Topical Protease Inhibitor Decreases Anal Carcinogenesis in a Transgenic Mouse Model of HPV Anal Disease

Abstract

Anal cancer is a major health problem. This study seeks to determine if the topical protease inhibitor Saquinavir (SQV), is effective at the prevention of anal cancer in transgenic mice with established anal dysplasia. K14E6/E7 mice were entered into the study when the majority spontaneously developed high-grade anal dysplasia. To ensure carcinoma development, a subset of the mice was treated with a topical carcinogen: 7,12-Dimethylbenz[a]anthracene (DMBA). Treatment groups included: no treatment, DMBA only, and topical SQV with/without DMBA. After 20 weeks of treatment, anal tissue was harvested and evaluated histologically. SQV was quantified in the blood and anal tissue, and tissue samples underwent analysis for E6, E7, p53, and pRb. There was minimal systemic absorption of SQV in the sera despite high tissue concentrations. There were no differences in tumor-free survival between SQV-treated and respective control groups but there was a lower grade of histological disease in the mice treated with SQV compared to those untreated. Changes in E6 and E7 levels with SQV treatment suggest that SQV may function independently of E6 and E7. Topical SQV decreased histological disease progression in HPV transgenic mice with or without DMBA treatment without local side effects or significant systemic absorption.

Keywords: HPV; anal cancer; anal dysplasia; chemoprevention; squamous cell carcinoma of the anus.

Figure 1

E6/E7 oncoprotein expression (optical density (O.D.)) in mice treated with SQV at doses, 5%, 2.5%, 1%, and 0.5% to establish a standard dosing concentration. (A) Mean E6 and E7 expression quantified from N = 4 mice per SQV dosage group, each distribution male and female; (B) Representative images of anal tissue from the assigned dosages immunohistochemically stained for E6 and E7.

Figure 1

E6/E7 oncoprotein expression (optical density (O.D.)) in mice treated with SQV at doses, 5%, 2.5%, 1%, and 0.5% to establish a standard dosing concentration. (A) Mean E6 and E7 expression quantified from N = 4 mice per SQV dosage group, each distribution male and female; (B) Representative images of anal tissue from the assigned dosages immunohistochemically stained for E6 and E7.

Figure 2

Tumor-free survival, tumor volume, and tumor onset: (A) Tumor-free survival and tumor volume over the 20-week treatment period; (B) Average initial tumor onset (in weeks) for mice that developed overt anal tumors during the treatment period; (C) Final tumor volume measured prior to sacrifice for mice that developed tumors within 20 weeks. In (B,C), “ns” is not significant.

Figure 3

Final anal-tissue histology for mice in each treatment group: (A) All mice anal histology (combined female and male mice), female mice only anal histology, and male mice only anal histology. (B) Representative histological images per treatment group. Significance was assessed as * p < 0.05, ** p < 0.01, *** p < 0.001, “ns” is not significant.

Figure 4

Expression of viral E6 and E7 oncoproteins: (A) Quantification of E6 and E7 expression in each treatment group. Significance was assessed as * p < 0.05, “ns” is not significant. (B) Representative images of immunohistochemically stained anal tissue from mice in each treatment group for E6 and E7.

Figure 4

Expression of viral E6 and E7 oncoproteins: (A) Quantification of E6 and E7 expression in each treatment group. Significance was assessed as * p < 0.05, “ns” is not significant. (B) Representative images of immunohistochemically stained anal tissue from mice in each treatment group for E6 and E7.

Figure 5

Expression of Rb and p53: (A) Quantification of Rb and p53 expression in each treatment group and separated by sex; (B) Representative images of immunohistochemically stained anal tissue from mice in each treatment group for Rb and p53. Significance was assessed as * p < 0.05, ** p < 0.01, and “ns” is not significant.

Figure 5

Expression of Rb and p53: (A) Quantification of Rb and p53 expression in each treatment group and separated by sex; (B) Representative images of immunohistochemically stained anal tissue from mice in each treatment group for Rb and p53. Significance was assessed as * p < 0.05, ** p < 0.01, and “ns” is not significant.