The PI3K/AKT/mTOR pathway is frequently deregulated in breast cancer. Specific inhibition of its key signaling nodes results in cell death and tumor growth arrest, but also in activation of feedback loops that ultimately limit therapeutic activity.
The subcellular localization and activity of Forkhead box O3 (FOXO3) transcription factor is heavily regulated by the PI3K pathway. Phosphorylation of FOXO3 by AKT inhibits its transcriptional functions leading to cell survival and growth.
Although our understanding of the transcription function of FOXO3 downstream of the PI3K pathway has increased significantly in recent years, it is still unclear how FOXO3 acts in concert with other cofactors of the transcription machinery.
We conjectured that inhibition of the PI3K/AKT/mTOR signaling by specific agents targeting the pathway at different nodes leads to unique FOXO3-mediated transcriptional programs unveiling a complex network of regulation. We therefore sought to identify proteins that differentially co-immunoprecipitate with FOXO3 upon PI3K, AKT or mTOR blockade.
To target the pathway at different levels, we used BYL719, a PI3Kα inhibitor; MK2206, an allosteric AKT inhibitor; and RAD001, an allosteric mTORC1 inhibitor. Upon treatment, we immunopreciptated endogenous FOXO3 and searched for FOXO3 interactors by mass spectrometry analysis. In parallel RNA samples from each treatment were analyzed using gene expression microarrays.
Gene expression analyses revealed that each specific compound originated distinct transcriptional signatures. Accordingly, mass spectrometry analysis revealed novel FOXO3 co-interactors engaging in different transcriptional complexes at each treatment conditions. We identified some of these proteins as CUL1, the core component of the SCF (SKP1-CUL1-F-BOX protein) E3 ubiquitin-protein ligase complex, the cyclin-dependent kinase 5 (CDK5), the upstream binding transcription factor (UBTF), and the core component of RNA polymerase I POLR1C (polymerase RNA I polypeptide C). We performed coimmunoprecipitation analyses to confirm that these cofactors are bona fide FOXO3-interacting proteins. We then analyzed the effects of overexpressing these proteins on FOXO3 transcriptional activity. Luciferase assays demonstrated an increase in FOXO3 transcriptional activity when CUL1, UBTF, CDK5 or POLR1C were overexpressed. The overexpression of cofactors also induced the expression of FOXO3 target genes such as TRAIL or IRS2. Furthermore, overexpression of these co-interactors enhanced the binding of FOXO3 to its target gene promoters. We are now engaged in both elucidating how these cofactors can modulate the transcriptional activity of FOXO3 upon PI3K/AKT/mTOR inhibition and understanding the therapeutic implications of these FOXO3-containing complexes.
Citation Format: Vasilis Hristidis, James Cownie, Ana Bosch, Joanne Soong, Pau Castel, Maurizio Scaltriti, Baselga José. Mechanisms of transcriptional regulation of FOXO3 by cofactors upon PI3K/AKT/mTOR inhibition. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2868.