Project Summary / Abstract High-risk human papillomaviruses (HPV) are a group of viral pathogens that infect and replicate in keratinocytes in the stratified squamous epithelium (SE) of the skin and mucosa, and are known to cause nearly 5% of all human cancers. HPV infection is highly prevalent, with >40 million currently infected individuals in the United States alone. Prevention of HPV-associated malignancies is limited by a lack of antivirals, and development of these therapeutics is in turn limited by the lack of knowledge on virus-host interactions preceding and leading to carcinogenesis in SE. The differentiation program of SE plays an important role in supporting the HPV life cycle. However, identification of distinct cell populations responsible for supporting this progression had been limited by the lack of single-cell resolution tools to define such populations. In a recent study, single-cell RNA sequencing was utilized to identify and compare keratinocytes populations present in HPV-naïve and HPV-infected SE. A novel keratinocyte cell population enriched in HPV-infected epithelium was identified, and termed HPV-induced differentiation dissonant epithelial non-conventional (HIDDEN) cells. These cells form a novel cellular compartment on the surface of HPV-infected, but not uninfected epithelium, and are implicated in carcinogenesis. The Erb-B2 receptor tyrosine kinase 2 (ERBB2) is a part of a core HIDDEN signature and is independently correlated with adverse patient outcomes in cervical squamous cell carcinomas (SCCs). ERBB2 can be clinically targeted in other cancers (e.g., breast cancer), and pre-clinical studies have suggested cooperative regulation between ERBB2 and HPV. However, the relationship between HPV and ERBB2 in the context of its relevance for the HPV life cycle and resulting SCC-related pathologies remains unexplored, and such studies require 3D models of SE. Based on preliminary data and the literature, in this proposal it is hypothesized that HPV infection enhances expression and activation of ERBB2 in the surface HIDDEN cell compartment, and that ERBB2 signaling promotes HPV pathologies in SE. To test this hypothesis, HPV+ and HPV– 3D organotypic epithelial rafts will be utilized to spatially resolve ERBB2 expression, activation, and downstream signaling, and define the viral gene responsible for ERBB2 upregulation (Aim 1). Rafts will also be utilized to define whether ERBB2 is required for HIDDEN formation, viral genome copy number and expression, and HPV-driven pathologies, using genetic inhibition of ERBB2 and FDA-approved drugs (Aim 2). The results will determine whether ERBB2 signaling promotes the HPV viral life cycle and dependent pathologies, and whether clinically relevant HPV phenotypes can be suppressed via existing FDA-approved drugs for the possible prevention of HPV-related cancer types.