Project 2-Three-dimensional modeling of EBV-HPV interactions leading to anogenital cell dysplasia

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ABSTRACT Infections with high-risk sub-types of Human Papilloma Virus (HPV) are responsible for more than 99% of all cervical carcinoma cases in the US. Our recent data in collaboration with Dr. Hagensee laboratory has shown that co-detection of Epstein-Barr virus (EBV) with HPV in cervical samples increases the risk of concurrent cervical dysplasia by 4-6 fold. In addition, HIV+ women from New Orleans (n=531) with detectable cervical HPV and EBV are at higher risk (69%) for concurrent squamous intraepithelial lesions (SIL) as compared to only 28% of women with only detectable HPV (p<0.001; OR 5.57, 95%CI 2.19-14.4). We hypothesize that EBV acts as a co-factor for progression of HPV-related cervical cancers. We will address this hypothesis directly using innovative 3-dimensional (3D) epithelial models of co-infection. First, using a well-characterized 3D model of terminally differentiated keratinocytes, we will determine whether co-infection with HPV and EBV adversely affects normal differentiation of the stratified multi-layer epithelium compared to HPV alone. Using targeted molecular methods, we will dissect the augmenting capacity of EBV's oncoprotein, LMP-1, for cellular transformation in the context of high-risk HPV types. Importantly, the proposed outcome measures will also address epithelial transformation in the context of the low-risk HPV6 because HPV6 is found more frequently in women shedding EBV, and since EBV's augmenting capacity might be greater in otherwise low-risk types. Parallel experiments will be performed in primary and/or HPV-immortalized human ectocervical epithelial cells. Utilizing expertise from all members of the COBRE team, the innovative 3D epithelial models utilized herein will facilitate a powerful and direct investigation of whether co-infection with EBV enhances malignant transformation. Specifically, these models allow for visualization of the stratified epithelial architecture and invasion of the underlying stromal layer, quantification of basal cell proliferation, and several outcomes not achievable with conventional 2D (monolayer) models. Also unique to the 3D model, we have the ability to produce and utilize infectious HPV thus allowing for co-infection with live viruses rather than modeling HPV infection by retroviral transduction of E6/E7 oncogenes. The mentoring duo of Drs. Alison Quayle and Augusto Ochoa, combined with consulting provided by Drs. Michelle Ozbun and Hagensee, will provide excellent scientific leadership to proposed COBRE mentee Dr. Chris McGowin. In line with the goals of the COBRE mechanism, the combined track record of the proposed mentors for training of junior investigators will provide Chris a rich environment conducive for translating his findings into independent R01 funding.