Loss of Dependence on Continued Expression of the Human Papillomavirus 16 E7 Oncogene in Cervical Cancers and Precancerous Lesions Arising in Fanconi Anemia Pathway-Deficient Mice

Abstract

Fanconi anemia (FA) is a rare genetic disorder caused by defects in DNA damage repair. FA patients often develop squamous cell carcinoma (SCC) at sites where high-risk human papillomaviruses (HPVs) are known to cause cancer, including the cervix. However, SCCs found in human FA patients are often HPV negative, even though the majority of female FA patients with anogenital cancers had preexisting HPV-positive dysplasia. We hypothesize that HPVs contribute to the development of SCCs in FA patients but that the continued expression of HPV oncogenes is not required for the maintenance of the cancer state because FA deficiency leads to an accumulation of mutations in cellular genes that render the cancer no longer dependent upon viral oncogenes. We tested this hypothesis, making use of Bi-L E7 transgenic mice in which we temporally controlled expression of HPV16 E7, the dominant viral oncogene in HPV-associated cancers. As seen before, the persistence of cervical neoplastic disease was highly dependent upon the continued expression of HPV16 E7 in FA-sufficient mice. However, in mice with FA deficiency, cervical cancers persisted in a large fraction of the mice after HPV16 E7 expression was turned off, indicating that these cancers had escaped from their dependency on E7. Furthermore, the severity of precancerous lesions also failed to be reduced significantly in the mice with FA deficiency upon turning off expression of E7. These findings confirm our hypothesis and may explain the fact that, while FA patients have a high frequency of infections by HPVs and HPV-induced precancerous lesions, the cancers are frequently HPV negative. IMPORTANCE : Fanconi anemia (FA) patients are at high risk for developing squamous cell carcinoma (SCC) at sites where high-risk human papillomaviruses (HPVs) frequently cause cancer. Yet these SCCs are often HPV negative. FA patients have a genetic defect in their capacity to repair damaged DNA. HPV oncogenes cause an accumulation of DNA damage. We hypothesize, therefore, that DNA damage induced by HPV leads to an accumulation of mutations in patients with FA deficiency and that such mutations allow HPV-driven cancers to become independent of the viral oncogenes. Consistent with this hypothesis, we found that cervical cancers arising in HPV16 transgenic mice with FA deficiency frequently escape from dependency on the HPV16 oncogene that drove its development. Our report provides further support for vaccination of FA patients against HPVs and argues for the need to define mutational profiles of SCCs arising in FA patients in order to inform precision medicine-based approaches to treating these patients.

FIG 1

Histopathology of representative female reproductive tracts. Low-magnification images of the cervices stained with hematoxylin and eosin are shown. A high-magnification image of cancers (Bi-L E7/K14–tTA/FancD2^+/+, Bi-L E7/K14-tTA/FancD2^−/−, and Bi-L E7/K14-tTA/FancD2^−/− [Dox]) or cervical epithelium (FancD2^+/+, FancD2^−/−, and Bi-L E7/K14–tTA/FancD2^+/+ [Dox]) is shown in the upper right corner of each image. Black boxes indicate the approximate locations of the enlarged areas.

FIG 2

Expression of MCM7, an E7-specific biomarker in cervical epithelium and cancers; evidence for temporal regulation of E7 expression. Representative immunohistochemistry images are shown at high magnification. The images for FancD2^+/+, Bi-L E7/K14–tTA/FancD2^+/+ (Dox), and FancD2^−/− are showing endocervical epithelium since they did not develop any cancer. The images for Bi-L E7/K14–tTA/FancD2^+/+, Bi-L E7/K14-tTA/FancD2^−/−, and Bi-L E7/K14-tTA/FancD2^−/− (Dox) are showing cervical cancers. Brown nuclei represent MCM7-positive cells, and hematoxylin (blue) was used to counterstain nuclei. Scale bar, 20 µm.

FIG 3

Cellular proliferation levels in cervical epithelium. To monitor newly synthesized DNA, the cervical epithelia were stained with BrdUrd (a complete description of the protocol used is provided in Materials and Methods). For each genotype, at least 3 mice were randomly selected and eight image frames of cells at the cervical epithelia (non-tumor-bearing regions) were quantified. The numbers of BrdUrd-positive cells and the total numbers of cells were plotted. Using a two-sided Wilcoxon rank sum test, statistical analyses were performed. *, FancD2^+/+ versus Bi-L E7/K14–tTA/FancD2 (P = 0.03571). **, Bi-L E7/K14–tTA/FancD2^+/+ versus Bi-L E7/K14–tTA/FancD2^+/+ (Dox) (P = 0.0556). #, Bi-L E7/K14-tTA/FancD2^−/− versus Bi-L E7/K14-tTA/FancD2^−/− (Dox) (P = 0.6286). For FancD2^+/+ versus FancD2^−/−, P = 0.5379.