C/EBPALPHA, G1 CHECKPOINT AND UVB-INDUCED DNA DAMAGE

• Sponsoring Institution: State Agricultural Experiment Station
• Project Status: COMPLETE
• Funding Source: STATE
• Reporting Frequency: Annual
• Accession No.: 0219959
• Project Start Date: Oct 1, 2009
• Project End Date: Jul 1, 2013
• Program Code: [(N/A)]- (N/A)

Recipient Organization
NORTH CAROLINA STATE UNIV
(N/A)
RALEIGH,NC 27695

Performing Department
Toxicology

Non Technical Summary
Solar (UVB) radiation is responsible for approximately one million nonmelanoma skin cancer (squamous cell carcinoma (SCC) and basal cell carcinoma (BCC)) cases in the US each year making it the most common cancer in the US. Understanding the role of C/EBPa in genome maintenance and skin cancer progression will provide new insights into the mechanisms of UVB-induced skin cancer and will have important implications for the pathogenesis of other epithelial cancers where C/EBPa expression in diminished. It is becoming increasingly apparent from recent studies involving the comprehensive genomic characterization of human tumors that the presence of mutations in specific genes can vary greatly within a given cancer type. These findings suggest that identifying the origins of point mutation genetic instability and its prevention may be a more effective strategy than the chemotherapeutic intervention aimed at a single specific cancer gene as mutations in other critical genes will be enhanced and selected for by the mutator phenotype.

Animal Health Component

(N/A)

Research Effort Categories

Basic

100%

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
723	6010	1150	100%

Knowledge Area
723 - Hazards to Human Health and Safety;

Subject Of Investigation
6010 - Individuals;

Field Of Science
1150 - Toxicology;

Keywords

skin cancer

solar radiation

ultraviolet radiation

Goals / Objectives
Human epidermis is routinely subjected to DNA damage induced by solar radiation and keratinocytes have developed intricate mechanisms to respond to UVB-induced DNA damage. Previous analysis of mouse and human keratinocytes as well as the epidermis of human subjects revealed that the bZIP transcription factor, CCAAT/enhancer binding protein alpha (C/EBPa), is induced by UVB and has a role in the G1 checkpoint. Cells deficient in C/EBPa?as well as genetically modified mice lacking C/EBPa in their epidermis display an impaired G1 checkpoint in response to UVB-induced DNA damage. These mice are highly susceptible to skin tumorigenesis induced by UVB and chemical carcinogens and the benign skin tumors that develop display an accelerated rate of malignant progression to squamous cell carcinoma. The expression of C/EBPa is diminished or ablated in human skin squamous cell carcinomas and basal cell carcinomas as well mouse skin squamous cell carcinoma. Collectively these data suggest a tumor suppressor function for C/EBPa?in skin cancer. There is recent substantial support for a mutator phenotype in many types of human cancers where the mutation rate in cancer cells greatly exceeds that in normal cells. We hypothesize that diminished or ablated C/EBPa expression results in an impaired G1 checkpoint resulting in the accumulation of somatic mutations and skin cancer progression. Specific Aims; 1) Determine the molecular mechanism through which C/EBPa regulates the G1 checkpoint. 2) Characterize the identity and function of UVB-induced post-translational modifications of C/EBPa downstream of DNA damage.3) Determine whether the loss of C/EBPa contributes to a mutator phenotype.4) Provide evidence for C/EBPa as tumor suppressor in human skin cancer.

Project Methods
For specific aim 1 we will use molecular, biochemical and cellular approaches to determine whether C/EBPa interacts with p21 and whether this interaction is important in the G1 checkpoint. In specific aim 2 we will use a liquid chromatography/mass spectrometry approach to identify post-translational modifications of C/EBPa. In specific aim 3 we will use the random mutation capture assay to determine whether the loss of C/EBPa results in a mutator phenotype. Finally in specific 4 we will use a grafting model in which human skin cancer cells are grafted on to mouse skin to determine if C/EBPa expression in these cancer cells can prevent tumorigenesis.

Progress 10/01/09 to 07/01/13

Outputs
Target Audience: Other scientists that are interested in skin cancer. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Graduate students and post-docs were trained under this project How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Keratinocytes respond to UVB-induced DNA damage by engaging the DNA damage response (DDR) which involves cell cycle arrest, DNA repair and apoptosis. Defects in any one of these responses can lead to mutations and cancer. Previously, we reported that UVB radiation is a potent inducer of the basic leucine zipper transcription factor, CCAAT/enhancer binding protein alpha (C/EBPα), in human and mouse keratinocytes and in the epidermis in vivo. C/EBPα-deficient keratinocytes in culture or in vivo in mouse epidermis fail to undergo cell cycle arrest in G1 in response to UVB-induced DNA damage, thus allowing cells with damaged DNA to inappropriately enter S-phase. We observe that protein levels of the of the cyclin dependent kinase (CDK) inhibitor p21CIP1/WAF1 are similar in untreated C/EBPa-deficient and wild-type keratinocytes, and that p21 protein levels are highly induced by UVB in wild-type keratinocytes in culture and in vivo in mouse epidermis. However, UVB failed to induce p21 protein levels in C/EBPa deficient keratinocytes in culture and in C/EBPα-deficient mouse epidermis in vivo, resulting in increased CDK activity and inappropriate entry into S phase and DNA synthesis. This defect in p21 protein accumulation in response to UVB in C/EBPa-deficient keratinocytes is not due to diminished induction of CDKN1A (p21) mRNA levels but rather to decreased p21 protein stability due to the increased proteasomal degradation of p21. Add any PTM or MASS SPEC discoveries here. Thus, despite the normal transcriptional regulation of CDKN1A (p21) in response to DNA damage, it is insufficient to up-regulate p21 protein levels in C/EBPa-deficient cells due to diminished p21 stabilization, indicating a crucial role for C/EBPa in the control of p21 stability in G1 DDR following exposure to UVB radiation.

Publications

• Type: Journal Articles Status: Accepted Year Published: 2009 Citation: Ranjan, R., E.A. Thompson, K. Yoon and R.C. Smart. C/EBP? expression is regulated by C/EBP? in response to DNA damage Oncogene 28: 3235-45 2009 House, J.S., S. Zhu, R. Ranjan, K. Linder and R.C. Smart C/EBP? and C/EBP? are required for sebocyte differentiation and stratified squamous differentiation in adult mouse skin PloS ONE 5;9837 2010 Lee, S., J.D. Shuman, T. Guszczynski, K. Sakchaisri, T. Sebastian, T.D. Copeland, M. Miller, M.S. Cohen, R.C. Smart, Z. Xiao, L.R. Lu, T.D. Veenstra and P.F. Johnson. RSK-mediated phophorylation in the C/EBP? leucine zipper regulates DNA binding, dimerizations and growth arrest. Mol. Cell Bio. 11:2621-2635 2010 Omori, E., K. Matsumoto, S. Zhu, R.C. Smart and J. Ninomiya-Tsuji. Ablation of TAK1 upregulates reactive oxygen species and selectively kills tumor cells. Cancer Res 70:8417-8425 2010 Kim, T.H., S.L. Chiera, K.E. Linder, C.S. Trempus, R.C. Smart and J.M. Horowitz. Overexpression of transcription factor Sp2 inhibits epidermal differentiation and increases susceptibility to wound- and carcinogen-induced tumorigenesis. Cancer Res 70:8507-8516 2010 Thompson, E.A., S. Zhu, J.R. Hall, J.S. House, R. Ranjan, J.A. Burr, Y-Y. He, D.M. Owens and R.C. Smart. C/EBP? expression is downregulated in human nonmelanoma skin cancers and inactivation of C/EBP? confers susceptibility to UVB-induced skin squamous cell carcinomas. J. Invest. Dermatol. 131:1339-1346 2011 Deterding, L.A., J.G. Williams, M.M. Humble, R.M. Petrovich, S-J. Wei, C.S. Trempus, M.B. Gates, R.C. Smart, R.W. Tennant and K.B. Tomer. CD34 antigen: Determination of specific sites of phosphorylation in vitro and in vivo Int J of Mass Spectrometry 301 1221 2011 Ming, M., L. Feng, C.R. Shea, K. Soltani, B. Zhao, W. Han, R.C. Smart, C.S. Trempus and Y.Y. He. PTEN positively regulates UVB-induced DNA damage repair. Cancer Res 71:5287-5295 2011 Gaddameedhi, S., C.P. Selby, W.K. Kaufmann, R.C. Smart and A. Sencar. Control of skin cancer by the circadian rhythm. Proc. Natl. Acad. Sci. USA 108:18790-18794 2011