Carcinogenesis vol.35 no.3 pp.

586596, 2014
doi:10.1093/carcin/bgt411
Advance Access publication December 11, 2013

The relative mRNA expression of p53 isoforms in breast cancer is associated with
clinical features and outcome
Kelly A.Avery-Kiejda1,2,*, BriannaMorten1,2,
Michelle W.Wong-Brown1,2, AndreaMathe1,2 and
Rodney J.Scott1,2,3
1

Centre for Information Based Medicine, Hunter Medical Research Institute,

New Lambton Heights, NSW 2305, Australia, 2Priority Research Centre for
Cancer, School of Biomedical Sciences and Pharmacy, Faculty of Health,
University of Newcastle, Newcastle, NSW 2308, Australia and 3Hunter Area
Pathology Service, John Hunter Hospital, New Lambton Heights, NSW 2305,
Australia

Mutation of p53 is a common feature of cancer. Breast cancer is

the most common malignancy that develops in women; however,
somatic mutation of p53 is rare, suggesting that p53 becomes
inactivated by other mechanisms. p53 is expressed as smaller isoforms, some of which inhibit wild-type p53. There are no studies
that have examined the relative expression of all isoforms in this
disease. We have analysed the relative messenger RNA expression
of the p53 isoforms, 40, 133, and in a panel of 6 breast
cancer cell lines, 148 breast cancers specimens and 31 matched
normal adjacent tissues by semi-quantitative real-time reverse
transcriptionPCR and analysed their relationship to clinical
features and outcome. We have identified several important clinical associations, particularly with 40p53, which was expressed
at levels that were ~50-fold higher than the least expressed isoform p53. 40p53 was significantly upregulated in tumour tissue when compared with the normal breast and was significantly
associated with an aggressive breast cancer subtypetriple negative. Additionally, p53 expression was significantly negatively
associated with tumour size and positively associated with disease-free survival, where high levels of p53 were protective, particularly in patients with a mutation in p53, suggesting p53 may
counteract the damage inflicted by mutant p53. In conclusion, the
relative expression of p53 isoforms is related to clinical features
of breast cancer and outcome. These results have implications for
the stratification of breast cancer based on p53 function and may
provide an alternate explanation for deregulated p53 signalling in
breast cancer.

Introduction
The tumour suppressor, p53, is critical for the response to DNA damage and has been classified as the guardian of the genome, due to its
ability to coordinate multiple and diverse signalling pathways involved
in this response (13). The importance of p53 in tumour suppression
has long been established, with mutations arising in more than half of
all human cancers (4). The role of p53 in suppressing tumour growth
is primarily due to its induction of cell cycle arrest and DNA repair or
apoptosis, following genotoxic stress (1,5). However, in recent years, it
has become apparent that mutations in the p53 gene can drive tumour
migration and metastasis, suggesting that not only is inactivation of
p53 critical for tumour initiation, it is also required for the acquisition
and maintenance of an invasive cancer phenotype (611).
Breast cancer is the most common malignancy that develops in
women, responsible for the highest cancer-associated death rates
Abbreviations: cDNA, complementary DNA; ER, oestrogen receptor; HER2,
human epidermal growth factor receptor negative; LN, lymph node; mRNA,
messenger RNA; MT, mutant; NAT, normal adjacent tissue; PR, progesterone
receptor; RTPCR, reverse transcriptionPCR; TNBC, triple-negative breast
cancer; SE, standard error; WTp53, wild-type p53.

Materials and methods

Studycohort
All patient samples used in this study were provided by the Australian Breast
Cancer Tissue Bank (Westmead, NSW, Australia). This included total RNA
extracted from 38 Grade 1, 38 Grade 2 and 72 Grade 3 fresh frozen invasive
ductal carcinomas. Matched DNA from invasive ductal carcinoma specimens
was available for 139/148 cases. Areas of matching histologically normal adjacent breast tissue (NAT), as determined by a pathologist, were provided as
frozen tissue cores and were available for 31 cases. The distribution of clinical

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*To whom correspondence should be addressed. Tel:+61 2 4042 0309;

Fax: +61 2 4042 0031;
Email: Kelly.Kiejda@newcastle.edu.au

(12). In breast cancer, somatic mutation of p53 is frequently associated with tumour progression, resistance to therapy and poor prognosis (4). However, its mutation frequency (~25%) is less than would
be expected for a protein that plays such a pivotal role in maintaining genomic integrity in the breast (4). Moreover, many studies have
shown a lack of association of breast cancer risk with polymorphisms
in p53 and/or components of this signalling pathway (e.g. MDM-2)
(13), and p53-dysregulated gene expression signatures have been
shown to be a better predictor of outcome and chemotherapy response
than p53 mutation (3,14,15), indicating that wild-type p53 (WTp53)
becomes inactivated by mechanisms other than mutation.
In recent years, it has been demonstrated that p53 is expressed as
12 distinct smaller isoforms: p53, p53, p53, 133p53, 133p53,
133p53, 40p53, 40p53, 40p53, 160p53, 160p53 and
160p53 (16,17). The isoforms are produced through a combination of alternative promoter usage (133, 160), alternative splicing
(40, , ) and alternative initiation of translation (40, 160). Their
functions and mechanisms of action are poorly understood and have
been derived primarily from studies utilising overexpression in cell
lines (16,1820). Nevertheless, reports suggest that they can enhance
or inhibit the ability of p53 to transactivate certain target promoters
and to induce apoptosis (16,18). Thus, regulated expression of p53
isoforms is critical for the biological outcome ofp53.
The expression of p53 isoforms has been shown to be dysregulated
in several human cancer types (16,2125), including breast cancer
(26,27). There has been little progress in establishing the endogenous
role of these isoforms in cancer due to a lack of available antibodies that can distinguish the isoforms from the WT protein and a lack
of clinical specimens to examine their messenger RNA (mRNA)
expression. However, there is emerging evidence that p53 isoforms
are associated with prognosis and treatment response in cancer. In
breast cancer patients with mutant (MT) p53, the presence of p53
mRNA was associated with lower recurrence rates and higher overall survival rates, compared with patients who did not express p53
(26). Increased expression of p53 mRNA in ovarian and renal cell
carcinoma was associated with worse prognosis and tumour grade,
respectively (23,24). High 40p53 mRNA expression was shown to
be independently associated with good prognosis in mucinous ovarian cancer (28). Furthermore, in leukaemia, the relative expression of
p53 has been inversely correlated with chemotherapy response (21),
whereas our own studies have shown that p53 was upregulated by
cisplatin in melanoma (25). This suggests that the ratio of p53 isoforms to WTp53 is important in cancer prognostication and may be
involved in the response to therapy.
There is a particular paucity of studies that have analysed the relative expression of p53 isoforms in breast cancer. All studies to date
have determined the expression of p53 isoforms using nested reverse
transcriptionPCR (RTPCR) (16,26,27), which is not a quantitative
technique. As a result, there is no quantitative information on which isoforms are most highly expressed and are therefore more likely to alter
p53 function and to have prognostic significance in breast cancer. This
study has analysed mRNA expression of the 40, 133, and p53
isoforms in 6 breast cancer cell lines and 148 invasive breast cancers.

p53 isoforms in breastcancer

diagnoses and patient information is described in Supplementary Table S1,

available at Carcinogenesis Online. An additional cohort of 14 Grade 3 oestrogen receptor negative (ER), progesterone receptor negative (PR), human
epidermal growth factor receptor negative (HER2) breast tumours was used
in validation studies. This study complies with the Helsinki Declaration with
ethical approval from the Hunter New England Human Research Ethics
Committee (approval number: 09/05/20/5.02). All patients have consented to
their tissue and clinical information being used in this study.
Celllines
Cell pellets were kindly provided for a panel of breast cancer cell lines MCF7, T-47D, ZR-75-1, MDA-MB-231, MDA-MB-468, Du4475 and HMT3522-T42 and the non-tumourigenic cell lines HMT-3522-S2 and HMEC
by Dr N.Verrills and Dr J.Weidenhofer, University of Newcastle, Newcastle,
NSW, Australia.

Semi-quantitative real-timePCR
Total RNA (660 ng) was reverse transcribed to generate complementary
DNA (cDNA) using the High Capacity cDNA Reverse Transcription Kit
(Life Technologies) according to the manufacturers instructions. Real-time
PCR analysis was performed in triplicate using TaqMan Universal PCR
mix (Life Technologies) on 20 ng (tumour samples) or 10 ng (cell lines) of
cDNA according to the manufacturers instructions, with results quantified
on a 7500 real-time PCR system (Life Technologies). The expression of the
following p53 isoform transcripts was analysed: p53, p53, 40p53 and
133p53 using the primer/probes shown in Supplementary Table S2, available
at Carcinogenesis Online. In addition, Taqman Gene Expression Assays for
-actin (Human ACTB Endogenous Control) and 2-microglobulin (assay ID:
Hs99999907_m1) were also used (Life Technologies). PCR reactions containing no cDNA and no reverse transcriptase were included in every PCR run.
cDNA from MCF-7 cells was used as a normalizer in each individual plate
to account for variation between PCR runs. Any gene that had a Ct value >35
was classed as not expressed in that sample. The relative expression of the
p53 isoform transcripts was normalized to -actin (Ct) and expressed as the
fold change calculated using the 2Ct method as described previously (29).
The relative expression of -actin in tumours was not significantly different
between the subgroups analysed and therefore served as an appropriate normalizer for this analysis. 2-Microglobulin was used as a normalizer in NAT
versus tumour comparisons.
Western blotting
Protein extraction, separation by sodium dodecyl sulphatepolyacrylamide
gel electrophoresis, and western blot analysis of cell lines to determine the
expression of WTp53 and its isoforms was performed as described previously
(29). The mouse monoclonal antibodies used for the detection of p53 (DO1,
1 g/ml; 1801, 7.5g/ml) were purchased from Merck Millipore (Darmstadt,
Germany). The rabbit monoclonal antibody used for the detection of glyceraldehyde-3-phosphate dehydrogenase (clone EPR6256) was purchased from
Abcam (Cambridge, England). Results were visualized and quantitated on an
Odyssey CLx fluorescent Imager (LI-COR, Lincoln, NE).
p53 mutation analysis
DNA derived from tumour tissue was used to determine the presence of
p53 mutations in cases where available (139 cases). All 11 exons (including
the intron/exon boundaries) of the TP53 gene were analysed by dideoxy
sequencing of the respective PCR products. PCR products were bi-directionally sequenced with the BigDye Terminator version 3.1 Cycle Sequencing
Kit and size separated by capillary electrophoresis using an ABI3730 DNA
Analyser (Life Technologies) as described previously (25,30). Sequencing
traces were analysed using Mutation Surveyor version 3.00 (SoftGenetics,
State College, PA). Mutations were described using the nomenclature guidelines of the Human Genome Variation Society (http://www.hgvs.org). The
DNA sequence numbering is based on the cDNA sequence for p53 (NCBI
RefSeq NM_000546.4). Non-synonymous and synonymous variants were
run through the SIFT predictive algorithm for amino acid substitution and
functional prediction (31,32) as described previously (30).

Survival analysis
KaplanMeier survival curves were generated to compare the development
of metastases following diagnosis in relation to p53 isoform expression and
p53 mutation. The primary outcome of this analysis was disease-free survival. Comparison of survival curves was performed using the log-rank
(MantelCox)test.
All statistical analyses were performed using GraphPad Prism (Version
5.04) software (GraphPad Software, La Jolla, CA) and SAS version 9.3 (SAS
Institute, Cary, NC). A P-value of <0.05 was considered to be statistically
significant.

Results
40p53 is the highest expressed p53 isoform in breast cancer
To determine which p53 isoform was the highest expressed in breast
cancer, the mRNA expression of 40p53, 133p53, p53 and p53
was analysed by real-time RTPCR in 148 breast cancer tissues. The
analysis showed that 40p53 was the highest expressed isoform in
breast cancer tissues (Figure 1A). 40p53 mRNA was expressed at
levels that were 49.8 higher than the median expression of the least
expressed isoform p53, and at levels that were 33.7 and 15.5 higher
than 133p53 and p53, respectively (Figure1A). Although the relative expression levels were remarkably reduced for the 133p53, p53
and p53 isoforms when compared with 40p53, Spearman rank
analysis showed that the relative expression of each isoform within
the tumours was highly correlated with one another (Supplementary
Table S3, available at Carcinogenesis Online). Analysis of p53 isoform mRNA expression in a panel of breast cancer cell lines confirmed
that 40p53 was the highest expressed isoform, with similar relative
expression patterns as found in breast cancer tissues (Figure1B).
40p53 is highly expressed in tumours when compared with normal breast tissue
Only one study to date has analysed p53 isoform mRNA expression
in breast cancer compared with normal breast tissues (16). However,
the analysis was not quantitative and the expression in tumour samples was compared with a pool of normal breast specimens not allowing the frequency of p53 isoform expression to be determined. From
these studies, the authors concluded that the expression of the p53 isoforms was not different in breast tumours compared with the normal

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Extraction ofRNA
Total RNA was extracted from fresh frozen normal adjacent tissue using the
RNeasy Mini Kit (Qiagen, Doncaster, VIC, Australia). RNA was quantified
using the Quant-it RiboGreen RNA Assay Kit (Life Technologies, Mulgrave,
VIC, Australia) and purity assessed by A260/A280 ratios (>1.8) using the
Nanodrop (Thermo Scientific, Wilmington, DE). The RNA integrity of tumour
samples was analysed using the 2100 Bioanalyser and the RNA 6000 Nano Kit
(Agilent Technologies, Mulgrave, VIC, Australia), and all samples used in this
analysis had an RNA integrity number of 8 or greater.

Statistical analysis
Analysis of p53 isoform expression in breast cancer tissues and cell lines. The
normality of the data distribution was tested using a DAgostino and Pearson
Omnibus test. The values showed that not all groups (depending on the comparison) had been sampled from a Gaussian distribution. Therefore, samples
were log transformed to distribute the data normally, prior to statistical analysis. Atwo-tailed t-test was used to determine if there was a statistically significant difference in the expression of p53 isoforms between any two subgroups.
One-way analysis of variance test followed by a Tukey multiple correction
test was used to determine the statistical significance of p53 isoform transcript expression between multiple (>2) subgroups. Analysis of the correlation of p53 isoform mRNA expression with one another was performed using
Spearmans correlation test. Pearsons chi-square test was used to evaluate the
distributions of the clinical parameters examined between tumours with and
without p53 mutations.
Analysis of p53 isoform expression in relation to clinical features. The relationship between p53 isoform expression and clinical parameters was interrogated using multiple linear regression, which adjusts for possible confounding
variables that may affect the relative expression of the p53 isoforms. To adjust
for heterogeneity in the sample distribution samples were ln transformed. For
the analysis of patients of all grades, where p53 isoform expression was the
response variable, the model took into account the following confounding variables: Age at diagnosis + Grade + p53 mutation + lymph node (LN) positivity
+ Tumour size. For the analysis of hormone receptor expression, only Grade 3
patients were analysed as this was the only grade that contained tumour specimens that were ER negative within the cohort, the model took into account the
following confounding variables: Age at diagnosis + p53 mutation + LN positivity + Tumour size + Receptor status. In the tables summarising the multiple
linear regression, the adjusted estimate indicates the mean change in isoform
expression for every unit change in the predictor, where a negative value indicates a decrease and a positive value indicates an increase.

K.A.Avery-Kiejda etal.

Fig.1. Expression of p53 isoforms in breast cancer tissues and cell lines.
Relative quantification of 40p53, 133p53, p53 and p53 by realtime RTPCR in (A) 148 breast cancer tissues and (B) a panel of 6 breast
cancer cell lines (MCF-7, T-47D, MDA-MB-231, MDA-MB 468, Du4475,
HMT-3522-T42). Results are shown as the relative normalized expression
(target/-actin) of the target gene (2Ct). Values represent the median
interquartile range. Relative quantification of 40p53 in (C) 31 tumour
samples and matched NATs. Results are shown as the relative normalized
expression (target/2-microglobulin) of the target gene in breast cancer
tissues/cells compared with matched NAT/cells (2Ct). Values represent the
median interquartile range (C). One-way analysis of variance test followed
by a Tukey multiple correction test (A and B only) was used to determine the
statistical significance of p53 isoform transcript expression. *P < 0.0001,
**P < 0.05. ***P = 0.0003.

588

p53 isoform mRNA expression is not associated with p53 mutation

status
To determine if p53 isoform expression was related to the mutation
status of p53, the entire coding region (including intron/exon boundaries) of p53 was sequenced in cases where tumour DNA was available
(139/148 cases). Deleterious mutations were present in 28.1% of the
cohort (Supplementary Table S4, available at Carcinogenesis Online,
and Table I), where they were significantly associated with grade, LN
status and hormone receptor status (Table I). As expected, tumours
positive for ER (20/105 cases analysed, 19%, Table I) and PR (17/96
cases analysed, 17.7%, Table I) were less likely to have p53 mutations, whereas those that were HER2 positive (12/24 cases analysed,
50%, Table I) or triple negative (ER, PR, HER2; 10/18 cases analysed, 55%, Table I) had a greater proportion of tumours containing
p53 mutations.
We analysed whether the relative expression of the p53 isoforms
was associated with deleterious mutations in p53 using multiple linear
regression. The relative mRNA expression of the p53 isoforms was
not associated with the mutation status of p53 (Table II).
The expression of p53 isoforms is associated with clinical features
The relationship of the relative mRNA expression of the p53 isoforms to clinical features, including age at diagnosis, tumour size,

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breast (16). Given that 40p53 was the highest expressed isoform in
breast cancer tissues and cell lines, we aimed to determine its relative
expression when compared with NAT. This analysis was performed
on 31 patients for which NAT was available. In tumours, 40p53
mRNA was expressed at levels that were significantly (2.6) higher
than the median expression of matched NAT samples, with levels up
to 240-fold higher in some tumour specimens (Figure1C).
To confirm that 40p53 was expressed at the protein level, we
analysed a panel of breast cancer cells containing WTp53 (MCF-7,
ZR-75-1) or MT p53 (MDA-MB-231, T-47D, HMT-3522-T42) and
two benign breast epithelial cell lines (HMT-3522-S2, HMEC) by
western blotting (Figure2). We used two different antibodies: DO1,
which detects an epitope in the N-terminus (amino acids 2025) and
cannot recognize the N-terminally deleted p53 isoforms and 1801,
which detects an epitope within the N-terminus (amino acids 4655)
and is capable of recognizing the 40p53 isoform. The 1801 antibody clearly detected p53 and a smaller molecular weight band of
~4446 kDa, consistent with 40p53 (Figure2A). This smaller band
was not detected by the DO1 antibody (a very faint smaller band was
detected by DO1 but its molecular weight was ~38 kDa). The 40p53
isoform was detected at the highest levels in the p53 MT cell lines
(MDA-MB-231, T-47D, HMT-3522-T42) and was detected at lower
levels in the WTp53 cell lines (MCF-7 and ZR-75-1). Its expression
in breast cancer cell lines was significantly higher than that seen in
the benign breast epithelial cell lines HMT-3522-S2 and HMEC, confirming our real-time PCR data that 40p53 is upregulated in breast
cancer when compared with normal breast tissue (Figure2A and B).
The ratio of 40p53 to full-length p53 protein was variable between
the cell lines examined, but 40p53 was shown to be higher than or
equivalent to p53 in four of the seven cell lines examined (Figure2A:
MCF-7, ZR-75-1, HMT-T42, HMT-S2).
Given that 40p53 can also be generated by alternative initiation of
translation (18,33), we next determined whether the mRNA expression of 40p53 was representative of its expression at the protein level.
The mRNA levels of 40p53 were comparable with that observed at
the protein level in the majority of the cell lines examined, suggesting
that alternative splicing (rather than alternative initiation of translation) represents a major route of production of this isoform in breast
cancer (Figure2B). In comparison, p53 mRNA expression was much
higher than its protein levels (Figure 2C). This was expected given
that full-length p53 is predominantly regulated post-translationally
and is much less stable than the 40p53 isoform, which lacks the
MDM-2 binding domain and escapes the rapid MDM-2-dependent
proteasomal degradation that full-length p53 is subject to (18,33,34).

p53 isoforms in breastcancer

LN positivity (Table II). However, p53 mRNA expression showed a

significant negative association with tumour size (Table II, P = 0.0326)
and p53 mRNA expression was associated with tumour grade (Table
II, P = 0.0264), with Grade 3 tumours expressing significantly less
p53 mRNA compared with Grade 1 and 2 tumours (Supplementary
Figure S1, available at Carcinogenesis Online).

Fig.2. Expression of 40p53 at the protein level in breast cancer cell lines.
(A) Protein (40g) from MCF-7, ZR-75-1, MDA-MB-231, T-47D and
HMT-3522-T42 breast cancer cell lines; and HMT-3522-S2 and HMEC
benign breast epithelial cell lines were analysed for the expression of
p53 and 40p53 by western blotting. 1801 detects both p53 and 40p53,
whereas DO1 detects p53 but will not detect 40p53. The expression of
glyceraldehyde-3-phosphate dehydrogenase was determined to ensure equal
loading. Relative quantification of (B) 40p53 and (C) p53 by real-time
RTPCR (grey bars) and western blot (black bars). Real-time PCR results are
shown as the relative normalized expression (target/-actin) of the target gene
(2Ct). Values represent the mean standard error (SE). Protein expression
(p53 or 40p53 normalized to glyceraldehyde-3-phosphate dehydrogenase)
has been quantified from the representative western blot using the 1801
antibody in (A). M, molecular weight marker.

LN status and grade was examined using multiple linear regression

analysis. There was no significant difference in the relative expression
of 40p53, 133p53, p53 and p53 in relation to age at diagnosis or

Metastasis-free survival and p53 isoform expression

We next examined the relationship between p53 isoform expression
and the development of metastases in 130 cases for which we had follow-up data. There was no difference in metastasis-free survival when
cases expressing high levels (higher than the median) of 40p53,
133p53 or p53 were compared with those expressing low levels
of these p53 isoforms (Figure 3A). However, cases expressing low
levels of p53 had significantly diminished metastasis-free survival
than cases expressing high levels of p53 (log-rank test, P = 0.0434),
suggesting that high levels of p53 may be protective against metastasis (Figure3A).
We next determined the additive effect of having multiple p53
isoforms highly expressed on breast cancer metastasis. Metastasisfree survival was not different in cases expressing high levels (higher
than the median) of multiple isoforms, compared with those expressing 0 or 1 isoform highly, suggesting that there is no additive effect

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The expression of 40p53 is associated with HER2 status and the

triple-negative phenotype
We next determined if there was an association between p53 isoform
expression and hormone receptor status. This analysis was performed
using multiple linear regression on Grade 3 tumours (n = 72) as this
was the only grade that contained tumour specimens that were ER
negative within the cohort. Of the predictors included in this model,
both age at diagnosis and tumour size were significantly negatively
associated with p53 (Tables III and IV).
We examined the relationship between the p53 isoforms and each
of the receptors (ER, PR and HER2) individually. None of the isoforms showed any significant association with ER or PR (Table III).
Additionally, 133p53, p53 and p53 were not significantly different in their relative expression level between tumours expressing
HER2 and those that did not (Table III). However, 40p53 was significantly negatively associated with HER2 positivity (P = 0.0021,
Table III), with its expression being significantly increased in HER2negative tumours when compared with HER2-positive tumours
(Supplementary Figure S2, available at Carcinogenesis Online).
The cohort was divided into eight distinct groups based on their
receptor status (positive or negative) for the combination of ER, PR
and HER2 (group 1: ER, PR, HER2; group 2: ER PR HER2+;
group 3: ER PR+ HER2; group 4: ER PR+ HER2+; group 5:
ER+ PR HER2; group 6: ER+ PR HER2+; group 7: ER+ PR+
HER2 and group 8: ER+ PR+ HER2+). 133p53, p53 and p53
showed no significant difference in their relative expression level
between the eight groups (Table IV). However, 40p53 was significantly associated with hormone receptor status (P = 0.0344), particularly in the triple-negative cohort (ER, PR, HER2), where it
was significantly increased by a factor of 1.114 when compared with
those that were hormone receptor positive (ER+, PR+, HER2+; Table
IV, Supplementary Figure S2, available at Carcinogenesis Online).
We compared an independent group of 14 Grade 3 ER PR HER2
cases against the other 7 subgroups (n = 53) in an effort to validate
the upregulation of 40p53 in this subgroup (Supplementary Table
S5, available at Carcinogenesis Online). We found that 40p53 was
not significantly different in ER PR HER2 breast cancer cases
(Supplementary Table S5, available at Carcinogenesis Online).
However, we did not have p53 mutation data on these additional cases
and, therefore, these cases could not be adjusted for p53 mutation
status as in our original analysis (Table IV). Given the high proportion
of p53 mutations in our original cohort (52.6% of ER PR HER2
cases), we suspect that not adjusting for p53 mutations would be the
main cause for this result.

K.A.Avery-Kiejda etal.

Table I. Association of TP53 truncating mutations with clinicopathological features

No mutation

p53 mutation

n=139

n=100 (72%)

n=39 (28%)

n (%)

n (%)

P valuea

0.672
11
42
86

7 (7)
32 (32)
61 (61)

4 (10.3)
10 (25.6)
25 (64.1)

68
71

64 (64)
36 (36)

4 (10.3)
35 (89.7)

77
62

58 (58)
42 (42)

19 (48.7)
20 (51.3)

65
53
21

51 (51)
39 (39)
10 (10)

14 (35.9)
14 (35.9)
11 (28.2)

105
34

85 (85)
15 (15)

20 (51.3)
19 (48.7)

96
43

79 (79)
21 (21)

17 (43.6)
22 (56.4)

24
115

12 (12)
88 (88)

12 (30.8)
27 (69.2)

18
121

8 (8)
92 (92)

10 (25.6)
29 (74.4)

<0.0001
0.3226
0.0224

<0.0001
<0.0001
0.0085
0.0054

analysis P value. Significant P values are shown in bold.

a 2

(Figure3A). Although metastasis-free and overall survival has been

reported to be lower in breast cancer patients with p53 mutations
(26,35), we observed no difference in metastasis-free survival when
cases with p53 mutations were compared with those that did not harbour deleterious mutations in this study (Figure3B). This is consistent
with the heterogeneity in the relationship between p53 mutations and
breast cancer prognosis (36).
Bourdon et al. (26) have shown previously that in breast cancer
patients with MT p53, the presence of p53 mRNA was associated
with lower recurrence rates and higher overall survival rates, compared with patients who did not express p53. Thus, we next determined the effect of high or low isoform expression in combination
with a p53 mutation on metastasis-free survival. The expression of
40p53, p53 or 133p53 did not alter metastasis-free survival in
either p53 WT or MT patients (Figure3B). In contrast, low expression
of p53 in combination with a mutation in p53 significantly decreased
metastasis-free survival (log-rank test, P = 0.0487; Figure 3B).
Metastasis-free survival of patients expressing high levels of p53
and carrying a mutation in p53 was similar to patients who did not
contain p53 mutations (Figure3B). This suggests that high levels of
the isoform in patients who have a mutation in p53 may counter the
damage inflicted by MT p53. We were unable to study the relationship
between overall survival and p53 isoform expression in this cohort,
as there were only five disease-related deaths in the 130 patients analysed during this time period.
Discussion
The tumour suppressor p53 is critical for the response to DNA damage (1,2,37). p53 isoforms represent critical regulators of p53 functional activity and there is emerging evidence that some isoforms play
critical roles in cell growth and tumour suppression that are independent of full-length p53 (38). However, there is a paucity of studies that

590

have analysed the relative expression of p53 isoforms in breast cancer.

Moreover, to the best of our knowledge, there have been no studies
that have analysed all the p53 mRNA variants in breast cancer within
the same study. This study has analysed the relative mRNA expression of the 40, 133, and p53 isoforms in 6 breast cancer cell
lines and 148 invasive breast cancers in relation to clinicopathological
features to determine those that are likely to have clinical significance
in this disease.
The relative expression of 40p53 in breast cancer has not previously been analysed at either the mRNA or the protein level in clinical
breast cancer specimens. Our study has shown for the first time that
40p53 was the most highly expressed isoform at the mRNA level in
breast cancer, expressed at levels almost 50-fold higher than the least
expressed isoform p53. Moreover, 40p53 was significantly higher
in breast tumour tissue when compared with that of matched NAT, suggesting that it may be involved in tumour development. We also showed
that 40p53 was the only isoform that was significantly associated with
hormone receptor expression. 40p53 expression was significantly
higher in triple-negative breast cancer (TNBC) and in HER2-negative
cases overall (regardless of ER/PR). TNBC is characterized by an
absence of ER, PR and HER2 (39,40). Although TNBCs comprise
only 1024% of all breast cancers, patients who are diagnosed with
TNBC are of younger age, tend to develop tumours of larger size and
have an increased likelihood of distant metastasis and death within 5
years (39,40). Thus, TNBC is a clinically aggressive subtype, for which
there are no current targeted therapies. The clinical relevance of high
40p53 expression in TNBC is unclear and we were unable to validate this finding in an independent set of TNBCs as we were unable to
obtain p53 mutation status from this set of patients. This result must be
validated in a larger cohort with p53 mutation status to truly determine
the significance of this finding. High 40p53 mRNA expression levels
have been shown to be an independent prognostic indicator in mucinous
ovarian cancer, associated with lower rates of recurrence, and this was

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Age (years)
Median=55
Range=2890
<40
4050
>50
Grade
1 or 2
3
Tumour size (mm), median=25
25
>25
No. of positive LNs
0
13
>3
ER
Positive
Negative
PR
Positive
Negative
HER2
Positive
Negative
TNBC
Yes
No

Total

p53 isoforms in breastcancer

Table II. Association of p53 isoforms with clinical features

Response variable: p53 isoforms

Predictor

Mean change in isoform for every unit change in the predictor

Adjusted estimate (SE)

133p53

40p53

p53

0.005 (0.006)
0.002 (0.005)
0.176 (0.241)
0.159 (0.238)
Ref
0.188 (0.183)
Ref
0.068 (0.223)
Ref
0.004 (0.004)
0.001 (0.003)
0.126 (0.144)
0.314 (0.143)
Ref
0.093 (0.109)
Ref
0.104 (0.134)
Ref
0.003 (0.005)
0.009 (0.004)
0.282 (0.193)
0.058 (0.191)
Ref
0.116 (0.146)
Ref
0.059 (0.179)
Ref
0.001 (0.004)
0.001 (0.004)
0.307 (0.161)
0.415 (0.159)
Ref
0.117 (0.122)
Ref
0.169 (0.149)
Ref

0.4033
0.7171
0.4130
0.3060
0.7629
0.2962
0.6613
0.0907
0.3966
0.4365
0.5432
0.0326
0.3213
0.4287
0.7409
0.8277
0.6910
0.0264
0.3398
0.2577

Results of multiple linear regression analysis with isoform expression as the response variable in 148 breast cancers. Significant P values and the associated
adjusted estimates are shown in bold.

not observed for other ovarian cancer subtypes (28). It is possible that
40p53 may be an independent prognostic indicator in the TNBC subtype specifically; however, we were unable to examine this in this study
due to the small number of TNBC cases (n = 19).
The functional role of high levels of 40p53 in breast cancer as
identified in this study remains to be determined and is the subject of
ongoing investigation. 40p53 is an N-terminal truncation of WTp53
that lacks the first transcriptional activation domain, but retains the
second transcriptional activation domain and is capable of homo- and
hetero-oligomerization (33,38). Initial reports suggested that 40p53
had no activity on its own (18,33), and subsequently, that it acted as a
dominant-negative inhibitor of p53-mediated transactivation, growth
suppression and apoptosis (18,33). It is now apparent that its functions are far more complex. 40p53 has been shown to alter p53 target
gene expression in both a positive and negative manner to modulate
the biological outcome of WTp53 activation. The functional impact of
40p53 appears to be intricately related to its relative levels and on the
cellular context (19,20,34,38,41). In a recent study conducted by Hasfi
etal. (34), it was shown that when expressed at levels higher than that
of p53, 40p53 exerts an inhibitory effect on p53 function. However,
at levels lower than or equivalent to p53, 40p53 acted as an enhancer
or an inhibitor of p53 transcriptional activity, depending on the cell
line under study (34). It was proposed that these contradictory effects
are due to 40p53 modulating p53 activity in two distinct ways: (i) by
altering the binding of basal transcription machinery to the 40p53/
p53 heterotetramer, thereby decreasing transcriptional activity and (ii)

by interfering with MDM-2 binding to p53, 40p53 can protect p53

from MDM-2-mediated proteasomal degradation, thus enhancing p53
activity (34). In this regard, it is clear that even at levels lower than
p53, as observed in this study (Figure 2A), 40p53 can still modulate p53 activity. The functional and somewhat paradoxical impact of
altered 40p53 levels has been demonstrated in several recent studies. Overexpression of 40p53 has recently been shown to increase
the amount of endogenous p53 and to increase apoptosis in human
melanoma cells, in part, by modulating the transcription of downstream target genes including downregulation of p21 and upregulation
of p53-induced death domain protein, to favour apoptosis rather than
cell cycle arrest (42). 40p53 has been shown to play critical roles
in the mouse at all developmental stages (43,44). In the developing
embryo, the expression of 40p53 is detected well before WTp53, and
in embryonic stem cells, 40p53 is required for the maintenance of a
highly proliferative pluripotent state (43). In contrast, overexpression
of 40p53 in somatic cells leads to p53-dependent senescence and
premature aging (44). Thus, although 40p53 has opposing roles in
embryonic and somatic cells, it is clear that proper control of the ratio
of WTp53 to 40p53 is absolutely required for normal cell growth
and proliferation. 40p53 also has functions that are independent of
WTp53, such as induction of G2 arrest, and has been shown to transcriptionally activate targets such as 14-3-3 to a greater extent than
WTp53 in response to cellular stress (20,38,45).
The p53 isoforms p53, p53 and 133p53 have been reported to
be expressed in a range of malignancies including melanoma, ovarian

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p53

Age
Tumour size
Grade 1
Grade 2
Grade 3
LN+
LN
No p53 mutations
p53 MT
Age
Tumour size
Grade 1
Grade 2
Grade 3
LN+
LN
No p53 mutations
p53 MT
Age
Tumour size
Grade 1
Grade 2
Grade 3
LN+
LN
No p53 mutations
p53 MT
Age
Tumour size
Grade 1
Grade 2
Grade 3
LN+
LN
No p53 mutations
p53 MT

P value

K.A.Avery-Kiejda etal.

Table III. Association of p53 isoforms with individual hormone receptors

Response variable: p53 isoforms

Predictor

Mean change in isoform for every unit change in the predictor

Adjusted estimate (SE)

133p53

p53

p53

0.012 (0.009)
0.003 (0.006)
0.097 (0.275)
Ref
0.200 (0.259)
Ref
0.133 (0.348)
Ref
0.072 (0.352)
Ref
0.384 (0.300)
Ref
0.002 (0.005)
0.005 (0.004)
0.071 (0.165)
Ref
0.135 (0.156)
Ref
0.271 (0.210)
Ref
0.166 (0.212)
Ref
0.578 (0.180)
Ref
0.015 (0.007)
0.010 (0.005)
0.031 (0.219)
Ref
0.089 (0.207)
Ref
0.131 (0.278)
Ref
0.557 (0.281)
Ref
0.029 (0.239)
Ref
0.004 (0.006)
0.002 (0.004)
0.066 (0.187)
Ref
0.062 (0.177)
Ref
0.093 (0.238)
Ref
0.027 (0.240)
Ref
0.325 (0.204)
Ref

0.1747
0.6055
0.7248
0.4429
0.7044
0.8386
0.2047
0.6611
0.2133
0.6680
0.3898
0.2018
0.4352
0.0021
0.0422
0.0421
0.8868
0.6693
0.6376
0.0514
0.9050
0.5158
0.6194
0.7255
0.7290
0.6959
0.9094
0.1172

Results of multiple linear regression analysis with isoform expression as the response variable in 72 Grade 3 breast cancers. Significant P values and the
associated adjusted estimates are shown in bold.

cancer, squamous cell carcinoma of the head and neck, renal cell carcinoma, acute myeloid leukaemia, colon cancer and breast cancer
(16,2125,28,46). Both p53 and p53 have been demonstrated to
enhance tumour growth of p53 null cells in vivo; although 133p53
can also perpetuate tumour growth, it appears to have additional roles
in stimulating angiogenesis and inflammation and can promote tumour
invasion and metastasis in mouse models (4749). In this study, we
examined whether the relative expression of these p53 isoforms was
associated with clinical features of breast cancer. We have shown that
the relative expression of p53 was associated with grade, whereas
p53 was negatively associated with tumour size. p53 was also associated with age of disease onset, but only when analysis was confined
to Grade 3 tumours. This is consistent with the findings of Fujita etal.
(22), who found decreased expression of p53 in colon cancers of
increasing severity (adenoma carcinoma), but in contrast to Song
etal. (24), who found p53 positively associated with tumour grade

592

in renal cell carcinoma (22,24). This suggests that the role of p53
in cancer may depend on the site of the cancer. Additionally, we did
not observe an association of p53 with mutation status, nor did we
observe an association of p53 with ER status as previously reported
by Bourdon etal. (26). This may be in part because of the differences
in study design. Although the proportion of breast tumours that were
Grade 3 (51%) or LN positive (53%) in our study was similar to the
proportion of breast tumours that were Grade 3 (48%) or LN positive (49.6%) in their study, Bourdon etal. (26) used nested PCR to
examine the entire coding region of the p53 and p53 transcripts,
classing transcript expression as either present or absent. In contrast,
our study used semi-quantitative PCR to examine N- and C-terminal
variations of p53 mRNA, resulting in a relative expression level of
each of the isoforms (40, 133, , ), which was used in our correlation analysis (the limitations of this are discussed below). 133p53
has previously been reported to be expressed in the majority of breast

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40p53

Age
Tumour size
LN+
LN
No p53 mutations
p53 MT
ER+
ER
PR+
PR
HER2+
HER2
Age
Tumour size
LN+
LN
No p53 mutations
p53 MT
ER+
ER
PR+
PR
HER2+
HER2
Age
Tumour size
LN+
LN
No p53 mutations
p53 MT
ER+
ER
PR+
PR
HER2+
HER2
Age
Tumour size
LN+
LN
No p53 mutations
p53 MT
ER+
ER
PR+
PR
HER2+
HER2

P value

p53 isoforms in breastcancer

Table IV. Association of p53 isoforms with combined hormone receptors

Response variable: p53 isoforms

Predictor

Mean change in isoform for every unit change in the predictor

Adjusted estimate (SE)

133p53

p53

p53

0.014 (0.010)
0.001 (0.007)
0.078 (0.284)
Ref
0.235 (0.269)
Ref
0.808 (0.649)
0.444 (0.678)
0.486 (1.003)
0.914 (0.967)
0.886 (0.751)
0.829 (0.866)
0.947 (0.623)
Ref
0.001 (0.006)
0.006 (0.004)
0.082 (0.168)
Ref
0.147 (0.159)
Ref
1.114 (0.385)
0.339 (0.402)
0.311 (0.595)
0.566 (0.573)
0.501 (0.445)
0.379 (0.514)
0.616 (0.369)
Ref
0.017 (0.007)
0.011 (0.005)
0.023 (0.222)
Ref
0.147 (0.210)
Ref
0.646 (0.507)
0.840 (0.529)
0.386 (0.783)
0.801 (0.755)
1.405 (0.586)
0.602 (0.676)
0.348 (0.486)
Ref
0.002 (0.006)
0.003 (0.004)
0.074 (0.186)
Ref
0.039 (0.176)
Ref
0.351 (0.425)
0.322 (0.444)
0.604 (0.657)
0.363 (0.633)
0.046 (0.492)
0.298 (0.568)
0.298 (0.408)
Ref

0.1572
0.8397
0.7838
0.3848
0.8115

0.8533
0.1599
0.6265
0.3601
0.0344

0.0296
0.0399
0.9168
0.4853
0.2586

0.8043
0.4533
0.6912
0.8245
0.2023

Results of multiple linear regression analysis with isoform expression as the response variable in 72 Grade 3 breast cancers. Significant P values and the associated
adjusted estimates are shown in bold.

cancers and to be inversely associated with the RNA helicase p68, but
its potential clinical significance is not known (16,27). In our study,
133p53 was one of the least expressed isoforms and no associations
with the clinical features examined were found, arguing against this
isoform having major clinical significance in breast cancer.
Perhaps one of the most clinically important findings from this
study was the association of p53 with disease-free survival. High
relative expression levels of p53 were associated with significantly

lower levels of recurrence compared with patients expressing low levels of p53. This is concordant with our finding of a negative association between p53 expression levels and tumour grade. Furthermore,
p53 expression has been positively associated with long-term survival in acute myeloid leukaemia, providing further support for our
findings (21). Additionally, we found that patients expressing low
levels of p53 and harbouring a mutation in p53 had the worst prognosis, whereas patients expressing high levels of p53 who also had

593

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40p53

Age
Tumour size
LN+
LN
No p53 mutations
p53 MT
ER PR HER2
ER PR HER2+
ER PR+ HER2
ER PR+ HER2+
ER+ PR HER2
ER+ PR HER2+
ER+ PR+ HER2
ER+ PR+ HER2+
Age
Tumour size
LN+
LN
No p53 mutations
p53 MT
ER PR HER2
ER PR HER2+
ER PR+ HER2
ER PR+ HER2+
ER+ PR HER2
ER+ PR HER2+
ER+ PR+ HER2
ER+ PR+ HER2+
Age
Tumour size
LN+
LN
No p53 mutations
p53 MT
ER PR HER2
ER PR HER2+
ER PR+ HER2
ER PR+ HER2+
ER+ PR HER2
ER+ PR HER2+
ER+ PR+ HER2
ER+ PR+ HER2+
Age
Tumour size
LN+
LN
No p53 mutations
p53 MT
ER PR HER2
ER PR HER2+
ER PR+ HER2
ER PR+ HER2+
ER+ PR HER2
ER+ PR HER2+
ER+ PR+ HER2
ER+ PR+ HER2+

P value

K.A.Avery-Kiejda etal.

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Fig.3. Metastasis-free survival and p53 isoform expression. KaplanMeier survival curves depicting the time to metastasis. (A) High (red) or low (black)
expression (compared with the median) of 40p53, 133p53, p53 or p53 in 130 cases for which follow-up data were available. The effect of multiple isoforms
expressed at high levels is shown in the bottom panel. (B) Analysis of cases with WT or MT p53 alone or in combination with high or low expression (compared
with the median) of 40p53, 133p53, p53 or p53 in 121 cases for which p53 had been sequenced and for which follow-up data were available. Censored
cases are represented by a . The log-rank (MantelCox) test P values are shown.

594

p53 isoforms in breastcancer

In conclusion, this study has analysed the relative expression of

p53 mRNA variants in breast cancer and examined their relationship to clinicopathological features to determine those that are likely
to have clinical significance in this disease. We have found several
potentially important clinical associations particularly with 40p53
mRNA, which was the most highly expressed isoform and was significantly associated with an aggressive breast cancer subtypeTNBC,
whereas p53 was significantly negatively associated with tumour
size and positively associated with metastasis-free survival. These
results have implications for the stratification of breast cancer based
on p53 functional activity.
Supplementary material
Supplementary Figures S1 and S2 and Supplementary Tables S1S5
can be found at http://carcin.oxfordjournals.org/
Funding
Hunter Medical Research Institute through Pink Frangipani Ball
and Bloomfield Group Foundation grants; National Breast Cancer
Foundation; Hunter Translational Cancer Research Unit Fellowship
from the Cancer Institute NSW (to K.A.A-K.); Australian Postgraduate
Award and Hunter Medical Research Institute Sawyer Scholarship (to
B.M.); Australian Postgraduate Award (to M.W.W-B.); University
Postgraduate Award (to A.M.).
Acknowledgements
The authors would like to thank Ms C.Barnes and Ms J.Carpenter for assistance with breast cancer specimens, Dr N.Verrills and Dr J.Weidenhofer for
providing cell pellets used in this study and Dr A.Bisquera for help with the
statistical analysis of isoform expression. We thank Ms T.Evans for proofreading of the manuscript.
Conflict of Interest Statement: None declared.

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a mutation in p53 had a similar prognosis to patients who did not

have a p53 mutation. This is in contrast to the results of Bourdon
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A limitation of our study design is that the p53 isoforms were analysed at the RNA level rather than the protein level in clinical specimens.
Currently, there are no antibodies that can specifically detect the p53
isoforms by immunohistochemistry and western blot analysis requires
a range of p53 antibodies to identify the isoforms; and even then, the
molecular weights are still very similar to that of the WT protein leaving some level of uncertainty with this type of analysis. RTPCR represents the only method by which the p53 isoforms can be specifically
detected with certainty. Although our analysis was highly advantageous
for detecting relative differences in the mRNA production of these Nand C- terminal variants, one disadvantage is that we are unable to differentiate between the full-length variant (i.e. 40p53, 133p53, p53,
p53) or its truncated variants (i.e. 40p53, 40p53, 133p53,
133p53) due to restrictions in amplicon length used for real-time
PCR. However, the fact that the relative expression levels of the individual isoforms were several fold different between one another (Figure1),
and in individual patients (data not shown), would argue against these
results being due to combinations of N- and C- terminal truncations and
would suggest that they are more likely to be the full-length variants
(i.e. an N- or C-terminal truncation, notboth).
Nevertheless, this report provides critical information on the
mRNA expression of the N- and C-terminal isoforms. Although the
expression of all the isoforms examined was highly correlated with
one another in individual tumours (Supplementary Table S3, available
at Carcinogenesis Online), the relative levels of the individual isoforms varied greatly, with 40p53 having significantly higher expression in the large majority of tumours compared with any other isoform
examined in this study. The regulation of 40p53 at the mRNA level
has not been intensively studied, with only one report to date. Marcel
etal. (51) have shown that G-quadruplex regulatory motifs in intron 3
can regulate the expression of the 40p53 transcript, with mutations
of this motif causing retention of intron 2 and leading to the production of 40p53 mRNA. We identified a polymorphism in intron 3 present in the majority of tumours in our study (Supplementary Table
S4, available at Carcinogenesis Online) that may alter this motif and
could be responsible for the high levels of 40p53 mRNA observed
in the tumours analysed. We cannot rule out the possibility that this
polymorphism is present in constitutional DNA and, therefore, is not
tumour specific. Although 40p53 can also be produced by alternative translation (18,33), our results confirmed that high levels of
40p53 were present at the protein level in breast cancer cell lines at
levels comparable with that of the mRNA (Figure2) suggesting that
alternative splicing is a major route of 40p53 production in breast
cancer cells. Furthermore, 40p53 protein was present at much higher
levels in breast cancer cells than observed in benign breast epithelial
cells suggesting that it is indeed relevant to the modulation of the p53
tumour suppressive properties.

K.A.Avery-Kiejda etal.

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Received May 22, 2013; revised November 14, 2013;
accepted December 5, 2013

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