Abstract C35: A pivotal role for FOXO4 in the regulation of cellular senescence and apoptosis

Unrepaired genomic insults can cause mutations that potentially drive malignant transformation of cells. Typically, tumorigenesis of damaged cells is restricted through activation of potent tumor suppressive mechanisms such as apoptosis or cellular senescence. Apoptotic cells are cleared from the organism, while senescent cells remain present in the organism and are permanently withdrawn from the cell cycle. Senescent cells accumulate with age, are resistant to apoptosis, and display many changes in gene expression. Unfortunately, when senescent cells acquire additional mutations (secondary hits) they can escape the growth arrest and thus essentially retain the ability to become tumorigenic. It is still unclear what makes cells decide to undergo apoptosis or enter senescence and identifying the responsible molecular pathways could aid in clinical targeting of cancer cells that have escaped senescence. Forkhead Box O (FOXO) transcription factors are tumor suppressors that are also known to promote lifespan extension. FOXOs are negatively regulated by growth factor signaling, but can be activated in response to oxidative stress, for instance caused by activated oncoges. Their role in DNA damage responses is only beginning to be unraveled. Here, we report that FOXO4 mRNA and protein expression rise significantly in response to senescence-inducing levels of DNA damage. Senescence caused by DNA damage is characterized by the formation of persistent nuclear foci termed DNA-SCARS (DNA Segments with Chromatin Alterations Reinforcing Senescence), which are required for the growth arrest. We observed that under DNA-damaging conditions FOXO4 is recruited to DNA-SCARS and that, strikingly, loss of FOXO4 expression following RNA interference induced apoptosis, rather than senescence. A major component of DNA-SCARS is the p53 tumor suppressor, which regulates cell fate after DNA damage. When phosphorylated on Ser46, p53 strongly favors apoptosis over cell cycle arrest, but Ser46 is also phosphorylated in response to several senescence-inducing stimuli such as active oncogenes. It is unclear how apoptosis is restrained in these cases. We observed that interference with the kinase responsible for Ser46-phosphorylation of p53 –HIPK2- impairs the apoptosis response caused by FOXO4 depletion in damaged cells. Melanoma is the main form of lethal skin cancer for which treatment is still limited. Oncogenic BRAF mutations (typically V600E) are found in ∼7% of all human tumors with especially high occurrence melanoma (∼70%). We have shown previously that BRAFV600E promotes FOXO4 activation, thereby promoting p21cip1-mediated senescence. Interestingly, we now observed that BRAFV600E also induces Ser46-p53 phosphorylation. Additionally, melanoma cells were found to generally have an elevated number of DNA-SCARS containing FOXO4 and, in line with the results from DNA-damaged cells, interference with FOXO4 expression in melanoma cells results in a marked increase of cell death due to apoptosis. Altogether, these results suggest that FOXO4 plays a pivotal role in restraining Ser46-p53-mediated apoptosis in favor of senescence. FOXO4 could thus provide an Achilles9 heel in the survival of melanoma cells and targeting FOXO4 expression may be clinically interesting as melanoma therapy. This work is supported by post-doctoral fellowship Buit-4649 of the Dutch Cancer Society awarded to PLJDK and NIH grant AG017242 awarded to JC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr C35.