Research Article

Critical Roles for Non-pRb Targets of Human Papillomavirus

Type 16 E7 in Cervical Carcinogenesis
1

2,3

Scott Balsitis, Fred Dick, Nicholas Dyson, and Paul F. Lambert

1
McArdle Laboratory for Cancer Research, University of Wisconsin Medical School, Madison, Wisconsin; 2Department of Biochemistry,
University of Western Ontario, London, Ontario, Canada; and 3Massachusetts General Hospital Cancer Center, Charlestown, Massachusetts

Abstract
High-risk human papillomaviruses (HPV) encode two oncogenes, E6 and E7, expressed in nearly all cervical cancers.
In vivo, HPV-16 E7 has been shown to induce multiple
phenotypes in the context of transgenic mice, including
cervical cancer. E7 is a multifunctional protein known best
for its ability to inactivate the tumor suppressor pRb. To
determine the importance of pRb inactivation by E7 in
cervical cancer, we pursued studies with genetically engineered mice. E7 expression in estrogen-treated murine cervix
induced dysplasia and invasive cancers as reported previously,
but targeted Rb inactivation in cervical epithelium was not
sufficient to induce any cervical dysplasia or neoplasia.
Furthermore, E7 induced cervical cancer formation even
when the E7-pRb interaction was disrupted by the use of a
knock-in mouse carrying an E7-resistant mutant Rb allele.
pRb inactivation was necessary but not sufficient for E7 to
overcome differentiation-induced or DNA damageinduced
cell cycle arrest, and expression patterns of the E2F-responsive
genes Mcm7 and cyclin E indicate that other E2F regulators
besides pRb are important targets of E7. Together, these data
indicate that non-pRb targets of E7 play critical roles in
cervical carcinogenesis. (Cancer Res 2006; 66(19): 9393-400)

Introduction
Human papillomaviruses (HPV) are small DNA viruses and the
causative agents of epithelial warts. The so-called high-risk HPVs
infect the anogenital tract epithelium and are associated with the
appearance of cervical dysplasia, almost all cases of cervical cancer,
many other genital cancers, and a subset of oral cancers (13). HPV
type 16 accounts for f60% of all cervical cancers as well as >90%
of HPV-positive oral cancers (2, 3). The HPV genome usually exists
extrachromosomally, but many HPV-associated cancers contain
HPV genomes integrated into the host DNA (4). These integrated
genomes invariably contain intact viral E6 and E7 genes, and
integration causes their increased expression (5). These data
suggest that E6 and E7 contribute to the development of cervical
cancers.
E7 binds to >20 cellular proteins (6). The most well characterized
target of E7 is the retinoblastoma tumor suppressor, pRb.
Interaction between E7 and pRb disrupts the ability of pRb to
bind cellular E2F transcription factors, and this inhibits pRbmediated repression of E2F-responsive genes (7, 8) and results in

Requests for reprints: Paul F. Lambert, McArdle Laboratory for Cancer Research,
University of Wisconsin Medical School, 1400 University Avenue, Madison, WI 53706.
Phone: 608-262-8533; Fax: 608-262-2824; E-mail: lambert@oncology.wisc.edu.
I2006 American Association for Cancer Research.
doi:10.1158/0008-5472.CAN-06-0984

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proteasomal degradation of pRb in cultured cells (911). E7 may

modulate E2F activity by other mechanisms; E7 also targets the
pRb-related p107 and p130 proteins for degradation, inhibits the
cyclin-dependent kinase (cdk) inhibitors p21 and p27, and may
directly activate both cyclin A/cdk2 and E2F1 (1214). Furthermore, E7 binds a wide variety of other cellular targets, although the
implications of these interactions are largely unclear (14).
Many studies have suggested that pRb inactivation is critical for
E7 function. pRb has been connected to all of the processes
disrupted by E7 in vivo, including cell cycle regulation, differentiation, DNA damage responses, centrosome synthesis, and
tumorigenesis (1518). Additionally, pRb inactivation is necessary
and sufficient for nearly all of the acute effects of E7 on the skin of
transgenic mice (17, 19). However, some studies have indicated that
targets of E7 other than pRb may also be important for the
phenotypes of E7. Two E7 mutants deficient in binding to pRb can
cooperate with E6 in the immortalization of primary human
keratinocytes (20), and the ability of E7 to transactivate certain
E2F-responsive promoters may depend on binding to p107 rather
than binding to pRb as shown with the B-myb promoter (21).
Furthermore, the E7D79-83 mutant exhibits a decreased ability to
transform baby rat kidney cells, although it efficiently binds and
degrades pRb (2225). Another E7 mutant, E7CVQ68-70AAA, which is
also able to bind and destabilize pRb but is deficient in p21
inactivation, fails to overcome differentiation-dependent cell cycle
withdrawal or DNA damageinduced cell cycle arrest in human
keratinocytes (24, 26). Additionally, E7 may contribute to
transformation of rat embryo fibroblasts via activation of c-Jun
independently of pRb inactivation (27), and E7 induces centrosome
abnormalities in pRb-deficient cells (28). Finally, E7 is able to
produce some phenotypes in murine skin independently of pRb
inactivation (17, 19). Thus, multiple studies indicate that the effects
of E7 on targets other than pRb make important contributions to
E7 function, although no consensus has emerged as to which
targets affect which phenotypes.
HPV-16 E7 expression alone is sufficient to induce invasive
cervical cancers in transgenic mice treated with chronic low doses
of estrogen (29, 30). In the present study, we assessed whether pRb
inactivation by E7 is necessary and/or sufficient for cervical
phenotypes in mice. The results show that E7 makes critical
contributions to cervical carcinogenesis independently of pRb
inactivation. Non-pRb targets of E7 were also required to overcome
differentiation or DNA damageinduced cell cycle arrest. Expression patterns of the E2F-responsive genes Mcm7 and cyclin E
showed that E7 modulates E2F-responsive gene expression through
multiple pathways in cervix, suggesting that other E2F regulators
besides pRb are important targets of E7.
A final interesting observation is that, when the E7-pRb
interaction was abolished in the context of the E7 transgenic
mice, cervical cancers arose in the absence of the dysplastic
cervical intraepithelial neoplasia (CIN) lesions that are otherwise

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observed throughout the cervix in estrogen-treated E7 transgenic

mice on a wild-type (WT) Rb background. The implications of
this finding in terms of the genesis of cervical cancers are
discussed.

Materials and Methods

Transgenic mice. K14E7, K14CreRbflox , and RbDL mice have been
described previously (17, 19, 31, 32). All mice were bred and maintained in
the American Association for Accreditation of Laboratory Animal Care
approved McArdle Laboratory Cancer Center Animal Care Facility
(Madison, WI). All Rbflox mice were analyzed as FVB backcross 6 from a
129-C57/BL6 background; all RbDLxCxE mice were analyzed as FVB
backcross 2 from a 129-C57/BL6 background. All mice were genotyped by
PCR as described previously (17, 19). One hour before sacrifice, all mice
were i.p. injected with bromodeoxyuridine (BrdUrd; 10 AL per g body weight
of 12.5 mg/mL solution). For the irradiation studies, mice were exposed to
0 or 12 Gy ionizing radiation from a 137Cs source 24 hours before BrdUrd
administration.
Estrogen treatment and cervical carcinogenesis. All analyses were
done in estrogen-treated mice as described previously (29). Each mouse was
assigned a diagnosis according to the worst cervical lesion detected.
Statistical analyses of the incidence of dysplasia and invasive cancer were
done using two-sided Fishers exact tests. Cross-sectional areas of invasive
cancers were determined using Zeiss Axiovision 3.1 software on a Zeiss
Axioskop microscope (Thornwood, NY). Cancer areas were compared using
a two-sided Wilcoxon rank sum test.
Immunohistochemical and immunofluorescence analysis of epidermis. Immunohistochemical stains were done for BrdUrd, pRb, cyclin E, and
Mcm7 as described previously (29) with the following modifications. For
BrdUrd, pRb, or cyclin E staining, further unmasking was achieved with
20 minutes of immersion in 2 N HCl. Primary antibodies were diluted in
blocking buffer and applied for 2.5 hours at room temperature: anti-pRb
(1:50, clone G3-245; BD PharMingen, San Diego, CA), anti-BrdUrd (1:40;
Oncogene, Carpinteria, CA), anti-Mcm7 (1:200; LabVision Neomarkers,
Fremont, CA), or anticyclin E (1:50, clone M-20; Santa Cruz Biotechnology,
Santa Cruz, CA).
Quantitation of pRb loss and cell proliferation. BrdUrd incorporation
into newly synthesized DNA was used as a measure of keratinocyte
proliferation by counting BrdUrd-stained cervical sections. All keratinocyte
nuclei in eight visual fields were scored as either positive (brown) or
negative (blue) for BrdUrd incorporation in both basal and suprabasal
layers of the epidermis. For counting purposes, even slightly brown cells
were counted as positive. Three to five mice were counted per genotype,
and results are presented as the mean F SD. Similarly, to quantify loss of
pRb expression, sections of epidermis were stained for pRb and counted.
Because pRb stains most robustly in basal layer cells, only basal
keratinocytes were counted in eight visual fields of both endocervical and
exocervical epithelium. Three to four mice were counted per genotype.
Statistical analyses of results were done using the two-sided Wilcoxon rank
sum test.

Results
Rb inactivation in murine cervix. To determine if pRb
inactivation by E7 was necessary for cervical phenotypes, mice
expressing HPV-16 E7 in the cervical stratified squamous
epithelium (K14E7 mice; ref. 31) were mated to mice carrying a
mutant Rb knock-in allele, RbDLxCxE (RbDL ), containing three
alanine mutations in pRb that interact with E7, thereby producing
a mutant pRb protein that fails to bind E7 (33). pRbDL retains the
ability to bind E2Fs, induces G1 arrest in pRb-negative SAOS2 cells,
and is phosphorylated and inactivated by cyclin D/cdk4 complexes
similarly to WT pRb (34). pRbDL also represses gene expression
from E2F-responsive promoter constructs, although incrementally
less effectively than WT pRb. Unlike WT pRb, pRbDL fails to bind

Cancer Res 2006; 66: (19). October 1, 2006

cellular LxCxE motif-containing proteins, and pRbDL-induced G1

arrest cannot be reversed by expression of HPV-16 or HPV-18 E7
(32, 34).
To determine if pRb inactivation is sufficient to reproduce any
of the phenotypes of E7, we used an Rb allele containing loxP
sites surrounding the third exon (Rbflox ) and expressed Cre using
the human keratin 14 promoter to abrogate pRb expression
selectively in stratified squamous epithelia. The unrecombined
Rbflox allele behaves exactly as the WT Rb allele in all tested
assays. After recombination, the now frameshifted Rbflox allele
produces no detectable protein and mimics a germ-line Rb-null
allele (17, 35). To verify the loss of pRb expression in K14CreRbflox/
flox
cervical epidermis, pRb immunohistochemical stains were
done on either Rbflox/flox or K14CreRbflox/flox cervical epidermis
from mice treated with estrogen for either 6 weeks or 6 months.
At both time points, Cre expression resulted in a dramatic 99%
loss of detectable pRb (95% confidence interval, 99.0-100; Fig. 1A;
data not shown).
Cervical carcinogenesis studies. Treatment of mice with
chronic, low doses of estrogen for 6 months results in severe
cervical dysplasia or invasive cervical cancers in 100% of treated
K14E7 mice, whereas little to no dysplasia and no cervical cancers
are seen in nontransgenic controls (30). We asked if Rb inactivation
was sufficient to reproduce this carcinogenic effect of E7 by
treating Rbflox/flox, K14CreRbflox/flox , or K14E7Rbflox/flox mice with
estrogen for 6 months and examining for the appearance of
cervical abnormalities (see Materials and Methods). As expected,
Rbflox/flox mice did not develop dysplasia or cervical cancers,
whereas K14E7Rbflox/flox mice always developed multiple CIN2 and
CIN3 lesions often accompanied by carcinoma in situ (CIS),
microinvasive cancer (MIC), or large invasive cancer (LIC; Fig. 2A;
Table 1). All invasive cervical cancers detected were squamous cell
carcinomas.
Surprisingly, the cervical epithelia of estrogen-treated pRbdeficient K14CreRbflox/flox mice were visually indistinguishable
from that of estrogen-treated pRb-sufficient Rbflox/flox mice
(Fig. 2A; Table 1). As controls, we also examined estrogen-treated
K14E7K14CreRbWT/WT mice to control for the possibility that Cre
has some unexpected effect on carcinogenesis independent of Rb
inactivation. These mice developed CIN2, CIN3, CIS, MIC, and LIC
lesions similarly to K14E7RbWT/WT mice, indicating that the Cre
protein does not itself inhibit cervical cancer formation (data not
shown).
To determine if pRb inactivation by E7 was necessary for the
carcinogenic effects of E7 in cervix, female RbWT/WT, K14E7RbWT/WT,
RbDL/DL , and K14E7RbDL/DL mice were also treated with estrogen
for 6 months. As expected, RbWT/WT epithelium exhibited little to
no dysplasia, whereas K14E7RbWT/WT epithelium always developed
multiple CIN2 and/or CIN3 lesions often with additional CIS, MIC,
or LIC (Fig. 2B; Table 2). The only difference seen between the
RbWT/WT and RbDL/DL epithelia was the occurrence of microscopic
ulcerated areas in the cervical epithelium in some (f50%) of the
RbDL/DL mice, indicating that the RbDL/DL mice may have decreased
epithelial integrity and/or a defect in wound healing (data not
shown). K14E7RbDL/DL mice developed invasive cancers with a
similar frequency to that seen in K14E7RbWT/WT mice (4 of 12
versus 5 of 14, respectively; Table 2). To determine if there was any
difference in the size of the invasive cancers in K14E7RbDL/DL
versus K14E7RbWT/WT mice, the largest cross-sectional area of each
cancer was measured. The median cancer area in K14E7RbWT/WT
mice (0.12 mm2) was larger than that in the K14E7RbDL/DL mice

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Mechanisms of Cervical Carcinogenesis by E7

Figure 1. Early cervical phenotypes.

Histologic cross sections of cervical
epithelium from mice treated for 6 weeks
with estrogen. Brown, positive staining; blue,
hematoxylin counterstain. Dashed line,
location of the basement membrane. A, pRb
inactivation in murine cervix. pRb staining.
B and C, BrdUrd incorporation in cervical
epithelia. BrdUrd staining labels newly
synthesized DNA. B, DNA synthesis is
restricted to the lowest two cell layers in
control epithelium, but E7 expression
induces suprabasal DNA synthesis. Rb
deletion in K14CreRbflox/flox cervix fails to
reproduce the effect of E7. C, E7 fails
to induce suprabasal DNA synthesis in the
presence of the RbDL allele. BrdUrd staining
pattern in K14E7RbWT/WT cervix is
indistinguishable from K14E7Rbflox/flox cervix
(data not shown).

(0.067 mm2), but this difference was not statistically significant

due to large variation within each genotype and the small number
of cancers observed (P = 0.1416). Furthermore, we did not
observe any consistent difference in tumor morphology or the
degree of differentiation in tumor cells between these two
genotypes.
pRb immunohistochemistry was done to determine if pRb
expression had been lost in the K14E7RbDL/DL cancers. pRb
staining was seen in the nuclei of invasive cells from all four
K14E7RbDL/DL cancers, whereas little to no pRb staining was seen
in K14E7RbWT/WT cancers, presumably reflective of the ability of E7
to degrade pRb (Fig. 2C). This indicates that the RbDL alleles were
not lost during the formation of K14E7RbDL/DL cancers. Furthermore, the fact that pRb levels are retained in the K14E7RbDL/DL
cancers confirms the prior observations that E7 cannot bind the
mutant pRbDL protein. Immunohistochemical detection of p16INK4a
was also done on all cancers to determine if pRb function was
indirectly compromised by p16 loss in the K14E7RbDL/DL cancers.
All cancers in both the K14E7RbWT/WT and K14E7RbDL/DL mice
exhibited similar levels of p16 staining, indicating that p16 loss did
not substitute for pRb inactivation by E7 in the K14E7RbDL/DL
cancers (data not shown).
Combined, these cervical carcinogenesis studies show that pRb
inactivation is not sufficient to induce cervical cancers in estrogentreated mice and that E7 is able to induce cervical cancer
formation independently of pRb inactivation in the K14E7RbDL/DL
mice, indicating that the effects of E7 on targets other than pRb are
critical for cervical carcinogenesis.
Acute cervical phenotypes. In previous work, we showed that
pRb inactivation is necessary and sufficient for most of the shortterm phenotypes of E7 in the cutaneous epithelium of mice (17, 19).

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Given the surprising results described above, we examined the

cervical epithelia of younger mice to determine if pRb inactivation
is able to reproduce the short-term effects of E7 in cervix as it is in
cutaneous epithelia. For these analyses, mice were treated with
estrogen for 6 weeks to ensure an estrus-like state in all mice, thus
avoiding background variation in the degree of epithelial
hyperplasia due to estrus cycling.
Suprabasal DNA synthesis. A hallmark of E7 is its ability to
reprogram suprabasal cells within stratified epithelia to support
DNA synthesis (3638). To determine if pRb inactivation is
necessary or sufficient for this effect, we injected mice with the
nucleotide analogue BrdUrd 1 hour before sacrifice to label newly
synthesized DNA. As observed in cutaneous epithelia, BrdUrd
incorporation in E7-negative RbWT/WT, Rbflox/flox , or RbDL/DL cervical
epithelia was mostly confined to the basal cell layer, with a few
BrdUrd-positive cells also observed in the parabasal layer (Fig. 1B
and C, quantified in Fig. 3A and B). As expected, E7 expression in
RbWT/WT or Rbflox/flox epithelia increased the frequency of BrdUrd
incorporation in suprabasal cells, including cells many layers above
the basement membrane (P = 0.021; Fig. 1B and C, quantified in
Fig. 3A and B). In contrast, Rb inactivation by Cre in K14CreRbflox/flox
epithelium did not induce any increase in suprabasal DNA
synthesis compared with control mice (Figs. 1A and 3A), and
E7 failed to induce suprabasal DNA synthesis in K14E7RbDL/DL
epithelium (Figs. 1C and 3B). These observations indicate that pRb
inactivation is necessary but not sufficient for E7 to induce
suprabasal DNA synthesis.
Surprisingly, E7 expression in the K14E7Rbflox/flox mice also had
the unexpected effect of reducing the frequency of BrdUrd
incorporation in the basal layer cells by f40% (P = 0.021;
Fig. 3A). A similar effect was produced by Rb inactivation in

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Figure 2. Cervical carcinogenesis studies.

Histologic cross sections of cervical
epithelium from mice treated with estrogen
for 6 months. A, E7 expression induces
severe cervical phenotypes, including
cancer. Control (Rbflox/flox ) cervix and
pRb-deficient K14CreRbflox/flox cervix show
no dysplasia or cancers. B, RbWT/WT and
RbDL/DL cervix do not develop dysplasia or
invasive cancer, but both K14E7RbDL/DL and
K14E7RbWT/WT cervixes develop invasive
cancers. C, pRb immunohistochemical stain
(brown ) of invasive cancers from
K14E7RbWT/WT and K14RbDL/DL mice.
Arrows, nuclei of invasive cells. Blue,
counterstained with hematoxylin.

the K14CreRbflox/flox mice (P = 0.021) or by E7 expression in

K14E7RbWT/WT mice (P = 0.055; Fig. 3A and B). However, E7 had no
effect on basal cell proliferation in the K14E7RbDL/DL epithelium
(P = 0.56; Fig. 3B). This counterintuitive result shows that pRb
inactivation by Cre or E7 inhibits proliferation in basal cervical
keratinocytes.
Inhibition of DNA damage response. E7 expression is able to
drive the synthesis of damaged DNA, which may contribute to the
accumulation of mutations and progression to cancer (39, 40). In
the epidermis of mouse skin, pRb inactivation is necessary and
sufficient to inhibit DNA damageinduced cell cycle arrest (17, 19).
To determine if pRb inactivation is sufficient to block DNA damage
responses in the cervix, K14CreRbflox/flox mice were exposed to
ionizing radiation from a 137Cs source, injected with BrdUrd 24
hours later, and sacrificed 1 hour after injection. As expected,
irradiation induced a prominent decrease in the frequency of DNA
synthesis in the cervical epithelium of Rbflox/flox mice, but E7
expression prevented this response (Fig. 3C). By contrast, Rb
inactivation in K14CreRbflox/flox epithelia did not inhibit this DNA
damage response in the cervical epithelium (Fig. 3C). Thus, pRb
inactivation is not sufficient to explain the ability of E7 to inhibit
DNA damage responses in the cervix.
K14E7RbDL/DL and RbDL/DL mice were irradiated to determine
if pRb inactivation is necessary for E7 to disrupt DNA damage
induced arrest. Irradiation decreased the frequency of BrdUrd
incorporation in both of these genotypes, indicating that pRb
inactivation is necessary for E7 to disrupt the DNA damage
induced cell cycle arrest response (Fig. 3D).
Induction of E2F-responsive genes. In several studies, other
groups have observed functional overlap between pRb and the
related pocket protein p107. These studies showed that pRb
inactivation alone was necessary but not sufficient to induce

Cancer Res 2006; 66: (19). October 1, 2006

phenotypes, including increased cell cycle progression, apoptosis,

resistance to senescence, and retinoblastoma. Only when both pRb
and p107 were inactivated were these phenotypes observed (33, 35,
4143). Because pRb inactivation was also necessary but not
sufficient for multiple phenotypes in our mice, we hypothesized
that pRb loss in the cervix may be functionally compensated for
by the action of other E2F regulators, resulting in little effect of
Rb inactivation alone. By contrast, the effects of E7 on a variety of
E2F pathway proteins, including p107, p130, p21, p27, cyclin A, and
E2F1, could overcome this compensatory effect (1214).
If Rb loss in K14CreRbflox/flox cervix is compensated for by the
action of other E2F regulators that are inactivated in K14E7 cervix,
greater E2F-responsive gene expression should be seen in K14E7
cervix than in K14CreRbflox/flox cervix. To test this, we looked for

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Table 1. Cervical histopathology summary for Rbflox/flox

mice
NH
Rbflox/flox
K14E7Rbflox/flox
K14CreRbflox/flox

CIN1

CIN2

CIN3

CIS

MIC

LIC

15
12

NOTE: Criteria for diagnosis are described in Materials and Methods.

Each number indicates the number of mice of the indicated genotype
for which the indicated lesion is the worst cervical lesion detected.
The lack of cervical dysplasia and cancers in K14CreRbflox/flox mice is
highly significant compared with K14E7Rbflox/flox mice (P < 0.05).
Abbreviation: NH, normal hyperplasia.

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Mechanisms of Cervical Carcinogenesis by E7

Table 2. Cervical histopathology summary for RbWT and

RbDL mice

RbWT/WT
K14E7RbWT/WT
RbDL/DL
K14E7RbDLDL

NH

CIN1

14

11
6

1
2

CIN2

CIN3

CIS

MIC

LIC

NOTE: Criteria for diagnosis are described in Materials and Methods.

Each number indicates the number of mice of the indicated genotype
for which the indicated lesion is the worst cervical lesion detected.
Note that all K14E7RbWT/WT mice developed multiple CIN2 and CIN3
lesions. The lack of CIN2 and CIN3 in K14E7RbDL/DL mice is significant
compared with K14E7RbWT/WT mice (P < 0.0001). The incidence of
invasive cancers in K14E7RbDL/DL mice is significantly higher than in
control mice (P < 0.05) and similar to that in K14E7RbWT/WT mice
(P = 0.555).

protein expression from two E2F-responsive genes, Mcm7 and

cyclin E, by immunohistochemistry. These were also chosen
because both Mcm7 and cyclin E are reported biomarkers for the
detection and diagnosis of cervical abnormalities (29).
Very little cyclin E was detected in Rbflox/flox cervix, but cyclin E
staining was greatly up-regulated in K14E7Rbflox/flox cervix in both
basal and suprabasal cells (Fig. 4A). By contrast, loss of pRb in
K14CreRbflox/flox cervix did not detectably induce cyclin E. This
result shows that E7 induces cyclin E to a greater extent than can
be explained by pRb inactivation alone.
Mcm7 staining in Rbflox/flox cervix was robust in nearly all of the
basal cells and in many of the parabasal cells immediately above
the basal layer. As cells differentiated further and became
quiescent, Mcm7 staining diminished rapidly (Fig. 4B ). In
K14E7Rbflox/flox and K14E7RbWT/WT cervix, strong Mcm7 staining

was observed in all suprabasal cell layers. Interestingly, though, E7

also increased the frequency of negatively staining basal cells,
consistent with E7 having caused a reduction in the proliferative
index in this compartment as scored by BrdUrd incorporation (Fig.
4B). In K14CreRbflox/flox cervix, an intermediate phenotype was
observed. Mcm7 staining was clearly increased in suprabasal cells
compared with Rbflox/flox mice, and sporadic Mcm7-negative basal
cells were observed. However, the intensity of suprabasal staining
was consistently diminished compared with K14E7Rbflox/flox mice in
the uppermost epithelial cell layers (Fig. 4B). This suggests that
Mcm7 induction by E7 may result largely from pRb inactivation, in
contrast to the result with cyclin E. To test this further, we
examined Mcm7 staining in K14E7RbDL/DL cervix. In RbDL/DL epithelium, Mcm7 staining was very similar to that seen in RbWT/WT
or Rbflox/flox epithelium (Fig. 4B). K14E7RbDL/DL cervix showed
much greater suprabasal Mcm7 staining than that seen without E7,
although staining intensity in the uppermost layers was still less
than in K14E7RbWT/WT (Fig. 4B). This indicates that E7 can induce
Mcm7 expression both via pRb inactivation and by another
mechanism. In sum, the Mcm7 and cyclin E expression data show
that different E2F-responsive promoters can be activated by
different mechanisms and that E7 activates E2F-responsive gene
expression both via pRb inactivation and via another mechanism.
Cervical cancer in the absence of CIN. One other striking
observation was made in the course of these studies. In the
K14E7RbDL/DL mice treated with estrogen for 6 months, we
observed invasive cervical cancers in the absence of noninvasive
dysplastic CIN or CIS lesions, which are commonly thought to be
the precursors of invasive cancer. Although four of the 12
K14E7RbDL/DL mice developed invasive cancers, we did not detect
any CIN2, CIN3, or CIS lesions in any of the mice in spite of
analyzing every tenth 5 Amol/L section throughout the entire
thickness of each cervix (Fig. 5; Table 2). In addition, we found no
evidence for dysplasia proximal to the tumors. No dysplasias could
be observed either in the regions directly neighboring the invasive
cancers in sections, in which the cancers were present, or in serial
sections surrounding the cancers (Fig. 5; data not shown). In

Figure 3. Quantitative effects on DNA

synthesis. Basal and suprabasal cells from
BrdUrd-stained cervical epithelia were
counted as BrdUrd positive or BrdUrd
negative, and the frequency of
BrdUrd-positive cells was graphed.
A, BrdUrd incorporation in Rbflox mice.
*, P < 0.05, statistically significant change
compared with Rbflox/flox. B, BrdUrd
incorporation in RbWT and RbDL mice.
*, P < 0.05, statistically significant increase in
suprabasal DNA synthesis in K14E7RbWT/WT
mice compared with all other genotypes.
C, E7 expression, but not Rb inactivation,
prevents DNA damageinduced cell cycle
arrest. Mice were exposed to 0 or 12 Gy
ionizing radiation 24 hours before BrdUrd
administration. BrdUrd staining frequency in
basal cells is graphed. BrdUrd incorporation
frequency is significantly reduced in
irradiated Rbflox/flox and K14CreRbflox/flox
mice versus unirradiated mice (P < 0.05).
D, pRb inactivation is necessary for E7 to
disrupt DNA damageinduced arrest. Mice
were irradiated and analyzed as in (C ).
BrdUrd incorporation frequency is
significantly reduced in irradiated RbWT/WT,
RbDL/DL , and K14E7RbDL/DL mice versus
unirradiated mice (P < 0.05).

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Figure 4. Expression of E2F-responsive

genes. A, cervical sections were stained for
cyclin E. Brown, positive staining; blue,
hematoxylin counterstain. Dashed line,
location of the basement membrane.
B, cervical cross sections of the indicated
genotypes were stained for Mcm7.
K14E7RbWT/WT cervix was identical to
K14E7Rbflox/flox , so is not shown.

contrast, 100% of K14E7RbWT/WT mice treated with estrogen for

6 months developed multiple CIN2 and CIN3 lesions in the cervix,
including those mice that developed invasive carcinoma (Fig. 5;
Table 2). Thus, whereas cervical cancers in the K14E7RbWT/WT mice
arose in the context of highly dysplastic epithelium, cancers in the
K14E7RbDL/DL mice arose in the context of otherwise histopathologically normal epithelium. The implications of this finding are
discussed below.

Discussion
In this study, we conducted the first genetic analysis of the
molecular mechanisms of E7 function in intact cervix. Our analysis
indicates that the effects of E7 on targets other than pRb are
critical for both the short-term and tumorigenic consequences of
E7 expression and that cervical cancers can develop independently
of CIN lesions.
pRb inactivation in cervical dysplasia and neoplasia. As
described previously (30), we saw that, after 6 months of estrogen
treatment, 100% of K14E7 mice developed numerous CIN2 and
CIN3 lesions and many developed CIS and/or invasive cancers
(Fig. 2; Tables 1 and 2). Surprisingly, Rb deletion in the cervix of
estrogen-treated K14CreRbflox/flox did not induce any CIN2, CIN3,
CIS, or invasive lesions (Fig. 2A; Table 1). Thus, pRb inactivation
alone cannot explain the ability of E7 to induce cervical dysplasia
or invasive cancers.
Invasive cancers were observed in K14E7RbDL/DL cervix at a
similar frequency to that seen in K14E7RbWT/WT cervix (Table 2),
and there were no consistent differences in cancer size or
morphology between these genotypes (Fig. 2; data not shown).

Cancer Res 2006; 66: (19). October 1, 2006

pRb expression was maintained in the K14E7RbDL/DL cancers,

showing that carcinogenesis in these mice did not require
mutational loss of the RbDL alleles (Fig. 2C). Furthermore,
immunohistochemical staining for p16INK4a detected p16 in all
the cancers in both K14E7RbWT/WT and K14E7RbDL/DL mice (data
not shown), indicating that pRb function was not compromised
indirectly in these cancers via loss of p16 expression. These data do
not imply that pRb inactivation is dispensable for cervical
carcinogenesis because partial defects in pRb function in the
mutant RbDL allele potentially cooperate with the effects of E7 on
other targets to induce cancers in K14E7RbDL/DL cervix. This
likelihood is reinforced by both in vivo and in vitro observations.
In vivo, we observed that RbDL/DL mice often developed ulcerations
of the cervical and vaginal epithelium in estrogen-treated mice,
implying that the RbDL/DL cervix may have reduced epithelial
integrity, defective wound healing, or some other defect. In vitro,
pRbDL exhibited diminished transcriptional repression function
compared with WT pRb, showing that this mutant allele is defective
in some aspects of pRb function, likely due to failure to bind LxCxE
motif-containing cellular proteins (34). More recently RbDL/DL mouse
embryo fibroblasts were found to exhibit aneuploidy associated with
defective centromere methylation and lagging mitotic chromosomes
(32). Thus, it remains reasonable to hypothesize that defects in at
least some functions of pRb contribute to cervical carcinogenesis.
Regardless, our data with the conditional null Rb mice clearly show
that other molecular activities of E7 make critical contributions to
cervical carcinogenesis.
pRb in acute cervical phenotypes. In previous work, we
showed that pRb inactivation is necessary and sufficient for
most of the short-term effects of E7 on murine skin and ear

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Mechanisms of Cervical Carcinogenesis by E7

epithelium (17, 19). In contrast, our analysis of K14E7RbDL/DL and

K14CreRbflox/flox mice showed that pRb inactivation is necessary
but not sufficient to induce suprabasal DNA synthesis and to block
DNA damageinduced cell cycle arrest in the cervical epithelium
(Figs. 1 and 3). Thus, the consequences of Rb inactivation are very
different in cervix than in cutaneous epithelia, indicating that the
mechanisms of E7 function may differ in one versus another
epithelial tissue. This may be important to human carcinogenesis
because HPV-16 is associated with cancers in multiple epithelial
tissues (3, 44). Future studies of E7 function in other physiologically
relevant sites, such as the oral cavity, will determine if the
mechanisms of E7 function also vary between different mucosal
epithelia.
Multiple mechanisms of E2F regulation by E7. Analysis of the
E2F-responsive cyclin E and Mcm7 genes showed that E7 induces
E2F-responsive genes via multiple mechanisms, such that full
induction of E2F-responsive genes requires both pRb inactivation
and some other activity or activities of E7 (Fig. 4). Furthermore,
Mcm7 was induced in K14CreRbflox/flox cervix but cyclin E was not
(Fig. 4), implying that different E2F-responsive genes can be
differentially regulated, which may explain why multiple mechanisms of E2F activation are necessary for full E7 function. Other
components of the E2F pathway bound by E7 in vitro include p107,
p130, p21, p27, cyclin A/cdk2, and E2F1 itself (1214), making these
excellent candidates for future studies of E7 function.
More specifically, published studies of p107 function suggest that
p107 may be of particular importance. The observation that pRb
inactivation is necessary but not sufficient for many cervical
phenotypes is strongly reminiscent of studies, which have shown
that p107 can functionally compensate for the loss of pRb under
some circumstances. These studies identified multiple phenotypes,
which do not occur following pRb or p107 inactivation alone but
are observed only when both genes are simultaneously inactivated
(35, 4143). If p107 is similarly compensating for the loss of pRb in
K14CreRbflox/flox cervix, then E7-mediated p107 inactivation could
explain why pRb inactivation is not sufficient to induce phenotypes
in the cervix. Unfortunately, K14CreRbflox/floxp107 / mice do not

Figure 5. Absence of CIN lesions in K14E7RbDL/DL cervix. Representative

sections of K14E7RbWT/WT and K14E7RbDL/DL cervixes. Arrows, sites of cancer
invasion into the dermis. In K14E7RbWT/WT cervix, cancer arises from epithelium
containing many dysplastic changes (arrowheads ), whereas in K14E7RbDL/DL
cervix, cancer is observed without any dysplastic changes elsewhere in the
cervical epithelium.

www.aacrjournals.org

survive to adulthood (S.B. and P.F.L.; data not shown; ref. 45), so
more refined mouse model studies will be necessary to test this
hypothesis.
Cervical carcinogenesis in the absence of detectable CIN. In
carrying out this study, we made the unexpected observation that
cervical cancers can develop in the absence of any detectable,
coexisting CIN or CIS lesions. Specifically, whereas cancers in
K14E7RbWT/WT mice always developed in the context of a severely
dysplastic epithelium, cancers in K14E7RbDL/DL mice developed in
the absence of detectable, coexisting CIN2, CIN3, or CIS lesions
(Fig. 5; Table 2). Importantly, the incidence and size of invasive
cancers were similar in K14E7RbDL/DL and K14E7RbWT/WT mice,
arguing that the rate at which the cancers arose is similar. These
unexpected results suggest that the profuse cervical dysplasia and
the formation of cervical cancer seen in K14E7 mice on the WT Rb
background are separable events.
Although both cervical dysplasia and cervical cancer are
associated with HPV infection (46, 47) and the likelihood of both
increase over time (47), there is no direct evidence that the
clinically apparent dysplastic lesions are obligate intermediates to
cervical cancer. Our mouse model data would argue otherwise.
That malignant cancer can arise without overt benign disease is
not unprecedented; in chemical carcinogenesis studies, invasive
skin carcinomas developed in p53-null mice without prior
development of detectable benign skin papillomas (48). It remains
to be determined in either model how these cancers arise.
One hypothesis is that, in the K14E7RbDL/DL mice, malignant
conversion arises so quickly as to obscure the preexistence of
dysplastic lesions. However, the fact that the incidence and size of
cancers in the K14E7RbDL/DL mice are no greater than that seen in
K14E7RbWT/WT mice argues against this possibility. Furthermore,
the cancers that arise on the K14E7RbDL/DL mice are not
sufficiently large as to obscure an ability to see underlying
dysplastic disease elsewhere in the cervix; yet, such dysplasia was
not apparent in these mice.
An alternative hypothesis that we favor is that both the profuse
dysplasia and the stochastic malignancies observed in the
K14E7RbWT/WT mice arise from a common progenitor tumor cell.
However, different derivative lineages develop from this common
progenitor, some of which lead to dysplasia, whereas others lead to
frank cancer without clinically apparent precursor benign disease.
Here, one has to argue further that the defects in Rb function
caused by the interaction of E7 with WT pRb (and therefore absent
in the K14E7RbDL/DL mice) contribute preferentially to the lineages
that cause dysplasias that are unable or unlikely to progress to
frank malignancy. This hypothesis is consistent with the observation that removal of dysplastic lesions in women reduces
significantly the risk of subsequent cancer, assuming that the
derivative cells that can cause frank cancers to reside proximal to
the dysplasia; this is a reasonable prediction given the focal nature
of HPV infections within the cervix.
There are several important implications to the favored
hypothesis. First is that treatments shown to eliminate dysplastic
lesions may or may not affect frank cancers, depending on whether
the therapy targets a feature shared by both the malignant as well
as dysplastic tumor lineages or a feature distinct only to the latter.
Likewise, genetic/epigenetic alterations found in dysplastic lesions
may or may not be predictive of alterations that contribute to frank
cancer, depending on whether the change arose at an early stage in
the genesis of the common progenitor cell or at a later stage in the
genesis of the derivative lineage that gave rise specifically to the

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Cancer Res 2006; 66: (19). October 1, 2006

Cancer Research

dysplasia. Further investigation is needed to determine whether

this hypothesis, which is derived from our study of HPV-associated
cancers in a mouse model, is relevant to understanding the genesis
of human cervical cancer.

Acknowledgments
Received 3/15/2006; revised 6/27/2006; accepted 7/31/2006.

Grant support: NIH grants CA64402, CA098428, and CA22443; NIH grant T32
GM00721 (S. Balsitis); and University of Wisconsin Comprehensive Cancer Center
grant P30 CA014520. F. Dick is a research scientist of the Canadian Cancer Society
through an award from the National Cancer Institute of Canada.
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 the University of Wisconsin Comprehensive Cancer Center histology
facility (Madison, WI) for expert histologic support and Ruth Sullivan for assistance
with histopathology.

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