Roles for Intracellular pH Dynamics in Cancer

细胞内 pH 动态在癌症中的作用

基本信息

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
项目类别：
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&apos 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

项目摘要

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病相关痴呆 (ADRD) 越来越被认为是与癌症和阿尔茨海默病相关的痴呆症 (ADRD) 尽管溶酶体功能严重依赖于约 5.0 的腔内酸性 pH 值。对于所含酸激活水解酶的活性，溶酶体 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。来自我们关于细胞内 pH 动态和癌症的母基金 CA197856，我们生成并验证了一个新的基因编码的 pHlys 生物传感器 pHLARE（pH 溶酶体活性报告仪），这是唯一的 pHlys 传感器可以在细胞中繁殖进行纵向研究，我们使用 pHLARE 来显示 pHlys 显着降低。与组织相比，来自不同组织来源且具有不同突变特征的人类癌细胞匹配未转化的细胞，并通过实验确定新的药理学和遗传学方法在我们家长拨款的资助下，我们正在使用这些新方法来确定如何改变 pHlys。 pHlys 能够促进癌细胞行为。我们的补充剂将使用这些开发的新方法。通过我们的家长资助来解决 ADRD 中有关 pHlys 的未解决问题，因此非常适合 NOT-CA-20-019 资金。我们的补充测试了 ADRD 增加 pHlys 的假设，该假设降低腔内水解酶和大分子分解代谢的活性，导致增加在目标 1 中，我们将量化表达 ADRD 模型中的 pHlys。 pHLARE，包括经过改造而失去颗粒体蛋白前体和早老蛋白 1 的神经上皮细胞，它们是 ADRD 中的决定因素并与溶酶体功能受损相关，并且神经元分化为 NHCDR 生成的 iPSC 源自已识别 ADRD 的患者和未受影响的家族突变在目标 2 中，我们将确定 ADRD 中 pHlys 失调的功能后果。通过使用药理学和遗传学方法的细胞模型，我们在母基金的研究中确定了使用目标 1 中描述的 ADRD 相关细胞模型，我们将确定 pHlys 的作用。蛋白质聚集的动态（ADRD 的标志）以及神经元形态和溶酶体定位和移动性在 ADRD 中受损，导致富含溶酶体的末端轴突增大我们还将使用延时显微镜通过基因编码的 pHLARE 来确定细胞存活率。允许使用新工具进行纵向研究，结果包括解决 pHlys 的功能后果。此外，我们还对癌症中的 pHlys 进行了平行研究。（家长资助）和 ADRD（补充）有望确定新的治疗方法 pHlys 失调可限制这些疾病的进展。