E6AP is Required for Human Papillomavirus type 16 E6 to Cause Cervical Cancer in Mice

Abstract

High-risk human papillomaviruses cause certain anogenital and head and neck cancers. E6, one of three potent HPV oncogenes that contribute to the development of these malignancies, is a multifunctional protein with many biochemical activities. Among these activities are its ability to bind and inactivate the cellular tumor suppressor p53, induce expression of telomerase, and bind to various other proteins including Bak, E6BP1, E6TP1, and proteins that contain PDZ domains such as hScrib and hDlg. Many of these activities are thought to contribute to E6’s role in carcinogenesis. E6’s interaction with many of these cellular proteins, including p53, leads to their destabilization. This property is mediated at least in part through E6’s ability to recruit the ubiquitin ligase, E6AP into complexes with these cellular proteins resulting in their ubiquitin–mediated degradation by the proteasome. In this study, we address the requirement for E6AP in mediating E6's acute and oncogenic phenotypes, including induction of epithelial hyperplasia, abrogation of DNA damage response and induction of cervical cancer. Loss of E6AP had no discernable effect on E6's ability to induce hyperplasia or abrogate DNA damage responses, akin to what we had earlier observed in the mouse epidermis. Nevertheless, in cervical carcinogenesis studies, there was a complete loss of E6’s oncogenic potential in mice nulligenic for E6AP. Thus, E6AP is absolutely required for E6 to cause cervical cancer.

Introduction

Human papillomaviruses (HPVs) are etiologically associated with cancers of the head and neck and the anogenital tract, which includes the cervix. Infection with HPVs is attributed with almost all cases of cervical cancer (1, 2). Cervical cancer accounts for approximately 5% of the world’s total tumor burden (3) and is the second leading cause of cancer-related deaths among women (4, 5). Approximately 70% of all cervical cancers are attributed to infection with the “high-risk” HPV types −16 or −18. In cervical cancers, two HPV genes, E6 and E7 are expressed (6, 7). HPV E6 and E7 have transforming and oncogenic properties in tissue culture (8, 9) and in animal models (10–12). E6 and E7 are primarily known for their abilities to target the tumor suppressors, p53 (13, 14) and pRb (15) respectively, for inactivation and ubiquitin-mediated degradation.

High-risk E6 is known to target p53 for ubiquitin-mediated degradation through its interaction with the E3 ligase, E6-associated protein (E6AP). Similarly, E6 has been found to induce the degradation of other cellular proteins and in many cases it is mediated through interactions with E6AP (16, 17). While the majority of data support the requirement of E6AP in E6-mediated degradation of p53 (18, 19), our previous studies in the mouse skin indicate that HPV16 E6 is able to target p53 for inactivation and degradation in the absence of E6AP (20). Results from other groups using either in vitro or in vivo systems also suggest the existence of E6AP-independent means for high-risk HPV E6 to target p53 for ubiquitin-mediated degradation (21, 22). Therefore, it remained unclear what role E6AP would have in E6-mediated acute and oncogenic phenotypes in the context of the cervix.

In mice, E6 and E7 cooperate to induce cervical carcinogenesis (23, 24). HPV E6 induces cervical cancer both through its ability to inactivate p53 and other cellular proteins including PDZ containing proteins that interact directly with E6 (24, 25). Similar requirements were needed for E6 to induce skin cancers in mice (26). Because HPV16 E6 can target p53 for inactivation in the skin through E6AP-independent means (20), it was of interest to determine the importance of E6AP in cervical carcinogenesis.

Further interest fueled our pursuits of E6AP in the cervix. In addition to its function as an E3 ubiquitin ligase, E6AP also functions as a steroid receptor coactivator (27) and may be differentially regulated in cancers of the breast and prostate (28, 29). Recent evidence demonstrate that the nuclear hormone receptor, ERα, is a necessary cofactor in HPV-mediated cervical carcinogenesis (30). Given the involvement of E6AP in the regulation of nuclear hormone receptors, its importance in cervical cancer warranted further investigation.

To determine E6AP’s role in the oncogenic effects of E6, we generated HPV transgenic mice on an FRAP-null (E6AP^−/−) background. Our results demonstrate that in the cervix, loss of E6AP ablates E6’s oncogenic activity. We conclude that E6AP is a critical mediator of HPV16 E6’s oncogenic activities in the cervix.

Materials and Methods

Mice

The mouse lines, K14E6^WT (31), K14E7^WT (10) and E6AP^−/− (20, 32) were maintained on the inbred FVB/n background. All mice were bred and maintained in the American Association for Accreditation of Laboratory Animal Care-approved McArdle Laboratory Animal Care Facility in accordance with an institutionally approved animal protocol.

Estrogen treatment and analysis of reproductive tracts

For acute studies, 5-week old virgin female mice were treated with 17β-estradiol for six weeks to achieve a state of persistent estrus (24, 25). Mice acutely treated with estrogen were either exposed to 10Gy of ionizing radiation from a ¹³⁷Cs source or left untreated and sacrificed 24 hours later. One hour prior to sacrifice, the nucleotide analog, 5-bromo-2’-deoxyuridine (BrdUrd, 12.5mg/mL in PBS) was injected intraperitoneally at 10µl /1g body weight to label any newly synthesized DNA.

For acute studies that require ovariectomy, 8-week old virgin female mice were ovariectomized and were allowed to finish the remaining cycle and recover for 10 days. The mice were then injected with either estrogen (30µg/kg weight) or ethanol (EtOH) vehicle and sacrificed 24 hours later. Animals were perfused with 4% paraformaldehyde and reproductive tracts were harvested for analysis.

For long-term cancer studies, five-week old virgin female mice were either untreated or treated with 17β-estradiol for a period of six to nine months after which, the reproductive tracts were harvested and fixed according to previously described methods (23) and sectioned. Every tenth, 5-µm section was stained with hematoxylin and eosin and evaluated histopathologically, with the worst lesion scored as the final diagnosis. The severity of disease was evaluated by taking the spectrum of lesions within a given group and given a score of 0–5. Specifically, a diagnosis of hyperplasia is given a value of 0; CIN1 (cervical intraepithelial neoplasia 1) a value of 1 and increases progressively with each higher lesion grade to a score of 5 for LIC (large invasive cancer).

Quantification of DNA synthesis and DNA damage response

To quantify the levels of DNA synthesis in the epithelium of the cervix, basal and suprabasal BrdUrd-positive cells were counted and divided by the total number of cells and multiplied by 100 to determine the percentage. To quantify the DNA damage response, the total number of BrdUrd-positive cells were counted and divided by the total number of cells in the epithelium and multiplied by 100. BrdUrd was counted in at least 8, 40× microscopic fields per mouse, with a total of at least 3 or more mice per genotype group.

Statistical Analysis

A two-sided Wilcoxon-Rank Sum test was used for determining statistical significance in the quantification of DNA synthesis, tumor multiplicity, tumor burden and severity of disease. A two-sided Fisher’s Exact test was used in determining significance for cancer incidence.

Immunohistochemistry and Immunofluorescence

Conditions for IHC or IF for the following antibodies BrdUrd (Calbiochem, #NA20), p53 (Novacastra, CM5), MCM7 (NeoMarkers, # ab47DC141) and ERα (Santa Cruz, MC20) were previously described (20, 24, 25).

Results

E6-induced cervical epithelial hyperplasia is not dependent on E6AP

Previous results indicate that the ubiquitin ligase, E6AP is not required for E6-induced epithelial hyperplasia in the skin (20). To determine whether E6AP is required for E6-induced cervical epithelial hyperplasia, we crossed K14E6^WT transgenic mice onto an E6AP^−/− background and monitored for proliferation in young female mice treated acutely with estrogen. Acute treatment with estrogen is necessary to avoid variability in the thickness of the cervical epithelium and synchronizes all mice. Animals were injected with the nucleotide analog, bromodeoxyuridine (BrdUrd) one-hour prior to sacrifice. Proliferation was subsequently measured by quantifying the incorporation of BrdUrd into nascently synthesized DNA via BrdUrd-specific immunohistochemistry (IHC). Similar to previous results, K14E6^WT transgenic mice were able to induce hyperplasia in the cervical epithelium as indicated by higher levels of suprabasal DNA synthesis relative to nontransgenic (NTG) mice (0.9% vs 0.4% respectively, p= 0.03, Fig. 1A inset). Loss of E6AP in K14E6^WT transgenic mice (K14E6^WTE6AP^−/−) did not result in a loss of epithelial hyperplasia (0.9%) and was not different than K14E6^WT mice (p=1). Surprisingly, the loss of E6AP in the absence of HPV E6 (E6AP^−/−) also resulted in significantly higher levels of suprabasal DNA synthesis relative to NTG mice (1.0% vs. 0.4% respectively, p=0.02). Thus E6AP is not the mediator of HPV-E6 induced hyperplasia.

Figure 1. Examination of acute effects in HPV E6 transgenic mice on an E6AP-deficient background in the cervix.

A. The graph shows the quantification of DNA synthesis in the cervical epithelium of female mice acutely treated with estrogen to synchronize mice in estrus. Groups of three or more mice per genotype were injected with BrdUrd one hour prior to sacrifice. Formalin fixed, paraffin embedded (FFPE) tissue was subjected to BrdUrd-specific immunohistochemistry and the percentage of BrdUrd-positive cells were counted both in the basal and suprabasal layers in at least eight 40× frames/mouse tissue, as a measure of DNA synthesis. Epithelial hyperplasia is defined by an increase in the percentage of suprabasal BrdUrd-positive cells. E6AP^−/− mice have a marginal increase in the number of basal BrdUrd-positive cells relative to NTG (p=0.08, #) mice and is significantly different than K14E6^WT mice (p=0.03, *). The percentage of suprabasal BrdUrd-positive cells was significantly higher in both E6AP^−/− and K14E6^WT groups (p<0.05) relative to NTG mice. Loss of E6AP in the presence of HPV E6 did not prevent the induction of epithelial hyperplasia (p=1). The inset is a magnification of the number of the BrdUrd-positive cells in the suprabasal layers.

B. The graph shows the quantification of DNA synthesis in the cervical epithelium of females acutely treated with estrogen and either given 10Gy of ionizing radiation or not treated. Groups of three or more mice per genotype were injected with BrdUrd 23 hours post irradiation and one hour later tissue was harvested. Like size groups of age matched mice not exposed to irradiation served as controls. Percentage BrdUrd-positive cells was quantified as described in A. Following irradiation, significant reductions in the percentage of cells supporting DNA synthesis were observed in both NTG (*, p=0.01) and E6AP^−/− (*, p=0.03) groups. Both K14E6 ^WT and K14E6 ^WTE6AP^−/− groups were abrogated in this DNA damage response, maintaining the levels of DNA synthesis seen in the control groups for each genotype (both p>0.05).

C. Shown are sections of cervical epithelium from mice acutely treated with exogenous estrogen and stained for BrdUrd or p53 by IHC. Mice were either unirradiated or irradiated and sacrificed 24hours later. Dotted lines in each picture demarcate the cervical epithelium from the underlying stroma. DNA synthesis, as represented by BrdUrd positive staining nuclei, is reduced upon radiation in nontransgenic mice on either an E6AP-sufficient or insufficient background (first and second columns). In contrast, both K14E6^WT and K14E6^WTE6AP^−/− retain the ability to synthesize DNA. p53 is undetectable in the absence of DNA damage (data not shown), however is induced in NTG and E6AP^−/− mice upon radiation (third column). K14E6^WT and K14E6^WTE6AP^−/− mice display variable levels of p53 upon irradiation. Arrows point to examples of p53-positive cells. Examples of a strong inhibition of the DNA damage response as exemplified by either a lack of (K14E6^WT) or a few (K14E6^WTE6AP^−/−) p53-positive cells in the epithelium are also shown (fourth column).

A trivial explanation for our observation that E6AP is not required for E6-induced cell proliferation is that E6 is itself causing lower steady state levels of E6AP. To determine if this is the case, we performed E6AP-specific western analysis. We observed no obvious differences in the levels of E6AP in NTG versus K14E6^WT, K14E7^WT or K14E6^WTE7^WT transgenic tissues (Fig. S1). Thus in our mouse model E6 does not alter levels of E6AP.

Inhibition of the DNA damage response by HPV E6 in the absense of E6AP

Previous results demonstrate that HPV E6 transgenic mice retain the ability to inhibit DNA damage responses in response to ionizing radiation in the epidermis of the mouse skin despite the absence of E6AP (20). Given these results, we wanted to determine whether HPV E6 maintains the ability to inhibit the DNA damage response in the absence of E6AP in the cervical epithelium. Mice acutely treated with estrogen were either given 10 Gy of ionizing radiation or left untreated and harvested 24-hours later. One hour prior to sacrifice, all of the mice were injected with BrdUrd to label nascently synthesized DNA. We assessed two different responses to DNA damage: (1) the ability to cease DNA synthesis and (2) the induction and subsequent accumulation of p53. As expected, DNA synthesis was significantly reduced in both NTG (2.0% to 0.3%, p=0.01) and E6AP^−/− (3.4% to 0.8%, p=0.03) mice upon radiation (Fig. 1B, C). Similar to previous results observed in the skin, DNA synthesis in K14E6^WT transgenic mice was not significantly changed upon radiation (2.6% vs 2.1%, p=0.09). Interestingly, K14E6^WTE6AP^−/− transgenic mice did not show a reduction in DNA synthesis upon DNA damage (2.3% vs 3.5%, p=0.16), suggesting that in the cervical epithelium, E6AP is not required for E6’s targeted inactivation of p53 in inhibiting the DNA damage response. This result is in kind to that observed previously in the skin (20).

To address whether E6 can prevent the accumulation of p53 after DNA damage in the absence of E6AP, IHC for p53 was performed on sections from both unirradiated and irradiated mice. In the absence of any DNA damage, p53 was undetectable in all genotypes (data not shown). As expected, p53 was highly induced in the cervical epithelium of NTG and E6AP^−/− mice upon radiation (Fig. 1C). In contrast to skin where E6 is able to largely prevent induction of detectable p53 following irradiation, p53 levels in the irradiated K14E6^WT cervix varied from nil to modest levels, with positive cells found predominantly in the basal layer. Thus in the cervix, E6 partially prevents the accumulation of p53 in response to irradiation, even though it efficiently prevents DNA damage-associated growth arrest (Fig. 1B). The cervical epithelia of irradiated K14E6^WTE6AP^−/− transgenic mice also displayed low to modest levels of p53 protein, similar to K14E6^WT mice, indicating that E6AP is not required for E6's ability to prevent accumulation of p53 following irradiation. This is similar to what was observed in the skin of E6AP-null mice (20).

The acute response to estrogen is affected in mice without E6AP

In addition to its function as an ubiquitin ligase, E6AP also functions as a steroid receptor coactivator (27). E6AP has been reported to target various nuclear hormone receptors (29) including estrogen receptor alpha (ERα) for ubiquitin-mediated degradation (33). Because E6AP is reported to play a role in estrogen signaling, we investigated whether loss of E6AP has any effect in the cervix, where estrogen signaling plays an important role. Effects from estrogen signaling can be observed quickly, and thus the acute response to estrogen was assessed in groups of NTG and E6AP^−/− mice. Mice were ovariectomized to eliminate endogenous levels of estrogen and treated 10 days later with either vehicle or estrogen for 24-hours and after which the reproductive tracts were harvested. Ovariectomy results in a thin epithelium throughout the reproductive tract due to a lack of proliferation signals from ovarian hormones. The normal response to estrogen is an increase in uterine wet weight (34) and the induction of epithelial hyperplasia. The epithelium from NTG and E6AP^−/− groups treated with ETOH vehicle were hypoplastic. Upon estrogen treatment, the majority of reproductive tracts from NTG mice were overtly (data not shown) and histologically thickened (Fig. 2). Acute treatment of E6AP^−/− mice with estrogen, on the contrary, did not result in either an overt or a histologically thickened epithelium. It is not clear why this is the case, because at the time of sacrifice, the proliferative index in the cervical epithelium of these mice was similar to NTG mice as assessed by BrdUrd incorporation over a one hour period prior to sacrifice, and there was no detectable programmed cell death (data not shown). One possible explanation is that there is a slight delay in the response of the E6AP-null tissue to estrogen, given that E6AP-null tissue does undergo hyperplasia when treated long term with estrogen (see below).

Figure 2. Acute responses to estrogen partially require E6AP.

Shown are H/E sections of cervix from mice that were ovariectomized and treated either with ETOH or estrogen (E₂) for 24hours. The first and third columns are sections taken at 1.25× magnification, while the second and fourth columns are sections taken at 40× magnification. Note the relative lack of epithelial hyperplasia in E6AP^−/− mice relative to NTG mice upon treatment with estrogen (far right column).

E6-induced cervical carcinogenesis is dependent upon E6AP

Given that ERα signaling is affected in E6AP^−/− mice, we investigated whether loss of E6AP would result in a reduced ability of HPV E6 to induce cervical cancer. We therefore compared mice nulligenic for E6AP and bred them to K14E6^WT mice. K14E6^WT mice develop cervical cancer after treatment with physiological levels of estrogen for nine months (24). Cervical lesions that arise in these mice are histopathologically similar to that found in HPV16-infected women. Specifically, we observe a progressive disease that includes the development of cervical intraepithelial neoplasia (CIN) grades 1–3, followed by the onset of frank cancer of the cervix. We therefore compared the severity of cervical disease by comparing the complete spectrum of lesions observed within any given group (see Methods for details). Similar to previous findings, K14E6^WT mice have an increase in the severity of disease, which was significantly different from NTG mice (p≪0.001). K14E6^WT transgenic mice have an increase in tumor incidence, multiplicity and tumor burden (as defined as the average of area of tumor mass) relative to NTG mice (Suppl. Fig. 2, Table 1 & Table 2). In K14E6^WTE6AP^−/− transgenic mice, however, the severity of disease was significantly reduced (p≪0.001) when compared to K14E6^WT. Specifically, K14E6^WTE6AP^−/− mice have reductions in the incidence of cervical cancer (6.3%, p=0.04), tumor multiplicity (0.13, p=0.06) and tumor burden (p=0.04). Indeed, the incidence of cervical disease in the K14E6 ^WTE6AP^−/− mice was not significantly different from that observed in nontransgenic mice. These results indicate that E6’s capacity to induce cervical cancer was completely abrogated on an E6AP-null background.

Table 1.

Summary of Disease in the Cervix and Vagina of Mice After 9 Months with Estrogen

	Mouse Genotype	Severity of Diseasea
		H	CIN1	CIN2	CIN3	MIC	LIC
cervix	NTG	17	3	1		2
	E6AP−/−	2	11	2	1	3
	K14E6WTb	3	9	7	8	5	7
	K14E6WTE6AP−/−c	1	12	2		1
vagina		H	VAIN1	VAIN2	VAIN3	MIC	LIC
	NTG	16	7
	E6AP−/−	1	18
	K14E6WTb	9	12	15	2	1
	K14E6WTE6AP−/−c	5	9	2

^aCohorts of female mice of the indicated genotypes were treated with estrogen for 9 months and the reproductive tracts from every mouse was scored for the worst state (severity) of cervical (top half) or vaginal (bottom half of table) disease. The overall severity of disease between different genotypes was compared using a two-sided Wilcoxon-rank sum test. Abbreviations: H, hyperplasia; CIN, cervical intraepithelial hyperplasia; MIC, microinvasive cancer; LIC, large invasive cancer; VAIN, vaginal intraepithelial neoplasia.

^bThe shift in the severity of disease in K14E6^WT transgenic mice is highly significant relative to NTG and E6AP^−/− mice (p ≪ 0.001).

^cLoss of E6AP results in a highly significant reduction in the severity of disease in K14E6^WTE6AP^−/− in the cervix (p≪0.001) and a significant reduction in the vagina (p = 0.05) relative to K14E6^WT mice.

Table 2.

Summary of Tumor Characteristics in the Cervix and Vagina

Genotype	Cancer Incidence# (%)	LIC Incidence (%)	Tumor Multiplicity (tumors/ mouse)	Tumor Burden (Area of Tumor Invasion/Mouse) (mm2)
NTG (n=23)	8.7	0	0.09	5 × 10−3
E6AP−/− (n=19)	15.8	0	0.21	2 × 10−3
K14E6WT (n=39)	30.8	17.9 b	0.51d	0.39 f
K14E6WTE6AP−/− (n=16)	6.3 a	0 b	0.13 e	3 × 10−3g
NTG (n=7)	0	0	0	0
K14E7WT (n=22)	50	9.1	1.8	0.17
K14E7WTE6AP−/− (n=7)	57.1	14.3 c	2.3	0.38
K14E6WTE7WT (n=17)	82.4	41	5.6	1.31
K14E6WTE7WTE6AP−/− (n=6)	50	0	3.2	0.15 h

NOTE: Animals from the top half of the table (above double line) were treated with estrogen for 9 months, while the bottom half of the table shows animals from a 6-month treatment. All statistical tests were two-sided.

^#Cancer incidence was defined as the incidence of cancer that occurred in the cervix and the lower reproductive tract combined.

^aLoss of E6AP resulted in a reduction in cancer incidence in K14E6^WTE6AP^−/− and was significantly different than K14E6^WT transgenic mice (p=0.04).

^bLoss of E6AP resulted in a marginally significant reduction in the formation of LIC in K14E6^WTE6AP^−/− relative to K14E6^WT transgenic mice (p=0.09).

^cLoss of E6AP did not result in a reduction in the percentage of LIC in K14E7^WTE6AP^−/− and was not different than K14E7^WT transgenic mice (p > 0.05).

^dTumor multiplicity is significantly higher in K14E6^WT transgenic mice relative to NTG mice (p <0.05).

^eLoss of E6AP results in a reduction in tumor multiplicity in K14E6^WTE6AP^−/− relative to K14E6^WT transgenic mice (p=0.06).

^fK14E6^WT transgenic mice have significantly larger tumors than NTG mice (p <0.05).

^gLoss of E6AP results in a significant reduction in tumor size in K14E6^WTE6AP^−/− relative to K14E6^WT transgenic mice (p < 0.05).

^hLoss of E6AP in K14E6^WTE7^WTE6AP^−/− transgenic mice resulted in smaller tumors relative to K14E6^WTE7^WT transgenic mice (p=0.07).

In human cervical cancers, E6 is always expressed with E7, a second HPV oncogene. HPV16 E7 is the more potent oncogene in the mouse cervix, as K14E7^WT mice develop cervical cancers after only treatment with six months of 17β-estradiol (23). In comparison, K14E6^WT mice only develop cervical cancers when treated for nine months (24). Double transgenic mice (K14E6^WTE7^WT) expressing both HPV16 E6 and E7, develop larger cancers than K14E7^WT mice when treated for six months with 17β-estradiol (23). To assess the importance of E6AP in cervical carcinogenesis in the presence of both E6 and E7, we assessed the severity and incidence of cervical disease in K14E6^WTE7^WT mice on an E6AP-sufficent or nulligenic background after treatment with estrogen for six months (Suppl. Fig. 2, Table 2 & Table 3). Histopathological diagnosis revealed that in the absence of E6AP (K14E6^WTE7^WTE6AP^−/−), the severity of disease is significantly reduced in the cervix of HPV mice (p=0.03). Moreover, K14E6^WTE7^WTE6AP^−/− transgenic mice did not show any incidence of large invasive cancers, as opposed to an incidence of 41% in K14E6^WTE7^WT mice on the E6AP-sufficient background. To determine if E6AP status had any influence on carcinogenesis when only E7 is expressed, we compared the severity and incidence of cervical disease in K14E7^WT mice on an E6AP-sufficent or nulligenic background (Table 3). No significant differences in cervical disease were observed. Based upon these results we conclude that E6AP is specifically a critical cofactor for HPV16 E6 in cervical carcinogenesis.

Table 3.

Summary of Disease State in the Cervix and Vagina of Mice After 6 Months with Estrogen

	Mouse Genotype	Severity of Diseasea
		H	CIN1	CIN2	CIN3	MIC	LIC
cervix	NTG	5	1	1
	K14E7WTb	1	1	2	7	9	2
	K14E7WTE6AP−/−b, c			2	1	3	1
	K14E6WTE7WTb, d				3	8	6
	K14E6WTE7WTE6AP−/− b,e		1	2		3
vagina		H	VAIN1	VAIN2	VAIN3	MIC	LIC
	NTG	5	1	1
	K14E7WTb	2	4	7	1	8
	K14E7WTE6AP−/− b,c		2	4		1
	K14E6WTE7WT b,d	1	2	2		11	1
	K14E6WTE7WTE6AP−/−b, e	1	2	2		1

^aCohorts of female mice of the indicated genotypes were treated with estrogen for 6 months and the reproductive tracts from every mouse was scored for the worst state (severity) of cervical (top half) or vaginal (bottom half of table) disease. The overall severity of disease between different genotypes was compared using a two-sided Wilcoxon-rank sum test. Abbreviations: H, hyperplasia; CIN, cervical intraepithelial hyperplasia; MIC, microinvasive cancer; LIC, large invasive cancer; VAIN, vaginal intraepithelial neoplasia.

^bRegardless of E6AP status, the shift in the severity of disease in transgenic mice expressing HPV E7 in the presence or absence of E6 were all highly significant relative to NTG mice (p ≪ 0.001).

^cLoss of E6AP did not result in a reduction in the severity of disease in K14E7^WTE6AP^−/− and was not significantly different than K14E7^WT.

^dThe severity of disease is significantly increased in K14E6^WTE7^WT transgenic mice in the cervix (p=0.001) and in the lower reproductive tract (p=0.07) relative to K14E7^WT mice.

^eLoss of E6AP resulted in a reduction in the severity of disease in K14E6^WTE7^WTE6AP^−/− transgenic mice both in the cervix (p=0.006) and lower reproductive tract (p=0.01) relative to K14E6^WTE7^WT mice; the resulting severity of disease is similar to K14E7^WTE6AP^−/− mice (p >0.05).

A progressive disease leading to cancer is also observed in the vaginal cavity or lower reproductive tract (LRT) in our HPV transgenic mouse model for cervical carcinogenesis, consistent with the role of high-risk HPVs in vaginal cancers (35, 36). We therefore assessed the influence of E6AP on vaginal carcinogenesis. Similar findings were observed as in the cervix. In K14E6^WT mice treated with estrogen for nine months, the absence of E6AP led to a complete abrogation of E6-dependent carcinogenesis in the vagina (Supp. Fig. 2, Table 1). Likewise, the contribution of E6 was lost in K14E6^WTE7^WT mice treated with estrogen for six months in K14E6^WTE7^WTE6AP^−/− mice (Supp. Fig. 2, Table 3).

It is interesting to note that in our analysis of the influence of E6AP-status on the acute response to estrogen in the cervical epithelia, we observed a defect in hyperplasia induced by estrogen in the E6AP-deficient cervix of ovariectomized mice. Nevertheless, in our 9-month estrogen treated E6AP^−/− mice we observed hyperplasia, dysplasia and even cancer at a low frequency, akin to what we observed in E6AP-sufficent mice (Table 1). These results indicate that any effect of E6AP-deficiency on estrogen's function in this tissue is not manifested in the long-term, and could simply reflect a slight delay in the acute response.

Reduced incidence of cervical cancer is not due to a lack of ERα expression

Given the acute and long-term results observed in mice on the E6AP^−/− background, we wanted to determine whether the reduced incidence of cancer in K14E6^WTE6AP^−/− mice is due to a lack of ERα expression. Previous findings show that ERα is a necessary cofactor in cervical carcinogenesis in our estrogen-dependent HPV model (30). Thus IHC for ERα was performed on long-term treated mice. There were varying levels of ERα in reproductive tract epithelium across all genotypes (data not shown). No differences were observed in the expression of ERα between NTG and E6AP^−/− cancers. In the presence of either HPV E6 or E7, the loss of E6AP resulted in minor reductions in the expression of ERα, however, the cancers still expressed appreciable levels of ERα (Figure 3A).

Figure 3. Immunohistochemical characterization of tumors from the reproductive tract.

A. Shown are sections of cancers taken from the reproductive tract from mice of the indicated genotypes and stained for ERα by IHC. All of the cancers from the various genotypes express ERα. All of the images are 40× magnification.

B. Shown are sections of epithelium (first & second column) or cancers (third column) taken from the reproductive tract from mice of the indicated genotypes and stained by IHC for either p53 or MCM7. Dotted lines in each picture demarcate the cervical epithelium from the underlying stroma. In the epithelium, p53 is not often detected, however, loss of E6AP in K14E6^WTE6AP^−/− mice results in the induction of p53 at either low or higher (second column) levels. p53 is detected both in the epithelium and cancers of K14E6^WTE7^WT and K14E6^WTE7^WTE6AP^−/− as p53 is stabilized by HPV E7. MCM7 (fourth column) is expressed at low levels in cancers from NTG and E6AP^−/− mice. Levels of MCM7 are increased in cancers that express HPV oncogenes, E6 and/or E7. All of the images are 40× magnification.

Reduced incidence of cancer is not a result of reduced proliferation or expression of the HPV biomarker, MCM7

To confirm that the reduction in the severity of disease and cancer incidence in HPV transgenic mice on the E6AP^−/− background is not due to an inability to proliferate, IHC for BrdUrd was performed on NTG, E6AP^−/−, K14E6^WT, K14E6^WE6AP^−/−, K14E7^WT, K14E7^WTE6AP^−/−, K14E6^WTE7^WT and K14E6^WTE7^WTE6AP^−/− mice treated with estrogen for either six or nine months (data not shown). As expected, proliferation was low in NTG mice. Similar to acute studies reported above, E6AP^−/− mice had higher levels of proliferation. No differences in the levels of BrdUrd were observed in comparing HPV transgenic (K14E6^WT or K14E6^WTE7^WT) mice either on an E6AP-sufficient or insufficient background.

Mcm7 is a biomarker for HPV-positive lesions both in clinical human and murine samples (37). Cancers from the above genotypes were evaluated for the expression of MCM7. Cancers from NTG and E6AP^−/− mice expressed low but detectable levels of MCM7. Mice expressing either HPV E6 and/or E7 generally had increased levels of MCM7 expression, driven mostly by E7 oncogene’s inactivation of pRb (38) or E6’s dysregulation of p16/pRb pathway (24, 39), both of which can lead to subsequent activation of E2F-target genes. Indeed, there were no significant differences in the expression of MCM7 between HPV transgenic mice on an E6AP-sufficient versus insufficient background (Fig 3B).

Loss of E6AP results in an increased frequency of detectable p53 in the reproductive tract

Due to the reduction in the incidence of cancer and the severity of disease in K14E6^WTE6AP^−/− mice, we wanted to determine whether the inability of E6 to degrade p53 accounted for these observations. To investigate this question, IHC for p53 was performed on mice treated with estrogen. Cancers from NTG, E6AP^−/−, K14E6^WT and K14E6^WTE6AP^−/− mice have little to no detectable p53 (Fig. 3B). However, focal expression of p53 is detectable in the reproductive epithelium of E6AP^−/− and K14E6^WT mice, with an appreciable increase in K14E6^WTE6AP^−/− mice. Expression of p53 was more likely to be detected in the epithelium of the LRT and thus may explain the rare occurrence of cancers in this region in mice that only express E6 and not the E7 transgene [(24, 36) and author’s observation]. Mice expressing the E7 transgene have detectable levels of p53 both in the dysplastic epithelium and cancers, likely due to E7’s ability to cause increases in the levels of p53 (40, 41). Accordingly, K14E6^WTE7^WTE6AP^−/− transgenic mice have an appreciable increase in the expression of p53 over that of K14E6^WE6AP^−/− mice, which is evident both in the epithelium and in the cancers. In sum, the loss of E6AP results in an increased frequency of detectable p53 in the reproductive tract that correlated with decreases in cancer incidence and the severity of disease in E6 transgenic mice.

Discussion

In this study, we found that the loss of E6AP had little effect on acute activities of E6 in the cervix, yet had dramatic effects on the ability of E6 to synergize with estrogen to form cancers in the reproductive tract, in the presence or absence of E7. The loss of E6AP resulted in reductions in both cancer incidence and the severity of disease. In sum, this study demonstrates that E6AP plays a critical role in mediating HPV16 E6’s contributions to cervical cancer.

E6AP is not required for HPV E6-induced acute effects

Similar to previous observations in the skin of HPV E6 transgenic mice (20), E6AP was found to be required neither for the induction of epithelial hyperplasia nor for the inactivation of the DNA damage response in the reproductive tract. E6AP is thought to potentially mediate E6-induced hyperplasia due to its predicted ability to target the PDZ partners of E6, Scribble and Dlg for degradation. Loss of either of these tumor suppressor genes in Drosophila results in the development of epithelial hyperplasia, loss of cell-cell contacts and cancer development (42–44). In human cervical cancers, hScrib and hDlg becomes mislocalized and their levels progressively reduced (45, 46). Observations from the skin and the reproductive tract suggest that E6AP is either not the mediator of E6-induced epithelial hyperplasia and/or that in its absence there is alternative ubiquitin ligase that can target these proteins for degradation. Evidence from others suggests that the latter hypothesis may be the case. Work from Massimi and colleagues show that E6 maintains the ability to target both Scribble and Dlg for ubiquitin-mediated degradation in the absence of E6AP (22) in contrast to reports that argue for a dependence of E6AP (16). Moreover, the ability of E6 to inhibit the DNA damage response in both the skin and reproductive tract (this study) in the absence of E6AP similarly suggests that there are alternative means to inactivating p53. Alternatively proposed mechanisms by which E6 inactivates p53 include repression of transactivation of p53 target genes either by acetylation (47) or by E6’s interaction with CBP/p300 (48), which may be relevant here.

Implications of our findings

Our studies provide clear evidence for an essential role of E6AP in mediating HPV16 E6’s oncogenic activity, but leave unanswered the mechanism underlying its requirement. Specifically, those acute phenotypes of E6 that were measured were retained on the E6AP-deficient background despite the complete loss of neoplastic disease. Our results suggest that E6AP's role in mediating E6's activities has yet to be fully appreciated.