A mouse model for human anal cancer

Abstract

Human anal cancers are associated with high-risk human papillomaviruses (HPV) that cause other anogenital cancers and head and neck cancers. As with other cancers, HPV16 is the most common high-risk HPV in anal cancers. We describe the generation and characterization of a mouse model for human anal cancer. This model makes use of K14E6 and K14E7 transgenic mice in which the HPV16 E6 and E7 genes are directed in their expression to stratified squamous epithelia. HPV16 E6 and E7 possess oncogenic properties including, but not limited to, their capacity to inactivate the cellular tumor suppressors p53 and pRb, respectively. Both E6 and E7 were found to be functionally expressed in the anal epithelia of K14E6/K14E7 transgenic mice. To assess the susceptibility of these mice to anal cancer, mice were treated topically with dimethylbenz[a]anthracene (DMBA), a chemical carcinogen that is known to induce squamous cell carcinomas in other sites. Nearly 50% of DMBA-treated HPV16 E6/E7 transgenic mice showed overt signs of tumors, whereas none of the like-treated nontransgenic mice showed tumors. Histopathologic analyses confirmed that the HPV16 transgenic mice were increased in their susceptibility to anal cancers and precancerous lesions. Biomarker analyses demonstrated that these mouse anal cancers exhibit properties that are similar to those observed in HPV-positive precursors to human anal cancer. This is the first mouse model for investigating the contributions of viral and cellular factors in anal carcinogenesis, and should provide a platform for assessing new therapeutic modalities for treating and/or preventing this type of cancer.

Figure 1. HPV oncoproteins are expressed in the anus of K14E6/K14E7 mice

Panel A. E7 western blot analysis. Note that E7 protein is expressed in E6/E7 anal tissue at levels similar to that found in an HPV16-positive human cervical cancer cell line (SiHa) whereas none was found in nontransgenic (NTG) mice. Panel B. E6 protein detected by Western blot analysis of protein lysates subjected to E6 pulldown assay using GST MAGI1-PDZ domain. In this experiment the HPV16-positive cervical cancer cell line CaSki was used as a positive control.

Figure 2. Acute phenotypes of HPV16 E6/E7 transgenic mouse anal epithelium

Panel A. Shown is MCM7-specific immunohistochemical staining of sections from the anus of nontransgenic (NTG) and K14E6/E7 transgenic mice. Note that in anal epithelium of E6/E7 mice, expression of MCM7, a biomarker for E7's inactivation of Rb, is strongly induced as compared to NTG mice. Brown nuclei indicate positive staining which is expected along the basement membrane (outlined in black) in NTG mice while it is seen throughout the epithelium in E6/E7 mice. Panel B. Shown is BrdUrd-specific immunohistochemical staining of sections from the anus of nontransgenic and K14E6/E7 transgenic mice. Panel C. Quantification of the percentage of suprabasal cells with BrdUrd-positive nuclei. Panel D. Response of anal epithelium to radiation-induced growth arrest. Shown is the quantification of the frequency of BrdUrd-positive basal cells in anal epithelia of nontransgenic and K14E6/E7 transgenic mice that were (+) or were not (-) treated with 12Gy (γ) external radiation.

Figure 3. Histopathology of anal tissue

Panel A. Shown are H&E stained sections of various samples categorized as normal (N) including inflammation and hyperkeratosis, hyperplasia, papilloma (P), atypia (A), and carcinoma (C). Panel B. Frequency of overt tumors. Note that overt tumors appeared at the highest frequency in the E6/E7 mice treated with 0.12 μmole DMBA. Tumors were a median of 2mm in size at the time of sacrifice with a range from 1mm to 17mm. The largest tumor occurred when 3 smaller tumors coalesced followed by aggressive growth; as such this mouse was euthanized four days prior to the original endpoint. Panel C. Time of onset of tumors. Note that overt tumors arose at a median of 22 weeks after 0.12 μmole DMBA treatment was initiated with a range from 11 to 27 weeks. Panel D. Expression of cancer biomarkers in mouse. Shown are tissue sections from mouse anal cancer. Top images are H&E stained sections showing keratin pearls and large atypical nuclei consistent with squamous cell carcinoma. Shown at the bottom are representative images of a cancer stained with MCM7 (left bottom) and p16 (right bottom). See Table S1 for a complete reporting of the IHC for all samples evaluated.