Roles for Intracellular pH Dynamics in Cancer

摘要 虽然癌症细胞内pH(Phi)的结构性升高使疾病进展，但 调节依赖Phi的癌细胞行为的分子机制研究得不够深入和不完全 明白了。在资助期间，我们将蛋白质静电学和细胞生物学结合起来，以确定分子 PHI调节肿瘤发生和代谢重编程以及致癌的机制-- 促进编码蛋白质电荷变化的体细胞突变的功能。我们的竞争续订适用于 我们在新方向上的专业知识解决了我们对Phi动力学和癌症理解中的两个空白。首先是 Phi动力学如何直接调控基因表达。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 尽管细胞核和细胞质的pH值相似，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 与之形成氢键的转录因子DNA结合域中组氨酸的滴定 核苷酸在赋予靶基因选择性中的作用。这一想法适用于至少65个转录因子 核苷酸结合组氨酸。关注来自不同家族的三个转录因子FOXC2、SOX4和 MAX在癌症中起作用，我们将检验癌细胞中调节失调的PHI动力学的假说 有助于转录因子-DNA结合的选择性，以支持癌细胞的行为。我们会 分解pH调节的DNA结合域与识别的DNA基序的亲和力，鉴定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，我们还应用了我们对phi调节的转录因子-DNA选择性的预测 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。基座 关于克隆癌细胞球体和细胞器异质性的初步数据 以及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人活检组织中的有机体的特性 结直肠肿瘤，通过单细胞RNA-seq和基因改变的phi产生的转录图谱。至 解决肿瘤发生过程中的phi动力学和异质性我们使用我们构建的果蝇系来表达 一种基因编码的Phi生物传感器，允许在体内纵向成像Phi动态，这不可能是 用鼠标模型实现的。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，对新出现的肿瘤细胞特性的新见解，以及临床 有望靶向癌细胞phi以预测预后价值和选择性地提供治疗药物。

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.