Regulation of Forkhead BOX O tumor suppressors by Reactive Oxygen Species

Abstract

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.