Abstract: Approximately 53,000 people in the United States are diagnosed with oral cancer yearly, and the 5- year survival rate remains poor due to late diagnosis and intervention. Recent evidence has suggested periodontal disease as a potential risk factor, and it is also associated with poor outcomes. Interestingly, periodontal disease and oral cancer share common phenomenon: disruption of epithelial barrier integrity and inflammation. More specifically, the periodontal pathogen Fusobacterium nucleatum has been associated with both barrier disruption and with oral cancer. Still, it is unclear whether disruption of the oral epithelial barrier by Fusobacterium nucleatum is a mechanistic promoter of pro-tumorigenic oral transformation, or just a consequence of it. To answer this question, we interrogate here a cellular complex that is essential for epithelial barrier integrity, called the adherens junction. More specifically, we have found that the adherens junction component PLEKHA7, which is an E-cadherin and p120 binding partner, recruits core components of the RNA interference machinery including the microprocessor, the RNA induced silencing complex, and sets of mRNAs and miRNAs to maintain epithelial homeostasis. PLEKHA7 depletion from cultured epithelial cells results in mis- localization of the RNAi machinery, decreased miRNA silencing activity, increased oncogene expression, and pro-tumorigenic cell transformation. However, such disruption of the RNAi interference machinery, particularly associated with barrier integrity, has not been studied to date in oral cancer, exposing a significant gap in knowledge. We hypothesize that PLEKHA7 recruits and regulates the RNAi machinery at oral epithelial adherens junctions to maintain homeostasis, and that this mechanism is disrupted in the presence of oral pathogens, which in turn promote expression of pro-tumorigenic and inflammatory markers. We will test our hypothesis through two Specific Aims: 1) determine whether oral epithelial adherens junctions recruit the RNAi machinery to regulate function and levels of miRNAs and of their target mRNAs, using two dimensional and three-dimensional epithelial cell models and by examining tissues from patients; 2) determine if the oral periodontal pathogen Fusobacterium nucleatum promotes upregulation of oncogenic and pro-inflammatory markers through miRNA dysregulation, using co-cultures with Fusobacterium nucleatum, immunofluorescence, protein, and RNA analyses. This project is significant, since it will identify a novel molecular mechanism of epithelial pro-tumorigenic transformation in the oral mucosa, advancing our understanding of the disease. The study is innovative, as it links pathogens, oral epithelial barrier function, and localized RNAi and miRNA regulation with oral cancer. We anticipate that the study will be impactful and contribute to future development of effective biomarkers oral cancer, which are critically needed for timely and successful intervention, as well as to the potential development of RNA-based therapeutics. Altogether, this fellowship will provide foundational training in oral epithelial biology to foster the development of the DMD/PhD trainee into a unique and critically needed academic dental scientist.

PROJECT NARRATIVE Oral cancer 5-year survival is poor, due to late diagnosis and intervention. Periodontal disease, caused by certain oral bacteria, compromises the oral tissue barrier and increases the risk of oral cancer, through still unclear mechanisms. This study will investigate a novel mechanism tethering microbes, the oral tissue barrier, and molecules called microRNAs (miRNAs), to explain this relationship with oral cancer risk and open the way to novel preventative and therapeutic approaches.