Abstract 27: The role of the mTORC2-dependent regulation of FOXO3a in UVB-induced apoptosis

The greatest environmental risk factor for developing non-melanoma skin cancer (NMSC) is ultraviolet B (UVB) radiation. Both complexes of the mammalian target of rapamycin (mTOR) signaling pathway, the rapamycin-sensitive mTOR complex 1 (mTORC1) and the rapamycin-resistant mTOR complex 2 (mTORC2), are stimulated in response to UVB. Cell survival in UVB-induced keratinocyte carcinogenesis has been shown to be mTORC2-depedent. The tumor suppressor FOXO3a, a member of the Forkhead box family of proteins, is an important transcription factor involved in regulating cell survival. FOXO3a activity is attenuated as a result of AKT activation, which is downstream of mTORC2. UVB has been shown to activate AKT through the mTORC2 pathway; however, the role of FOXO3a in UVB-induced apoptosis in keratinocytes has yet to be studied. To investigate the role of mTORC2 and FOXO3a in UVB-induced apoptosis, we utilized both human keratinocytes (HaCaT cells) as well as inducible Rictor-deficient mouse embryonic fibroblasts (iRictKO cells) following treatment with 4-hydroxy-tamoxifen (4OHT). We also employed the use of inhibitors and shRNA targeting Rictor or mSIN1. The Rictor and mSIN1 proteins are key structural components unique to mTORC2 and their removal dramatically lowers mTORC2 activity. Finally, we compared the effects of shRNA targeting FOXO3a with the expression of a constitutively active mutant FOXO3a. For all the experiments described, cells were exposed to an apoptotic dose of UVB and allowed to incubate for defined experimentally established intervals. Our results show that there is an mTORC2-dependent regulation of FOXO3a expression, localization, and activity upon UVB-irradiation. Through western analysis we demonstrate that UVB exposure promotes the phosphorylation of FOXO3a by AKT in cells with intact mTORC2 signaling, which is repressed when mTORC2 is inhibited. This phosphorylation has been shown in other systems to reduce FOXO3a activity by causing a cytoplasmic localization of the protein. In agreement with this, we observed a rapid cytoplasmic localization of FOXO3a and decreased FOXO3a promoter activation in response to UVB, which is rescued following disruption of mTORC2 signaling. Additionally, we find that FOXO3a protein expression is reduced upon UVB-irradiation when mTORC2-signaling is reduced. These data support previous findings that suggest the mTORC2-driven FOXO3a cytoplasmic sequestration protects FOXO3a from dephosphorylation and degradation. Taken together, these studies provide strong evidence that mTORC2 plays a critical role in regulating FOXO3a expression, localization and activity following UVB-irradiation. In addition, our results support the hypothesis that the mTORC2-dependent UVB-induced apoptosis is due at least in part to FOXO3a. Supported by ES19242 (LMS) Citation Format: Robert P. Feehan, Lisa M. Shantz. The role of the mTORC2-dependent regulation of FOXO3a in UVB-induced apoptosis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 27. doi:10.1158/1538-7445.AM2015-27