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.

摘要 癌症和阿尔茨海默病相关痴呆(ADRD)越来越被认为是一种 调节失调的溶酶体。尽管溶酶体的功能严重依赖于管腔酸性pH~5.0 对于所含的酸激活水解酶的活性，溶酶体的pH(PHlys)是否失调，以及 癌症和ADRD中溶酶体功能受损的决定因素受到的关注有限。有了支持 从我们的父母授予的关于细胞内pH动态和癌症的CA197856，我们产生并验证了一个新的 基因编码的PHLYS生物传感器，PHLARE(pH溶酶体活性报告)，这是唯一的PHLYS传感器 可以在细胞中繁殖以进行纵向研究。我们使用PHLARE显示了显著较低的PHLYS 人类癌细胞来自不同的组织来源，与组织相比具有不同的突变特征- 匹配未转化的细胞，并确定新的药理学和遗传学方法来进行实验 换个PHLYS。在我们母公司的资助下，我们正在使用这些新的方法来确定如何 PHLys的降低使癌细胞的行为得以实现。我们的增刊将使用这些开发的新方法 通过我们的母公司拨款解决ADRDS中尚未解决的PHLYS问题，因此非常适合 不是-CA-20-019资金。我们的补充检验了ADRDS使PHlys升高的假设，这一假设 降低管腔水解酶活性和大分子分解代谢，导致增加 蛋白质聚集和神经退行性变。在目标1中，我们将量化ADRD模型中的PHLY PHLARE，包括专为丢失前颗粒蛋白和早老素1而设计的神经上皮细胞，它们是 ADRD中的决定因素与溶酶体功能受损相关，以及神经元分化为 NHCDR产生的IPSCs来自已确诊的ADRD患者和未受影响的家系突变 承运人和非承运者。在目标2中，我们将确定ADRD中调节失调的PHLys的功能后果 通过使用我们在父母资助的研究中确定的药理学和遗传学方法建立细胞模型 换个PHLYS。使用目标1中描述的ADRD相关细胞模型，我们将确定pHlys的作用 ADRDS的标志蛋白质聚集的动力学，以及神经元形态和溶酶体 ADRDS的定位和移动性受损，导致富含溶酶体的终末轴突增大 肿胀。我们还将使用延时显微镜来确定细胞存活，并使用基因编码的PHLARE 为纵向研究提供了一种新的工具。结果包括解决PHLYS的功能后果 ADRDS的动力学和溶酶体功能失调。此外，我们对PHLys在癌症中的平行研究 (父母资助)和ADRDS(补充)承诺确定新的治疗方法 调节失调的PHLys，以限制这些疾病的进展。