Cervical cancers require the continuous expression of the human papillomavirus type 16 E7 oncoprotein even in the presence of the viral E6 oncoprotein

Abstract

High-risk human papillomaviruses (HPV), such as HPV-16, are etiologic agents of a variety of anogenital and oral malignancies, including nearly all cases of cervical cancer. Cervical cancers arising in transgenic mice that express HPV-16 E7 in an inducible manner require the continuous expression of E7 for their maintenance. However, in HPV-associated cancers in vivo, E6 and E7 invariably are coexpressed. In this study, we investigated whether cervical cancers rely on the continuous expression of E7 in the context of constitutively expressed E6. We placed the inducible HPV-16 E7 transgene onto a background in which HPV-16 E6 was constitutively expressed. In transgenic mice with high-grade cervical dysplastic lesions and cervical cancer, repressing the expression of E7 led to the regression of all cancers and the vast majority of high-grade dysplastic lesions. In addition, cervical cancers were occasionally observed in transgenic mice in which E7 was repressed and then reexpressed. Our findings indicate that even in the presence of constitutively expressed E6, the continuous expression of E7 is required for the maintenance of cervical cancers and most precancerous lesions. These data have important implications for the potential clinical use of drugs designed to inhibit the expression and/or function of E7 to treat HPV-associated cancers.

Figure 1

The expression of luciferase and E7 in the stratified squamous epithelia of Bi-L E7/K14-tTA bitransgenic mice is eliminated on treatment with doxycycline. A, luciferase assays done on lysates from the dorsal skin, ear, or lower female reproductive tracts of mice from the indicated genotypes and treatment groups. 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. B, Western blot to examine the expression of E7 in lysates from the lower female reproductive tract. To compare the level of expression of E7 in Bi-L E7/K14-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

The endocervical epithelium of Bi-L E7/K14-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 or negative for BrdUrd. Columns, mean; bars, SD. ^*, P < 1 × 10⁻⁴ 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 3

Expression of E6 in the lower reproductive tracts of Bi-L E7/K14E6/K14-tTA triply transgenic mice. A, lateral flow assays for the detection of high-risk HPV E6 proteins. Left, duplicate runs for CaSki cell lysates (positive control, top) and lysis buffer (negative control, bottom). The top and bottom panels to the right of the control strips represent duplicate runs of the assay for each of six experimental samples. Along the top, the total amount of cellular protein, in μg, used from each sample is displayed. Samples were taken from Bi-L E7/K14E6/K14-tTA mice treated for 10 months with estrogen that were (+Dox) or were not (No Dox) treated with doxycycline. Shown in the margins are the relative positions for detection of HPV-16 (16), HPV-18 (18) and HPV-45 (45) E6 proteins as well as an internal control (C) that accounts for any interference that lysates may have with the assay’s method for detecting bound protein. Note the signal in each assay that reflects the presence of HPV-16 E6. Under each sample is provided the estimate of amount of E6 per ug of sample based upon a standard curve established using a serial dilution of total protein lysate from the HPV-16-positive CaSki cervical cancer cell line. B. quantification of p53 levels in the same tissues evaluated for E6. To learn whether the 2 fold reduction in E6 led to an impairment in its overall function we analyzed levels of 53, a known target of E6 that is destabilized through E6’s recruitment of the ubiquitin ligase, E6AP (ube3a). No changes in the levels of p53 were noted. To account for the amount of epithelial cell content in the tissue samples, Westerns were reprobed for the epithelial specific marker, keratin 14 (K14).

Figure 4

Expression of E7-specific biomarkers in Bi-L E7/K14E6/K14-tTA triply transgenic mice treated chronically with estrogen. Shown are representative microscopic images at ×40 magnification of tissue sections from triply transgenic mice stained with antibodies to either MCM7 (left) or p16 (right). Top, cervical cancers from mice treated for 10 months with estrogen that were not treated with doxycycline. Middle, endocervical epithelia from mice treated for 10 months with estrogen that were treated during the final month with doxycycline. Bottom, cervical cancers from mice treated for 10 months with estrogen that were treated with doxycycline during the 10^th month, then taken off of doxycycline and maintained on estrogen for an additional 2 months.