[Cancer Research 60, 6875-6877, December 15, 2000]
© 2000 American Association for Cancer Research
Preferential Retention of Codon 72 Arginine p53 in Squamous Cell Carcinomas of the Vulva Occurs in Cancers Positive and Negative for Human Papillomavirus1
Louise A. Brooks,
John A. Tidy,
Barry Gusterson,
Louise Hiller,
Jenny O’Nions,
Milena Gasco,
Maria Carmen Marin,
Paul J. Farrell,
William G. Kaelin, Jr. and
Tim Crook2
Ludwig Institute for Cancer Research, Imperial College School of Medicine, St Mary’s Hospital, London W2 1PG, England [L. A. B., J. O., P. J. F., T. C.]; Department of Gynaecological Oncology, University of Sheffield, Northern General Hospital, Sheffield S5 7AU, England [J. A. T.]; University of Glasgow, Pathology Department, Western Infirmary, Glasgow G11 6NT, Scotland [B. G.]; Birmingham Clinical Trials Unit, University of Birmingham, Birmingham B15 2TT, England [L. H.]; UO Oncologia Medica, Azienda Ospedaliera S. Croce e Carle, 12100 Cuneo, Italy [M. G.]; and Dana-Farber Cancer Institute, Boston, Massachusetts 02115 [M. C. M., W. G. K.]
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ABSTRACT
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We
have sought to determine the basis for preferential loss of the codon
72 proline (72P) rather than the arginine (72R)
allele in squamous cell carcinoma of the vulva with loss of
heterozygosity (LOH) in p53. The proportion of cases containing human
papillomavirus (HPV) 16 was not statistically different among
individuals with either 72RR or 72RP in the
germ line (P > 0.99), but p53 LOH was
significantly more common in individuals heterozygous 72RP
than in 72RR individuals (P = 0.04). LOH more commonly involved the 72P allele in both
HPV-positive and HPV-negative cancers. Our results imply that
preferential loss of the 72P allele in vulval squamous cell
carcinoma occurs by HPV-dependent and -independent mechanisms.
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Introduction
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The frequent mutation of p53 in human cancer attests to its
critical importance in carcinogenesis (1)
. p53 exists in
two principal polymorphic forms that have either arginine
(72R) or proline (72P) at codon 72
(2)
. This polymorphism is balanced, although the selective
pressure maintaining this is not known (3)
. Representation
of each allele within populations varies according to latitude, with a
strong correlation between the R allele and increasing latitude
(3)
. Functional differences between the two polymorphic
forms of the wild-type protein have been described previously
(4)
. Furthermore, the 72R polymorphic form of
wild-type p53 is more sensitive to proteolysis mediated by the E6
protein encoded by oncogenic types of
HPV3
than the wild-type
72P (5)
. Consistent with this observation,
increased representation of individuals homozygous 72RR in
the germ line was observed in patients with HPV-related SCC of the
cervix (5)
. Other studies have, however, failed to observe
such an effect (6
, 7)
. Although the etiological
association between HPV infection and cancer is most well established
for cervical SCC, other cancers such as SCC of the vulva, head and
neck, and esophagus may also be associated with HPV. We previously
observed no significant differences in germ line representation of
72R and 72P between individuals with vulval or
head and neck SCC, which suggests that germ-line possession of
wild-type 72R/R is unlikely to represent a significant risk
factor for development of these cancers (8)
. However,
analysis of vulval and head and neck SCC arising in individuals with
germ-line 72R/P revealed that p53 mutations preferentially
target the 72R allele, whereas LOH in p53 more frequently
targeted the 72P allele (8)
. p73, a gene with
structural and functional homology to p53, was recently cloned
(9)
, but mutations do not appear to be common genetic
events in human cancer (10)
. However,
methylation-dependent transcriptional silencing of p73 in some
hematological malignancies suggests that loss of function in p73 may be
an important event in oncogenesis, at least in some tissues
(11)
. Some tumor-associated p53 mutants are able to
associate with, and inhibit the function of, p73, raising the
possibility that this interaction may be important in carcinogenesis in
tissues that express p73 (12)
. Recently, we have shown
that the ability of these mutants to form heterodimers with, and
thereby inactivate, p73 is strongly influenced by the identity of the
amino acid at codon 72 (8)
. Specific p53 mutations with R
at codon 72 efficiently form complexes with p73 and inhibit its DNA
binding and proapoptotic activity, but this property is absent or
markedly reduced if the same mutants have P at position 72.
Moreover, we detected such mutants in SCC of both vulva and head and
neck (8)
. Taken together, these observations led us to
hypothesize that the preferential mutation of the R allele
of p53 in vulval and head and neck SCC might reflect, at least in part,
the selective interaction of mutant p53 72R but not p53
72P proteins with p73. Apparently nonrandom allele loss in
72R/P germ-line heterozygotes has also been reported in
esophageal SCC, in which the lost allele in six cancers with LOH was
exclusively 72P (13)
. In this study, the
authors suggested that loss of P reflected the preferential
retention of the 72R form of wild-type p53, which has been
shown to be more sensitive to HPV E6-dependent degradation
(5)
.
In the present study, we have investigated whether the preferential
loss of the 72P allele in SCC of the vulva (8)
is associated with HPV status, as observed in esophageal cancer
(13)
, or whether other selective pressure(s) might operate
to favor loss of the 72P allele and/or mutation of the
72R allele.
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Materials and Methods
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Tumors.
Tissues were obtained at surgical removal of cancers with matched
normal tissue and snap-frozen in liquid N2 on harvesting
prior to nucleic acid isolation. Diagnosis and presence of tumor within
the resected tissue was confirmed by routine histopathological
analysis.
Nucleic Acid Isolation and p53 and HPV Analysis.
Genomic DNA was isolated from frozen tissues by proteinase K digestion
and RNA using RNAzol B according to the manufacturer’s instructions
(Biogenesis Ltd). p53 status of the vulval SCC has been described
previously (8)
. HPV DNA sequences were sought using
established PCR methodology (14)
. HPV typing was by
sequencing of amplified products.
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Results
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Loss of 72P Allele Occurs in HPV-Negative and -Positive
SCC.
We previously reported (8)
that the germ-line frequencies
of 72RR and 72RP were similar in individuals with
SCC of the vulva and that, in these cancers, LOH in p53 occurred more
commonly in the P allele in individuals germ-line
72R/P. To ascertain whether the preferential loss of
72P was associated with HPV status, genomic DNA was analyzed
for the presence of HPV DNA sequences. The characteristics of each
cancer are shown in Table 1
. HPV DNA was
detected in 13 of 36 cancers (Table 2A)
,
and sequencing revealed that this was HPV 16 in each case. The
proportion of HPV-positive cancers was not significantly different in
72RR and 72RP germ-line individuals (5 of 14
versus 8 of 21; P > 0.99; Table 2B
). The frequency of p53 LOH was similar in HPV-positive and
HPV-negative vulval SCC (7 of 13 versus 14 of 23;
P = 0.95) consistent with a previous report
(15)
. However, LOH was significantly more common in
72R/P germ-line heterozygotes than in 72R/R
homozygotes (16 of 21 versus 5 of 14; P = 0.04) (Table 2A)
. In the R/P germ-line heterozygotes
with LOH, the lost allele was 72P in 4 of 5 HPV-positive SCC
and in 10 of 11 HPV-negative SCC (Table 2C)
.
View this table:
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Table 2 p53 and HPV status of vulval cancers
One HPV-negative cancer occurred in a patient with germ-line 72 PP.
Figures in brackets are proportions
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p53 Status in Vulval Cancers.
Because preferential loss of the 72P allele occurs in both
HPV-positive and -negative cancers, we next considered the hypothesis
that the apparently nonrandom loss of the 72P allele
actually represents selection for mutation in R, with
subsequent loss of the wild-type 72P allele. We, therefore,
analyzed each cancer for p53 mutation. Missense mutations were
identified in 17 of 23 HPV-negative and 4 of 13 HPV-positive cancers.
These results are consistent with previous reports of p53 mutation
occurring more commonly in HPV-negative vulval SCC (16)
,
and with a high incidence of mutations in clinically aggressive and/or
recurrent vulval SCC (17)
. HPV p53 mutations occurred
significantly more commonly in the 72R allele rather than
the 72P allele in individuals R/P in the germ line
(8)
.
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Discussion
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We and others have previously demonstrated that the 72P
allele of p53 is preferentially deleted in squamous cell cancers
arising in individuals with germ-line 72R/P heterozygosity
(8
, 13)
. In the present study, we have sought to clarify
which selective pressure underlies the preferential loss of the
72P allele. Kawaguchi et al. (13)
reported that allele loss exclusively involved the 72P
allele in esophageal cancer and proposed that this was attributable to
the relatively increased sensitivity of the 72R wild-type
p53 protein to proteolysis mediated by HPV E6 (13)
. In the
present study, we show that preferential loss of the 72P
allele occurs in both HPV-positive and HPV-negative vulval SCC. There
are several possible explanations as to why this is the case, and these
are not mutually incompatible. Firstly, it is possible that cancers
that lack detectable HPV DNA are initially HPV positive and that the
HPV genome is lost during tumorigenesis. In this scenario, selection
would favor the retention of the more easily degraded 72R
p53, as proposed for esophageal cancer. Subsequent loss of the HPV
genome would then require mutation of the p53 allele to
functionally substitute for loss of HPV E6-dependent p53 inactivation,
and this would inevitably occur in the remaining (72R)
allele. Such a "hit and run" mechanism has been proposed previously
in HPV-negative vulval SCC (17)
and in cervical SCC in
which p53 mutation was convincingly demonstrated after loss of HPV DNA
(18)
. It is also consistent with the observation that p53
mutation in cervical and anal SCC occurs almost exclusively in
HPV-negative cases (19
, 20)
. A second plausible
explanation is that the apparent nonrandom loss of the 72P
allele actually represents selection for mutation in the 72R
allele. Such a hypothesis is consistent with functionally significant
interactions between some p53 mutants and the p53-related protein p73
(12)
and the demonstration that, at least for some p53
proteins, this activity is significantly greater when R is
the amino acid at codon 72 (8)
. On the basis of our
previous work (8)
, mutant p53 proteins that occur in
72R rather than 72P would be predicted to have
greater ability to be complexed with and to inactivate p73 
and ß.
Both isoforms of p73 are expressed in normal vulval epithelium and are
often abundantly overexpressed in vulval cancers (data not shown). It
is of interest that many of the p53 72R mutants that
preferentially inactivate p73 are those with "gain of function" and
that four of five metastatic vulval cancers from the present series
expressed 72R p53 mutant proteins.
In conclusion, our data suggest the possibility that distinct selective
pressures operate in HPV-negative and HPV-positive cancers. In
HPV-positive vulval cancers, loss of 72P may occur as a result of the
increased sensitivity of the wild-type 72R p53 protein to
degradation that is mediated by HPV E6, as suggested for esophageal
carcinomas. In HPV-negative cancers, pressure may be for mutation in
the 72R allele (rather than for selective loss of the
72P). The frequent presence of p53 mutations in metastatic
vulval cancers reported in this and other studies is consistent with
the hypothesis that gain of function, perhaps mediated via interaction
with p53 family members, more readily occurs when mutations occur in
the 72R allele.
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FOOTNOTES
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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.
1 L. B. was funded by the Medical Research
Council and J. O. by the Leukaemia Research Fund. W. K.
is an assistant investigator of the Howard Hughes Institute. 
2 To whom requests for reprints should be
addressed, at The Ludwig Institute for Cancer Research, Imperial
College School of Medicine, St Mary’s Hospital, Norfolk Place, London
W2 1PG, England. Phone: 0207-563-7721; Fax: 0207-724-8586; E-mail: t.crook{at}ic.ac.uk
3 The abbreviations used are: HPV, human
papillomavirus; SCC, squamous cell carcinoma; LOH, loss of
heterozygosity.
Received 7/14/00.
Accepted 10/31/00.
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Top
ABSTRACT
Introduction
