Abstract 1032: Novel mouse models of high-risk HPV-related oral cancers
Authors: Carper Miranda B. , Troutman Scott , Byrd Kevin M. , Wagner Bethany , Henry Erin C. , Montgomery Stephanie A. , Williams Scott E. , Kissil Joseph L. , Amelio Antonio L. 
Published: August 2018

Abstract
The rate of HPV-induced head and neck squamous cell carcinoma (HNSCC) is steadily increasing and implicated in approximately 60% of all oropharyngeal carcinomas. The advent of whole genome, transcriptome, and proteome analyses have aided in identifying altered signaling pathways in HPV-induced HNSCCs, however, additional tools such as mouse models are needed to study the role of HPV oncogenes in oral tumor initiation and progression. Current inducible models of HPV-driven oral cancer do not accurately recapitulate the levels, stoichiometric ratios, or anatomic location of oncoprotein expression. To address these limitations, we developed a tractable genetically engineered mouse model (GEMM) that enables directed expression of high-risk HPV16 E6 and E7 oncogenes (Rosa26-loxP-STOP-loxP-E7iresE6; “H”) in the oral epithelium. Analysis of mouse embryonic fibroblasts treated with Cre recombinase confirmed E6 and E7 expression and changes in mRNA expression of known E6 and E7 targets. We crossed our H mice to several tissue-specific transgenic Cre-driver mouse strains including the EBV lytic promoter (ED-L2-Cre recombinase; “L”) to drive robust expression in the oropharyngeal squamous epithelia. Analysis of epithelial tissues isolated from LH mice displayed increased oral volumes and cutaneous epithelial thickening associated with hyperplasia, dysplasia, accumulation of neutrophils and recruitment of cytotoxic and regulatory T cells. A second cross to the keratin 14 promoter (KRT14-Cre; “K”) line enabled targeted expression to the basal epithelial layer (KH) and confirmed increased oral volumes and cutaneous epithelial thickening associated with increased suprabasal proliferation and expression of canonical E7 targets. Lastly, a third cross to a tamoxifen-inducible Cre (CreERT2) line driven by the keratin 14 promoter (KRT14-CreERT2; “iK”) enabled conditional and inducible post-natal E6 and E7 expression in basal epithelia (iKH). To prevent systemic oncogene expression in K14+ cells, we validated a method for submucosal delivery of tamoxifen to the tongue. We confirmed that this approach prevents tamoxifen spread and anatomically restricts activation oncogene expression to intra-lingual regions using an optical reporter mouse recently generated by the Amelio lab (Rosa26-loxP-STOP-loxP-LumiFluor; “F”) crossed to the iK line (“iKF”). Notably, direct intra-lingual injection yields higher recombination and reporter activation using 40% less overall tamoxifen than traditional i.p. administration routes. Moreover, immunofluorescent staining revealed mosaic transgene activation in the oropharynx, an important feature for modeling HPV-induced OSCC. In fact, intra-lingual injection of tamoxifen in iKH mice coupled with 4-nitroquniloline 1-oxide (4NQO) administration led to E6 and E7 expression and dysplasia in the tongue epithelia. Ongoing studies are evaluating cooperating mutations in driving HNSCC development.
Citation Format: Miranda B. Carper, Scott Troutman, Kevin M. Byrd, Bethany Wagner, Erin C. Henry, Stephanie A. Montgomery, Scott E. Williams, Joseph L. Kissil, Antonio L. Amelio. Novel mouse models of high-risk HPV-related oral cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1032.