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：
10659948
负责人：
DIANE L BARBER
金额：
$ 40.38万
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 2028-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10659948
关键词：
3-Dimensional Address Adult Affinity Binding Binding Proteins Biophysics Biopsy Biosensor Cell membrane Cells Cellular biology ChIP-seq Charge Clinical Colorectal Cancer Colorectal Neoplasms DNA DNA Binding DNA Binding Domain DNA-Binding Proteins Data Disease Progression Dissociation Drosophila genus Electrostatics Epithelium Evolution FOXC2 gene Family Fluorescence Anisotropy Funding Gene Expression Gene Expression Regulation Genes Genetic Heterogeneity Histidine Human Hydrogen Bonding Immunologic Surveillance Invaded Ligands Malignant Neoplasms Mediating Metabolic Molecular Normal Cell Nuclear Nucleic Acids Nucleotides Organoids Outcome Pathway interactions Phenotype Phospholipids Population Post-Translational Protein Processing Property Proteins Recombinant DNA Reporter Reporting Role SOX4 gene Somatic Mutation Specificity Testing Therapeutic Time Tissues Titrations Tumor Suppressor Proteins Work anticancer research cancer cell cell behavior colon cancer patients deprotonation design genome-wide in vivo in vivo imaging insight mouse model neoplastic cell novel therapeutic intervention preference prognostic value protonation serial imaging single-cell RNA sequencing stem cell differentiation stem cells transcription factor transcriptomics tumor tumor heterogeneity tumor metabolism tumor progression tumorigenesis

项目摘要

Abstract Although cancers have a constitutively increased intracellular pH (pHi) that enables disease progression, the molecular mechanisms mediating pHi-dependent cancer cell behaviors are understudied and incompletely understood. During the funding period we bridged protein electrostatics and cell biology to identify molecular mechanisms for pHi regulating tumorigenesis and metabolic reprogramming, and for conferring cancer- promoting functions of somatic mutations encoding charge changes in proteins. Our competing renewal applies our expertise in new directions to address two gaps in our understanding of pHi dynamics and cancer. First is how pHi dynamics can directly regulate gene expression. Our previous work resolved how pHi dynamics can regulate protein-phospholipid and protein-protein binding; however, how pHi dynamics can regulate protein-DNA binding, despite nuclear and cytosolic pH being similar, remains unknown. In Aim 1 we test a new idea on pHi titration of a histidine in the DNA-binding domain of transcription factors that forms hydrogen bonds with nucleotides in conferring target gene selectivity. This idea is applicable to at least 65 transcription factors with a nucleotide-binding histidine. Focusing on three transcription factors from different families, FOXC2, SOX4 and MAX that have roles in cancers, we will test the hypothesis that dysregulated pHi dynamics in cancer cells contributes to transcription factor-DNA binding selectivity for enabling cancer cell behaviors. We will resolve pH regulated affinities of DNA binding domains to recognized DNA motifs, identify pH-dependent genome-wide binding preferences, and determine pH regulated transcription factor-DNA binding selectivity in cancer cells. Additionally, we apply our predictions on pHi regulated selectivity of transcription factor-DNA binding by testing targeting the nucleotide-binding histidine to reactivate a tumor suppressor pathway in cancer cells. A second gap in our understanding is the role of pHi dynamics and heterogeneity in tumorigenesis. Based on our preliminary data of intercellular pHi heterogeneity in clonal cancer cell spheroids and organoids of cancer cells from human biopsies, and established roles for pHi dynamics in epithelial plasticity, cell migratory capacity, and stem cell differentiation, in Aim 2 we will test the hypothesis that pHi heterogeneity distinguishes tumor cell phenotypic heterogeneity. We will determine whether pHi heterogeneity reflects distinct cell phenotypes and identities in spheroids of clonal colorectal cancer cells and organoids derived from biopsies of human colorectal tumors, by transcriptomics profiles generated by single cell RNA-seq and genetically changed pHi. To resolve pHi dynamics and heterogeneity during tumorigenesis we use a Drosophila line we generated expressing a genetically encoded pHi biosensor that allows longitudinal imaging of pHi dynamics in vivo, which cannot be achieved with mouse models. Significant outcomes of our studies include showing for the first time that pHi can regulate transcription factor-DNA binding selectivity, new insights on emergent tumor cell properties, and clinical promise for targeting cancer cell pHi for prognostic value and selective delivery of therapeutics.

抽象的尽管癌症的细胞内 pH (pHi) 持续升高，导致疾病进展，但介导 pHi 依赖性癌细胞行为的分子机制尚未得到充分研究且不完整明白了。在资助期间，我们将蛋白质静电学和细胞生物学联系起来，以识别分子 pHi 调节肿瘤发生和代谢重编程以及赋予癌症的机制促进编码蛋白质电荷变化的体细胞突变的功能。我们的竞争性续订适用我们在新方向上的专业知识可以弥补我们对 pHi 动力学和癌症理解上的两个差距。首先是 pHi 动力学如何直接调节基因表达。我们之前的工作解决了 pHi 动力学如何调节蛋白质-磷脂和蛋白质-蛋白质结合；然而，pHi 动力学如何调节蛋白质-DNA 尽管核和胞质 pH 值相似，但结合仍然未知。在目标 1 中，我们测试了关于 pHi 的新想法滴定转录因子 DNA 结合域中的组氨酸，与形成氢键核苷酸赋予靶基因选择性。这个想法适用于至少 65 个转录因子核苷酸结合组氨酸。重点关注来自不同家族的三种转录因子：FOXC2、SOX4 和 MAX 在癌症中发挥作用，我们将检验癌细胞中 pHi 动态失调的假设有助于转录因子-DNA 结合选择性，从而实现癌细胞行为。我们将解析 DNA 结合域与已识别 DNA 基序的 pH 调节亲和力，识别 pH 依赖性全基因组结合偏好，并确定 pH 调节的转录因子-DNA 结合选择性癌细胞。此外，我们将我们的预测应用于 pHi 调节转录因子-DNA 的选择性通过测试靶向核苷酸结合组氨酸来重新激活癌症中的肿瘤抑制途径细胞。我们理解中的第二个差距是 pHi 动力学和异质性在肿瘤发生中的作用。基于基于克隆癌细胞球体和癌症类器官细胞间 pHi 异质性的初步数据人类活组织检查中的细胞，并确定了 pHi 动力学在上皮可塑性、细胞迁移能力、和干细胞分化，在目标 2 中，我们将检验 pHi 异质性区分肿瘤的假设细胞表型异质性。我们将确定 pHi 异质性是否反映了不同的细胞表型克隆性结直肠癌细胞球体和源自人类活组织检查的类器官的特性结直肠肿瘤，通过单细胞 RNA-seq 和基因改变的 pHi 生成的转录组学图谱。到解决肿瘤发生过程中的 pHi 动力学和异质性，我们使用我们生成的表达表达的果蝇系一种基因编码的 pHi 生物传感器，可以对体内 pHi 动态进行纵向成像，这是无法实现的通过小鼠模型实现。我们研究的重大成果包括首次表明 pHi 可以调节转录因子-DNA 结合选择性，对新兴肿瘤细胞特性和临床的新见解有望针对癌细胞 pHi 提供预后价值和选择性治疗。