Roles for Intracellular pH Dynamics in Cancer

摘要 尽管癌症具有使疾病进展成为可能的组成性增加的细胞内pH（pHi）， 介导pH依赖性癌细胞行为的分子机制研究不足且不完全 明白在资助期间，我们将蛋白质静电学和细胞生物学联系起来， pHi调节肿瘤发生和代谢重编程的机制，以及赋予癌症- 促进编码蛋白质电荷变化的体细胞突变的功能。我们的竞争性续约适用于 我们在新方向的专业知识，以解决我们在pHi动力学和癌症的理解两个差距。一是 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相似，但结合仍然未知。在目标1中，我们测试了一个关于pHi的新想法 在转录因子的DNA结合结构域中的组氨酸滴定，该组氨酸与 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。聚焦于来自不同家族的三种转录因子FOXC 2、SOX 4和 MAX在癌症中的作用，我们将测试癌细胞中失调的pHi动力学的假设， 有助于转录因子-DNA结合的选择性，使癌细胞的行为。我们将 解析pH调节的DNA结合结构域与识别的DNA基序的亲和力， 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调节的转录因子-DNA的选择性， 通过测试靶向核苷酸结合组氨酸以重新激活癌症中的肿瘤抑制途径的结合 细胞我们理解的第二个差距是pHi动力学和异质性在肿瘤发生中的作用。基于 根据我们关于克隆癌细胞球体和癌症类器官中细胞间pHi异质性的初步数据 细胞从人类活检，并建立了作用的pHi动力学上皮可塑性，细胞迁移能力， 和干细胞分化，在目标2中，我们将检验pHi异质性区分肿瘤细胞的假设。 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的球体和来自人活检组织的类器官的特性 结直肠肿瘤，通过单细胞RNA-seq和遗传改变的pHi产生的转录组学谱。到 为了解决肿瘤发生过程中的pHi动力学和异质性，我们使用了我们产生的表达 一种基因编码的pHi生物传感器，其允许体内pHi动态的纵向成像，这不能被 用小鼠模型实现。我们研究的重要成果包括首次显示pHi可以 调节转录因子-DNA结合的选择性，对紧急肿瘤细胞特性的新见解， 有望靶向癌细胞pHi，用于预后价值和选择性递送治疗剂。

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.