The greatest environmental risk factor for developing nonmelanoma 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 mTORC2, are stimulated in response to UVB. Cell survival in UVB-induced keratinocyte carcinogenesis has been shown in our previous studies 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. AKT can phosphorylate FOXO3a and this phosphorylation motif causes the chaperone protein 14-3-3 to dimerize and bind to FOXO3a. The bound complex is then sequestered into the cytoplasm, which reduces FOXO3a nuclear activity. 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. We hypothesize that UVB generates a unique anti-apoptotic response in keratinocytes as a result of post-translational modifications of FOXO3a that are dependent on mTORC2. To investigate the role of mTORC2 and FOXO3a in UVB-induced apoptosis, we will use both human keratinocytes (HaCaT cells) as well as inducible Rictor-deficient mouse embryonic fibroblasts (iRictKO cells). Disruption of mTORC2-signaling will be accomplished using mTOR pathway inhibitors and shRNA targeting mSIN1 in HaCaT cells and treatment with 4-hydroxy-tamoxifen (4OHT) in iRictKO cells. The Rictor and mSIN1 proteins are key structural components unique to mTORC2 and their removal dramatically lowers mTORC2 activity. FOXO3a protein expression will also be knocked down using shRNA. The work proposed in Aim 1 will examine the mechanisms behind mTORC2-dependent FOXO3a regulation by studying 14-3-3 dimerization, binding to FOXO3a, and localization of the 14-3-3/FOXO3a complex in response to a previously optimized apoptotic dose of UVB.
Aim 2 will investigate UVB-induced FOXO3a-dependent apoptosis using Fluorescence-activated cell sorting analysis, as well as measuring FOXO3a-specific binding and subsequent transcription of pro-apoptotic genes through Chromatin Immunoprecipitation and Real-Time Quantitative PCR, respectively. Confirmation of our hypotheses will provide strong evidence that mTORC2 plays a critical role in regulating FOXO3a expression, localization and activity following UVB-irradiation, in addition to demonstrating that the UVB-induced apoptotic response in keratinocytes is largely dependent on signaling through mTORC2 and FOXO3a.
Nonmelanoma skin cancer (NMSC), the most common of all cancers, has become an epidemic in this country primarily due to increased sun exposure. The goal of this research is to enhance our understanding of how sun exposure triggers processes within the skin that may lead to NMSC, specifically by activating signaling pathways driven by mTOR. Our research will attempt to unravel the biological mechanisms involved in mTOR activation in order to develop therapies aimed at skin cancer prevention and treatment.
|Feehan, Robert P; Shantz, Lisa M (2016) Molecular signaling cascades involved in nonmelanoma skin carcinogenesis. Biochem J 473:2973-94|
|Feehan, Robert P; Shantz, Lisa M (2016) Negative regulation of the FOXO3a transcription factor by mTORC2 induces a pro-survival response following exposure to ultraviolet-B irradiation. Cell Signal 28:798-809|