A Modular Polymer Platform That Delivers Recombinant Cytokines and Cisplatin Allows for De-escalation of Radiation Therapy in an Animal Model of Head and Neck Squamous Cell Carcinoma

Abstract

The human papillomavirus (HPV) expresses the viral oncogene E7 that inhibits the retinoblastoma protein (RB1). RB1 mediates contradictory cell growth and cell death pathways via E2F family members. Here, we assessed the extent to which HPV oncogenes caused toxicity as measured by mouse survival and tumor growth. Materials/Methods: iHPV mice contained a LoxP-Stop-LoxP (LSL)-iE6E7 transgene in which conditional E6E7 expression is regulated by Cre recombinase. We constitutively expressed HPV oncogenes by breeding iHPV transgenic mice to CMV-Cre transgenic mice expressing Cre recombinase under a CMV promoter (CMV-HPV mice). We induced HPV oncogene expression in adult mice using RosaHPV mice containing the iHPV transgene and a Rosa-CreER tam transgene expressing a tamoxifenregulated (TAM) regulated Cre recombinase in all tissues. We studied primary oral tumors using triple transgenic KHR mice containing a K14-CreER tam transgene, a LSL-iE6E7 transgene and a LSL-Kras G12D transgene that formed HPV-positive oral tumors after TAM treatment. We assessed the role of E2f1 on survival and oral tumor formation using RosaHPV-E2f1-/mice and KHR-E2f1-/mice, respectively, that contained a homozygous E2f1deletion. HPV oncogene expression and E2f target gene expression was assessed by quantitative RT-PCR. Results: Induction of HPV oncogenes caused embryonic lethality as CMV-HPV double transgenic mice were born at significantly lower frequencies compared to mice carrying single transgenes (P<.0001). Tamoxifen treatment of adult RosaHPV (RosaHPV+TAM) mice caused recombination of the LSL-E6E7 transgenes and HPV oncogene expression in all organs tested. Furthermore, RosaHPV+TAM mice had decreased survival compared to vehicle treated RosaHPV mice (median survival: 50d for RosaHPV+TAM vs not reached for RosaHPV-TAM; P<.0001). Decreased survival in RosaHPV+TAM mice was associated with focal necrosis in hepatocytes and pancreatic tissues and the activation of the E2f target genes. Deletion of E2f1 increased survival of RosaHPV+TAM mice indicating that E2f1 mediated HPV oncogene toxicity (median survival: not reached for RosaHPV-E2f1-/mice vs 49 days for RosaHPV-E2f1 +/mice vs 30 days for RosaHPV-E2f1 +/+ ; P<.0001). Compared to tumors with heterozygous loss of E2f1, KHR tumors with homozygous loss of E2f1 grew faster and had more proliferating tumor cells as measured by Pcna immunohistochemistry (tumor volume at d18: 453.7 mm 3 for KHR-E2f1-/vs 139.7 mm 3 for KHR-E2f1 +/-; PZ.0004). Conclusion: Our results indicate that HPV oncogenes activated the E2f1 pathway to cause toxicity in normal mice and to suppress oral tumor growth. These results suggest that selective modulation of the E2f1 pathway, which is activated in HPV tumors, may facilitate tumor regression.