Increased Intracellular pH Promotes Cancer Cell Behaviors

Project Summary Constitutively increased intracellular pH (pHi) is common to most cancers regardless of tissue origin or genetic background. Increased pHi is sufficient to induce oncogenic phenotypes, including dysregulated tissue growth, dysplasia, and invasive cell migration. pH-regulated cell behaviors are mediated by changes in the protonation state of pH sensitive proteins, termed pH sensors. At the molecular level, changes in pHi can alter the protonation state of amino acid residues with pKa values near neutral, which can markedly affect protein conformation and function. By changing the protonation of multiple proteins in unison, pHi dynamics can coordinate complex cell processes. Although understudied, emerging evidence suggests that increased pHi contributes to tumorigenesis. In this proposal, we will explore how increased pHi regulates the proto-oncogene Myc, and determine how autophagic cell death is enhanced under these conditions. Aim 1 will explore pH dependent regulation of Myc. Over-expression of Myc rescues the size and patterning errors induced by increased pHi, suggesting decreased Myc abundance or activity. We will identify the domains in Myc protein that mediate pH-sensitivity, and the effects of increased pHi on Myc function. We will explore transcriptional effects of increased pHi to determine affected pathways and molecules. Aim 2 will determine regulation of autophagy at increased pHi. We will test if Myc directly regulates autophagy in Drosophila tissues using established molecular markers. We will develop our clonal mammalian cell system to test whether increased autophagy at increased pHi is conserved, and identify affected proteins. Finally, we will measure autophagic flux in tumors with dysregulated pHi to test the hypothesis that increased pHi promotes tumor growth via recycling of cellular building blocks generated by autophagy. Using our established methods in the genetic model organism Drosophila and in clonal mammalian cells, we bridge scales from tissue-level phenotypic analyses to individual pH- sensitive proteins that regulate cancer cell behaviors. Significant outcomes from our studies include new insights on how pH-regulated cellular behaviors enable tumorigenesis. Our strengths and experience linking tissue-level phenotypes to individual pH-sensitive molecules permit unique insights in this under-studied area of biology. Insights into the molecular mechanisms of pH- sensitive proteins may inform therapeutic approaches to limit tumorigenesis.