Only ~3% of human genome encodes protein. The remaining 97% of the human genome is referred to as noncoding DNA. Initially, much of the intergenic noncoding sequence was referred to as ����������������junk DNA��������������� as it was considered to have no function. While some intergenic sequences contain DNA elements important in gene regulation, many intergenic sequences can be transcribed into RNA. In fact, ~85% of the human genome is transcribed into RNA. RNAs that lack protein coding function are referred to as noncoding RNAs (ncRNAs) and of these the long noncoding RNAs (lncRNAs >200 nt) represent the majority. LncRNAs are one of the largest and more diverse classes of cellular transcripts with over 10,000 lncRNA transcripts reported in the human genome; in most cases their biological function is unknown. Emerging evidence indicates they play an important role in regulating gene expression and are associated with human diseases such as cancer, Alzheimer������������������s and heart disease. There is a critical need to determine whether associations of lncRNAs with specific disease are functionally significant and to define and characterize the function of lncRNAs using in vivo disease model systems. Given that the etiology of most chronic human diseases involves interactions with environment, it is also important to determine how environmental factors impact the expression, activity and function of lncRNAs. Nonmelanoma skin cancer (NMSC) is the most common cancer in the United States. The majority of NMSCs is caused by solar UVB radiation. p53 plays a key role in the response of skin keratinocytes to UVB-induced DNA damage by inducing cell cycle arrest and apoptosis. In skin cancer, the incidence of p53 mutations ranges from 50-90%. UVB-induced mutation of p53 allows keratinocytes upon successive UVB exposures to evade apoptosis and cell cycle arrest and these defects have a critical role in skin cancer development. LincRNA-p21 is a lncRNA and was recently discovered to be a direct transcriptional target of p53 where it serves as a mediator of p53-dependent transcriptional repression. We observed that; i) lincRNA-p21 is highly inducible by UVB in the mouse skin in vivo and in human/mouse keratinocytes, ii) UVB-induction of lincRNA-p21 is p53-dependent and iii) lincRNA-p21 has a key role in UVB-induced apoptotic cell death in keratinocytes. Our plan is to characterize the regulation and function of lincRNA-p21 in keratinocytes and skin in vivo in response to UVB and to define the role of lincRNA-p21 in UVB-induced skin cancer. The central hypotheses are i) lincRNA-p21 is induced by UVB in keratinocytes through a p53-dependent pathway to produce apoptotic cell death and ii) lincRNA-p21 functions as a tumor suppressor in NMSC whereby the loss of lincRNA-p21 expression allows mutant p53 and non-mutant p53 keratinocytes to evade UVB-induced apoptotic death leading to skin cancer. The proposed studies are significant as they represent the first characterization of a lncRNA function in an in vivo disease model with a highly relevant environmental component; moreover if the hypothesis is correct this will be the first demonstration of a lncRNA functioning as a tumor suppressor in vivo.

The transcription factor p53 can be activated by several environmental stressors including UVB solar radiation, to regulate the transcription of genes involved in senescence, apoptosis and cell cycle arrest. These anti-tumor activities of p53 are essential to prevent cancer. p53 is a tumor suppressor and approximately half of all human cancers have p53 alterations that results in loss of p53 transcriptional activity. Recently, we discovered that deletion of the C/EBP�������� transcription factor increased p53 protein levels and enhances p53 pro-apoptotic activity/apoptosis greater than 3-fold following UVB exposure. We have also found that UVB treatment induces a C/EBP��������-p53 complex and knockdown of C/EBP�������� in UVB-treated keratinocytes increases p53 protein stability. These results suggest C/EBP�������� is a negative regulator of p53 pro-apoptotic activity, through an unknown mechanism. C/EBP�������� has a key role in an environmentally-induced disease as C/EBP��������-/- mice are resistant to UVB-induced skin cancer, however it is unknown if the enhancement of p53 pro-apoptotic activity prevents the onset of UVB-induced skin cancer in C/EBP��������-/- mice. Through utilization of ChIP-seq and RNA-seq we will map both C/EBP�������� and p53 chromatin interactions and determine the effects of deletion of C/EBP�������� on the p53 transcriptional network. These studies will provide mechanistic insight into an uncharacterized function of C/EBP�������� in regulating p53 prop-apoptotic activity following UVB exposure and will enhance the foundation and fundability for our long-term goal to determine how C/EBP�������� regulates p53 activity, stability and protein-protein interactions and the importance of this function of C/EBP�������� in an environmentally-induced disease, UVB-induced skin cancer.

Cancer is a disease that arises from genomic alterations in somatic cells and it is the accumulation of genetic alterations that drives tumorigenesis. Moreover, it is the rate at which a developing tumor cell acquires these genetic alterations that ultimately determines the onset of cancer. Human skin is routinely subjected to DNA damage induced by solar radiation and keratinocytes have developed intricate pathways to response to UVB-induced DNA damage. Recently, we provided the first genetic evidence CCAAT/enhancer binding protein á (C/EBPá), a member of the basic leucine zipper family of transcription factors, functions as an epithelial tumor suppressor through utilization of mice with an epidermal-targeted ablation of C/EBPá. These mice are highly susceptible to UVB- and carcinogen-induced squamous papilloma development and these benign skin tumors display a highly accelerated rate of malignant progression to squamous cell carcinomas. Human skin squamous cell carcinomas and basal cell carcinomas as well as mouse skin squamous carcinomas display weak or ablated expression of C/EBPá. Together these findings suggest a tumor suppressor function of C/EBPá in skin cancer through maintenance of the genome. We hypothesize that reduced or ablated expression of C/EBPá results in an impaired DNA damage-induced G1 checkpoint, resulting in the accumulation of somatic mutations and promoting skin cancer progression. The overall objective of this proposal is to understand how the loss of C/EBPá contributes to an increased rate of malignant tumor progression focusing on the role of C/EBPá in the DNA damage-induced G1 checkpoint. Understanding how C/EBPá influences the rate of cancer progression and the acquisition of mutations will provide further insights to the mechanisms of carcinogen- and UVB-induced skin cancer as well as numerous cancers where C/EBPá expression is diminished. Recent analysis of the genomes from human cancers discovered that mutations in specific cancer associated genes can vary dramatically within a give tumor type and suggest the identification of the origin of genomic point mutations may be a more effective strategy for cancer treatment than targeting a specific cancer gene as mutations in other essential genes will be enhanced and selected by the mutator phenotype. Drugs that target point mutation genetic instability may delay the accumulation of mutations and subsequently prevent cancer onset.