Cancer Research Annual Meeting 2010 2010 Workshops
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
Cancer Research 68, 9928, December 1, 2008. doi: 10.1158/0008-5472.CAN-08-2051
© 2008 American Association for Cancer Research
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chung, S.-H.
Right arrow Articles by Lambert, P. F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chung, S.-H.
Right arrow Articles by Lambert, P. F.

Molecular Biology, Pathobiology, and Genetics

Requirement for Estrogen Receptor {alpha} in a Mouse Model for Human Papillomavirus–Associated Cervical Cancer

Sang-Hyuk Chung1, Kerri Wiedmeyer1, Anny Shai1, Kenneth S. Korach2 and Paul F. Lambert1

1 McArdle Laboratory for Cancer Research, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin and 2 Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina

Requests for reprints: Paul F. Lambert, McArdle Laboratory for Cancer Research, University of Wisconsin School of Medicine and Public Health, 1400 University Avenue, Madison, WI 53706. Phone: 608-262-8533; Fax: 608-262-2824; E-mail: lambert{at}oncology.wisc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
The majority of human cervical cancers are associated with the high-risk human papillomaviruses (HPV), which encode the potent E6 and E7 oncogenes. On prolonged treatment with physiologic levels of exogenous estrogen, K14E7 transgenic mice expressing HPV-16 E7 oncoprotein in their squamous epithelia succumb to uterine cervical cancer. Furthermore, prolonged withdrawal of exogenous estrogen results in complete or partial regression of tumors in this mouse model. In the current study, we investigated whether estrogen receptor {alpha} (ER{alpha}) is required for the development of cervical cancer in K14E7 transgenic mice. We show that exogenous estrogen fails to promote either dysplasia or cervical cancer in K14E7/ER{alpha}–/– mice despite the continued presence of the presumed cervical cancer precursor cell type, reserve cells, and evidence for E7 expression therein. We also observed that cervical cancers in our mouse models are strictly associated with atypical squamous metaplasia (ASM), which is believed to be the precursor for cervical cancer in women. Consistently, E7 and exogenous estrogen failed to promote ASM in the absence of ER{alpha}. We conclude that ER{alpha} plays a crucial role at an early stage of cervical carcinogenesis in this mouse model. [Cancer Res 2008;68(23):9928–34]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
Cervical cancer, a virally caused cancer, is the second most common cancer and the second most frequent cause of death by cancer in women worldwide (1). The vast majority of cervical cancers are associated with the so-called high-risk human papillomaviruses (HPV), among which HPV-16 is most common, being found in ~60% of all cervical cancers (2). Compelling epidemiologic and experimental evidence has clearly established a causative role of HPV in this human malignancy (2, 3). Specifically, E6 and E7 oncoproteins expressed by high-risk HPV can immortalize primary human keratinocytes and cause cancers in transgenic mouse models in a cofactor-dependent manner (4–7). In addition, E6 and E7 are required for the continued proliferation of cervical cancer cell lines (8, 9). The tumorigenic potential of HPV E6 and E7 oncoproteins depends, at least in part, on their ability to inactivate p53 and pRb tumor suppressor protein, respectively (10–12).

Despite the robust carcinogenic potential of E6 and E7, HPV infection alone is not sufficient for the development of cervical cancer because only a minor fraction of patients infected with HPV develop cervical cancer (13). Indeed, several cofactors, including long-term use of oral contraceptives and high parity, have been implicated in the genesis of HPV-associated cervical cancer, suggesting a potential role of female steroid hormones, such as estrogen, in cervical carcinogenesis (14, 15). Other studies, however, have concluded otherwise. For instance, one observational study argues that estrogen does not increase risk of cervical cancer (16). This study, however, did not control for HPV, a major factor for cervical cancer. Another clinical study shows that antiestrogen tamoxifen has no beneficial effect on cervical cancer (17). This result is not surprising because tamoxifen has an agonistic rather than antagonistic effect on estrogen function in the human cervix (18). Thus, there remains a poor understanding as to the estrogen dependence of cervical cancers in humans.

An essential role of estrogen in cervical cancer, however, has been clearly defined in mouse models for HPV-associated cervical cancer that make use of transgenic mice expressing HPV-16 E6 or E7, or both, under the control of human keratin 14 (K14) promoter, which drives transgene expression in stratified squamous epithelia, natural HPV infection sites. In these mouse models, either a HPV oncogene or estrogen alone is insufficient to cause cervical cancers, whereas a HPV oncogene in conjunction with physiologic levels of exogenous estrogen can promote the development of cervical cancer (4, 11, 19, 20). The progressive cervical disease that arises in these mouse models recapitulates various aspects of human cervical disease, including the multiple stages of cervical carcinogenesis, anatomic location and histopathologic nature of the cancers, and expression patterns of various biomarkers (4, 21). In both HPV-infected women and these mouse models, cervical cancer is preceded by cervical intraepithelial neoplasia (CIN) of increasing severity that arises preferentially in the transformation zone of the endocervix, at which is found the normal transition from columnar epithelium to stratified squamous epithelium (4, 22). The transformation zone is hypothesized to be the preferential site of carcinogenesis by HPV because therein lie the reserve cells, which are thought to be multipotential progenitor cells from which cervical cancer is argued to arise (23).

It is hypothesized that estrogen can contribute to the development of cancers by estrogen receptor (ER)-dependent and ER-independent mechanisms. Estrogen is best known for exerting its physiologic effects by binding and activating its receptors, ER{alpha} and/or ERβ (24). The mitogenic effects of estrogen that are mediated through ER{alpha} are crucial for the development and maintenance of most breast cancers (25). ER{alpha}-positive breast cancers are highly responsive to the therapy with antiestrogen drugs such as tamoxifen and fulvestrant that directly bind and thus inhibit function for ER{alpha} (26). The function for ERβ in breast cancer and other estrogen-dependent cancers is less well understood. The potential ER-independent mechanism involves estrogen metabolites that can function as a direct carcinogen inducing detrimental genetic mutations (27). However, it remains unclear to what extent this mechanism contributes to the role of estrogen in estrogen-dependent cancers.

In the present study, we used ER{alpha} knockout (ER{alpha}–/–) mice to assess whether ER{alpha} is crucial for cervical carcinogenesis in the K14E7 transgenic mouse model. Our results clearly show that ER{alpha} is absolutely necessary for the development of estrogen-dependent cervical cancer in this mouse model.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 References
 
Mice. All transgenes in mice used in this study were derived from HPV-16. K14E7 transgenic mice and ER{alpha} knockout (ER{alpha}–/–) mice were described previously (28, 29). Experimental mice were generated by intercrossing F1 generations of K14E7 (FBV) and ER{alpha}+/– (C57BL/6) matings. Female progenies were genotyped by PCR. A slow-releasing 17β-estradiol tablet (0.05 mg/60 days; Innovative Research of America) was inserted s.c. under the dorsal skin every 2 mo beginning at 4 to 6 wk of age. Mice were injected i.p. with 0.3 mL bromodeoxyuridine (BrdUrd; 12.5 mg/mL) 1 h before euthanasia to measure cellular proliferation. Female reproductive tracts were harvested and processed as previously described (20). Mice were housed in McArdle Laboratory Animal Care Unit of the University of Wisconsin Medical School approved by the Association for Assessment of Laboratory Animal Care. All procedures were carried out according to an animal protocol approved by the University of Wisconsin Medical School Institutional Animal Care and Use Committee.

Antibodies and cervical cancer specimens. Antibodies were purchased from Santa Cruz Biotechnology (ER{alpha}, E7), Abcam (ERβ), Developmental Studies Hybridoma Bank (K8), NeoMarkers (p63, Mcm7), Sigma (β-actin), Calbiochem (BrdUrd), Vector Laboratories (biotinylated horse anti-mouse/rabbit IgG), and Invitrogen (Alexa Fluor 350/488/595–conjugated secondary antibody against rabbit, mouse, or rat IgG). Human cervical cancer specimens used in this study were previously described and genotyped for HPVs (30).

Immunohistochemistry and immunoblot. Immunohistochemical analyses for the detection of Mcm7 and BrdUrd were performed as described previously (7, 10). For staining ER{alpha}, ERβ, p63, and K8, standard procedures were followed as previously described (21). Briefly, deparaffinized/rehydrated sections were blocked and incubated with an antibody at appropriate dilution ({alpha}-ER{alpha}, 1:100 in 5% nonfat milk/5% horse serum; {alpha}-ERβ, 1:100 in 3% horse serum; {alpha}-K8, 1:50; and p63, 1:150 in 3% bovine serum albumin/10% goat serum). Proteins were visualized by 3,3'-diaminobenzidine (Vector Laboratories) or fluorescent microscopy. Vaginal tissues were lysed in radioimmunoprecipitation assay buffer [50 mmol/L Tris-HCl (pH 8.0), 150 mmol/L NaCl, 1% IGEPAL CA-630, 0.5% sodium deoxycholate, 0.1% SDS] supplemented with protease inhibitors, and immunoblot analyses were performed as described previously (11).

H&E staining. H&E staining was performed as previously described (20).

Statistical analyses. Two-sided Fisher's exact test and Wilcoxon rank sum test were carried out with MSTAT software version 12.0.0.3


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 References
 
ER{alpha} but not ERβ is detectable in cervical cancers of mice and women. Exogenous estrogen is required for the development and maintenance of cervical cancer in mouse models (19, 20). If ERs are necessary for cervical carcinogenesis in mice, then ER{alpha} and/or ERβ should be expressed in the cancers and/or the surrounding stroma. To test this prediction, we stained archival paraformaldehyde-fixed and paraffin-embedded female reproductive tracts of K14E6 or K14E7 single or K14E6/K14E7 double transgenic mice treated with exogenous estrogen for 6 or 9 months for ER{alpha} or ERβ (11, 19). Expression of ER{alpha} was evident in the basal and suprabasal cells of normal cervical epithelia (Fig. 1A ) and stromal cells (Fig. 1A, inset). Results also showed that 94% of cancer cells and 57% of stromal cells surrounding the cancers were positive for ER{alpha} in both K14E6 and K14E7 single transgenic mice (Fig. 1A). In contrast, we failed to detect ERβ in cancers and normal cervical epithelia as well as the surrounding stroma (Fig. 1A). ERβ was readily detected in mouse ovary (data not shown). We obtained similar results with female reproductive tracts of K14E6/K14E7 double transgenic mice (data not shown).


Figure 1
View larger version (53K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 1. ER{alpha} but not ERβ is detected in cervical cancers. A, ER{alpha} is expressed in murine cervical cancers. Archival paraformaldehyde-fixed and paraffin-embedded female reproductive tracts of indicated mice treated with 17β-estradiol were stained for ER{alpha} (top) or ERβ (bottom). Inset, stromal cells stained for ER{alpha}. Shown are representatives of more than six cancers in each transgenic mouse. More than 700 cancer cells in at least three different areas of each cancer were examined for positive staining. Nuclei were counterstained with hematoxylin. Black lines, basement membrane separating epithelium from underlying stroma. B, ER{alpha} is expressed in human cervical cancers. Formalin-fixed and paraffin-embedded human cervical tissue sections were stained as described in A. Shown is representative of three HPV-positive and four HPV-negative cervical cancers.

 
To evaluate the potential relevance of ERs in human cervical cancer, we stained formalin-fixed and paraffin-embedded human cervical cancers for ER{alpha} or ERβ. As observed in the mouse tissues, ER{alpha} was expressed in cervical cancers and adjacent normal epithelia as well as the surrounding stroma regardless of HPV status (Fig. 1B). Although the majority of cancer cells expressed detectable levels of ER{alpha}, the percentage of ER{alpha}-positive cells (85 ± 6.9%) in human cervical cancers was significantly less than that (94 ± 11.4%) observed in murine cervical cancers (P = 0.02, Wilcoxon rank sum test). Nevertheless, the vast majority of cells in both human and mouse cancers were ER{alpha} positive, consistent with a potential role for this receptor in cervical cancers in both species. ERβ was not detected in any areas of the human cervix (Fig. 1B) but could be detected in the human endometrium (data not shown). Based on these results and the knowledge that estrogen is a critical cofactor for cervical carcinogenesis in HPV transgenic mice, we hypothesized that ER{alpha} is required for cervical carcinogenesis.

Normal cervical structure and cervical reserve cells are retained in ER{alpha} knockout mice. To test this hypothesis, we used ER{alpha} knockout (ER{alpha}–/–) mice. Due to the well-known function for ER{alpha} in female reproductive tract biology (29, 31), we first investigated whether overall cervical structure is preserved in ER{alpha}–/– mice and specifically whether reserve cells, the cell type from which cervical cancers are thought to arise, are retained in these mice. Female ER{alpha}–/– mice were found to retain a normal cervical structure, including a well-defined endocervix (endocervical septum and endocervical canal) and ectocervix, as found in wild-type (wt) ER{alpha}+/+ littermates (Fig. 2A ). Reserve cells can be identified by their anatomic location just underneath the columnar epithelial lining of the cervix and expression patterns of various marker proteins. Reserve cells express both squamous and columnar epithelial makers: p63, K5, and K14 that are absent in columnar epithelia, and K8 and K18 that are not expressed in squamous epithelia (32, 33). We found cells that are double positive for K14 and K8 (data not shown) or p63 (nuclear) and K8 (cytoplasmic/membrane) underneath the p63-negative columnar epithelia of the cervix of ER{alpha}–/– as well as ER{alpha}+/+ mice (Fig. 2B). It was noted that K8 expression was enhanced in ER{alpha}–/– cervical epithelia. Finding that p63+/K8+ reserve cells express ER{alpha} is consistent with the hypothesis that both reserve cells and ER{alpha} are critical for cervical carcinogenesis (Fig. 2C).


Figure 2
View larger version (49K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 2. ER{alpha}–/– mouse retains intact cervical structure and reserve cells. A, endocervix and ectocervix are preserved in ER{alpha}–/– mouse. Female reproductive tracts from mice of the indicated genotypes were stained with H&E. Asterisks in black, red, and blue denote the endocervical septum, endocervical canal, and ectocervix, respectively. B, ER{alpha}–/– mice retain cells positive for both p63 and K8. Shown are high-power microscopic images of the transformation zone from paraffin-embedded sections of female reproductive tracts from mice of the indicated genotype that were doubly stained for p63 (red) and K8 (green). Dotted lines, basement membrane separating epithelium from underlying stroma. Note the presence of p63/K8 double-positive reserve cells positioned underneath the K8-positive, p63-negative columnar epithelium in both the ER-sufficient and ER-deficient mouse tissues. C, high-power microscopic images of the transformation zone from a paraffin-embedded section of the female reproductive tract of an ER{alpha}+/+ mouse triply stained for p63 (blue), ER{alpha} (red), and K8 (green). Dotted lines, basement membrane separating epithelium from underlying stroma. Left, note that the reserve cells (arrowheads) underlying the K8-positive, p63-negative columnar epithelium again are double positive for p63 and K8; middle, note that the reserve cells (arrowheads) are positive for both K8 and ER{alpha}; right, note (merge of K8, p63, and ER{alpha} staining) that the nuclei of the reserve cells (arrowheads) are purple indicative of double positivity for both p63 and ER{alpha} (these cells again are also positive in the cytoplasm for K8).

 
HPV-16 E7 and exogenous estrogen fail to promote cervical cancers in ER{alpha}–/– mice. In a prior study making use of K14E6 and K14E7 transgenic mice treated with exogenous estrogen, the HPV-16 E7 oncogene was determined to be more potent in promoting cervical carcinogenesis (11, 20). Therefore, to test whether ER{alpha} contributes to cervical carcinogenesis in mice, we bred K14E7/ER{alpha}+/– mice to ER{alpha}+/– mice. Offspring were genotyped for the E7 transgene and ER{alpha} status. Nontransgenic (NTG) and K14E7 transgenic female offspring that were either ER{alpha}–/– or ER{alpha}+/+ were treated with 17β-estradiol (using slow-release pellets that deliver 0.05 mg/60 days, a physiologic dose sufficient to induce continuous estrus) for 6 months, a treatment period sufficient for the development of cervical cancers in the majority of K14E7 transgenic mice (4, 20). Reproductive tracts were harvested, fixed in paraformaldehyde, and embedded in paraffin. Every tenth 5-µm section was stained with H&E and histopathologically scored to identify the worst grade of cervical/vaginal disease present in each animal. Consistent with previous results (20), the majority (67%) of K14E7/ER{alpha}+/+ mice treated with estrogen developed cervical cancer and the remainder developed high-grade dysplasias (CIN3). As expected, none of NTG/ER{alpha}+/+ mice treated with estrogen had cervical cancers and only one developed a low-grade dysplasia (CIN1; Table 1 ). This difference in cervical cancer incidence was statistically significant (P = 0.001) and recapitulated our prior findings that E7 synergizes with exogenous estrogen to induce cervical cancer (20). A strikingly different result was observed on the ER{alpha}–/– background. K14E7/ER{alpha}–/– mice treated with estrogen failed to develop cervical cancers or any grade of dysplasia (Table 1). This result shows that ER{alpha} is absolutely necessary for cervical carcinogenesis in HPV transgenic mice. Not surprisingly, no cervical disease was evident in the NTG/ER{alpha}–/– mice treated with estrogen. Vaginal disease, including vaginal cancer, was also absent in K14E7/ER{alpha}–/– mice unlike in K14E7/ER{alpha}+/+ mice (P = 0.00003, Wilcoxon rank sum test).


View this table:
[in this window]
[in a new window]

  		Table 1. State of lower reproductive tract disease in K14E7/ER{alpha}–/– mice

 
Cervical cancers are associated with atypical squamous metaplasia in mice. Because human cervical cancers are always accompanied by atypical squamous metaplasia (ASM), it has been proposed that such an aberrant metaplasia is a precursor for cervical cancer (2). For this reason, we investigated whether K14E7/ER{alpha}–/– mice treated with estrogen display ASM. We found that K14E7/ER{alpha}+/+ and NTG/ER{alpha}+/+ mice treated with estrogen have ASM (K14E7/ER{alpha}+/+ versus NTG/ER{alpha}+/+, P = 0.49, Fisher's exact test), but not K14E7/ER{alpha}–/– or NTG/ER{alpha}–/– mice, as evidenced by the presence of multiple patches of squamous epithelium within the columnar epithelium of the endocervix (Fig. 3A ). These results clearly show that ER{alpha} is necessary for the development of ASM. We further analyzed archival paraformaldehyde-fixed and paraffin-embedded female reproductive tracts of mice either untreated or treated with estrogen for the presence of ASM (19, 20). Similar to mice treated with estrogen on the ER{alpha}+/+ background described above, all examined tissues of NTG, K14E6, and K14E7 mice having wt ER{alpha} and treated with estrogen for 6 months showed atypical metaplastic changes (Fig. 3B, top). In contrast, none of untreated NTG mice had ASM and 54% of K14E6 and 100% of K14E7 untreated mice developed ASM, which are statistically significantly different (NTG versus K14E6, P = 0.044; NTG versus K14E7, P = 0.0003; K14E6 versus K14E7, P = 0.046, Fisher's exact test; Fig. 3B, middle). We also observed that ASM was absent in ovariectomized K14E7 mice (P = 0.006, compared with intact K14E7, Fisher's exact test; Fig. 3B, bottom). These results strongly suggest that HPV oncogenes and endogenous estrogen cooperate to promote the development of ASM in an ER{alpha}-dependent manner. It was noted that ASM was more prevalent in K14E7 than K14E6 mice, correlating with more potent tumorigenic potential of E7 than E6 in the mouse cervix (11, 20) and thus further supporting the hypothesis that ASM is a prerequisite for the development of cervical cancer in mice (4, 20). As such, this mouse model recapitulates the progressive cervical disease observed in women. We conclude that the absence of ASM in K14E7/ER{alpha}–/– mice treated with estrogen is primarily responsible for the lack of subsequent stages in the progressive disease leading to cervical cancers.


Figure 3
View larger version (46K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 3. Cervical cancers are associated with ASM in mice. A, estrogen-treated K14E7/ER{alpha}–/– mice lack ASM. H&E-stained reproductive tracts of mice described in Table 1 were examined for the presence of ASM. Representatives of each genotype are shown. Arrowheads, ASM. B, E6 and E7 contribute to ASM independently. H&E-stained archival reproductive tracts of indicated mice that are treated with 17β-estradiol (+E2) for 6 mo (top) or untreated (–E2; middle) were analyzed for the presence of ASM (20). Bottom, reproductive tracts of ovariectomized and untreated K14E7 mice were also examined. Arrowheads, ASM. Representatives of each group of mice are shown. Number of ASM-positive mice over total number of mice analyzed is shown at right lower corner of each image.

 
E7 oncoprotein enhances Mcm7 expression and cellular proliferation in cervical epithelium of ER{alpha}–/– mice. As E7 expression in the K14E7 transgenic mouse is driven by the human K14 promoter (28), the presence of K14-positive cells in ER{alpha}–/– cervix (data not shown) implies expression of E7 therein, which was verified by immunoblot with an E7-specific antibody (Fig. 4A ). A well-known cellular target of E7 is the pRb tumor suppressor that negatively regulates E2F transcription factors (34). Therefore, induction of E2F target genes, such as Mcm7, is indicative of expression of functional E7 (7, 21). Whereas Mcm7 expression was primarily restricted to the basal cells in NTG/ER{alpha}+/+ cervical epithelium, virtually all of the epithelial cells in K14E7/ER{alpha}+/+ cervix were found to express Mcm7 (Fig. 4B), as previously described (35). More importantly, whereas only some cells in cervical epithelium of NTG/ER{alpha}–/– mice were positive for Mcm7, nearly all of the epithelial cells in K14E7/ER{alpha}–/– cervix expressed high levels of Mcm7, consistent with E7 retaining the ability to inactivate the tumor suppressor pRb in ER{alpha}–/– cervical epithelial cells.


Figure 4
View larger version (85K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 4. Functional E7 is expressed in K14E7/ER{alpha}–/– cervical epithelium. A, E7 is expressed in the cervix of ER{alpha}–/– mice. Vaginal tissue lysates from mice of indicated genotype were immunoblotted with anti-HPV-16 E7 or anti-actin antibodies. B, Mcm7 expression is enhanced in K14E7/ER{alpha}–/– cervix compared with NTG/ER{alpha}–/– cervix. Paraffin sections of female reproductive tracts from estrogen-treated mice of the indicated genotypes were stained for Mcm7 and nuclei were counterstained with hematoxylin. Shown are representatives of three mice in each genotype. Black lines, basement membrane separating epithelium from underlying stroma. C, BrdUrd incorporation into DNA is increased in K14E7/ER{alpha}–/– cervix compared with NTG/ER{alpha}–/– cervix. Paraffin sections of female reproductive tracts from estrogen-treated mice of indicated genotypes were stained for BrdUrd. Nuclei were counterstained with hematoxylin. D, results shown in C were quantified (ER{alpha}+/+ background, n = 3 for each genotype; ER{alpha}–/– background, n = 10 for each genotype). More than 500 cells in five different areas of cervix per mouse were examined. P values for two-sided Wilcoxon rank sum test are shown.

 
Another readout for E7 function is its ability to induce hyperproliferation of epithelial cells within the cervix of K14E7 mice (19). To investigate whether E7 also induces hyperproliferation in the ER{alpha}–/– cervix, we carried out BrdUrd staining on paraffin sections of female reproductive tracts. In the absence of E7, the proliferation index of the ER{alpha}–/– cervical epithelia was significantly lower than that of the ER{alpha}+/+ cervical epithelia (NTG/ER{alpha}+/+ versus NTG/ER{alpha}–/–, P = 0.007, Wilcoxon rank sum test), suggesting that ER{alpha} is required for the normal proliferative state of the cervical epithelium (Fig. 4C and D). Nevertheless, E7 retained an ability to induce cell proliferation in the absence of ER{alpha} (compare the proliferation index of K14E7/ER{alpha}–/– with NTG/ER{alpha}–/– cervical epithelia, P = 0.047, Wilcoxon rank sum test; Fig. 4C and D).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 References
 
ER{alpha} has been shown to either promote or inhibit the development of cancers in mouse models. For instance, ER{alpha} inhibits APC-dependant colon carcinogenesis (36), whereas it is required for hormone-induced prostatic carcinogenesis (37). Similar to the latter, we showed in the present study that ER{alpha} is absolutely necessary for cervical carcinogenesis in the context of HPV transgenic mice (Table 1), which require estrogen for the development of cervical cancer (4, 11, 19, 20, 38). We also observed that ASM is associated with cervical carcinogenesis in our HPV transgenic mouse models like in women and that HPV oncogenes as well as estrogen and its receptor ER{alpha} contribute to ASM development (Fig. 3; ref. 2). We speculate that HPV oncogenes alter host gene expression in such a way as to induce squamous metaplasia that itself is reliant on estrogen and ER{alpha}, and this metaplasia renders the tissue more prone to carcinogenesis.

A model for the role of ER{alpha} in cervical carcinogenesis. Based on our and other studies, we propose a model, in which the cooperation between the HPV-16 oncogenes, estrogen, and its receptor ER{alpha} leads to the progressive disease that initiates with ASM and ends in cervical cancer (Fig. 5 ). In this model, reserve cells are the origin of ASM. This is consistent with the fact that cervical cancers preferentially arise at the transformation zone of the endocervix, the site of reserve cells, and the hypothesis that reserve cells are the progenitor cell type for cervical carcinogenesis (4, 23, 39, 40). Our results showed that ER{alpha} and likely estrogen are necessary for the development of ASM and that HPV oncogenes also contribute to this process (Fig. 3). Role of HPV E6 and E7 in the development of ASM is also supported by studies showing the ability of HPV-16 to induce squamous metaplasia of colon and lung adenocarcinoma cells in vivo (41, 42). We also argue that the same three factors, HPV oncogenes, estrogen, and ER{alpha}, are essential for the subsequent steps in the progressive cervical disease and the maintenance of cervical cancer. This is supported in part by the fact that HPV transgenic mice develop progressively worse disease as treated for longer period of time with exogenous estrogen and its withdrawal from cancer-bearing HPV transgenic mice leads to reduction not only in the cancers but also in the severity of dysplastic lesions in the cervix (4, 19). In addition, HPV-16 oncogenes (E6 and E7) are required for the continued proliferation of cell lines derived from human cervical cancer (8, 9). The continued role of E6/E7 and ER{alpha} in the later steps of progressive disease downstream of ASM has not been directly shown but could be with the evaluation of mice in which the expression of these viral oncogenes and ER{alpha} can be temporally regulated. Nonetheless, facts that exogenous estrogen, the ligand for ER{alpha}, is required for the later steps in cervical carcinogenesis as well as cancer maintenance and that HPV oncogenes are expressed in all stages of human cervical cancer are reasonable basis for predicting that these factors also are required (19, 22, 39).


Figure 5
View larger version (8K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 5. Model for cervical carcinogenesis. See the text for details.

 
This model for cervical cancer (Fig. 5) puts forth that the reserve cells are the progenitor cell from which HPV-associated cervical cancers arise. HPV-associated cancers, however, can also be found in other sites of the female reproductive tract both in the HPV transgenic mouse models and in women, where reserve cells are not known to be present. Nevertheless, these cancers seem to rely on the same three factors. In mice, the development and maintenance of cancers in the vagina are also dependent on HPV oncogenes and exogenous estrogen (19, 20). Furthermore, based on the current study, these cancers must also be dependent on ER{alpha}, as we failed to see any tumors or dysplasia in the lower reproductive tracts of K14E7/ER{alpha}–/– mice treated with estrogen (Table 1). It remains to be understood what the progenitor cell is for cancers arising in these tissues. Perhaps, it remains to be reserve cells and such reserve cells reside within these tissues but simply have not been detected to date, or the initiated cancer precursor cells for these lower reproductive tract cancers actually are the reserve cells at the transformation zone and then migrate. Alternatively, these cancers might arise not from multipotent precursor cells, such as reserve cells, but some other, perhaps more committed, cell type (e.g., a basal cell). Were this the case, then one might predict that these lower reproductive tract cancers, which are rarer than cervical cancer in women and in mouse models, might require different and/or additional genetic/epigenetic changes that arise less frequently.

Potential ER{alpha} target genes that are crucial for cervical carcinogenesis. It will be difficult to identify which estrogen-responsive genes are critical for cervical carcinogenesis, as ER{alpha} is known to activate or repress a myriad of genes, many of which have been implicated in tumorigenesis. In addition, it is not clear whether known ER{alpha} target genes [up-regulated or down-regulated by treatment with estrogen for a short period of time (hours to several days)] are likewise regulated by this ER in mice treated with estrogen for 6 months. Nonetheless, it is interesting to note that ER{alpha} up-regulates proto-oncogenes, such as c-myc, cyclin D1, epidermal growth factor receptor (EGFR), and insulin-like growth factor-I (IGF-I), and represses proapoptotic genes (43, 44). With regard to a potential role of these genes in HPV-associated cervical carcinogenesis, it has been shown that normal human cervical keratinocytes expressing HPV E6 and E7 acquire tumorigenic potential on c-myc overexpression and cyclin D1 is overexpressed in cervical cancers (45, 46). It is also relevant that EGFR inhibitor treatment is marginally effective for controlling recurring cervical cancers and high serum IGF-I levels are correlated with higher risk for CIN (47, 48).

About the possible role of the second ER, ERβ in cervical carcinogenesis, we failed to detect this isoform in cervical cancers and surrounding stroma in both mice and human (Fig. 1). In addition, the level of ERβ in ER{alpha}–/– mice is comparable with that in ER{alpha}+/+ mice (24), yet K14E7/ER{alpha}–/– mice did not develop any cervical disease (Table 1), indicating no major role of ERβ in cervical carcinogenesis. Furthermore, other lesions in mouse models induced by estrogen or diethylstilbestrol are also dependent on ER{alpha} but not ERβ (49, 50). Therefore, it is unlikely that ERβ plays a crucial role in cervical carcinogenesis.

The demonstration herein that ER{alpha} is required for cervical carcinogenesis provides support for the hypothesis that drugs that can interfere with the function of this specific nuclear receptor will be effective therapeutic agents in preventing and/or treating cervical cancers. Preclinical studies directed toward testing this hypothesis could provide a strong basis for the use of such drugs in treating human cervical disease.


   Disclosure of Potential Conflicts of Interest
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
References
 
No potential conflicts of interest were disclosed.


   Acknowledgments
 
Grant support: NIH grants CA120847, CA098428, and CA113297 (P.F. Lambert) and Division of Intramural Research of the National Institute of Environmental Health Sciences (K.S. Korach).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Elaine Alarid for critically reviewing the manuscript and providing paraffin-embedded human endometrial tissue sections.


   Footnotes
 
Note: Current address for K. Wiedmeyer: University of Wisconsin School of Veterinary Medicine, Madison, WI 53706.

3 http://www.mcardle.wisc.edu/mstat Back

Received 5/30/08. Revised 8/16/08. Accepted 8/20/08.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 

  1. Sankaranarayanan R, Ferlay J. Worldwide burden of gynaecological cancer: the size of the problem. Best Pract Res Clin Obstet Gynaecol 2006;20:207–25.[CrossRef][Medline]
  2. Burd EM. Human papillomavirus and cervical cancer. Clin Microbiol Rev 2003;16:1–17.[Abstract/Free Full Text]
  3. zur Hausen H. Papillomaviruses and cancer: from basic studies to clinical application. Nat Rev Cancer 2002;2:342–50.[CrossRef][Medline]
  4. Elson DA, Riley RR, Lacey A, Thordarson G, Talamantes FJ, Arbeit JM. Sensitivity of the cervical transformation zone to estrogen-induced squamous carcinogenesis. Cancer Res 2000;60:1267–75.[Abstract/Free Full Text]
  5. Munger K, Phelps WC, Bubb V, Howley PM, Schlegel R. The E6 and E7 genes of the human papillomavirus type 16 together are necessary and sufficient for transformation of primary human keratinocytes. J Virol 1989;63:4417–21.[Abstract/Free Full Text]
  6. Song S, Pitot HC, Lambert PF. The human papillomavirus type 16 E6 gene alone is sufficient to induce carcinomas in transgenic animals. J Virol 1999;73:5887–93.[Abstract/Free Full Text]
  7. Strati K, Pitot HC, Lambert PF. Identification of biomarkers that distinguish human papillomavirus (HPV)-positive versus HPV-negative head and neck cancers in a mouse model. Proc Natl Acad Sci U S A 2006;103:14152–7.[Abstract/Free Full Text]
  8. DeFilippis RA, Goodwin EC, Wu L, DiMaio D. Endogenous human papillomavirus E6 and E7 proteins differentially regulate proliferation, senescence, and apoptosis in HeLa cervical carcinoma cells. J Virol 2003;77:1551–63.[CrossRef][Medline]
  9. Wells SI, Aronow BJ, Wise TM, Williams SS, Couget JA, Howley PM. Transcriptome signature of irreversible senescence in human papillomavirus-positive cervical cancer cells. Proc Natl Acad Sci U S A 2003;100:7093–8.[Abstract/Free Full Text]
  10. Balsitis S, Dick F, Lee D, et al. Examination of the pRb-dependent and pRb-independent functions of E7 in vivo. J Virol 2005;79:11392–402.[Abstract/Free Full Text]
  11. Shai A, Brake T, Somoza C, Lambert PF. The human papillomavirus E6 oncogene dysregulates the cell cycle and contributes to cervical carcinogenesis through two independent activities. Cancer Res 2007;67:1626–35.[Abstract/Free Full Text]
  12. Simonson SJ, Difilippantonio MJ, Lambert PF. Two distinct activities contribute to human papillomavirus 16 E6's oncogenic potential. Cancer Res 2005;65:8266–73.[Abstract/Free Full Text]
  13. Schiffman M, Castle PE, Jeronimo J, Rodriguez AC, Wacholder S. Human papillomavirus and cervical cancer. Lancet 2007;370:890–907.[CrossRef][Medline]
  14. Moreno V, Bosch FX, Munoz N, et al. Effect of oral contraceptives on risk of cervical cancer in women with human papillomavirus infection: the IARC multicentric case-control study. Lancet 2002;359:1085–92.[CrossRef][Medline]
  15. Munoz N, Franceschi S, Bosetti C, et al. Role of parity and human papillomavirus in cervical cancer: the IARC multicentric case-control study. Lancet 2002;359:1093–101.[CrossRef][Medline]
  16. Persson I, Yuen J, Bergkvist L, Schairer C. Cancer incidence and mortality in women receiving estrogen and estrogen-progestin replacement therapy-long-term follow-up of a Swedish cohort. Int J Cancer 1996;67:327–32.[CrossRef][Medline]
  17. Bigler LR, Tate Thigpen J, Blessing JA, Fiorica J, Monk BJ. Evaluation of tamoxifen in persistent or recurrent nonsquamous cell carcinoma of the cervix: a Gynecologic Oncology Group study. Int J Gynecol Cancer 2004;14:871–4.[CrossRef][Medline]
  18. Friedrich M, Mink D, Villena-Heinsen C, Woll-Hermann A, Schmidt W. Tamoxifen and proliferation of vaginal and cervical epithelium in postmenopausal women with breast cancer. Eur J Obstet Gynecol Reprod Biol 1998;80:221–5.[CrossRef][Medline]
  19. Brake T, Lambert PF. Estrogen contributes to the onset, persistence, and malignant progression of cervical cancer in a human papillomavirus-transgenic mouse model. Proc Natl Acad Sci U S A 2005;102:2490–5.[Abstract/Free Full Text]
  20. Riley RR, Duensing S, Brake T, Munger K, Lambert PF, Arbeit JM. Dissection of human papillomavirus E6 and E7 function in transgenic mouse models of cervical carcinogenesis. Cancer Res 2003;63:4862–71.[Abstract/Free Full Text]
  21. Brake T, Connor JP, Petereit DG, Lambert PF. Comparative analysis of cervical cancer in women and in a human papillomavirus-transgenic mouse model: identification of minichromosome maintenance protein 7 as an informative biomarker for human cervical cancer. Cancer Res 2003;63:8173–80.[Abstract/Free Full Text]
  22. Woodman CB, Collins SI, Young LS. The natural history of cervical HPV infection: unresolved issues. Nat Rev Cancer 2007;7:11–22.[CrossRef][Medline]
  23. Ledwaba T, Dlamini Z, Naicker S, Bhoola K. Molecular genetics of human cervical cancer: role of papillomavirus and the apoptotic cascade. Biol Chem 2004;385:671–82.[CrossRef][Medline]
  24. Couse JF, Korach KS. Estrogen receptor null mice: what have we learned and where will they lead us? Endocr Rev 1999;20:358–417.[Abstract/Free Full Text]
  25. Palmieri C, Cheng GJ, Saji S, et al. Estrogen receptor β in breast cancer. Endocr Relat Cancer 2002;9:1–13.[Abstract]
  26. Heldring N, Pike A, Andersson S, et al. Estrogen receptors: how do they signal and what are their targets. Physiol Rev 2007;87:905–31.[Abstract/Free Full Text]
  27. Cavalieri E, Chakravarti D, Guttenplan J, et al. Catechol estrogen quinones as initiators of breast and other human cancers: implications for biomarkers of susceptibility and cancer prevention. Biochim Biophys Acta 2006;1766:63–78.[Medline]
  28. Herber R, Liem A, Pitot H, Lambert PF. Squamous epithelial hyperplasia and carcinoma in mice transgenic for the human papillomavirus type 16 E7 oncogene. J Virol 1996;70:1873–81.[Abstract/Free Full Text]
  29. Lubahn DB, Moyer JS, Golding TS, Couse JF, Korach KS, Smithies O. Alteration of reproductive function but not prenatal sexual development after insertional disruption of the mouse estrogen receptor gene. Proc Natl Acad Sci U S A 1993;90:11162–6.[Abstract/Free Full Text]
  30. Nelson JH, Hawkins GA, Edlund K, et al. A novel and rapid PCR-based method for genotyping human papillomaviruses in clinical samples. J Clin Microbiol 2000;38:688–95.[Abstract/Free Full Text]
  31. Curtis SW, Washburn T, Sewall C, et al. Physiological coupling of growth factor and steroid receptor signaling pathways: estrogen receptor knockout mice lack estrogen-like response to epidermal growth factor. Proc Natl Acad Sci U S A 1996;93:12626–30.[Abstract/Free Full Text]
  32. O'Connell JT, Mutter GL, Cviko A, et al. Identification of a basal/reserve cell immunophenotype in benign and neoplastic endometrium: a study with the p53 homologue p63. Gynecol Oncol 2001;80:30–6.[CrossRef][Medline]
  33. Smedts F, Ramaekers F, Troyanovsky S, et al. Basal-cell keratins in cervical reserve cells and a comparison to their expression in cervical intraepithelial neoplasia. Am J Pathol 1992;140:601–12.[Abstract]
  34. Felsani A, Mileo AM, Paggi MG. Retinoblastoma family proteins as key targets of the small DNA virus oncoproteins. Oncogene 2006;25:5277–85.[CrossRef][Medline]
  35. Balsitis S, Dick F, Dyson N, Lambert PF. Critical roles for non-pRb targets of human papillomavirus type 16 E7 in cervical carcinogenesis. Cancer Res 2006;66:9393–400.[Abstract/Free Full Text]
  36. Cho NL, Javid SH, Carothers AM, Redston M, Bertagnolli MM. Estrogen receptors {alpha} and β are inhibitory modifiers of Apc-dependent tumorigenesis in the proximal colon of Min/+ mice. Cancer Res 2007;67:2366–72.[Abstract/Free Full Text]
  37. Ricke WA, Wang Y, Cunha GR. Steroid hormones and carcinogenesis of the prostate: the role of estrogens. Differentiation 2007;75:871–82.[CrossRef][Medline]
  38. Arbeit JM, Howley PM, Hanahan D. Chronic estrogen-induced cervical and vaginal squamous carcinogenesis in human papillomavirus type 16 transgenic mice. Proc Natl Acad Sci U S A 1996;93:2930–5.[Abstract/Free Full Text]
  39. Doorbar J. Molecular biology of human papillomavirus infection and cervical cancer. Clin Sci (Lond) 2006;110:525–41.[Medline]
  40. Tsutsumi K, Sun Q, Yasumoto S, et al. In vitro and in vivo analysis of cellular origin of cervical squamous metaplasia. Am J Pathol 1993;143:1150–8.[Abstract]
  41. Kinjo T, Kamiyama K, Chinen K, Iwamasa T, Kurihara K, Hamada T. Squamous metaplasia induced by transfection of human papillomavirus DNA into cultured adenocarcinoma cells. Mol Pathol 2003;56:97–108.[Abstract/Free Full Text]
  42. Nakazato I, Hirayasu T, Kamada Y, Tsuhako K, Iwamasa T. Carcinoma of the lung in Okinawa, Japan: with special reference to squamous cell carcinoma and squamous metaplasia. Pathol Int 1997;47:659–72.[Medline]
  43. Shang Y, Brown M. Molecular determinants for the tissue specificity of SERMs. Science 2002;295:2465–8.[Abstract/Free Full Text]
  44. Carroll JS, Brown M. Estrogen receptor target gene: an evolving concept. Mol Endocrinol 2006;20:1707–14.[Abstract/Free Full Text]
  45. Cheung TH, Yu MM, Lo KW, Yim SF, Chung TK, Wong YF. Alteration of cyclin D1 and CDK4 gene in carcinoma of uterine cervix. Cancer Lett 2001;166:199–206.[CrossRef][Medline]
  46. Narisawa-Saito M, Yoshimatsu Y, Ohno S, et al. An in vitro multistep carcinogenesis model for human cervical cancer. Cancer Res 2008;68:5699–705.[Abstract/Free Full Text]
  47. Wu X, Tortolero-Luna G, Zhao H, Phatak D, Spitz MR, Follen M. Serum levels of insulin-like growth factor I and risk of squamous intraepithelial lesions of the cervix. Clin Cancer Res 2003;9:3356–61.[Abstract/Free Full Text]
  48. Goncalves A, Fabbro M, Lhomme C, et al. A phase II trial to evaluate gefitinib as second- or third-line treatment in patients with recurring locoregionally advanced or metastatic cervical cancer. Gynecol Oncol 2008;108:42–6.[CrossRef][Medline]
  49. Couse JF, Dixon D, Yates M, et al. Estrogen receptor-{alpha} knockout mice exhibit resistance to the developmental effects of neonatal diethylstilbestrol exposure on the female reproductive tract. Dev Biol 2001;238:224–38.[CrossRef][Medline]
  50. Prins GS, Birch L, Couse JF, Choi I, Katzenellenbogen B, Korach KS. Estrogen imprinting of the developing prostate gland is mediated through stromal estrogen receptor {alpha}: studies with {alpha}ERKO and βERKO mice. Cancer Res 2001;61:6089–97.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Proc. Natl. Acad. Sci. USAHome page
S.-H. Chung and P. F. Lambert
Prevention and treatment of cervical cancer in mice using estrogen receptor antagonists
PNAS, November 17, 2009; 106(46): 19467 - 19472.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chung, S.-H.
Right arrow Articles by Lambert, P. F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chung, S.-H.
Right arrow Articles by Lambert, P. F.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online