Recently, the FOXO (Forkhead box, class O) subfamily of Forkhead transcription factors has been identified as direct targets of phosphoinositide 3-kinase-mediated signal transduction. The AFX (acute-lymphocytic-leukaemia-1 fused gene from chromosome X), FKHR (Forkhead in rhabdomyosarcoma) and FKHR-L1 (FKHR-like 1) transcription factors are directly phosphorylated by protein kinase B, resulting in nuclear export and inhibition of transcription. This signalling pathway was first identified in the nematode worm Caenorhabditis elegans, where it has a role in regulation of the life span of the organism. Studies have shown that this evolutionarily conserved signalling module has a role in regulation of both cell-cycle progression and cell survival in higher eukaryotes. These effects are co-ordinated by FOXO-mediated induction of a variety of specific target genes that are only now beginning to be identified. Interestingly, FOXO transcription factors appear to be able to regulate transcription through both DNA-binding-dependent and -independent mechanisms. Our understanding of the regulation of FOXO activity, and defining specific transcriptional targets, may provide clues to the molecular mechanisms controlling cell fate decisions to divide, differentiate or die.

The binding of insulin to its receptor initiates multiple signal transduction pathways regulating such diverse processes as proliferation, di erentiation, glucose transport, and glycogen metabolism. The STAT-family of transcription factors has been demonstrated to play a critical role in gene induction by a variety of hemopoietic cytokines and hormones. Furthermore, constitutive activation of STATs is observed in transformed cells. Here we describe activation of a transcriptional complex binding to a consensus STAT-transcriptional element in response to insulin challenge. This complex is induced rapidly after tyrosine autophosphorylation of the insulin receptor, and is sustained for several hours. Supershift analysis of the insulin-induced complex reveals that it speci®cally contains the transcription factor Stat3. DAN binding of this complex is inhibited by pre-incubation with tyrosine, but not serine/threonine protein kinase inhibitors, whereas transcriptional activation is inhibited by both. Utilising a dominant negative mutant of p21ras we demonstrate that both insulin-induced Stat3 DNAbinding and also transactivation do not require p21ras. Furthermore, although previous studies have suggested a role for MAP kinases (ERKs) and PI-3K in STAT activation, utilising the speci®c MEK inhibitor PD098059 and the PI-3K inhibitor wortmannin, we demonstrate that activation of ERKs or PI-3K are not required for insulin induced Stat3 phosphorylation or transactivation.

Transcriptional activity of Forkhead box transcription factor class O (FOXO) proteins can result in a variety of cellular outcomes depending on cell type and activating stimulus. These transcription factors are negatively regulated by the phosphoinositol 3-kinase (PI3K)-protein kinase B (PKB) signaling pathway, which is thought to have a pivotal role in regulating survival of tumor cells in a variety of cancers. Recently, it has become clear that FOXO proteins can promote resistance to anti-cancer therapeutics, designed to inhibit PI3K-PKB activity, by inducing the expression of proteins that provide feedback at different levels of this pathway. We questioned whether such a feedback mechanism may also exist directly at the level of FOXO-induced transcription. To identify critical modulators of FOXO transcriptional output, we performed gene expression analyses after conditional activation of key components of the PI3K-PKB-FOXO signaling pathway and identified FOXP1 as a direct FOXO transcriptional target. Using chromatin immunoprecipitation followed by next-generation sequencing, we show that FOXP1 binds enhancers that are pre-occupied by FOXO3. By sequencing the transcriptomes of cells in which FOXO is specifically activated in the absence of FOXP1, we demonstrate that FOXP1 can modulate the expression of a specific subset of FOXO target genes, including inhibiting expression of the pro-apoptotic gene BIK. FOXO activation in FOXP1-knockdown cells resulted in increased cell death, demonstrating that FOXP1 prevents FOXO-induced apoptosis. We therefore propose that FOXP1 represents an important modulator of FOXO-induced transcription, promoting cellular survival.