MEDIATING THE mTORC2-DEPENDENT SURVIVAL RESPONSE TO UVB EXPOSURE THROUGH NEGATIVE REGULATION OF THE TUMOR SUPRESSOR FOXO3a IN KERATINOCYTES
Open Access
- Author:
- Feehan, Robert Patrick
- Graduate Program:
- Biomedical Sciences
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- February 22, 2017
- Committee Members:
- Lisa M. Shantz, PhD, Dissertation Advisor/Co-Advisor
Lisa M. Shantz, PhD, Committee Chair/Co-Chair
Scot R. Kimball, PhD, Committee Member
Sarah K. Bronson, PhD, Committee Member
Diane M. Thiboutot, MD, Outside Member
Charles H. Lang, PhD, Committee Member - Keywords:
- UVB
Skin Cancer
mTOR
FOXO3a
Keratinocyte
Apoptosis - Abstract:
- Ultraviolet B (UVB) radiation, the primary cause of non-melanoma skin cancer (NMSC), activates the rapamycin-sensitive mammalian target of rapamycin complex 1 (mTORC1) and the rapamycin-resistant mTORC2. Our lab has demonstrated that UVB-induced activation of mTORC2 controls tumor initiation by enhancing the survival of damaged cells. FOXO3a is an important regulator of apoptosis and tumor suppressor in several types of cancer and is negatively regulated by mTORC2. Importantly, 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 inhibitors and shRNA targeting FOXO3a and components of mTORC2 in cells exposed to an apoptotic dose of UVB. We established that loss of mTORC2 causes FOXO3a to be localized to the nucleus and sensitizes cells to UVB-induced apoptosis. Furthermore, this sensitization was rescued by knockdown of FOXO3a. Additionally, we showed that inhibiting mTORC2 increases the FOXO3a-dependent protein expression of TRAIL and the BH3-only protein NOXA, suggesting activation of both extrinsic and intrinsic apoptosis. Using CRISPR/CAS9 technology to target TRAIL, as well as lentiviral shRNA against both TRAIL and NOXA, we found that only knockdown of NOXA is able to rescue the increased apoptosis following mTORC2-inhibition in a manner similar to that of FOXO3a. The Regulated in DNA damage and Development 1 (REDD1) protein is a stress response protein that is activated by DNA damage and negatively regulates mTORC1, though the exact mechanism remains unclear. Importantly, the role of REDD1 in UVB-induced apoptosis has not been studied. To investigate this question, we used human keratinocyte HaCaT cells with REDD1 knockout via CRISPR-CAS9 and exposed them to an apoptotic dose of UVB. Chemical inhibitors of mTOR signaling were used to study the relationship of mTOR/AKT to REDD1. The UVB-induced apoptotic response was analyzed a by immunoblot analysis of apoptotic proteins and levels of apoptosis were quantified via annexin V/7-AAD FACS analysis. Notably, REDD1 knockout sensitized cells to UVB-induced apoptosis, which was surprisingly independent of both mTOR complexes. Taken together, these studies add to our understanding of the multi-faceted roles of both mTORC2 and REDD1 in UVB-induced apoptosis.