Long-term goal of our research is to discover novel biomarkers and effective therapies against oral cancer. The current project will investigate the role of Grainyhead-like 2 (GRHL2) in mediating oral carcinogenesis by high risk human papillomavirus (HPV) through epigenetic mechanisms. GRHL2 regulates keratinocyte cell fate through multiple target genes, including human telomerase reverse transcriptase (hTERT) gene, forkhead box protein 1 (FoxM1), and epidermal differentiation complex (EDC) genes. Our recent published data revealed that GRHL2 is a master regulator of keratinocyte proliferation and differentiation. GRHL2 elicits these effects through epigenetic mechanisms that involve altered DNA methylation and histone modifications at target gene promoters. We found that GRHL2 is notably induced in human oral keratinocytes (HOKs) harboring high risk HPV and HPV+ oral squamous cell carcinomas (OSCCs). Furthermore, high level of GRHL2 was detected in OSCC tumor spheroids, which are enriched with cancer stem cells (CSCs). When GRHL2 was knocked down in OSCCs, self-renewal of tumor spheroids in vitro and tumorigenicity in vivo were significantly impaired. Thus, HPV may cause deregulation of GRHL2 to promote its oncogenic activity by inducing the FoxM1 oncogene and enhance CSC self-renewal. Recent studies underscore the importance of non-coding RNAs in regulation of pluripotency and CSC self-renewal. GRHL2 upregulates miR-21, an oncomir associated with HPV and CSC phenotype. Thus, differential regulation of non-coding RNAs by GRHL2 may underlie the mechanism of enhancing CSC phenotype in OSCCs. To investigate the pathobiology of HPV in oral carcinogenesis, we have constructed infectious HPV16 virions capable of expressing the viral oncogenes. These novel HPV virions will allow us to test the effects of HPV infection in primary NHOK in well-controlled experiments. In the current project, we will test the hypothesis that GRHL2 is necessary for HPV-associated cellular aberrations by altered epigenetic regulation of its target genes and by enhancing CSC self-renewal, resulting in the maintenance of oral cancer phenotype. This novel hypothesis will be tested by investigating the pathophysiologic role of GRHL2 in HPV-associated cellular aberrations; determining the epigenetic mechanisms by which GRHL2 modulates epithelial proliferation and differentiation, and how GRHL2-associated epigenomic profile is altered by HPV; and investigating whether GRHL2 determines CSC phenotype in HPV-associated oral cancer by regulation of non-coding RNAs. This project will delineate the epigenetic mechanisms by which GRHL2 mediates pathobiology of high risk HPV in oral carcinogenesis, and will validate the role of GRHL2 in CSC renewal through regulation of non-coding RNAs, which may be used for discovery of novel diagnostic marker and therapeutic targets.