Analysis of FOXO transcriptional networks

Abstract

The PI3K-PKB-FOXO signalling module plays a pivotal role in a wide variety of cellular processes, including proliferation, survival, differentiation and metabolism. Inappropriate activation of this network is frequently observed in human cancer and causes uncontrolled proliferation and survival. In this thesis we have identified novel phosphorylation and transcriptional targets of the PI3K-PKB-FOXO signal transduction pathway. We identified eIF4B as a novel PKB substrate by phospho-proteome analysis after activation of PKB, thereby revealing a novel mechanism by which PKB can regulate translation. We demonstrate that phosphorylation of eIF4B by PKB on Ser 422 activates eIF4B, resulting in increased translation initiation. Furthermore, we focussed on identification of novel transcriptional targets of the FOXO transcription factors through global microarray analyses. We identified Id1, and JAK2 as FOXO transcriptional targets, which might be involved in oncogenic transformation. We demonstrate that FOXO3-induced downregulation of Id1 results in erythroid differentiation of a chronic myeloid leukemia cell line. These results indicate that inactivation of FOXO3 and consequent increased Id1 expression is critical for maintenance of the leukemic phenotype. In addition, we performed microarray analyses after inducible activation of constitutively active PI3K, PKB, FOXO3 and FOXO4 to identify novel transcriptional targets involved in survival and stress resistance. Utilising this unique “pathway” approach, we identified glutamine synthetase (GS) as being transcriptionally regulated by PI3K-PKB-FOXO signalling. Activation of PI3K or PKB results in downregulation of GS expression, while activation of either FOXO3 or FOXO4 induces an upregulation. The increase of GS expression correlates with increased GS activity and increased glutamine levels. After activation of FOXO3 the increase in glutamine levels induces autophagy, which is important for survival of the cells. These data show that the PI3K modulates glutamine metabolism and reveal a novel mechanism by which the PI3K-PKB-FOXO signalling module regulates autophagy. This might contribute to the tumour suppressive function of FOXO transcription factors through protection of cells from the build-up of cellular damage. Overall, the characterisation of these novel targets reveals previously undiscovered signalling pathways and biological functions regulated by PI3K-PKB-FOXO signalling and provides novel targets for the development of anti-cancer therapeutics.