; Hydrolase; Image; Impairment; Individual; Kymography; Longitudinal Studies; Lysosomes; Malignant Neoplasms; Measurement; Measures; Metabolic; Microscopy; Modeling; Morphology; Mutation; Neoplasm Metastasis; Nerve Degeneration; Neurodegenerative Disorders; Neuroepithelial Cells; Neurons; Normal tissue morphology; Nutrient; Oncogenic; Organelles; Outcome; Outcome Study; PGRN gene; Pathology; Patients; Pharmacology; Presynaptic Terminals; Proteins; Proteolysis; Quality Control; Reagent; Reporter; Reporting; Role; Somatic Mutation; Specificity; Stains; Structure of retinal pigment epithelium; Swelling; Testing; Therapeutic; Time; Tissues; Work; biomass fuel; cancer cell; cell behavior; cell motility; cellular engineering; genetic approach; induced pluripotent stem cell; longitudinal analysis; loss of function; macromolecule; misfolded protein; mutation carrier; neuron loss; neurotoxic; novel strategies; novel therapeutic intervention; parent grant; presenilin-1; protein aggregation; ratiometric; sensor; time use; tool; trafficking; tumorigenic

Abstract Cancers and Alzheimer’s Disease Related Dementias (ADRDs) are increasingly recognized as diseases with dysregulated lysosomes. Although lysosome functions are critically dependent on a lumenal acidic pH of ~ 5.0 for the activity of contained acid-activated hydrolases, whether lysosome pH (pHlys) is dysregulated and a determinant for impaired lysosome functions in cancers and ADRDs has received limited attention. With support from our parent grant CA197856 on intracellular pH dynamics and cancer we generated and validated a new genetically encoded pHlys biosensor, pHLARE (pH Lysosomal Activity REporter), which is the only pHlys sensor that can be propagated in cells for longitudinal studies. We used pHLARE to show a significantly lower pHlys in human cancer cells from different tissue origins and with different mutational signatures compared with tissue- matched untransformed cells, and to identify new pharmacological and genetic approaches to experimentally change pHlys. With funding from our parent grant we are using these new approaches to determine how decreased pHlys enables cancer cell behaviors. Our supplement will use these new approaches developed through our parent grant to address unresolved questions on pHlys in ADRDs, and hence, is ideally suited for NOT-CA-20-019 funding. Our supplement tests the hypothesis that ADRDs have increased pHlys, which decreases the activity of lumenal hydrolases and macromolecular catabolism, leading to increased protein aggregation and neurodegeneration. In Aim 1 we will quantify pHlys in ADRD models expressing pHLARE, including neuroepithelial cells engineered for loss of progranulin and presenilin 1, which are determinants in ADRDs and associated with impaired lysosome function, and neurons differentiated from NHCDR-generated iPSCs derived from patients with identified ADRDs and from unaffected family mutations carriers and non-carriers. In Aim 2 we will determine functional consequences of dysregulated pHlys in ADRD cell models by using pharmacological and genetic approaches we identified in studies in our parent grant that change pHlys. Using the ADRD-associated cell models described in Aim 1 we will to determine the role of pHlys dynamics in protein aggregation, a hallmark of ADRDs, as well as neuronal morphology and lysosome localization and mobility, which are impaired in ADRDs resulting in lysosome-rich enlarged terminal axon swellings. We also will use time-lapse microscopy to determine cell survival, with genetically-encoded pHLARE allowing a new tool for longitudinal studies. Outcomes include resolving the functional consequences of pHlys dynamics and dysregulated lysosome functions in ADRDs. Additionally, our parallel studies on pHlys in cancer (parent grant) and ADRDs (supplement) have promise to identify new therapeutic approaches targeting dysregulated pHlys to limit the progression of these diseases.

抽象的 癌症和阿尔茨海默氏病有关的痴呆症（ADRD）越来越被认为是 失调的溶酶体。尽管溶酶体功能严重取决于〜5.0的液体酸性pH 对于含有酸激活的水解酶的活性，溶酶体pH（Phlys）是否失调并且A 癌症和ADRD中溶酶体功能受损的决定因素受到了有限的关注。支持 从我们的父母赠款CA197856关于细胞内pH动力学和癌症的授予 遗传编码的Phlys生物传感器Phlare（pH溶酶体活性报告基因），这是唯一的phlys传感器 可以在细胞中传播纵向研究。我们使用phlare在 与组织相比 匹配未转换的细胞，并确定实验的新药物和遗传方法 更改phlys。在我们父母赠款的资助下，我们正在使用这些新方法来确定如何 减少的phlys促进了癌细胞行为。我们的补充将使用这些新方法开发 通过我们的父母赠款来解决有关ADRDS中的phlys的未解决问题，因此非常适合 非CA-20-019资金。我们的补充测试了ADRD增加的假设，这是 降低腔水解酶和大分子分解代谢的活性，导致增加 蛋白质聚集和神经退行性。在AIM 1中，我们将在表达ADRD模型中量化phlys Phlare，包括针对损失程序努力蛋白和Presenilin 1的神经上皮细胞，是 确定ADRD并与溶酶体功能受损相关，而神经元与 NHCDR生成的IPSC来自具有鉴定ADRD的患者和未受影响的家庭突变的患者 运营商和非载体。在AIM 2中，我们将确定ADRD失调的PHLYS的功能后果 通过使用药物和遗传学方法，我们在父母授予的研究中发现的细胞模型 更改phlys。使用AIM 1中描述的ADRD相关细胞模型，我们将确定Phlys的作用 蛋白质聚集的动力学，ADRD的标志，以及神经元形态和溶酶体 本地化和迁移率，在ADRD中受损，导致富含溶酶体的末端轴突扩大 肿胀。我们还将使用延时显微镜确定细胞存活，并用遗传编码的Phlare 允许使用纵向研究的新工具。结果包括解决Phlys的功能后果 ADRD中的动力学和失调的溶酶体功能。此外，我们对癌症phlys的平行研究 （父母赠款）和ADRD（补充）有望确定针对目标的新治疗方法 失调的phlys以限制这些疾病的进展。