Induction of Mxi1-SRα by FOXO3a contributes to repression of Myc-dependent gene expression

Forkhead transcription factors of the O class (FOXOs) are important targets of the phosphatidylinositol 3-kinase (PI3-kinase)/Akt pathway. FOXOs have been implicated in the regulation of cell cycle progression, oxidative stress resistance, and apoptosis. Using DNA microarrays, we analyzed the transcriptional response to FOXO3a activation by gene expression analysis in DLD-1 colon cancer cells stably expressing a FOX03a. A3-ER fusion protein. We found that activation of FOX03a resulted in repression of a number of previously identified Myc target genes. Furthermore, FOX03a activation induced expression of several members of the Mad/Mxd family of transcriptional repressors, most notably Mxi1. The induction of Mxi1 by FOX03a was specific to the Mxi1-SRa isoform and was mediated by three highly conserved FOXO binding sites within the first intron of the gene. Activation of FOX03a in response to inhibition of Akt also resulted in activation of Mxi1-SRa expression. Silencing of Mxi1 by small interfering RNA (siRNA) reduced FOX03a-mediated repression of a number of Myc target genes. We also observed that FOX03a activation induced a switch in promoter occupancy from Myc to Mxi1 on the E-box containing promoter regions of two Myc target genes, APEX and FOXM1. siRNA-mediated transient silencing of Mxi1 or all Mad/Mxd proteins reduced exit from S phase in response to FOX03a activation, and stable silencing of Mxi1 or Mad1 reduced the growth inhibitory effect of FOX03a. We conclude that induction of Mad/Mxd proteins contributes to the inhibition of proliferation in response to FOX03a activation. Our results provide evidence of direct regulation of Mxi1 by FOX03a and imply an additional mechanism through which the PI3-kinase/Akt/FOXO pathway can modulate Myc function.