Persistence of high-grade cervical dysplasia and cervical cancer requires the continuous expression of the human papillomavirus type 16 E7 oncogene

Abstract

Several mucosotropic human papillomaviruses (HPV), including HPV type 16 (HPV-16), are etiologic agents of a subset of anogenital cancers and head and neck squamous cell carcinomas. In mice, HPV-16 E7 is the most potent of the papillomaviral oncogenes in the development of cervical disease. Furthermore, interfering specifically with the expression of E7 in HPV-positive cell lines derived from human cervical cancers inhibits their ability to proliferate, indicating that the expression of E7 is important in maintaining the transformed phenotype in vitro. To assess the temporal role of E7 in maintaining HPV-associated tumors and precancerous lesions in vivo, we generated Bi-L E7 transgenic mice that harbor a tetracycline-inducible transgene that expresses both HPV-16 E7 and firefly luciferase. When we crossed Bi-L E7 mice to a K5-tTA transgene-inducing line of mice, which expresses a tetracycline-responsive transactivator selectively in the stratified squamous epithelia, the resulting Bi-L E7/K5-tTA bitransgenic mice expressed E7 and luciferase in the skin and cervical epithelium, and doxycycline repressed this expression. Bitransgenic mice displayed several overt and acute epithelial phenotypes previously shown to be associated with the expression of E7, and these phenotypes were reversed on treatment with doxycycline. Repressing the expression of E7 caused the regression of high-grade cervical dysplasia and established cervical tumors, indicating that they depend on the continuous expression of E7 for their persistence. These results suggest that E7 is a relevant target not only for anticancer therapy but also for the treatment of HPV-positive dysplastic cervical lesions.

Figure 1

The expression of luciferase and E7 in the dorsal skin and female reproductive tracts of Bi-L E7/K5-tTA bitransgenic mice is eliminated on treatment with doxycycline (dox). A and B, luciferase assays done on lysates from dorsal skin (A) or female reproductive tracts (B) from mice of the indicated genotypes. Columns, mean; bars, SD. *, P < 0.05 versus nontransgenic mice by a two-sided Wilcoxon rank-sum test, and n ≥ 3 for all groups of mice. C and D, Western blots to examine the expression of E7 in lysates from dorsal skin (C) or female reproductive tracts (D). To compare the level of expression of E7 in line 406 Bi-L E7/K5-tTA bitransgenic mice to that seen in K14E7 mice, equivalent amounts of lysates from three K14E7 mice were pooled, and the indicated amounts of this pool were analyzed in parallel with lysates from mice of the indicated genotypes. Detection of β-actin was used as a loading control.

Figure 2

Bi-L E7/K5-tTA bitransgenic mice display striking overt phenotypes that are reversible on treatment with doxycycline. Shown are photographs of the head (top) and flank (bottom) of the same mouse, either before treatment with doxycycline (Pre-dox) or after the indicated times on doxycycline. Although images from only one mouse are shown, similar results were obtained with all 10 mice treated and followed for 20 d.

Figure 3

The endocervical epithelium of Bi-L E7/K5-tTA bitransgenic mice displays aberrant proliferation. A, immunohistochemical analysis of the incorporation of BrdUrd in sections of the endocervical epithelium from mice of the indicated genotypes. Brown nuclei represent cells positive for BrdUrd, and the black line delineates the basement membrane. B, quantification of nuclei positive for BrdUrd in sections shown in A. Eight visual fields at ×40 magnification were scored for nuclei positive (brown) or negative (blue) for BrdUrd. Data are presented as the mean ± SD. *, P < 0.005 versus nontransgenic mice by a two-sided Wilcoxon rank-sum test, and n ≥ 3 for all groups of mice. C, immunohistochemical analysis of the expression of MCM7 in sections of the endocervical epithelium from mice of the indicated genotypes. Brown nuclei represent cells positive for MCM7, and the black line delineates the basement membrane.

Figure 4

The expression of E7 is required for the maintenance of high-grade cervical dysplasia and cervical cancer in Bi-L E7/K5-tTA bitransgenic mice. Shown in all panels are images of sections of the endocervices from bitransgenic mice expressing E7 for the indicated duration. A, composite images at ×10 magnification of sections of the endocervix stained with H&E. Insets, ×40 magnification of cancers in mice receiving no doxycycline (left) or 3 d of doxycycline (middle). B, images at ×40 magnification of sections from the endocervical epithelium stained with H&E, illustrating a region of high-grade dysplasia observed in a bitransgenic mouse receiving no doxycycline (left) and the regression of high-grade dysplasia on treatment of bitransgenic mice with doxycycline for 3 d (middle) or 1 mo (right). C and D, immunohistochemical analysis of BrdUrd (C) and MCM7 (D) in the endocervical epithelium of mice receiving no doxycycline (left), 3 d of doxycycline (middle), or 1 mo of doxycycline (right). The black line delineates the basement membrane. Insets, results from tumors from the same animals.