Regulation of Forkhead BOX O tumor suppressors by Reactive Oxygen Species

The risk on developing cancer increases with age. Moreover, many processes that affect the onset of aging, such as altered proliferation, metabolism and stress resistance, are also frequently deregulated in cancer. The molecular mechanisms that prevent the onset of aging may therefore be partially related to those that suppress carcinogenesis. Reactive Oxygen Species (ROS) are produced as a byproduct of energy production. ROS are essential for cell cycle progression, however, when ROS levels rise above a threshold, the cellular interior can be damaged through oxidation of proteins and lipids and through induction of DNA breaks. Inherently, excessive ROS accelerate the onset of aging and increase the chance on tumorigenesis. FOXO (Forkhead Box O) transcription factors counteract the excess in cellular ROS by transactivating transcription of the ROS scavenging enzymes. As such, FOXO activity is associated with longevity in model organisms and humans. FOXO signaling in turn can be triggered by ROS, inducing a feed-back loop through which FOXO activation is negatively regulated. In the past, it has been recognized that in response to elevated ROS levels FOXOs can become post translationally modified (i.e. phosphorylation, acetylation and ubiquitination) and specifically interact with other proteins (e.g. ?-Catenin and p53). Nonetheless, how FOXO activity is controlled under conditions of elevated ROS is still only partially unraveled. In this thesis, we have explored the molecular mechanisms behind FOXO signaling, particularly FOXO4, in response to elevated cellular ROS. Predominantly with regard to tumor suppression, new modes of regulation are uncovered. Initially, we show that elevated cellular ROS promotes phosphorylation of FOXO4, thereby providing a docking surface for the peptidyl-prolyl isomerase Pin1. ROS-induced phosphorylation of FOXO4 enhances its activity and subsequent cell cycle arrest. However, we observed that Pin1 binding counteracts this effect. Pin1 is frequently expressed at high levels in breast tumors and indeed we observed a negative correlation with expression of a critical FOXO target gene used as a prognostic marker, p27kip1. Later on, we identify FOXOs as mediators of oncogene induced senescence (OIS), a process critical to prevent tumorigenesis upon hyperactivation of oncogenes such as BRAF. We show that signaling by oncogenic BRAF induces a chronic increase in cellular ROS levels and as a consequence promotes FOXO4 phosphorylation by the stress kinase JNK. Modification of FOXO4 by this pathway resulted in increased interaction with p53 and a p21cip1-dependent cell cycle arrest. Importantly, whereas FOXOs had thus far mainly been implicated in temporal cell cycle arrest, this mode of FOXO regulation results in OIS, a permanent state of arrest. These data characterize a role for FOXOs in the antagonistic pleiotropy between tumor suppression and aging. Finally, we show that a transcription factor related to FOXOs, FOXM1, is also a downstream target of oncogenic BRAF signaling through ROS. In contrast to FOXOs however, we show that FOXM1 is activated by Pin1. FOXM1 is associated with proliferation and our data suggest that differential regulation of FOXO4 and FOXM1 activity in response to oncogenic BRAF determines the fate of cell cycle progression

The DNA damage repair protein Ku70 interacts with FOXO4 to coordinate a conserved cellular stress response

In this study, we searched for proteins regulating the tumor suppressor and life-span regulator FOXO4. Through an unbiased tandem-affinity purification strategy combined with mass spectrometry, we identified the heterodimer Ku70/Ku80 (Ku), a DNA double-strand break repair component. Using biochemical interaction studies, we found Ku70 to be necessary and sufficient for the interaction. FOXO4 mediates its tumor-suppressive function in part through transcriptional regulation of the cell cycle arrest p27kip1gene. Immunoblotting, luciferase reporter assays, and flow cytometry showed that Ku70 inhibited FOXO4-mediated p27kip1transcription and cell cycle arrest induction by >40%. In contrast, Ku70 RNAi but not control RNAi significantly increased p27kip1transcription. In addition, in contrast to wild-type mouse embryonic stem (ES) cells, Ku70-/-ES cells showed significantly increased FOXO activity, which was rescued by Ku70 reexpression. Immunofluorescence studies demonstrated that Ku70 sequestered FOXO4 in the nucleus. Interestingly, the Ku70-FOXO4 interaction stoichiometry followed a nonlinear dose-response curve by hydrogen peroxide-generated oxidative stress. Low levels of oxidative stress increased interaction stoichiometry up to 75%, peaking at 50 μM, after which dissociation occurred. Because the Ku70 ortholog in the roundworm Caenorhabditis elegans was shown to regulate life span involving C. elegans FOXO, our findings suggest a conserved critical Ku70 role for FOXO function toward coordination of a survival program, regulated by the magnitude of oxidative damage

Activation of forkhead box O transcription factors by oncogenic BRAF promotes p21cip1-dependent senescence

Oncogene-induced senescence (OIS) is a potent tumor-suppressive mechanism that is thought to come at the cost of aging. The Forkhead box O (FOXO) transcription factors are regulators of life span and tumor suppression. However, whether and how FOXOs function in OIS have been unclear. Here, we show a role for FOXO4 in mediating senescence by the human BRAFV600E oncogene, which arises commonly in melanoma. BRAFV600E signaling through mitogen-activated protein kinase/extracellular signal-regulated kinase kinase resulted in increased reactive oxygen species levels and c-Jun NH 2 terminal kinase-mediated activation of FOXO4 via its phosphorylation on Thr223, Ser226, Thr447, and Thr451. BRAFV600E-induced FOXO4 phosphorylation resulted in p21cip1-mediated cell senescence independent of p16 ink4a or p27kip1. Importantly, melanocyte-specific activation of BRAFV600E in vivo resulted in the formation of skin nevi expressing Thr223/Ser226-phosphorylated FOXO4 and elevated p21cip1. Together, these findings support a model in which FOXOs mediate a trade-off between cancer and aging

Targeted Apoptosis of Senescent Cells Restores Tissue Homeostasis in Response to Chemotoxicity and Aging

The accumulation of irreparable cellular damage restricts healthspan after acute stress or natural aging. Senescent cells are thought to impair tissue function, and their genetic clearance can delay features of aging. Identifying how senescent cells avoid apoptosis allows for the prospective design of anti-senescence compounds to address whether homeostasis can also be restored. Here, we identify FOXO4 as a pivot in senescent cell viability. We designed a FOXO4 peptide that perturbs the FOXO4 interaction with p53. In senescent cells, this selectively causes p53 nuclear exclusion and cell-intrinsic apoptosis. Under conditions where it was well tolerated in vivo, this FOXO4 peptide neutralized doxorubicin-induced chemotoxicity. Moreover, it restored fitness, fur density, and renal function in both fast aging XpdTTD/TTD and naturally aged mice. Thus, therapeutic targeting of senescent cells is feasible under conditions where loss of health has already occurred, and in doing so tissue homeostasis can effectively be restored