• Estimating the Rate of FOXO1 Phosphorylation and Dephosphorylation Using Live Cell Imaging

      Paek, Andrew L.; Bellomo, Dante Anthony; Weinert, Ted A.; Capaldi, Andrew P. (The University of Arizona., 2020)
      FoxO1 is a signaling transcription factor regulated by the growth factor/PI3K/AKT pathway. Phosphorylation of FOXO1 by the serine/threonine kinase AKT, sequesters FOXO1 in the cytoplasm by blocking the interaction of FOXO1’s nuclear localization signal (NLS) with nuclear transport receptors and promoting FOXO1 binding to the cytoplasmic 14-3-3 proteins. Dephosphorylation of FOXO1 by the phosphatase PP2A restores NLS function and leads to accumulation of FOXO1 in the nucleus. Here, we use fluorescently labeled FOXO1 to characterize its nuclear trafficking dynamics under conditions of AKT and PP2A inhibition in order to describe the relative cytoplasmic dephosphorylation rate by PP2A and relative nuclear phosphorylation rate by AKT on FOXO1 respectively. Measured results affirm previous data that indicates AKT is less active in the nucleus than the cytoplasm and suggests that FOXO1 may undergo rapid shuttling into and out of the nucleus even during AKT activation.
    • Understanding the Mechanism of Cancer Therapeutic SAH5-EJ1 in Targeting Breast Cancer Metastasis

      Schroeder, Joyce A.; Soyfer, Eli Michael; Paek, Andrew L.; McEvoy, Justina D. (The University of Arizona., 2020)
      While many breast cancer subtypes overexpress members of the ERBB family of receptor tyrosine kinases (including the Epidermal Growth Factor Receptor/EGFR, HER2, ERBB3 and ERBB4), only HER2 has been effectively targeted. Evidence indicates that established therapeutics against EGFR targeting either the extracellular domain or the kinase domain fail due to unique biological activities of the receptor in breast cancer. In light of this, a stapled peptide mimicking the EGFR juxtamembrane domain (SAH5-EJ1) was developed and found to induce complete tumor regression in a model of inflammatory breast cancer (SUM149/NODSCID). SAH5-EJ1 was found to induce both necrosis and apoptosis through calcium and ROSdependent mechanisms, but the mechanism by which this was achieved was unknown. In the current study, we have evaluated EGFR-dependent calcium signaling as a means to promote cell death. We have discovered that SAH5-EJ1-induced cell death is dependent upon expression of both EGFR and the TRPV1 (transient receptor potential cation channel subfamily V member 1) calcium channel located on the plasma membrane. Mechanistically, SAH5-EJ1induces the activation of TRPV1, resulting in a dramatic influx of extracellular calcium. This is followed by a sharp rise in cellular reactive oxygen species (ROS) into the cell and induction of cell death. These data demonstrate a reliance of EGFR on calcium signaling in breast cancer survival, one which can be effectively targeted in breast cancer.