Monday, July 5, 2021
7/5/2021
PROJECT SUMMARY/ABSTRACT FOXO transcription factors are regulators of cellular homeostasis that are activated in response to a variety of environmental stresses. Upon activation, FOXOs control the expression of genes in multiple and often conflicting cellular processes. For example, FOXOs activate the expression of transcriptional programs involved in cell death but also in cytoprotective processes like DNA repair and cell-cycle arrest. How FOXOs determine which of these outcomes a cell will undergo is not known. Cells often utilize a single transcription factor to “decide” between multiple, often opposing cellular outcomes, as seen with FOXO transcription factors. Previous single cell studies in the p53 and NF-kB transcription factor systems have revealed that the dynamics of these transcription factors, or how their levels or location change over time, following a stimulus controls which outcome the cells enact. Like p53 and NF-kB, FOXO has the same multiple input and multiple output motif, making it a good candidate for a dynamically regulated system. FOXO transcription factors are largely regulated by subcellular location. In unstressed conditions FOXOs are located in the cytoplasm rendering them inactive. Cell stresses like growth factor depletion or DNA damage lead to shuttling of FOXOs into the nucleus where they actively transcribe target genes. In this proposal, we will use live fluorescence microscopy of single cells to elucidate whether FOXO nuclear/cytoplasmic shuttling dynamics can induce different cellular outcomes in cancer cells. In the first aim, we will take advantage of the multiple cellular phenotypes (arrest and apoptosis) that arise after treating cancer cells with Epidermal Growth Factor Receptor (EGFR) tyrosine kinase inhibitors (TKIs). EGFR TKIs are clinically approved drugs used to treat patients with advanced Non-Small Cell Lung Cancers with hyperactivating mutations in EGFR; Foxo3a has been strongly implemented in both cancer cell death and arrest after treatment. We will stimulate both arrest and death outcomes in cancer cells using EGFR TKIs and determine whether each fate is correlated with a specific Foxo3a dynamic pattern. Our preliminary data has suggested that Foxo3a is pulsatile following EGFR TKI treatment. We will focus on development of an experimentally validated mathematical model to elucidate the structure of the PI3K/Akt/Foxo3a signaling network that causes Foxo3a pulses. Overall, this research will further our understanding of how protein dynamics regulate cellular fate outcomes.
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