Human head and neck cancers that develop from the squamous cells of the oropharynx (Oropharyngeal Squamous Cell Carcinomas or OPSCC) are commonly associated with the papillomavirus infection. A papillomavirus infection-based mouse model of oropharyngeal tumorigenesis would be valuable for studying the development and treatment of these tumors. We have developed an efficient system using the mouse papillomavirus (MmuPV1) to generate dysplastic oropharyngeal lesions, including tumors, in the soft palate and the base of the tongue of two immune-deficient strains of mice. To maximize efficiency and safety during infection and endoscopy, we have designed a nose cone for isoflurane-induced anesthesia that takes advantage of a mouse’s need to breathe nasally and has a large window for oral manipulations. To reach and infect the oropharynx efficiently, we have repurposed the Greer Pick allergy testing device as a virus delivery tool. We show that the Pick can be used to infect the epithelium of the soft palate and the base of the tongue of mice directly, without prior scarification. The ability to induce and track oropharyngeal papillomavirus-induced tumors in the mouse, easily and robustly, will facilitate the study of oropharyngeal tumorigenesis and potential treatments.

Recurrent respiratory papillomatosis (RRP), caused by laryngeal infection with low-risk human papillomaviruses, has devastating effects on vocal communication and quality of life. Factors in RRP onset, other than viral presence in the airway, are poorly understood. RRP research has been stalled by limited preclinical models. The only known papillomavirus able to infect laboratory mice, Mus musculus papillomavirus (MmuPV1), induces disease in a variety of tissues. We hypothesized that MmuPV1 could infect the larynx as a foundation for a preclinical model of RRP. We further hypothesized that epithelial injury would enhance the ability of MmuPV1 to cause laryngeal disease, because injury is a potential factor in RRP and promotes MmuPV1 infection in other tissues. In this report, we infected larynges of NOD scid gamma mice with MmuPV1 with and without vocal fold abrasion and measured infection and disease pathogenesis over 12 weeks. Laryngeal disease incidence and severity increased earlier in mice that underwent injury in addition to infection. However, laryngeal disease emerged in all infected mice by week 12, with or without injury. Secondary laryngeal infections and disease arose in nude mice after MmuPV1 skin infections, confirming that experimentally induced injury is dispensable for laryngeal MmuPV1 infection and disease in immunocompromised mice. Unlike RRP, lesions were relatively flat dysplasias and they could progress to cancer. Similar to RRP, MmuPV1 transcript was detected in all laryngeal disease and in clinically normal larynges. MmuPV1 capsid protein was largely absent from the larynx, but productive infection arose in a case of squamous metaplasia at the level of the cricoid cartilage. Similar to RRP, disease spread beyond the larynx to the trachea and bronchi. This first report of laryngeal MmuPV1 infection provides a foundation for a preclinical model of RRP.

Approximately 25% of head and neck squamous cell carcinomas (HNSCC) are associated with human papillomavirus (HPV) infection. In these cancers as well as in HPV-associated anogenital cancers, PI3K signaling is highly activated. We previously showed that IQ motif-containing GTPase activating protein 1 (IQGAP1), a PI3K pathway scaffolding protein, is overexpressed in and contributes to HNSCC and that blocking IQGAP1-mediated PI3K signaling reduces HPV-positive HNSCC cell survival and migration. In this study, we tested whether IQGAP1 promotes papillomavirus (PV)-associated HNSCCs. IQGAP1 was necessary for optimal PI3K signaling induced by HPV16 oncoproteins in transgenic mice and MmuPV1 infection, a mouse papillomavirus that causes HNSCC in mice. Furthermore, we found that, at 6 months post-infection, MmuPV1-infected Iqgap1^−/− mice developed significantly less severe tumor phenotypes than MmuPV1-infected Iqgap1^+/+ mice, indicating a role of IQGAP1 in MmuPV1-associated HNSCC. The tumors resulting from MmuPV1 infection showed features consistent with HPV infection and HPV-associated cancer. However, such IQGAP1-dependent effects on disease severity were not observed in an HPV16 transgenic mouse model for HNC. This may reflect that IQGAP1 plays a role in earlier stages of viral pathogenesis, or other activities of HPV16 oncogenes are more dominant in driving carcinogenesis than their influence on PI3K signaling.

Laryngeal infection with low-risk human papillomaviruses can cause recurrent respiratory papillomatosis (RRP), a disease with severe effects on vocal fold epithelium resulting in impaired voice function and communication. RRP research has been stymied by limited preclinical models. We recently reported a murine model of laryngeal MmuPV1 infection and disease in immunodeficient mice. In the current study, we compare quantitative and qualitative measures of epithelial proliferation, apoptosis, differentiation, and barrier between mice with MmuPV1-induced disease of the larynx and surrounding tissues and equal numbers of uninfected controls. Findings supported our hypothesis that laryngeal MmuPV1 infection recapitulates many features of RRP. Like RRP, MmuPV1 increased proliferation in infected vocal fold epithelium, expanded the basal compartment of cells, decreased differentiated cells, and altered cell–cell junctions and basement membrane. Effects of MmuPV1 on apoptosis were equivocal, as with RRP. Barrier markers resembled human neoplastic disease in severe MmuPV1-induced disease. We conclude that MmuPV1 infection of the mouse larynx provides a useful, if imperfect, preclinical model for RRP that will facilitate further study and treatment development for this intractable and devastating disease.

Latent infection of Epstein-Barr virus (EBV) is associated with lymphoid and epithelial cell cancers, including 10% of gastric carcinomas. We previously reported that hypoxia inducible factor-1α (HIF-1α) induces EBV’s latent-to-lytic switch and identified several HIF-1α-stabilizing drugs that induce this viral reactivation. Here, we tested three classes of these drugs for preferential killing of the EBV-positive gastric cancer AGS-Akata cell line compared to its matched EBV-negative AGS control. We observed preferential killing with iron chelators [Deferoxamine (DFO); Deferasirox (DFX)] and a prolyl hydroxylase inhibitor (BAY 85-3934 (Molidustat)), but not with a neddylation inhibitor [MLN4924 (Pevonedistat)]. DFO and DFX also induced preferential killing of the EBV-positive gastric cancer AGS-BDneo and SNU-719 cell lines. Preferential killing was enhanced when low-dose DFX (10 μM) was combined with the antiviral prodrug ganciclovir. DFO and DFX induced lytic EBV reactivation in approximately 10% of SNU-719 and 20-30% of AGS-Akata and AGS-BDneo cells. However, neither DFO nor DFX significantly induced synthesis of lytic EBV proteins in xenografts grown in NSG mice from AGS-Akata cells above the level observed in control-treated mice. Therefore, these FDA-approved iron chelators are less effective than gemcitabine at promoting EBV reactivation in vivo despite their high specificity and efficiency in vitro.

The artemisinin family of compounds is cytopathic in certain cancer cell lines that are positive for human papillomaviruses (HPV) and can potentially drive the regression of dysplastic lesions. We evaluated the efficacy of topical dihydroartemisinin (DHA) on cervical dysplasia and anal dysplasia in two papillomavirus mouse models: K14E6/E7 transgenic mice, which express HPV16 oncogenes; and immunodeficient NOD/SCID gamma (NSG) mice infected with Mus musculus papillomavirus (MmuPV1). Mice started treatment with DHA at 25 weeks of age (K14E6/E7) or 20 weeks post infection (MmuPV1-infected), when the majority of mice are known to have papillomavirus-induced low- to high-grade dysplasia. Mice were treated with or without topical DHA at the cervix or anus and with or without topical treatment with the chemical carcinogen 7,12 dimethylbenz(a)anthracene (DMBA) at the anus of in transgenic mice to induce neoplastic progression. Mice were monitored for overt tumor growth, and tissue was harvested after 20 weeks of treatment and scored for severity of histological disease. For MmuPV1-infected mice, anogenital lavages were taken to monitor for viral clearance. Tissues were also evaluated for viral gene expression at the RNA and/or protein levels. Treatment with topical DHA did not reduce dysplasia in the anogenital tract in either papillomavirus-induced mouse model and did not prevent progression to anal cancer in the DMBA-treated K14E6/E7 mice.