Regulation of ZEB1 by Porphyromonas gingivalis in gingival epithelial cells

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Project Summary Oral cancer is the 11th most frequently diagnosed cancer in the world, and due to its late diagnosis, it has an approximately 50% survival rate after 5 years. Metastasis is meditated through Epithelial to Mesenchymal Transition (EMT), which is the process by which epithelial cells lose their tight junctions and apical/basal polarity and shift to a more invasive mesenchymal phenotype giving the cancer cells systemic access through invasion of the bloodstream. EMT is mediated through several transcriptional factors, however we will focus on ZEB1. Porphyromonas gingivalis is an oral obligate anaerobe that when present shifts the host/microbe relationship from synergy to a more dysbiotic environment, which contributes to periodontal disease. P. gingivalis has been shown to invade gingival epithelial cells, and once inside the cells can inhibit apoptosis, ramp up the cell cycle, and more recently by our lab has been shown to upregulate ZEB1. Although we are beginning to understand the potential oncogenic role of P. gingivalis, we do not currently know how ZEB1 is regulated by P. gingivalis in gingival epithelial cells. Our preliminary data suggests that this regulation is occurring through SMAD signaling regulation and through the long non-coding RNA ZEB1-AS1. SMAD signaling is a branch of the TGF-b signaling pathway, which during embryonic development is responsible for producing mesenchymal cells from the initial epithelium. SMAD signaling occurs by phosphorylation of SMAD2/3 by the TGF-b receptor, followed by the formation of a transcriptional regulatory trimer by SMAD2/3 and SMAD4, and finally nuclear localization and upregulation of ZEB1. Additionally, ZEB1 can bind to SMAD2/3 and SMAD4 to form a ZEB1/SMAD trimer to regulate ZEB1 transcription. We have found that P. gingivalis upregulates the regulatory SMAD3, which suggests P. gingivalis is able to intercept this pathway to upregulate ZEB1. We have also found that P. gingivalis upregulates ZEB1-AS1, which has been shown in hepatocarcinoma and esophageal squamous cell carcinoma to directly upregulate ZEB1, and our preliminary data supports this as knocking down ZEB1-AS1 leads to abrogation of ZEB1 regulation by P. gingivalis in gingival epithelial cells. We will investigate mechanisms of regulation by ZEB1-AS1 including direct promotion of gene expression in the nucleus through binding to P300, and/or control of ZEB1 mRNA stability. EMT regulation is a complex process that involves integration of several host signaling pathways, so we also suspect that ZEB1-AS1 will be integrated with SMAD signaling by potentially acting as a scaffold for SMAD3. Developing a better understanding of P. gingivalis mediated EMT will lay the foundation for future studies to target these regulatory mechanisms for diagnosis and treatment, and ultimately reduce the 50% chance of survival current patients have 5 years after diagnosis.