The Human Papillomavirus E6 protein and its contribution to malignant progression

Abstract

The human papillomavirus (HPV) E6 protein is one of three oncoproteins encoded by the virus. It has long been recognized as a potent oncogene and is intimately associated with the events that result in the malignant conversion of virally infected cells. In order to understand the mechanisms by which E6 contributes to the development of human malignancy many laboratories have focused their attention on identifying the cellular proteins with which E6 interacts. In this review we discuss these interactions in the light of their respective contributions to the malignant progression of HPV transformed cells.

Full text

The Human Papillomavirus E6 protein and its contribution to malignant
progression
Fiamma Mantovani
1
and Lawrence Banks*
,1
1
International Centre for Genetic Engineering and Biotechnology Padriciano 99, I-34012 Trieste, Italy
The Human Papillomavirus (HPV) E6 protein is one of
three oncoproteins encoded by the virus. It has long been
recognized as a potent oncogene and is intimately
associated with the events that result in the malignant
conversion of virally infected cells. In order to under-
stand the mechanisms by which E6 contributes to the
development of human malignancy many laboratories
have focused their attention on identifying the cellular
proteins with which E6 interacts. In this review we
discuss these interactions in the light of their respective
contributions to the malignant progression of HPV
transformed cells. Oncogene (2001) 20, 78 74 ± 7887.
Keywords: HPV; E6; transformation; proteasome;
PDZ domains
Introduction
Human Papillomaviruses (HPVs) infect keratinocytes
in the basal layers of stra ti®ed epithelia at a variety of
anatomical sites and their replicative cycle is intimately
linked with the dierentiation of the infected cell.
Mucosal HPVs are the best characterized, and include
the high-risk types, such as HPV-16 and HPV-18,
which cause lesions that can progress to cervical
carcinoma. In contrast, the low-risk types, such as
HPV-6 and HPV-11, induce benign genital warts and
are very rarely associated with malignancies (zur
Hausen and Schneider, 1987). Less is known about
the cutaneous HPV types but, once again, a subset of
these types, including HPV- 5 and HPV-8, is linked
with the development of human cancers, particularly
squamous cell carcinoma (SCC) at sun exposed sites in
immunocompromised individuals (for review see Ben-
ton and Arends, 1996).
Due to their limited coding capacity, HPVs have to
use the cellular DNA synthesis machinery in order to
replicate their genomes. However, while low-risk HPVs
begin replication in cells that are still proliferating, the
replicative phase of high-risk HPV infection is con®ned
to more dierentiated cells that have already exited the
cell cycle and are non-permissive for DNA synthesis
(Doorbar et al., 1997). In order to overcome this
problem, the high-risk HPV E7 protein targets a
number of cell cycle regulatory proteins, includin g the
`pocket protein' family of pRb, p107 and p130, thereby
upregulating genes required for G1/S transition and
DNA synthesis (see Mu
È
nger et al., this issue).
However, the host cell's normal response to this
unscheduled induction of proliferation would be to
trigger apoptosis and/or growth arrest. To overcome
these obstacles, the high-risk E6 protein targe ts a
variety of cellular proteins involved in regulating these
defence mechanisms, as well as those involved in
terminal dierentiation and antiviral defence. Under
normal circumstances, viral repli cation would then
continue, resulting in production and release of
infectious virions. On rare occasions, however, the
viral life cycle is interrupted and processes are initiated
that lead to immortalization and ultimately to full
transformation of the cell.
In this review we shall address the functions of E6
which appear most relevant for cell transformation,
highlighting those aspects which appear to be con-
served across many dierent viral oncoproteins, as well
as those re¯ecting unique aspects of E6 function. Prior
to this we will review the studies which de®ne the high-
risk HPV E6 as a potent viral oncoprotein.
Transformation by HPV E6
The HPV E6 proteins are small polypeptides of
approximately 150 amino acids and contain two zinc-
®nger motifs (Cole and Danos, 1987; Barbosa et al.,
1989), whose integrity is essential for E6 function
(Kanda et al., 1991; Sherman and Schlegel, 1996). The
®rst indirect evidence that E6 was a viral oncoprotein
came from studies on cervical tumours and derived cell
lines, where E6 was found to be retained and expressed
many years after the initial transforming events
(Schwarz et al., 1985; Androphy et al., 1987; Banks
et al., 1987). Subsequently E6 was found to possess
intrinsic transforming activity in a variety of dierent
assay systems. Although E6 has weak transforming
activity in established rodent cells (Sedman et al.,
1991), high-risk but not low-risk E6 proteins can
eciently cooperate with an activated ras oncogene in
the transformation of primary rodent cells (Storey and
Banks, 1993; Pim et al., 1994; Liu et al., 1994). In
addition, E6 has also been found to immortalize
Oncogene (2001) 20, 7874 ± 7887
ã
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*Correspondence: L Banks; E-mail: Banks@icgeb.trieste.it

primary human mammary epithelial cells at late
passage (Band et al., 1991; Wazer et al., 1995),
although this activity is also exhibited by the low-risk
HPV E6 proteins (Band et al., 1993).
Perhaps the most relevant system for evaluating the
transforming potential of the HPV oncoproteins is
immortalization of primary human keratinocytes,
which are the natural host cells of the virus in vivo.
Numerous studies have shown that high-risk E6 and
E7 are together sucient to induce immortalization of
primary human keratinocytes (Mu
È
nger et al., 1989;
Hawley-Nelson et al., 1989), while the low-risk HPV
proteins are completely inactive in this assay (Wood-
worth et al., 1989; Pecoraro et al., 1989). It is also
interesting to note that these keratinocytes, although
immortalized, will not form tumours in nude mice.
Only following expression of activated oncogenes
(DiPaolo et al ., 1989) or after extended passage in
tissue culture (Du
È
rst et al., 1989; Hurlin et al., 1991) do
these cells become fully transformed. This nicely
resembles the process of HPV induced tumorigenicity
in vivo, where there are long periods between the initial
immortalization events and the ultimate progression to
cervical cancer, thereby highlighting the multistep
nature of the disease progression.
More recently, ecient models of HPV-induced
carcinogenesis have been obtained through the genera-
tion of transgenic mice. E6 and E7 together can induce
various types of tumours, depending on the particular
tissue in which they are expressed (Arbeit et al., 1993;
Griep et al., 1993; Comerford et al., 1995): when
targeted to the basal cells of the squamous epithelium,
under control of the human keratin 14 promoter
(K14), the transgenic mice developed progressive
squamous epithelial neoplasia (Arbeit et al., 1994).
Individual expression of each oncogene induced
epithelial hyperplasia and skin tumours (Herber et
al., 1996 ; Song et al., 1999), however E7 was found to
primarily cause benign, highly dierentiated tumours,
whereas those promoted by E6 were mostly malignant.
This suggests that the two oncoproteins play dierent
roles in the process of carcinogenesis, and also
supports the notion that they act cooperatively to
induce transformation. Further investigation was
performed on E6- and E7-transgenic mice, following
treatment with speci®c carcinogens known to aect
distinct stages of tumour formation. Interestingly, E7
was found to primarily cause tumour promotion,
whereas E6 contributed weakly to the early stages,
acting more strongly during tumour progression,
accelerating the malignant conversion of benign
tumours (Song et al., 2000): this ®nding was
particularly important, since it suggests that E6 may
be responsible for the malignant progression of HPV
induced tumours in vivo.
Interactions between HPV E6 and p53
Analysis of the cellular targets of the HPV E6 proteins
has provided a wealth of information on how E6
contributes to malignant transformation. The ®rst
cellular target of E6 to be identi®ed, and probably
still the most important, is p53.
The p53 tumour suppressor represents a major
constraint to viral replication, since, once activated
by the unscheduled induction of DNA replicati on, it
can promote cell cycle arrest or apoptosis of the
infected cell (El-Deiry et al., 1993; Harper et al., 1993;
Lowe et al., 1994; Wu and Levine, 1994). To overcome
this obstacle, several viruses encode proteins that
functionally inactivate p53. SV40 LT prevents transac-
tivation of p53 targe t genes through association with
its DNA binding domain (Ruppert and Stillmann,
1993), Ad E1B-55K abolishes the same function by
binding to the transactivating domain of p53 (Lin et
al., 1994), yet in association with E4orf6 it can also
lead to p53 degradation (Steegeng a et al., 1998,
Querido et al., 2001; Ta
È
uber and Dobner, this issue),
while the HBV X protein sequesters p53 in the
cytoplasm (Elmore et al., 1997). A major strategy
employed by the high-risk HPV E6 proteins to
abrogate p53's oncosuppressive functions is to induce
its degradation through the ubiquitin-proteasome path-
way (Schener et al., 1990). As a consequence, p53
levels are extremely low in cervical tumour cells
(Matlashewski et al., 1986), and p53-induced growth
arrest and apoptosis in response to DNA damage are
abolished (Kessis et al., 1993; Foster et al., 1994).
Under normal growth conditions in the absence of
HPV, p53 is also turned over by the ubiquitin
proteasome pathway, and this is mediated by the
ubiquitin ligase Mdm2 (Honda et al., 1997). However
under stress conditions, e.g. the expression of viral
oncogenes, this degradative pathway is inhibited and
p53 is both stabilized and activated (Ashcroft and
Vousden, 1999, for review). Recent experiments have
indeed shown that in HPV-positive cancer cells the
Mdm2 pathway is completely inactive, while p53
degradation depends entirely on E6 (Hengstermann et
al., 2001). This indicates that E6 can target p53 for
degradation under conditions when this would be
normally inhibited, e.g. after DNA damage, thereby
allowing the accumulation of genomic mutations in the
infected keratinocytes, and thus contributing tow ards
malignant progression. E6 reactivates degradation of
p53 by recruiting E6AP, a ubiquitin ligase. E6AP
belongs to the HECT-domain family of ubiquitin
ligases, whose large and divergent N-terminal domains
mediate substrate recognition, while ubiquitination of
bound substrates is catalysed by the conserved C-
terminal HECT domain (Schwarz et al., 1998). High-
risk HP V E6 binds to E6AP within its N-terminal
substrate recognition domain (Huibregtse et al., 1993a),
and formation of a stable E6 ± E6AP complex precedes
association with p53, thereby redirecting the substrate
speci®city of E6AP towards p53 (Huibregtse et al.,
1993b). Indeed, approaches aimed at blocking E6AP
activity, either by the use of antisense oligonucleotides
(Beer-Romero et al., 1997) or dominant negative
mutants (Talis et al., 1998), increased the levels of
p53 in HPV-positive, but not in HPV-negative cells,
Oncogene
HPV E6
F Mantovani and L Banks
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con®rming that E6AP plays an essential role in E6
directed degradation of p53 in vivo, but has no eect
on p53 levels in cells lacking E6.
The eciency in mediating degradation of p53 varies
among dierent E6 proteins, depending on their ability
to interact with both p53 and E6AP. Both high- and
low-risk mucosal HPV E6 proteins are able to bind the
p53 C-terminus, however such interactions do not
induce degradation. Binding to the core region of p53
is much greater for high-risk E6 proteins and is
enhanced by the presence of E6AP, and it is this
interaction that allows ecient degradation of p53 (Li
and Cono, 1996). In addition, HPV-16 E6 binds
E6AP more strongly, and concomitantly degrades p53
more eectively, than HPV-18 E6. HPV-11 E6 has
minimal levels of binding to E6AP (Huibregtse et al.,
1993b), and de grades p53 in vivo only weakly (Storey et
al., 1998). Interestingly, the E6 proteins of both high-
and low-risk cutaneous HPV types do not associate with
either E6AP or p53 and are incapable of aecting p53
stability (Elbel et al., 1997), therefore the mechanism by
which these viruses evade the constraints that p53 places
on viral replication remain to be determined.
Although targeting p53 for degradation is the major
route by which E6 overcomes its eects, several reports
indicate that E6 makes use of additional pathways to
abrogate p53's growth suppressive activities. Both low-
and high-risk HPV E6 proteins are capable of
abolishing p53-mediated transcript ional repression in
vivo (Lechner et al., 1992) and this is likely to occur
through binding to the p53 C-terminus (Li and
Cono, 1996). Moreover, the capacity of the high-risk
E6 proteins to abrogate transactivation of p53 target
genes does not only depend on p53 destabilization,
since E6 mutants defective for degradation retain the
ability to abrogate transcriptional activation by p53 in
vivo (Pim et al., 1994). Several mechanisms can be
invoked to explain this property of E6. First, E6 can
interfere with binding of p53 to its DNA recognition
site (Lechner and Laimins, 1994; Thomas et al., 1995).
In addition, repres sion of p53-responsive promoters
could be mediated through the interaction of high-risk
HPV-16 E6 with the transcriptional coactivator s p300/
CBP (Patel et al., 1999; Zimmermann et al ., 1999),
similarly to what has been reported for Ad E1A
(Somasundaram and El-D eiry, 1997). Finally, cyto-
plasmic sequestration of p53 is another common
strategy adopted by dierent viral proteins, such as
Ad E1B 55K (Ko
È
nig et al., 1999), HBV protein X
(Elmore et al., 1997) and HPV E6. It has been reported
that in HPV-positive cancer cells, the nuclear localiza-
tion of p53 in response to DNA damage is blocked
even if proteasome degradation is inhibited (Mantovani
and Banks, 1999). Cytoplasmic retention may be due
to masking p53's nuclear localization signal by E6
binding to the p53 C-terminus, or to enhanced nuclear
export of p53. The latter mechanism is supported by
the observation that inhibition of nuclear export in
HPV-positive tumour cells by the drug leptomycin B
results in accumulation of p53, indicating that E6-
mediated degradation of p53 is, at least in part,
dependent on nuclear export (Freedman and Levine,
1998). Although these data strongly suggest that it
occurs in cytoplasmic proteasomes, colocalization of
the two proteins in the cytoplasm is not alone sucient
to trigger degradation. In neuroblastoma cells p53 is
constitutively cytoplasmic and appears to be resistant
to proteolysis induced by either Mdm2 or E6 (Isaacs et
al., 1999), implying the requirement for nucleocyto-
plasmic shuttling to allow degradation. Whether this
process is mediated directly by E6, its cellular partner
E6AP, or another protein, however remai ns to be
determined, since E6, E6AP and p53 all contain
putative nuclear export signals.
It is quite clear, however, that during viral infection
and in HPV-induced cervical lesions not all p53 is
degraded, as several studies have reported detectable
levels of p53 in HPV-infected cells (Mantovani and
Banks, 1999; Cooper et al., 1993; Lie et al., 1999). A
possible viral pathway for regulating E6 activity with
respect to p53 relies on a series of polypeptides termed
E6*, which are expressed by the high-risk HPV types
through alternative splicing of E6 mRNA (Schneider-
Ga
È
dicke et al., 1988). Interestingly, HPV-18 E6*I was
found to interact with both the full-length E6 and
E6AP, thereby blocking degradation of p53 (Pim et al.,
1997): this might allow a ®ne-tuning of the activity of
E6 with respect to p53 during viral infection.
Interestingly, while p53 has been shown to speci®cally
inhibit HPV ampli®cational DNA replication in vivo ,it
did not aect episomal maintenance, which occurs in
synchrony with the cell cycle (Lepik et al., 1998). In
order to elic it a productive infection, viral DNA
ampli®cation needs to be controlled and it is plausible
that the activity of E6* could ensure the presence of a
limited amount of p53 at the replication sites, where it
could both prevent overreplication of the viral genome
and, possibly, assist DNA synthesis by means of its
proofreading capacity. Indeed, HPV recruits DNA
polymerase a for DNA replication, and the 3' ±5'
exonuclease activity of p53 could enhance the
replicative ®delity of this enzyme (for review, Albrecht-
sen et al., 1999). It is interesting to note that p53 has
been found in the replication centre s of Herpes Simplex
Virus (Wilcock and Lane, 1991), Cytomegalovirus
(Fortunato and Spector, 1998) and Adenovirus (Ko
È
nig
et al., 1999), and recent studies reported an interaction
between the HPV ori-complex binding protein E2 and
p53, further suggest ing a potential positive role for p53
in viral replication (Massimi et al., 1999).
It is quite clear that the E6 ± p53 interaction
represents one of the key even ts in E6 induced
malignancy: continuous degradation of p53 can lead
to the accumulation of genetic mutations in the
infected cell. Indeed, loss of p53 leads to early tumour
development (Donehower et al., 1992), and enh ances
the malignant progression of chemically induced skin
cancers in mice (Kemp et al., 1993), which is consistent
with the observation that E6 accelerates the malignant
conversion of tumours promoted by E7 (Song et al.,
2000). In addition, E6 has been shown to induce gross
chromosomal alterations in cell culture, including
HPV E6
F Mantovani and L Banks
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Oncogene

translocations and aneuploidy (Rezniko et al., 1994;
White et al., 1994). These chromosomal changes are
also common in malignant cells that have lost p53
function, indicating that E6 can induce genomic
instability through inactivation of p53.
p53-independent activities of E6
Although p53 is a vital aspect of E6 function, analysis
of E6 mutants has shown that activities other than
targeting p53 are required for its full transforming
potential (Pim et al., 1994; Nakagawa et al., 1995; Liu
et al., 1999). Moreover, the oncogenic potential of
cutaneous HPVs mainly relies on their E6 proteins
which, however, do not interact with p53 (Elbel et al.,
1997). It is now clear that, in common with many viral
oncoproteins, E6 is multifunctional and, in line with
the above observations, numerous cellular targets have
been identi®ed, some of which are listed in Tabl e 1 and
Figure 1. There is now active debate as to which of
these other activities of E6 are relevant for the
development of malignancy.
De-regulation of transcription and DNA replication by
HPV E6
The E6 protein s of both high- and low-risk HPV types
have long been known to modulate transcription from
many cellular and viral promoters (Sedman et al.,
1991; Desaintes et al., 1992; Etscheid et al., 1994;
Veldman et al., 2001). It is only recently, however,
that clues to the mechanisms by which this may occur
have come, with the demonstration that E6 interacts
with p300/CBP (Patel et al., 1999; Zimmermann et al.,
1999). The p300/CBP transcriptional co-activators
play important roles in activating a great number of
genes involved in the regulation of cell cyle,
dierentiation and immune response. Indeed, many
viral oncoproteins including SV40 LT, Py LT,
EBNA2, Ad E1A, and HTLV-1 Tax have been shown
to require the interaction with p300 for optimal
transforming activity (Goodman and Smolik, 2000,
for review), highlighting its central importance in
regulating cellular homeostasis. HPV-16 E6 was
shown to directly bind three regions of p300/CBP,
namely C/H1, C/H3 and the carboxy terminus, while
low-risk HPV-6 E6 was found to only weakly bind to
the C/H1 region (Patel et al., 1999). Moreover, HPV-
16 E6 was also shown to inhibit the intrinsic
transcriptional activity of p300/CBP on both p53-
and NF-kB-responsive promoter elemen ts. Obviously,
p300/CBP aects expression of many dierent cellular
promoters, including those regulating dierentiation
(Bannister and Kouzarides, 1995; Goodman and
Smolik, 2000), and at present it is not clear which
are the true targets for E6 mediated inhibition via the
p300/CBP interaction. However, possible E6 inhibition
of NF-kB-responsive promoters is particularly in-
triguing, since dysregulation of the NF-kB pathway
can result in hyperproliferation of the stratum
spinosum, the epithelial layer in which HPV DNA
ampli®cation occurs (Hu et al., 1999). NF-kBis
activated upon viral infection and promotes transcrip-
tion of a number of genes involved in the local
immune response such as class I MHC, interleukins
and GM ± CSF, some of whi ch are synthesized directly
by the keratinocytes (for review see Baldwin, 1996),
therefore inhibition of NF-kB target genes may help
the virus to escape immune recognition. Moreover,
NF-kB activation by viral infection stimulates the
IFN-b promoter (Thanos and Maniatis, 1995), and
thus E6 inhibition of NF-kB could help overcome the
interferon-mediated antiviral response. Interestingly,
HPV-16 E6 has also been found to bind the interferon
regulatory factor 3 (IRF-3) (Ronco et al., 1998),
which is an important transactivator of interferons
and binds to the regulatory elements of the IFN-b
promoter (Wathelet et al., 1998). As a consequence of
this interaction, E6 was also found to inhibit IFN-b
induction (Ronco et al., 1998). Interestingly, a recent
screen of cDNA microarrays demonstrated that
transfection of HPV-16 E6 in dierentiating cervical
keratinocytes also downregulates the expression of
interferon-responsive genes (Nees et al., 2001). Since
viral infection stimulates the assembly and activation
Table 1 Cellular targets of HPV E6
Target protein High-risk E6 Low-risk E6 Degradation Cellular function E6AP involved
p53 yes yes yes Transcription factor, apoptosis inducer, tumour suppressor yes
E6AP yes no yes Ubiquitin ligase yes
hMcm7 yes yes yes DNA replication initiation yes
E6TP1 yes yes yes Putative GAP protein yes
Bak yes yes yes Apoptosis inducer yes
c-Myc yes no yes Transcription factor yes
p300/CBP yes yes no Transcriptional coactivators no
AMF-1/Gps2 yes yes yes Transcriptional coactivator ?
IRF-3 yes (HPV-16) no no Transcription factor no
E6BP/ERC-55 yes no no Ca
2+
binding protein yes
paxillin yes no no Signal transduction no
hDlg yes no yes Control of cell polarity and growth no
MAGI-1 yes no yes Putative signal transduction no
MUPP-1 yes no yes Scaffolding protein/putative signal transduction no
hScrib yes no yes Control of cell polarity and growth yes
Oncogene
HPV E6
F Mantovani and L Banks
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of a large transcription factor complex that includes
IRF-3 and CBP/p300, which in turn activates a/b
interferon-responsive genes (Weaver et al., 1998), it is
likely that the combined targeting of all these
components by E6 will con tribute to the disruption
of the cellular antiviral response.
Finally, it should also be borne in mind that the
E6/p300 interaction, and its possible contribution
towards survival of the transformed cell, could be a
by-product of the regulation of viral gene expression.
Thus, the HPV major transcriptional activator, E2,
also interacts with p300/CBP (Lee et al., 2000a;
Marcello et al., 2000). This appears to involve a
cellular protein, AMF-1/Gps2, which in turn enhances
p300 acti vity but which, intriguingly, is also a target
for E6 mediated degradation (Peng et al., 2000;
Degenhardt and Silverstein, 2001). Therefore the
interaction between E6 and p300 may represent a
means of downregulating E2 transcriptional activity,
and thereby controlling the levels of E6 expression by
a feed-back mechanism.
An additional link with tumour progression and
HPV E6 activity has come from studies showing
deregulation of the cellular DNA replication machin-
ery. Normal somatic cells terminate their replicati ve
life span through a pathway leading to cellular
senescence, which is triggered in response to
critically shortened telomere DNA. Telomerase
activity is absent from most normal somatic cells
and neoplastic cells must ®rst overcome the senes-
cence checkpoint mechanisms, and subsequently
activate telomerase to propagate inde®nitely. In
senescent cells the levels of p16
ink
are elevated, and
its inactivation has been shown to abrogate a late
step during senescence of human epithelial cells
(Kiyono et al., 1998). Interestingly, the immortaliza-
tion of human uroepithelial cells by HPV-16 E6
correlated with undetectable levels of p16
ink
due to
gene inactivation. In addition, activati on of telomer-
ase is detected in cervical carcinomas and in a
subset of high grade cervical lesions associated with
high-risk HPV (Snijders et al ., 1998). Telomerase can
be induced by HPV-16 E6 in primary epithelial cells
through a p53-independent mechanism (Klingelhutz
et al., 1996; Veldman et al., 2001) via E6-mediated
transcriptional activation of the gene encoding the
telomerase catalytic subunit, hTERT, and the
minimal promoter region involved in induction by
E6 was found to require an intact E box (Veldman
et al., 2001; Gewin and Galloway, 2001). Interest-
ingly, this activation of the hTERT promoter was
found to be dependent on the ability of E6 to
interact with E6AP, suggesting that the activity of
the E6-E6AP complex may target a regulator of the
hTERT promoter (Gewin and Galloway, 2001).
E6 proteins of both benign and oncogenic HPV
types have also been found to interact with hMcm7, a
component of the DNA replication licensing complex,
suggesting that this interaction might be required for
viral genome replication (Kukimoto et al., 1998;
Ku
È
hne and Banks, 1998). However, hMcm7 is a
substrate for E6AP-dependent ubiquitination, and
HPV-18 E6 was able to enhan ce its proteasome
degradation in vivo (Ku
È
hne and Banks, 1998): this
might also be expected to cause p53-independent
chromosomal abnormalities in HPV-positive cells.
Figure 1 The high-risk HPV E6 protein. Schematic diagram of E6 showing the two zinc ®ngers, together with the regions involved
in interactions with cellular proteins that are targeted by the oncoproteins of other viruses. Also shown is the C-terminal PDZ
binding motif, ETQV, and the overlapping site of PKA phosphorylation is arrowed
HPV E6
F Mantovani and L Banks
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Oncogene

Mitogenic activities of E6
It has been shown that expression of oncogenic E6 can
aect the early stages of carcinogenesis in transgenic
mice, although to a much lesser extent than E7 (Song
et al., 2000), and this probably relies on the ability of
E6 to induce cellular hyperproliferation and ep idermal
hyperplasia (Song et al., 1999). Interestingly, this
appears to be largely independent of p53, since similar
levels of proliferation are induced by E6 in p53-null
mice (Song et al., 1999). At present , little is known
about the pathways by which E6 could stimulate
proliferation but a recent study identi®ed E6TP1 in a
two-hybrid screen. This protein shows high hom ology
with a family of GTPase activating proteins (GAPs)
that are negative regulators of Rap (Gao et al., 1999).
Therefore E6TP1 might be involved in the inhibition of
Rap-mediated mitogenic signalling. Moreover, its gene
has been mapped to a putative tumour suppressor
locus on chromosome 14 (Menon et al., 1997). High-
risk and, to a lesser extent, low-risk HPV E6 proteins
can bind E6TP1, while only the oncogenic E6s can
promote its degradation. Inter estingly, E6AP was
reported to participate in the interaction, and it might
therefore be responsible for ubiquitination of E6TP1
(Gao et al., 1999). There is a strong correlation
between the ability of HPV-16 E6 mutants to degrade
E6TP1 and to immortalize human mammary epithelial
cells (Gao et al., 2001), however more data concerning
the GAP function of E6TP1 are necessary to under-
stand its role in E6-induced malignancy.
Inhibition of apoptosis
Oncogenic HPV E6 proteins counteract the induction
of apoptosis by activated p53 by inducing its
degradation. However, there is increasing evidence that
E6 can also inhibit p53-independent apoptotic path-
ways, promoted by dierent stimuli. Indeed, in
transgenic mice expressing HPV-16 E6 in the ocular
lens, E6 was found to prevent apoptosi s both in wt and
in p53-null animals (Pan and Griep, 1995), and it has
also been reported to inhibit drug-induced apoptosis in
cells lacking p53 (Steller et al., 1996). Among the more
recently discovered cellular targets of E6, there are
several proapoptotic factors. One of them is Bak,
whose high levels of expression in the upper epithelial
layers (Krajewski et al., 1996) appear to represent a
common obstacle for a broad range of HPV types that
replicate in dierentiating keratinocytes, since the E6
proteins of both high and low-risk mucosal HPVs, plus
those of high-risk cutaneous types, have been shown to
inhibit Bak-induced ap optosis (Thomas and Banks,
1998, 1999; Jackson and Storey, 2000). HPV-18 E6
stimulates the ubiquitin-dependent degradation of Bak
catalysed by E6AP, probably by accelerating a normal
cellular process, since Bak appears to be a substrate of
E6AP in the absence of E6 (Thomas and Banks, 1998).
Degradation of Bak by HPV-11 E6 is less eective, and
this correlates with a weaker anti-ap optotic activity of
the low-risk mucosal HPV types (Thomas and Banks,
1999). Recently, a link was demonstrated between
cutaneous HPV infection and the induction of HPV-
associated skin cancer by UV radiation. The cutaneous
E6 proteins were shown to abrogate both p53-
dependent and independent apoptosis in response to
UV-induced DNA damage. p53 protein levels were
unaected in cells expressing cutaneous E6s, and its
transcriptional activity with respect to p21 also did not
change (Jackson and Storey, 2000). The mechanism
used by these E6 types to inhibit p53-dependent
apoptosis is not known, although one possibility is
that they mediate the speci®c inhibition of p53 target
genes directly involved in apoptosis, such as the PIG
genes induced by DNA damage (Polyak et al., 1997).
The cutaneou s HPV E6 proteins also abrogate Bak
function by promoting its ubiquitin-mediated degrada-
tion and Bak protein is undetectable in HPV-positive
skin cancers, in contrast to HPV negative cancers
which express it (Jackson et al., 2000). Interestingly,
Ad E1B 19K also inhibits Bak induced apoptosis
(Farrow et al., 1995; White, this issue ), demonstrating
a high degree of conservation of function among these
viruses.
The c-Myc transcription factor is normally degraded
through the proteasome pathway, and it was shown
that high-risk, but not low-risk HPV E6, accelerates
degradation of c-Myc by recruiting E6AP (Gross-
Mesilaty et al., 1998). It may seem unusual that E6
targets a protein which would normally stimulate cell
proliferation, but this ®nding can be also viewed in the
light of c-Myc proapoptotic activity. TGFb is a potent
growth inhibitor for many cell types, including most
epithelial cells (Lyons and Moses, 1990), and its eects
are mediated at least in part by suppression of c-myc
transcription (Pietenpol et al., 1990a). It has been
demonstrated that HPV-16 E7 and other viral proteins
binding pRb, such as SV40 LT and Adenovirus E1A,
are able to block TGFb-mediated downregu lation of c-
Myc, in order to sustain proliferation of the infected
keratinocytes (Pietenpol et al., 1990b; Mu
È
nger et al.,
this issue). However, deregulated expression of c-Myc
in dierentiating cells induces apoptotic cell death
(Askew et al., 1991). It is therefore possible that
degradation of c-Myc by E6 contributes towards
maintaining an equilibrium between E7-promoted
proliferation and cell survival.
Interference with epithelial organization and
differentiation
High-risk HPV types have evolved to replicate in the
dierentiated layers of the squamous epithelium, in an
environment that is non-permissive for DNA rep lica-
tion. A characteristic of the high-risk E6 proteins is
their ability to inhibit terminal dierentiation of
epithelial cells, which normally leads to keratinization
and cell death. HPV-16 E6 impairs cell dierentiation
in the ocular lens of transgenic mice (Pan and Griep,
1994) and causes expansion of the undierentiated
compartment of the epithelia in K14-E6 transgenic
mice and, interest ingly, this activity of E6 appears to
Oncogene
HPV E6
F Mantovani and L Banks
7879

be p53-independent (Song et al., 1999). Consistent with
this, HPV-16 E6 was also found to increase the
resistance of human keratinocytes to serum and
calcium induced dierentiation through p53-indepen-
dent pathways (Sherman et al., 1997; Sherman and
Schlegel, 1996), although little is known about the
molecular mechanisms by which E6 does this. How-
ever, HPV-16 E6 was reported to interact with E6BP/
ERC-55 (Chen et al ., 1995) which is a putative calcium
binding protein localized in the endoplasmic reticulum
(Weis et al., 1994). E6BP was found to form a complex
with both E6 and E6AP in vivo, however direct binding
to E6AP and E6 targeted degradation were not shown
(Chen et al., 1995). Epithelial dierentiation is
responsive to Ca
2+
mediated signalling, and it might
be speculated that targeting E6BP contributes to E6's
impairment of terminal dierentiation. Ca
2+
signalling
also has a role in blocking apoptosis, and it is
interesting that the antiapoptotic Ad E1B 19K protein
also interacts with a putative calcium binding protein
localized in the nuclear envelope and ER (Boyd et al.,
1994). Hence, the E6 ± E6BP interaction might similarly
be involved in the p53-independent inhibition of
apoptosis in HPV infected cells.
Cellular adhesion to the extracellular matrix aects
many dierent cellular processes including cell mor-
phology, pro liferation and migration. The restriction of
cell proliferation to matrix ± interacting cells serves to
prevent dysplasia, and the circumvention of anchorage
dependence plays an important role in tumorigenesis
(Sastry and Horwitz, 1996). Cell ± matrix adhesion is
mediated by specialized structures called focal adhe-
sions, which contain integrins, vinculin, focal adhesion
kinase and paxillin (Sastry and Burridge, 2000).
Paxillin is involved in mediating signalling from the
plasma membrane to focal adhesions and to the actin
cytoskeleton, and its activity is regulated by tyrosine
phosphorylation in response to various stimuli, includ-
ing integrin crosslinking and treatment with growth
factors (Turner, 2000). HPV-16 E6 has also been
shown to bind paxillin and this interaction correlates
with E6 transforming activity, although it does not
lead to paxillin degradation (Tong and Howley, 1997).
However, since most of the biological eects of this
interaction have been determined for the more highly
abundant BPV-1 E6 protein, its relevance for high-risk
HPV E6 proteins remains to be determined.
Interactions with PDZ proteins: interference with
cell ± cell adhesion, polarity and proliferation control
A striking feature of all E6 proteins derived from the
high-risk HPV types is the presence of a highly
conserved C-terminal domain (see Figure 1), which is
not involved in p53 binding and degradation (Crook et
al., 1991; Pim et al., 1994), but which nonetheless
contributes to E6 transforming activity, since its
deletion impairs E6's ability to transform rodent cells
(Kiyono et al., 1997) and immortalize keratinocytes (C
Meyers, personal communication). This region con-
tains a PDZ-binding motif (XT/SXV), a short stretch
of amino acids which mediates the speci®c interaction
with proteins containing PDZ domains (Doyle et al.,
1996; Songyang et al., 1997). These are 80 ± 90 amino-
acid long motifs, present on a variety of proteins
involved in clustering ion c hannels, signalling enzymes,
and adhesion molecules to speci®c structures at the
membrane-cytoskeleton interface of polarized cells
(reviewed in Kim, 1997). The ®rst PDZ-protein shown
to be a target for high-risk E6 was hDlg (Kiyono et al.,
1997; Lee et al., 1997). This is the human homologue
of the Drosophila tumour suppressor Dlg, required for
formation of adherens junctions and for regulation of
cell adhesion, apicobasal polarity and proliferation in
epithelial tissues. Indeed, loss of Dlg function causes
aberrant morphology and invasive growth of epithelial
cells, resulting in embryonic lethality (Woods et al.,
1996; Bilder et al., 2000). A recent study also described
a Dlg truncation mutant that resulted in impaired
morphogenesis and perinatal death during murine
development (Caruana and Bernstein, 2001). Human
hDlg colocalizes with E-cadherin at adherens junctions
of epithelial cells, (Reuver and Garner, 1998; Ide et al.,
1999) and interacts through dierent domains (see
Figure 2) with several proteins, including Shaker-type
K
+
channels (Kim et al., 1995), cytoskeletal protein 4.1
(Lue et al., 1994; Marfatia et al., 1996), and the APC
tumour suppressor protein (Matsumine et al., 1996),
which is mutated in the majority of colon cancers
(Kinzler and Vogelstein, 1996). Indeed, complex
formation between hDlg and APC was reported to
block cell cycle progression (Ishidate et al., 2000). HPV
E6 can target hDlg for ubiquitin mediated degradation
(Gardiol et al., 1999), probably by enhancing a
physiological process, since hDlg appears to be
ubiquitinated and degraded by the proteasome in cells
even in the absence of E6 (Gardiol et al., 1999;
Mantovani et al., 2001, in press). This interaction
between E6 and hDlg might be nece ssary at a de®ned
point during the viral life cycle, in order to disrupt cell
junctions and to abolish cell polarity, thereby altering
the normal maturation and inducing proliferation of
the infected keratinocytes. However, the consequences
for the metastatic potential of tumour s harbouring
high-risk HPVs are clear. While HPV-18 E6 has a
canonical PDZ-binding moti f (ETQV), HPV-16 E6 has
only a sub-optimal consensus site (ETQL). Conse-
quently, HPV-18 E6 binds hDlg with higher anity
than HPV-16 E6 (Pim et al., 2000) and can degrade it
more eciently (Gardiol et al., 1999; Pim et al., 2000).
Interestingly, this also correlates with the dierent
malignant potential of the viruses, since it has been
reported that cervical tumours associated with HPV-18
are more invasive and recurrent than those caused by
HPV-16 (Burnett et al., 1992; Kurman et al., 1988;
Zhang et al., 1995); this is in marked contrast with the
reported lower eciency with which HPV-18 E6
degrades p53 (Schener et al., 1990). Interestingly,
low-risk E6 proteins lack PDZ-binding motifs, and
indeed they can neither bind hDlg (Kiyono et al., 1997;
Lee et al., 1997) nor induce its degradation (Gardiol et
al., 1999; Pim et al., 2000), however they acquire this
HPV E6
F Mantovani and L Banks
7880
Oncogene

ability if provided with a C-terminal PDZ binding
domain derived from a high-risk E6 protein (Pim et al.,
2000). In contrast, cutaneous HPV E6 proteins are
unable to degrade hDlg even when the interaction is
allowed by adding a PDZ-binding motif to their C-
termini (Pim et al., submitted). This suggests that the
mucosal HPV E6 proteins, whether derived from high
or low-risk HPV types, can interact similarly with the
cellular proteolytic machinery, whereas the high-risk
cutaneous HPV E6 proteins appear to signi®cantly
dier in this aspect.
An increasing number of PDZ domain-containing
proteins involved in the organization of epithelial
architecture have now been reported to be targeted
by E6 through their PDZ domains. Among them is
hScrib, a protein expressed at epithelial tight junctions,
which has recently been shown to be a substrate for
ubiquitination by the E6-E6AP complex in vitro,and
for proteasome degradation mediated by high-risk E6
in vivo (Nakagawa and Huibregtse, 2000). hScrib is the
human homologue of Drosophila tumour suppressor
Scrib, which cooperates with Dlg and Lgl to control
both formation of cell junctions and inhibition of
epithelial cell growth, possibly through controlling the
localization of growth factor receptors and signalling
molecules (Bilder and Perrimon, 2000; Bilder et al.,
2000). Interestingly, expression of oncogenic HPV E6
in mammalian cells was reported to disrupt the
integrity of epithelial tight junctions, while E6 mutated
in the PDZ-binding motif did not (Nakagawa and
Huibregtse, 2000). This further suggests that combined
degradation of dierent PDZ-proteins by E6 might be
responsible for the loss of epithelial cell adhesion and
polarity of HPV-positive cancer cells. E6 proteins from
high-risk, but not low-risk HPV types were also shown
to bind another tight junction protein, MAGI-1
(Dobrosotskaya et al., 1997; Ide et al., 1999), through
the ®rst of its ®ve PDZ domains (Thomas et al., 2001),
and to reduce its steady-state levels and half-life
(Glaunsinger et al., 2000). MAGI-1 forms a complex
with b-catenin (Dobrosotskaya and James, 2000), the
expression of which is deregulated in many human
cancers (reviewed in Polakis, 1999). Interestingly, the
closely related proteins MAG I-2 and MAGI-3 are
involved in the regulation of the PTEN tumour
suppressor, a component of the Akt kinase signalling
pathway that promotes cell survival and proliferation
(Marte and Downward, 1997): MAGI proteins are
required to enhance PTEN's ability to suppress Akt
activation, probably through assembling a multiprotein
complex at the cell membrane (Wu et al., 2000a,b).
Therefore it is plausible that MAGI-2/3 degradation,
promoted by high-risk HPV E6 (M Thomas, personal
communication), might also aect Akt signalling and
thereby inhibit apoptosis independently of targeting
p53, as well as representing an alternative mitogenic
activity of E6 (see above).
HPV-18 E6 has also been shown to bind MUPP1, a
large multi-PDZ scaold protein with a putative role in
signal transduction (Ullmer et al., 1998). Indeed,
MUPP1 interacts selectively through its 10th PDZ
domain with the cytoplasmic portion of the 5-HT
2c
serotonin receptor (Ull mer et al., 1998; Becamel et al.,
2001), and with the unphosphorylated c-Kit tyrosine
Figure 2 The hDlg protein. Schematic diagram showing the location of hDlg at the membrane-cytoskeleton interface of epithelial
cells at regions of cell ± cell contact. hDlg contains 3 PDZ domains, an SH3 signalling domain and a Guanylate Kinase (GUK)
homology domain. Here it co-localizes with E-cadherin and binds the APC tumour suppressor, which in turn regulates b-catenin.
The interaction between hDlg and APC is via PDZ domain 2 and this is the same domain targeted by the high risk HPV E6
proteins. Also shown is the interaction with protein 4.1, which connects hDlg to the actin cytoskeleton
Oncogene
HPV E6
F Mantovani and L Banks
7881

kinase receptor (Mancini et al., 2000). HPV-18 E6 was
shown to reduce MUPP1 half-life in vitro and in vivo
(Lee et al., 2000b), and there is evidence that this also
occurs through proteasome mediated degradation (F
Mantovani, personal observations). Destruction of
MUPP1 by E6 could thereby interfere with the
assembly of signalling complexes at the epithelial cell
membranes.
It has been frequently reported that dierent
oncogenic viruses inactivate the same cellular targets
to overcome common obs tacles to their replication:
thus, there are viral oncoproteins other than HPV
E6 which bind PDZ-proteins, and this contributes to
their transforming potential. hDlg has been shown
to interact, through its PDZ domains, with both Ad
9 E4-ORF1 (Lee et al., 1997; Ta
È
uber and Dobner,
this issue) and HT LV-1 Tax, and these interactions
interfere with the binding of APC, thereby perturb-
ing cell growth control (Lee et al., 1997; Suzuki et
al., 1999). MUPP1 and MAG I-1 are also bound by
the Ad 9 E4-ORF1, which abolishes their activities
by sequestrating them into cytoplasmic bodies (Lee
et al., 2000b; Glaunsinger et al., 2000). However,
ubiquitin mediated degradation of PDZ-proteins is,
to date, an exclusive pro perty of the high-risk HPV
E6 proteins. It has recently been shown that E6 can
bridge the interaction between hScrib and the E6AP
ubiquitin ligase, which normally would not recognize
hScrib (Nakagawa and Huibregtse, 2000 ). This is
reminiscent of p53 degradation, yet a clear role for
E6AP in hScrib degradation in vivo has not been
con®rmed. In contrast, proteasome degradation of
hDlg appears to be independent of E6AP (Pim et
al., 2000), as deduced from the basis of E6's ability
to degrade hDlg in ex tracts lacking E6AP, and from
the fact that E6 mutants defect ive for E6AP binding
and p53 degradation can still degrade hDlg.
Similarly, low-risk E6 proteins can also target hDlg
for degradation, with an eciency similar to that of
the high-risk E6 proteins, when they are provided
with a PDZ-binding motif (Pim et al., 2000).
However, these low risk E6 proteins show only
minimal levels of interaction with E6AP (Huibregtse
et al., 1991, 1993b).
E6 binding motifs: a matter of speci®city
It is clear from the above discussion that E6 is a
multifunctional protein which eciently interferes with
diverse cellular pathways. However, it is logical to ask
how such a small and low abundance viral protein
could evolve to interact with so many dierent cellular
proteins, both with respect to the speci®city of the
interactions and to the relatively high abundance of
the partners. The analysis of E6 targets has identi®ed
conserved E6 binding motifs, thereby characterizing
clusters of proteins which are bound by E6 through
similar domains. The PDZ domain, for instance,
de®nes a family of proteins with common functions,
which are targeted by high-risk E6 proteins through
their C-terminal PDZ-binding motifs. E6 interaction
with proteins containing multiple PDZ domains is,
however, highly speci®c: thus, despite their high
overall homology, only single PDZ domains on hDlg
and MAGI-1 (Kiyono et al., 1997; Thomas et al.,
2001) are recognized by the E6 protein. Another
conserved binding site, the L2G box which comprises
a a-helical motif, has been foun d on a number of
cellular targets of E6, including E6AP, hMcm7, E6BP
and paxillin. The binding speci®city of E6AP is
mediated by this moti f, present on both enzyme and
substrate, and this in turn is used by E6 to interact
with both (Chen et al., 1998; Elston et al., 1998;
Ku
È
hne and Banks, 1998). Not surprisingly, E6 has
also been reported to induce self-ubiquitination of
E6AP (Kao et al., 2000), nonetheless, there are
proteins which interact with E6 through an L2G
box without being targeted for degradation, such as
E6BP (Chen et al., 1995) and paxillin (Tong and
Howley, 1997).
The amount of the E6 protein produced during a
viral infection is very low, and the problem of targeting
cellular proteins which are much more abundant within
the cell is eciently solved by inducing their ubiquitin-
mediated degradation. Moreover, it seems plausible
that the virus does not require the complete destruction
of its targets, rather, even a transient decrease of their
local concentration within the cell is likely to perturb
the physiological conditions in favour of viral replica-
tion. This also implies that E6 does not need to interact
with all of its putative targets at the same time, since
probably only a limited subset will be available at
de®ned stages during cell dierentiation and in speci®c
compartments within the cell. It is therefore possible
that the intracellular localization of E6 might change,
either as a consequence of dierentiation or as a result
of exogenous stimuli. Indeed, the PDZ-binding speci-
®city of high-risk E6 has also been shown to be
regulated by a cellu lar pathway: the E6/hDlg interac-
tion is inhibited by PKA phosphorylation of the
conserved threonine residue within the PD Z binding
domain on E6, and this was also shown to inhibit E6-
mediated degradation of hDlg. Indeed, high levels of
hDlg could be restored in HPV-18 positive cervical
cancer cells by induction of PKA activity (Ku
È
hne et al.,
2000). Interestingly, PKA regulation of PDZ domain
binding was also reported to control the interaction
between the K
+
channel Kir2.3 and the PDZ protein
PSD-95 (Cohen et al., 1996), and this would suggest
that the high-risk E6 proteins might have acquired the
PDZ binding domain, with its PKA-associated regula-
tion, from the cellular genome. Its strict conservation
among all high-risk E6 proteins is intrigui ng, and
implies a requirement for the virus to ®nely balance its
eects upon hDlg and other PDZ domain-contai ning
proteins. Recent studies have also shown that E6 is
phosphorylated by PKN (Gao et al., 2000) and,
although the site of phosphorylation has not been
mapped, it will nonetheless be extremely interesting to
evaluate the eects of this phosphorylation upon E6's
other activities.
HPV E6
F Mantovani and L Banks
7882
Oncogene

E6 as a therapeutic target
Since E6 is invariably expressed in cervical lesions and
would appear to be responsible for their malignant
progression, this protein represents an attractive
candidate for developing therapeutic strategies against
cervical cancer. Many approaches have been tested to
block the expression of E6 in HPV-positive cervical
cancer cells, e.g. by selectively inhibiting viral tran-
scription (Goodwin and DiMaio, 2000), or by using
antisense constructs (Hamada et al., 1996; von Knebel
Doeberitz et al., 1992) or ribozymes (Alvarez-Salas et
al., 1998) directed against the polycistronic E6/E7
mRNA. All these approaches cause growth suppression
and concomitant reduction of tumorigenicity in vivo,
implying the feasibility of reactivating functional
tumour suppressor pathways in HPV-positive cells. It
can be reasoned that blocking the activity of E6, while
retaining that of E7 as a proapoptotic stimulus, might
provide a higher therapeutic potential. Since this would
be dicult to obtain by targeting the viral mRNAs,
since the E6 and E7 genes are expressed together as
polycistronic transcripts, this could be done by using
peptide aptamers to block the E6 protein in vivo.
Indeed, administration of such peptides was found to
induce apoptosis of HPV-positive cancer cells (Butz et
al., 2000). Thus blocking the E6-mediated degradation
of p53 is a major therapeutic goal, since there is strong
evidence that the p53-responsive pathways are fully
functional in cervical tumour cell lines (Butz et al.,
1995, 1999), and reactivating p53 would then bring
about growth arrest and/or apoptosis of the HPV
transformed cells. However this is unlikely to be
universally applicable, since inhibition of E6 induced
degradation does not always lead to increa sed p53
levels. In several cervical cancer cell lines p53 can be
stabilized only after additional genotoxic insult,
indicating a lack of intrinsic activation of p53 despite
the presence of the viral oncogenes (Mantovani and
Banks, 1999). A note of caution should also be added,
since targeting the p53/E6AP/E6 complex co uld
interfere with normal E6AP functions, and mutations
of E6AP have been implicated in a serious develop-
mental disorder, the Angelman Syndrome (Kishino et
al., 1997; Matsuura et al., 1997).
The inter actions of E6 with PDZ-proteins are also
excellent therapeutic targets, because of their co rrela-
tion with the malignant progression of HPV-associated
disease. Moreover, the binding motif on E6 is small
and exposed, and the structures of a number of PDZ
domains have been solved (Doyle et al., 199 6; Tochio
et al., 2000). In add ition, the interaction between E6
and its PDZ domain-containing substrates is highly
speci®c, with E6 binding only single PDZ domains on
both hDlg and MAGI-1. Therefore it might be feasible
to design chemotherapeutic agents, capable of speci®-
cally inhibiting the interaction between E6 and this
class of targets.
Concluding remarks
During HPV infection E6 plays multiple roles,
interfering with several cellular pathways in order to
create a favourable environment for viral replication,
and neutralizing the cellular surveillance controls that
are turned on as the infected cell is unnaturally forced
to restart DNA replication . Thus, high-risk E6 blocks
apoptosis by targeting p53, Bak and Myc proteins for
degradation, however this leaves the cell unprotected
from the detrimental eects of DNA mutations. This is
con®rmed by biochemical and epidemiological studies
reporting a synergy between HPV oncogenes and
chemical carcinogens in the developm ent of malignancy
Figure 3 Contribution of HPV E6 to dierent stages of tumour progression. The role of E6 in tumour promotion is weak
compared with that of E7 which actively stimulates cell proliferation. In contrast E6 promotes malignant progression: degradation
of p53 overcomes growth arrest and/or apoptosis allowing accumulation of DNA damage, and induction of telomerase contributes
towards immortalization. Finally, targeting PDZ-containing proteins through the C-terminal XT/SXV sequence of E6 causes loss of
cell polarity and contact, strongly contributing to the malignant phenotype
Oncogene
HPV E6
F Mantovani and L Banks
7883

(Song et al., 2000; Daling et al., 1992). Moreover, E6
inhibits terminal dierentiation and senescence, thereby
stimulating cell immortalization. Finall y, by targeting a
class of PDZ domain containing proteins, E6 can also
aect cell contact and polarity and, when the pathways
regulating these interactions are impaired, either due to
mutation or exogenous stimuli, this ®nal loss of control
in the immortal ized cell can ®nally lead to an invasive
and metastatic phenotype (see Figure 3).
It should be clear, however, that the above picture is
far from the `physiological' outcome of an HPV
infection, representing instead the most unfortunate
result of a progressive and multifactorial process that
only occurs on some occasions. Nonetheless, cervical
cancer is an extremely seri ous disease, representing the
third major cause of cancer-related death in women
worldwide (Parkin et al., 1999), and the study of the
E6 cellular targets has important implications for the
development of eective therapeutic strategies.
Acknowledgements
We are grateful to C Meyers, D Pim and M Thomas for
allowing us to cite their unpublished work. We are also
grateful to M Thomas for valuable comments on the
manuscript. L Banks gratefully acknowledges research
support provided by the Associazione Italiana per la
Ricerca sul Cancro.
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