PROJECT SUMMARY Disorders of the esophagus are significant health problems in the U.S. and throughout the world, and esophageal cancer, of which more than 80% is esophageal squamous cell cancer (ESCC), is the 6th most common cause of cancer death worldwide. Exposure of esophageal squamous epithelial cells (keratinocytes) to injurious agents such as cigarette smoke and alcohol predisposes to ESCC, and yet malignant transformation of a single esophageal keratinocyte even in response to such stressors is rare. Thus, important cytoprotective mechanisms must exist in normal esophageal keratinocytes to respond to these insults and prevent malignant transformation of these cells. To date, these mechanisms are not well understood. Here, we propose to delineate important cytoprotective pathways in esophageal keratinocytes, focusing on the key transcriptional regulator Krüppel-like factor 5 (KLF5) and the tumor suppressor p53 in the response to physiologic stress. In normal epithelia, KLF5 functions to promote proliferation and migration and to inhibit inflammation, and in new preliminary data, we define critical functions for KLF5 and wild-type p53 in the cellular responses to exogenous stress in non-transformed esophageal keratinocytes and demonstrate that mutant p53 modulates genome-wide binding of KLF5, thereby altering the targets and pathways governed by KLF5 in this context. Our overarching hypothesis is that KLF5 and p53 are a molecular rheostat, coordinately regulating esophageal squamous epithelial responses to exogenous stressors, and that disruption of this regulation underlies defective cell repair and ESCC. To test this hypothesis, we will pursue the following interrelated Specific Aims: 1. We will define KLF5-p53 targets and function in normal keratinocytes with exogenous stress; 2. We will determine mutant p53 alterations of genome-wide KLF5 binding in homeostasis and stress; 3. We will delineate the functions of KLF5 and p53 in esophageal mucosal injury resulting from smoking and alcohol. Overall, the proposed studies provide a framework to understand the mechanisms by which normal esophageal keratinocytes respond to environmental stresses and the perturbations of these responses that underlie malignant transformation and progression in the squamous esophagus.