Roles for Intracellular pH Dynamics in Cancer

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.