Identification of biomarkers that distinguish human papillomavirus (HPV)-positive versus HPV-negative head and neck cancers in a mouse model

Abstract

Head and neck squamous cell carcinoma (HNSCC) is a leading cause of cancer mortality worldwide. Recent reports have associated a subset of HNSCC with high-risk human papillomaviruses (HPVs), particularly HPV16, the same subset of HPVs responsible for the majority of cervical and anogenital cancers. In this study we describe a mouse model for HPV-associated HNSCC that employs mice transgenic for the HPV16 oncogenes E6 and E7. In these mice, E6 and E7 induce aberrant epithelial proliferation and, in the presence of a chemical carcinogen, they increase dramatically the animal's susceptibility to HNSCC. The cancers arising in the HPV16-transgenic mice mirror the molecular and histopathological characteristics of human HPV-positive HNSCC that distinguish the latter from human HPV-negative HNSCC, including overexpression of p16 protein and formation of more basaloid cancers. This validated model of HPV-associated HNSCC provides the means to define the contributions of individual HPV oncogenes to HNSCC and to understand the molecular basis for the differing clinical properties of HPV-positive and HPV-negative human HNSCC. From this study, we identify minichromosome maintenance protein 7 (MCM7) and p16 as potentially useful biomarkers for HPV-positive head and neck cancer.

Fig. 1.

HPV16 oncogenes can induce DNA synthesis in the normally quiescent compartment of oral and esophageal epithelia. Adult mice (7 weeks old) were injected with the nucleotide analog BrdU 1 h before euthanasia. The tongue and esophagus were harvested from each mouse, fixed, and embedded in paraffin. BrdU-specific immunohistochemistry was performed on slides containing sections of tongue (A and B) and esophagus (C and D) from nontransgenic (A and C) and K14E6/K14E7-transgenic (B and D) mice. The results from three animals of each genotype were quantified (suprabasal positive nuclei/total nuclei) (E). In both the tongue and esophagus of HPV16-transgenic mice (E6E7), the increase in suprabasal DNA synthesis compared with that seen in nontransgenic (NTG) counterparts was statistically significant (P = 0.02; Wilcoxon rank-sum test).

Fig. 2.

Carcinogen-treated mice develop a progressive neoplastic disease. Shown are sections of tissues harvested from 4NQO-treated mice. The tissues were fixed, embedded in paraffin, sectioned, and stained with H&E. (A–C) Nontransgenic animals. (D–F) Bitransgenic animals. (A) The epithelium in tissues from untreated animals remained normal. (B) Histopathologic analysis of the treated mice indicated that they developed areas of hyperplasia with embedded dysplasia (an example of dysplastic epithelium is demarcated by arrowheads). Hyperplasia was not observed in the absence of dysplasia. This phenotype was focal in nontransgenic mice but profuse in HPV-transgenic mice. More severe neoplastic disease was also noted, ranging from benign papillomas or polyps (C) that had not invaded the underlying stroma to grade I (D), grade II (E), and grade III (F) invasive squamous-cell carcinomas. Note that the grade I cancer is well differentiated, showing areas of keratinization (keratin in C–E is demarcated by inverted arrowheads). In contrast, most of the cells within the grade III cancer have a basaloid morphology, with few differentiated cells evident and virtually no keratin observed.

Fig. 3.

Evidence for metastatic disease. Shown are cross-sections of tumors collected from a 4NQO-treated K14E6/K14E7-transgenic mouse that had tumors in the esophagus (A and B), forestomach (not shown), and liver (C and D). Normal liver is shown in E and F. (A, C, and E) Sections were stained with H&E. (B, D, and F) Sections were stained with antibodies specific to K14. Note the similarity in cellular morphology of the tumor from the esophagus (A) and liver (C), which is distinct from the morphology of the liver hepatocytes (E). Note the abundant cytoplasmic staining for K14 in the tumor from the esophagus (B) and liver (D) and the absence of cytoplasmic staining for K14 in the surrounding hepatocytes in the liver (F).

Fig. 4.

Cancers from nontransgenic and K14E6/K14E7-transgenic mice express different markers, results consistent with those seen in human HPV-negative versus HPV-positive HNSCC. Shown are histological sections of tumors from 4NQO-treated nontransgenic (A, C, and E) and K14E6/K14E7-transgenic (B, D, and F) mice that were immunohistochemically stained for Ki67 (A and B), p16 (E and F), or MCM7 (C and D). Note the similar frequency of Ki67-positive cells in the tumors from nontransgenic and HPV-transgenic mice. In contrast, note the increased frequency and intensity of staining for p16 and MCM7 in the tumor from the HPV-transgenic mice compared with the tumor from the nontransgenic mouse.