We obtained the majority of commercially available esophageal adenocarcinoma cell lines. With minimal data about the relative contribution of glycolysis and mitochondrial respiration, we are utilizing the Seahorse machine (which measures oxygen utilization and acid production by a living cell in real time) to characterize the oxygen consumption rate (OCR) and the extracellular acidification rate (ECAR) of each cell line. The ratio of OCR to ECAR shows the cellular dependence on mitochondrial respiration versus the glycolytic pathway. The relative dependence on either pathway can be targeted for metabolic inhibition, however, the drugs targeting each pathway are different so knowledge of these differences is critical to defining the most important mechanism to target. We are further characterizing these pathways by treatment with either metformin or 2-dexyglucose (2-DG) which targets the mitochondrial transport chain or glucose uptake for glycolysis, respectively. We have shown that the inhibition of proliferation is different with metformin or 2-DG based on the ration of OCR to ECAR. The relative levels of inhibition by metformin or 2-DG can be utilized to determine the most effective synergistic strategy when the cells are treated with standard therapies such as cisplatin. p53-mut tumors have bee reported to alter the ratio of OCR to ECAR and induce a Warburg effect. To reverse the metabolic derangement, targeting the p53-mut tumors with p53 inhibitors may sensitize these cells to standard therapeutics. In order to determine whether p53-mut should be targeted, we started by sending all of the esophageal cell lines for p53 mutational analysis. With this data, we have discovered that 100% of esophageal adenocarcinoma cell lines have reported p53 mutations which is in contrast to patient specimens. In addition, there are several different p53 mutations. Therefore, we are developing a p53 inducible system by removing two different p53-mut genes with CRISPR technology. We will reintroduce the wildtype p53 genes into the knockout cells. Once this model is developed, we will explore the effects of the wildtype and mutant p53 cells on cellular respiration. Additionally, we will assess the efficacy of p53 inhibition on these cells.
