Estrogen contributes to the onset, persistence, and malignant progression of cervical cancer in a human papillomavirus-transgenic mouse model

Abstract

Cervical cancer is a leading cause of death by cancer among women worldwide. High-risk human papillomaviruses (HPVs) are the major etiological agents for cervical cancer, but other factors likely contribute to cervical cancer, because these cancers commonly arise decades after initial exposure to HPV. Estrogen is thought to be one such cofactor; however, its temporal requirements in human cervical cancer are not known. Here we evaluate the temporal requirements of estrogen in cervical carcinogenesis in a mouse model for HPV-associated cervical cancer. Tumors arising in HPV16 transgenic mice treated with estrogen for 9 months were greatly increased in their size compared with tumors developing after 6 months of estrogen treatment. HPV16 transgenic mice treated 6 months with estrogen followed by 3 months without exogenous estrogen had significantly fewer tumors and the tumors were smaller and less aggressive than those arising in mice treated the full 9 months. Importantly, cervical cancers that arose in the mice treated the first 6 of 9 months with estrogen must have regressed, based upon the reduced incidence of cancers in these mice compared with those treated for 6 months with estrogen, then immediately analyzed. We conclude that estrogen plays a critical role not only in the genesis of cervical cancer but also in its persistence and continued development in this mouse model. These findings raise the clinically relevant possibility that, if human cervical cancer has a similar dependence on estrogen for continued tumor growth, then antiestrogen therapy may be effective in the treatment of cervical cancer.

Figures

Fig. 1.

Cervical squamous epithelium of untreated mice. (A, C, and E) H&E-stained cross sections of untreated nontransgenic (A), K14E7 transgenic (C), and K14E6/K14E7 transgenic (E) cervical squamous epithelium. Transgenic mice show extensive papillomatosis, more cells with basal-like appearance, and more double nuclei than nontransgenic mice. (B, D, and F) Immunohistochemical staining for 5-bromodeoxyuridine in control nontransgenic (B), K14E7 transgenic (D), or K14E6/K14E7 transgenic (F) sections. Bromodeoxyuridine-positive cells (indicated by brown nuclear staining) are restricted to the basal and parabasal layers of the epithelium in nontransgenic mice, whereas they extend into the suprabasal layers in transgenic mice.

Fig. 2.

Abnormalities resulting from 9 months of chronic estrogen treatment. Shown are histological cross sections of the cervical transformation zone of an untreated K14E6/K14E7 mouse (representative of all untreated mice; A), with black arrow indicating the junction between squamous and columnar epithelium. In nontransgenic mice treated for 9 months with estrogen, alternating squamous and columnar regions (B; indicated by white arrows) are found below the transformation zone (black arrow). Additionally, hyperplastic uterine glands extend into the entire cervical septum. Transgenic mice treated for 9 months with estrogen (C) show metaplastic squamous cells growing past the transformation zone (black arrow) and replacing columnar cells within the glands, indicated by blue arrows.

Fig. 3.

Classification of tumors by location. Diagram in center shows a schematic of the mouse reproductive tract and demarcation of approximate boundaries used in classifying location of malignancies. Pie charts on the left (for K14E7) or right (for K14E6/K14E7) show, by location, the percentage of the total tumors arising in each region (upper) and the percentage of the total tumor area these tumors encompassed (lower).

Fig. 4.

Cancer phenotype alterations in mice withdrawn from exogenous estrogen treatment. Shown are H&E-stained histological cross sections of tumors arising in the K14E6E7 mice after differing estrogen treatments. Withdrawal of exogenous estrogen led to a much more organized phenotype in the tumor tissue (A) than in mice given 9 months of continuous estrogen treatment (B). Notice the more organized structure in A, where most masses are confined by a basement membrane. Additionally, B shows more fibrous stromal reaction that is indicative of malignancy. At higher magnification, mice withdrawn from exogenous estrogen treatment (C) show less nuclear atypia and a lower number of individually invasive cells than mice given 9 months of continuous estrogen treatment (D).

Fig. 5.

Cervical squamous epithelial phenotypes differ among estrogen treatment regimens. Shown are cross sections of cervical squamous epithelium from K14E6/K14E7 mice stained with H&E from mice treated with estrogen for 6 months (A), 9 months (B), or 6 months followed by 3 months of aging (C). One-hundred percent of mice given 6 months of estrogen treatment showed severe dysplasias (A) or microinvasive cancers, a phenotype that became more aggressive after 9 months of treatment with estrogen (B). In contrast, mice exposed to estrogen for 6 months and then withdrawn from exogenous estrogen treatment showed a lower degree of dysplasia than either of the continuous estrogen treatments (C), with only 38% of these mice diagnosed with severe dysplasia or beyond.