Complex regulation of Forkhead Box O transcription factors

Abstract

A complex network of signaling pathways mediates the balance between FOXO activation and inactivation. Loss of the tumor suppressive function of FOXOs as a result of proteasomal-mediated degradation and cytoplasmic localization often occurs in cancer cells. Therefore, deciphering the signaling pathways that regulate FOXO localization and degradation is important and may lead to the identification of new therapeutic targets for cancer. This thesis provides further molecular insight in the signaling pathways that regulate FOXO activity and localization. In Chapter 2, we show how the small GTPase RALA is involved in FOXO activation. We describe RALA-dependent formation of a scaffold complex consisting of RLF-JIP1-MLK3-MKK4 and JNK, which is necessary to mediate JNK-dependent FOXO activation. We also show evolutionary conservation of RALA/JIP1-mediated FOXO activation in C. elegans. Furthermore in Chapter 3 we show that next to JNK-mediated phosphorylation of FOXO, RALA is also important in regulation of its ubiquitination. It mediates ubiquitination of both FOXO4 and p53 and in addition RALA seems involved in HDMX protein abundance. HDMX functions together with HDM2 as an E3 ligase complex to regulate ubiquitination of p53. Our data suggest that RALA plays a role in regulation of the HDMX-HDM2 complex formation. In Chapter 4 we used a quantitative proteomics approach to determine the interactomes of FOXO1, FOXO3 and FOXO4 to shed light on their different behavior. This approach allows comparison of the different interaction partners and we discuss possible improvements on the method we used, one of which we applied in Chapter 6. In Chapter 5 we discuss the current literature on CCM1 and propose a dual role for CCM1 in regulation of both adhesion and transcription. In Chapter 6 we show that indeed CCM1 can act as a transcriptional co-regulator. We demonstrate that CCM1 can bind to FOXO1, FOXO3 and FOXO4 and that localization of FOXO correlates with CCM1 localization to the same compartment. In addition, this nuclear CCM1-FOXO complex interacts with a SWI/SNF chromatin remodeling complex. This SWI/SNF complex contains ARID1A and the histone deacetelyse HDAC1, suggesting a role in chromatin condensation and subsequent transcriptional repression. Indeed, our experiments in Chapter 6 show an inhibitory role of CCM1 towards FOXO induced changes in transcription. In Chapter 7 we summarize and discuss the work described in this thesis and propose future research directions.