Deficiency in Ever2 does not increase susceptibility of mice to pathogenesis by the mouse papillomavirus, MmuPV1.

Epidermodysplasia verruciformis (EV) is a rare genetic skin disorder that is characterized by the development of papillomavirus-induced skin lesions that can progress to squamous cell carcinoma (SCC). Certain high-risk, cutaneous ß-genus human papillomaviruses (ß-HPVs), in particular HPV5 and HPV8, are associated with inducing EV in individuals who have a homozygous mutation in one of three genes tied to this disease: EVER1, EVER2, or CIB1. EVER1 and EVER2 are also known as TMC6 and TMC8, respectively. Little is known about the biochemical activities of EVER gene products or their roles in facilitating EV in conjunction with ß-HPV infection. To investigate the potential effect of EVER genes on papillomavirus infection, we pursued in vivo infection studies by infecting Ever2-null mice with mouse papillomavirus (MmuPV1). MmuPV1 shares characteristics with ß-HPVs including similar genome organization, shared molecular activities of their early, E6 and E7, oncoproteins, the lack of a viral E5 gene, and the capacity to cause skin lesions that can progress to SCC. MmuPV1 infections were conducted both in the presence and absence of UVB irradiation, which is known to increase the risk of MmuPV1-induced pathogenesis. Infection with MmuPV1 induced skin lesions in both wild-type and Ever2-null mice with and without UVB. Many lesions in both genotypes progressed to malignancy, and the disease severity did not differ between Ever2-null and wild-type mice. However, somewhat surprisingly, lesion growth and viral transcription was decreased, and lesion regression was increased in Ever2-null mice compared with wild-type mice. These studies demonstrate that Ever2-null mice infected with MmuPV1 do not exhibit the same phenotype as human EV patients infected with ß-HPVs.IMPORTANCEHumans with homozygous mutations in the EVER2 gene develop epidermodysplasia verruciformis (EV), a disease characterized by predisposition to persistent ß-genus human papillomavirus (ß-HPV) skin infections, which can progress to skin cancer. To investigate how EVER2 confers protection from papillomaviruses, we infected the skin of homozygous Ever2-null mice with mouse papillomavirus MmuPV1. Like in humans with EV, infected Ever2-null mice developed skin lesions that could progress to cancer. Unlike in humans with EV, lesions in these Ever2-null mice grew more slowly and regressed more frequently than in wild-type mice. MmuPV1 transcription was higher in wild-type mice than in Ever2-null mice, indicating that mouse EVER2 does not confer protection from papillomaviruses. These findings suggest that there are functional differences between MmuPV1 and ß-HPVs and/or between mouse and human EVER2.

The human papillomavirus 16 E5 gene potentiates MmuPV1-Dependent pathogenesis.

The papillomavirus E5 gene contributes to transformation and tumorigenesis; however, its exact function in these processes and viral pathogenesis is unclear. While E5 is present in high-risk mucosotropic HPVs that cause anogenital and head and neck cancers, it is absent in cutaneous HPVs and the recently discovered mouse papillomavirus (MmuPV1), which causes papillomas and squamous cell carcinomas of the skin and mucosal epithelia in laboratory mice. We infected K14E5 transgenic mice, which express the high-risk mucosotropic HPV16 E5 gene in stratified epithelia, with MmuPV1 to investigate the effects of E5 on papillomavirus-induced pathogenesis. Skin lesions in MmuPV1-infected K14E5 mice had earlier onset, higher incidence, and reduced frequency of spontaneous regression compared to those in non-transgenic mice. K14E5 mice were also more susceptible to cervicovaginal cancers when infected with MmuPV1 and treated with estrogen compared to non-transgenic mice. Our studies support the hypothesis that E5 contributes to papillomavirus-induced pathogenesis.

Human papillomavirus type 16 E7 oncoprotein causes a delay in repair of DNA damage.

BACKGROUND AND PURPOSE: Patients with human papillomavirus related (HPV+) head and neck cancers (HNCs) demonstrate improved clinical outcomes compared to traditional HPV negative (HPV-) HNC patients. We have recently shown that HPV+ HNC cells are more sensitive to radiation than HPV- HNC cells. However, roles of HPV oncogenes in regulating the response of DNA damage repair remain unknown. MATERIAL AND METHODS: Using immortalized normal oral epithelial cell lines, HPV+ HNC derived cell lines, and HPV16 E7-transgenic mice we assessed the repair of DNA damage using γ-H2AX foci, single and split dose clonogenic survival assays, and immunoblot. The ability of E7 to modulate expression of proteins associated with DNA repair pathways was assessed by immunoblot. RESULTS: HPV16 E7 increased retention of γ-H2AX nuclear foci and significantly decreased sublethal DNA damage repair. While phospho-ATM, phospho-ATR, Ku70, and Ku80 expressions were not altered by E7, Rad51 was induced by E7. Correspondingly, HPV+ HNC cell lines showed retention of Rad51 after γ-radiation. CONCLUSIONS: Our findings provide further understanding as to how HPV16 E7 manipulates cellular DNA damage responses that may underlie its oncogenic potential and influence the altered sensitivity to radiation seen in HPV+ HNC as compared to HPV- HNC.

Enhanced radiation sensitivity in HPV-positive head and neck cancer.

Patients with human papillomavirus (HPV+)-associated head and neck cancer (HNC) show significantly improved survival outcome compared with those with HPV-negative (HPV-) tumors. Published data examining this difference offers conflicting results to date. We systematically investigated the radiation sensitivity of all available validated HPV+ HNC cell lines and a series of HPV- HNC cell lines using in vitro and in vivo techniques. HPV+ HNCs exhibited greater intrinsic radiation sensitivity (average SF2 HPV-: 0.59 vs. HPV+: 0.22; P < 0.0001), corresponding with a prolonged G2-M cell-cycle arrest and increased apoptosis following radiation exposure (percent change 0% vs. 85%; P = 0.002). A genome-wide microarray was used to compare gene expression 24 hours following radiation between HPV+ and HPV- cell lines. Multiple genes in TP53 pathway were upregulated in HPV+ cells (Z score 4.90), including a 4.6-fold increase in TP53 (P < 0.0001). Using immortalized human tonsillar epithelial (HTE) cells, increased radiation sensitivity was seen in cell expressing HPV-16 E6 despite the effect of E6 to degrade p53. This suggested that low levels of normally functioning p53 in HPV+ HNC cells could be activated by radiation, leading to cell death. Consistent with this, more complete knockdown of TP53 by siRNA resulted in radiation resistance. These results provide clear evidence, and a supporting mechanism, for increased radiation sensitivity in HPV+ HNC relative to HPV- HNC. This issue is under active investigation in a series of clinical trials attempting to de-escalate radiation (and chemotherapy) in selected patients with HPV+ HNC in light of their favorable overall survival outcome.

Development and characterization of HPV-positive and HPV-negative head and neck squamous cell carcinoma tumorgrafts.

PURPOSE: To develop a clinically relevant model system to study head and neck squamous cell carcinoma (HNSCC), we have established and characterized a direct-from-patient tumorgraft model of human papillomavirus (HPV)-positive and HPV-negative cancers. EXPERIMENTAL DESIGN: Patients with newly diagnosed or recurrent HNSCC were consented for donation of tumor specimens. Surgically obtained tissue was implanted subcutaneously into immunodeficient mice. During subsequent passages, both formalin-fixed/paraffin-embedded as well as flash-frozen tissues were harvested. Tumors were analyzed for a variety of relevant tumor markers. Tumor growth rates and response to radiation, cisplatin, or cetuximab were assessed and early passage cell strains were developed for rapid testing of drug sensitivity. RESULTS: Tumorgrafts have been established in 22 of 26 patients to date. Significant diversity in tumorgraft tumor differentiation was observed with good agreement in degree of differentiation between patient tumor and tumorgraft (Kappa 0.72). Six tumorgrafts were HPV-positive on the basis of p16 staining. A strong inverse correlation between tumorgraft p16 and p53 or Rb was identified (Spearman correlations P = 0.085 and P = 0.002, respectively). Significant growth inhibition of representative tumorgrafts was shown with cisplatin, cetuximab, or radiation treatment delivered over a two-week period. Early passage cell strains showed high consistency in response to cancer therapy between tumorgraft and cell strain. CONCLUSIONS: We have established a robust human tumorgraft model system for investigating HPV-positive and HPV-negative HNSCC. These tumorgrafts show strong correlation with the original tumor specimens and provide a powerful resource for investigating mechanisms of therapeutic response as well as preclinical testing.

HPV-associated head and neck cancer: molecular and nano-scale markers for prognosis and therapeutic stratification.

Over the last 10 years, it has become clear that patients with head and neck cancer can be stratified into two distinct subgroups on the basis of the etiology of their disease. Patients with human papillomavirus-related cancers have significantly better survival rates and may necessitate different therapeutic approaches than those with tobacco and/or alcohol related cancers. This review discusses the various biomarkers currently in use for identification of patients with HPV-positive cancers with a focus on the advantages and limitations of molecular and nano-scale markers.