Download Regulation of p63 Isoforms by Snail and Slug Transcription Factors

The American Journal of Pathology, Vol. 176, No. 4, April 2010
Copyright © American Society for Investigative Pathology
DOI: 10.2353/ajpath.2010.090804
Tumorigenesis and Neoplastic Progression
Regulation of p63 Isoforms by Snail and Slug
Transcription Factors in Human Squamous Cell
Carcinoma
Michael Herfs,* Pascale Hubert,*
Meggy Suarez-Carmona,* Anca Reschner,*
Sven Saussez,† Geert Berx,‡§ Pierre Savagner,¶
Jacques Boniver,* and Philippe Delvenne*
for the therapeutic modulation of neoplastic cell
invasiveness. (Am J Pathol 2010, 176:1941–1949; DOI:
From the Laboratory of Experimental Pathology,* GIGA-Cancer
(Centre for Experimental Cancer Research), University of Liege,
Liege, Belgium; the Laboratory of Anatomy, Faculty of Medicine
and Pharmacy,† University of Mons-Hainaut, Mons, Belgium; the
Department for Molecular Biomedical Research, Unit of
Molecular and Cellular Oncology,‡ VIB, Ghent, Belgium; the
Department for Biomedical Molecular Biology,§ University of
Ghent, Ghent, Belgium; and the Centre de Recherche en
Cancerologie,¶ CRLC Val d’Aurelle-Paul Lamarque, Montpellier,
France
p63 is a group of six different transcription factors that
exhibit a high sequence and structural homology to the
well-known p53 tumor suppressor protein. Because of
the use of alternative promoters and transcription start
sites, the TP63 gene gives rise to transcripts that encode
proteins with (TAp63) or without (⌬Np63) an amino-transactivating domain. Both TA and ⌬N transcripts are alternatively spliced at the 3⬘ end to yield further carboxylterminal isotypes (␣, ␤, ␥).1 The extensive defect in a high
number of epithelial structures including skin, breast, and
prostate exhibited by p63-null mice previously suggested
an essential role for p63 isoforms in epithelial development.2,3 Subsequently, additional studies demonstrated
that p63 proteins are implicated not only in the stratification of squamous epithelia4 but also in the differentiation
of mature keratinocytes5 and in the maintenance of the
proliferative potential of epithelial stem cells.6 Recently, in
vitro studies have also shown that ⌬Np63 isoforms inhibit
TAp63 isoforms in a dose-dependent manner.7 In addition to their role in normal development, a potential role
for p63 proteins in tumorigenesis is supported by the
finding that p63 immunoreactivity is observed in more
than 90% of squamous epithelial malignancies.8 However, because of the lack of reliable antibodies for ⌬N
TP63 is a p53-related gene that contains two alternative promoters, which give rise to transcripts that
encode proteins with (TAp63) or without (⌬Np63) an
amino-transactivating domain. Whereas the expression of p63 is required for proper development of
epithelial structures, the role of p63 in tumorigenesis
remains unclear. Here, we investigated the role of
Snail and Slug transcription factors, known to promote epithelial-to-mesenchymal transitions during
development and cancer, in the regulation of p63
isoforms in human squamous cell carcinoma (SCC).
In the present study, we observed that the expressions of ⌬N and TAp63 isoforms were, respectively,
down- and up-regulated by both Snail and Slug. However, the induction of TAp63 was not directly caused
by these two transcription factors but resulted from
the loss of ⌬Np63, which acts as dominant-negative
inhibitor of TAp63. In SCC cell lines and cancer tissues, high expression of Snail and Slug was also significantly associated with altered p63 expression.
Finally, we showed that ⌬Np63 silencing reduced cell–
cell adhesion and increased the migratory properties
of cancer cells. These data suggest that the disruption
of p63 expression induced by Snail and Slug plays a
crucial role in tumor progression. Therefore, p63 and
its regulating factors could constitute novel prognosis
markers in patients with SCC and attractive targets
10.2353/ajpath.2010.090804)
Supported by the Marshall Program of the Walloon Region (Neoangio N°
616476), the Belgian Fund for Medical Scientific Research, the Centre
Anti-Cancereux près l’Université de Liège, and the Faculty of Medicine of
the University of Liege. P. Delvenne is a Senior Research Associate of the
Belgian National Fund for Scientific Research. M.H. is a Research fellow
of the Belgian National Fund for Scientific Research.
M.H. and P.H contributed equally to this study.
Accepted for publication December 11, 2009.
Supplemental material for this article can be found on http://ajp.
amjpathol.org.
Address reprint requests to Michael Herfs, Ph.D., Laboratory of Experimental Pathology, GIGA-Cancer, University of Liege, 4000 Liege, Belgium.
E-mail: [email protected].
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AJP April 2010, Vol. 176, No. 4
and TAp63, the p63 isoforms expressed in these malignant lesions were not determined in most studies. Despite some data on their implication in apoptotic pathways,9 –12 the role of p63 proteins in cancer is still unclear
and accumulating evidence suggests that p63 proteins
could exert both oncogenic and tumor suppressor functions (reviewed by Mills13).
First described in Drosophila melanogaster, Snail represents the founding member of a superfamily of zincfinger transcriptional regulators. Members of this family
are involved in the formation of mesoderm and neural
crest as well as in the malignant progression of epithelial
tumors. In mammals, the best characterized members of
the Snail superfamily, Snail and Slug, have been each
implicated in the loss of epithelial features associated
with the acquisition of a fibroblast-like motile and invasive
phenotype by tumors (reviewed by Nieto14).
The purpose of this study was to examine the implication of Snail and Slug transcription factors in the regulation of p63 isoform expression. We showed that these two
transcriptional regulators repress ⌬Np63 expression,
which leads to an upregulation of TAp63. These in vitro
data were congruent with results obtained in tissue samples from patients with cervical, esophageal, or head and
neck squamous cell carcinoma (SCC). We also observed
that the loss of ⌬Np63 associated with the induction of
TAp63 reduces cell– cell adhesion and increases the migration of squamous malignant cells.
Materials and Methods
Patients and Tissue Samples
One hundred sixty specimens of SCC including 53 cervical SCC (mean age: 53 ⫾ 7 years), 58 head and neck
SCC (50 men, 8 women, mean age: 56 ⫾ 9 years), and 39
esophageal SCC (24 men, 15 women, mean age: 48 ⫾ 9
years) were obtained from patients who underwent surgery at the University Hospital Center of Liege or Brussels
in the period 2002 to 2008. These tissue samples were
collected at the Tumor Bank of the University of Liege.
Tissues were either frozen or fixed in 10% formalin and
embedded in paraffin. The protocol was approved by the
Ethics Committee of the University Hospital of Liege.
Cell Cultures
Four genital SCC cell lines (A431, C4II, SiHa, CaSki) were
grown in a 3:1 mixture of Dulbecco’s modified Eagle’s
medium (Gibco-Invitrogen, Carlsbad, CA) and Ham’s
F12 (Gibco) containing 10% fetal calf serum (FCS) and
supplemented with 1% nonessential amino acid (Gibco)
and 1% sodium pyruvate (Gibco). Three head and neck
(FaDu, Detroit 562, RPMI 2650) and two esophageal
(Te-1 and Te-13) SCC cell lines were respectively maintained in minimal essential medium (Gibco) and in RPMI1640 (Gibco) containing 10% FCS and supplied with 1%
L-glutamine (Gibco). All of the cell lines were incubated at
37°C in a humidified CO2 atmosphere until a 50% to 60%
confluence was reached.
Immunohistochemistry
Immunohistochemical analysis of frozen and paraffin-embedded specimens was performed as previously described.15 Briefly, paraffin sections were deparaffinized,
rehydrated in graded alcohols, and antigens were retrieved in EDTA or in citrate buffer, whereas frozen sections were fixed with 4% paraformaldehyde and nonspecific binding sites were blocked by a 2% BSA solution.
Antibodies anti-p63 (clone 7JUL; Novocastra, Newcastle,
UK) recognizing all p63 isoforms, anti-Snail (Abcam,
Cambridge, UK), and anti-Slug (Abcam) were used for
the primary reaction. Immunoperoxidase staining was
performed using the Envision kit (Dako, Glostrup, Denmark) according to the supplier’s recommendations. Positive cells were visualized using a 3,3⬘-diaminobenzidine
(DAB) substrate, and the sections were counterstained
with hematoxylin.
Immunostaining Assessment
The immunolabeled tissues were evaluated by using a
semiquantitative score of the intensity and extent of the
staining according to an arbitrary scale. For staining intensity, 0 represented samples in which the immunoreactivity was undetectable, whereas 1, 2, and 3 denoted
samples with, respectively, a low, moderate, and strong
staining. For staining extent, 0, 1, 2, and 3 represented
samples in which the immunoreactivity was detectable,
respectively, in ⬍5%, 6% to 25%, 26% to 75%, and
⬎75% of the tumor cells. To provide a global score for
each case, the results obtained with the two scales were
multiplied, yielding a single scale of 0, ⫹1, ⫹2, ⫹3, ⫹4,
⫹6, et ⫹9.16,17 The biopsies were classified into four
groups: high expression (score ⬎3) for either Snail or
Slug as well as high expression and low expression
(score ⬍3) for both Snail and Slug.
Laser Capture Microdissection
Serial frozen sections (6 ␮m thick) of SCC were obtained
using a Microm HM 500 M cryostat (Microm International,
Francheville, France) and mounted on glass slides covered with a thin membrane (Carl Zeiss Microscopy, Munich, Germany). Sections were then stained with Gill III
hematoxylin (RNase free) (Merck, Darmstadt, Germany)
for 1 minute, washed in distilled water, and dried on ice
for a minimum of 30 minutes. Microdissection was performed using a P.A.L.M. microdissector (Carl Zeiss Microscopy) and was supervised by a histopathologist to
ensure ⬎95% specificity of capture tumor cells.
RT-PCR Analysis
One ␮g of total RNA extracted from either cell cultures or
frozen microdissected biopsies (RNeasy mini kit, Qiagen,
Valencia, CA) and quantified with a ND-1000 spectrophotometer (NanoDrop, Wilmington, DE) was reverse transcribed using Superscript II reverse transcriptase (Invitrogen) according to the manufacturer’s instructions.
Snail/Slug-Mediated p63 Regulation in SCC
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The reactions were performed at 42°C for 50 minutes,
followed by inactivation of the enzyme at 75°C for 15
minutes. The cDNA was stored at ⫺20°C. For PCR reactions, primer sequences and annealing temperatures
were as follows: Pan-p63 forward, 5⬘-TCCTCAGGGAGCTGTTATCC-3⬘; Pan-p63 reverse, 5⬘-ATTCACGGCTCAGCTCATGG-3⬘, 56°C; TAp63 forward, 5⬘-TGTATCCGCATGCAGGACT-3⬘; TAp63 reverse, 5⬘-CTGTGTTATAGGGACTGGTGGAC-3⬘, 56°C; ⌬Np63 forward, 5⬘-GAAAACAATGCCCAGACTCAA-3⬘; ⌬Np63 reverse, 5⬘-TGCGCGTGGTCTGTGTTA-3⬘, 56°C18; Snail forward, 5⬘AATCGGAAGCCTAACTACAGCGAG-3⬘; Snail reverse,
5⬘-CCTTCCCACTGTCCTCATCTGACA-3⬘, 65°C; Slug
forward, 5⬘-CCTTCCTGGTCAAGAAGCATTTCA-3⬘; Slug
reverse, 5⬘-AGGCTCACATATTCCTTGTCACAG-3⬘,
65°C19; HPRT forward, 5⬘-TTGGATATAAGCCAGACTTTGTTG-3⬘; HPRT reverse, 5⬘-AGATGTTTCCAAACTCAACTTGAA-3⬘, 60°C. Thirty (36 for TAp63 detection)
cycles, including denaturation at 94°C for 30 s, annealing
for 30 seconds and extension at 72°C for 1 minute, were
used for the analysis. Samples were run on 1.8% agarose
gels containing ethidium bromide and visualized with an
UV transilluminator.
Quantitative Real-Time RT-PCR Analysis
Total RNA was extracted, and cDNA was generated by
reverse transcription as described above. For quantitative real-time PCR experiments, 25 ng of cDNA were
amplified in 50 ␮l of 1⫻ SYBR-Green I qPCR master mix
plus (Eurogentec, Seraing, Belgium), containing 200
nmol/L of each primer for Pan-p63, TAp63, ⌬Np63 (described above) or 300 nmol/L of following primers: Snail
reverse, 5⬘-GTGGGATGGCTGCCAGC-3⬘; Snail forward,
5⬘-TGCAGGACTCTAATCCAAGTTTACC-3⬘20; Slug reverse, 5⬘-TCCGGAAAGAGGAGAGAGG-3⬘; Slug forward,
5⬘-TGTGTGGACTACCGCTGC-3⬘21; N-cadherin reverse,
5⬘-CTCCTATGAGTGGAACAGGAACG-3⬘; N-cadherin forward, 5⬘-TTGGATCAATGTCATAATCAAGTGCTGTA-3⬘22;
HPRT reverse, 5⬘-GGTCCTTTTCACCAGCAAGCT-3⬘;
HPRT forward, 5⬘-TGACACTGGCAAAACAATGCA-3⬘.23
Thermal cycling conditions were: 50°C for 2 minutes, 95°C
for 10 minutes, 40 cycles of denaturation at 95°C for 15
seconds and annealing/extension at 60°C for 1 minute. All
of the experiments were performed in triplicate, using the
ABI-Prism 7700 Sequence Detection System (Applied Biosystems, Foster City, CA) and negative controls (master mix
without any cDNA or RNA) were added in each run. Each
quantitative real-time PCR experiment was normalized to
the amount of HPRT mRNA from the same sample. The
acquired data were analyzed by Sequence Detector software, Version 1.9 (Applied Biosystems).
Western Blotting Analysis
Cells were lysed in a buffer containing 50 mmol/L Tris, pH
7.5, 300 mmol/L NaCl, 1 mmol/L ethylenediaminetetraacetic acid, 1% Igepal CA-630 (Sigma, St. Louis, MO),
1 mmol/L phenylmethyl sulfonyl fluoride (Sigma), and
protease inhibitors (Roche, Bale, Switzerland). After
quantification (BCA protein assay; Pierce, Rockford, IL),
20 ␮g of proteins were separated by electrophoresis on
4% to 12% NuPAGE polyacrylamide gels (Invitrogen) and
transferred onto polyvinylidene difluoride membranes.
The membranes were subsequently blocked with 5%
skim milk for 30 minutes and incubated overnight at 4°C
with anti–␤-actin (Sigma), anti-⌬Np63 (anti-p40, Calbiochem, Gibbstown, NJ), anti-TAp63 (Biolegend, San Diego, CA), anti-Snail (Abcam), anti-Slug (Clone G-18,
Santa Cruz Biotechnology), anti–E-cadherin (BD Transduction Laboratories, Franklin Lakes, NJ), anti–N-cadherin (Zymed Laboratories, San Francisco, CA), and antivimentin (Clone V9, Dako) antibodies. The membranes
were then washed with Tris-Buffered Saline Tween-20
(TBS-T) and incubated with appropriate secondary antibodies. After washing, the protein bands were detected
using an enhanced chemiluminescence system (ECL
Plus; Amersham Biosciences, Piscataway, NJ).
siRNA Transfection and Gene Silencing
Small interfering RNA (siRNA) targeting human ⌬Np63
was designed (5⬘-UGCCCAGACUCAAUUUAGU-3⬘) and
purchased from Eurogentec. The sense and the antisense strands were annealed to obtain duplexes with
identical 3⬘ overhangs. The sequence was submitted to a
BLAST search against the human genome to ensure the
specificity of the siRNA. The day before transfection, 105
cells per well of a six-well plate were seeded in 3 ml of
appropriate growth medium. For each transfection, 50 ng
of siRNA duplexes and 3 ␮l of Transfectin (Bio-Rad,
Hercules, CA) were diluted in 1 ml of Optimem (Invitrogen). The mixture was then incubated at room temperature for 20 minutes to allow the formation of siRNA–
liposome complexes. Growth medium was aspirated
from the cells and the transfecting solution was added
drop by drop. The cells were incubated with the complexes for 4 hours at 37°C in a CO2 incubator. After
incubation, 1 ml of growth medium (containing 20% of
serum) was added without removing the transfection mixture. Twenty-four hours after transfection, the medium
was replaced with normal growth medium. The transfection of an ATTO 647N-labeled control siRNA (Eurogentec,
Seraing, Belgium) was also performed and revealed a
siRNA uptake in more than 95% of the cells.
Transient Transfections of Slug, Snail, and p63
cDNAs
To study the regulation of the endogenous p63 gene by
Snail and Slug transcription factors, 1.5 ⫻ 105 cells
plated in six-well plates were transiently transfected with
a pcDNA3.1 Zeo expression vector (Invitrogen) containing a full-length human Slug sequence and/or a pEF6/
Myc-His version A expression vector (Invitrogen) containing a human Snail sequence using Exgene transfection
reagent (Fermentas, Burlington, Canada). Similar conditions were used to transfect expression vectors (pcDNA3)
encoding each p63 isoform (provided by Dr. Caron de
Fromentel, INSERM U590, Lyon, France). Twenty-four
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Figure 1. Expression of the p63 isoforms, Snail
and Slug in human SCC cell lines. RT-PCR (A)
and Western blot (B) analysis of Snail, Slug,
⌬Np63, and TAp63 isoform expression were
performed on nine human SCC cell lines. HPRT
and ␤-actin were used as controls for RNA and
protein loading, respectively. A representative
experiment is shown of three independent experiments performed.
hours after plating, transfection was performed as recommended by the manufacturer by adding, in each well,
a mixture containing 200 ␮l of 150 mmol/L NaCl, 9 ␮l of
Exgene and 2 ␮g of the Slug and/or Snail expression
vector. As a control, cells were transfected with the corresponding empty vector. At 24, 48, and 72 hours after
transfection, cells were collected for RT-PCR or for Western blotting analyses. A control transfection condition
using a plasmid encoding GFP (pEGFP-IRESpuro, Clontech, CA) was performed in parallel to determine the
transfection efficiency. All experiments were set up to
obtain at least 60% of transfected cells.
Boyden Chamber Migration Assay
The migratory properties of cells transfected with the
si⌬Np63 were assessed using the Boyden chamber assay. 104 cells were suspended in 55 ␮l of serum-free
medium supplemented with 0.1% BSA and placed in the
upper compartment of a 48-well Boyden microchamber
(Neuroprobe, Cabin John, MD). The lower compartment
was filled with 27 ␮l of medium containing 10% FCS and
1% BSA. After 18 hours of incubation at 37°C in a CO2
incubator, the cells that had migrated to the underside of
the filter (Poretics Corp., Livermore, CA) were fixed and
stained with Diff Quick Stain set (Baxter Diagnostics AG,
Düdingen, Switzerland). The upper side of the filter was
scraped to remove residual nonmigrating cells. One random field was counted per well using an eyepiece with a
calibrated grid to evaluate the number of fully migrated
cells. Experiments were performed at least three times in
sixplicate.
Cell–Cell Adhesion Assay
This assay was performed as previously described by
Vessey et al.24 A single cell suspension of 2 ⫻ 106 cells
in 2 ml polystyrene tubes was magnetically stirred at
37°C in a humidified CO2 atmosphere. The number of
single cells was determined using a hemocytometer at
time 0, 20, 40, and 60 minutes. The degree of aggregation was represented by the aggregation index Nt/N0,
where N0 is the total number of single cells before incubation and Nt is the total number of single cells after
incubation for t min.
Statistical Analysis
Statistical analysis was performed with Instat 3 software
(Graph-Pad Software, San Diego, CA). The statistical
significance of the results was calculated by using a
Student t test. Differences were considered as statistically significant when P values were less than 0.05.
Results
TA- and ⌬Np63 Expressions Show,
Respectively, a Positive and Negative
Correlation to Snail and Slug Levels in
Human SCC Cell Lines
To examine the possible relationship between Snail and
Slug transcription factors and p63 isoforms, we first analyzed the expression of these proteins in four genital
(A431, C4-II, CasKi, SiHa), three head and neck (FaDu,
Detroit 562, RPMI 2650), and two esophageal (Te-1, Te13) SCC cell lines. All of the cells expressed both Snail
and Slug at the mRNA (Figure 1A) and protein (Figure 1B)
levels. However, several SCC cell lines (SiHa, CasKi,
RPMI 2650) exhibited extremely high levels of Snail
and/or Slug expression. Interestingly, in contrast to other
cell lines, SiHa, CasKi, and RPMI 2650 weakly expressed
⌬Np63 isoforms whereas TAp63 isoforms were up-regulated. These results were observed by RT-PCR (Figure 1A),
Western blot (Figure 1B), and quantitative real-time RT-PCR
(Supplemental Figure 1 at http://ajp.amjpathol.org). As a
reference for identifying p63 isoforms, cDNA corresponding
to each isoform was transfected in SiHa cells and analyzed
by Western blot (data not shown).
Snail and Slug Regulate p63 Isoform Expression
To determine the exact role of Snail and Slug in the
regulation of p63 isoform expression, we transfected
Snail and Slug cDNA sequences in the A431 cell line. As
shown in Figures 2B and 3B, Western blot analyses indicated that ⌬Np63␣ and TAp63␥ are, respectively, the
major ⌬N and TA isotypes expressed in these cancerous
cells. By using primers designed to amplify all p63 transcripts, we showed that the transient transfection of Snail
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Figure 2. ⌬N- and TAp63 isoform expressions
are, respectively, down- and up-regulated by
Snail transfection. RT-PCR (A) and Western blot
(B) analysis of Snail and p63 isoform expression
in A431 cells transiently transfected with Snail
cDNA for 24, 48, or 72 hours. A representative
experiment is shown of three independent experiments performed. C: Real-time RT-PCR analysis of p63 isoform expression in A431 cells
transiently transfected with Snail cDNA for 24,
48, or 72 hours. Each real-time RT-PCR experiment was normalized to the amount of HPRT
mRNA from the same sample. Results are the
means ⫾ SD of four independent transfection experiments performed in duplicate. Asterisks indicate statistically significant differences (*P ⬍ 0.05,
**P ⬍ 0.01, ***P ⬍ 0.001).
and Slug cDNA for 24, 48, and 72 hours globally reduces
p63 expression (Figures 2A and 3A). However, when the
different p63 isoforms were specifically analyzed, the
expressions of ⌬Np63 and TAp63 were, respectively,
down- and up-regulated by both Snail and Slug transcription factors (Figures 2 and 3). These results were obtained both at the mRNA and protein levels. No synergistic effect was detected when cells were transfected with
both Snail and Slug cDNA (data not shown). Similar results were observed with the cervical C4-II cell line (data
not shown). To investigate whether the increase in TAp63
expression was caused by Snail and Slug transcription
factors or whether it resulted from the loss of ⌬Np63
isoforms, which have a dominant-negative function on
TAp63 isoforms, SiHa cells were transfected with Snail
and/or Slug cDNA. As shown in Figure 1, this cervical
SCC cell line does not express ⌬Np63. No up-regulation
of TAp63 isoforms was observed in SiHa cells transfected with Snail or Slug cDNA (Supplemental Figure 2
at http://ajp.amjpathol.org).
Snail and Slug Immunoreactivity Is Associated
with a Global Loss of p63 in Cervical, Head and
Neck, and Esophageal SCC
We next investigated the expression of Snail, Slug, and
p63 (by using an antibody against all p63 isoforms) in 38
cervical, 32 head and neck, and 25 esophageal paraffinembedded SCC specimens. The immunostaining results
are shown in Figure 4, A and B. Positive staining for Snail
and Slug was observed in 88 tissue samples (93%). As
Figure 3. ⌬N- and TAp63 isoform expressions
are, respectively, down- and up-regulated by
Slug transfection. RT-PCR (A) and Western blot
(B) analysis of Slug and p63 isoform expression
in A431 cells transiently transfected with Slug
cDNA for 24, 48, or 72 hours. A representative
experiment is shown of three independent experiments performed. C: Real-time RT-PCR analysis of p63 isoform expression in A431 cells
transiently transfected with Slug cDNA for 24, 48,
or 72 hours. Each real-time RT-PCR experiment
was normalized to the amount of HPRT mRNA
from the same sample. Results are the means ⫾ SD
of four independent transfection experiments performed in duplicate. Asterisks indicate statistically
significant differences (*P ⬍ 0.05, **P ⬍ 0.01).
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AJP April 2010, Vol. 176, No. 4
Figure 4. A loss of p63 immunoreactivity is observed in human SCC overexpressing Snail
and/or Slug. A: Snail, Slug, and p63 expressions
in paraffin-embedded sections of human SCC
surgical specimens were assessed by immunohistochemistry. Variable degrees of Snail, Slug,
and p63 expression were detected. B: Semiquantitative evaluation of p63 expression in 38
cervical, 32 head and neck, and 35 esophageal
paraffin-embedded SCC specimens. The tissue
samples were classified into four groups according to Snail and Slug immunoreactivity (high:
⫹⫹, low: ⫹/⫺). Asterisks indicate statistically
significant differences (*P ⬍ 0.05, **P ⬍ 0.01,
***P ⬍ 0.001). Original magnifications: ⫻200.
shown in Figure 4A, variable degrees of nuclear Snail and
Slug expression were detected. High expression of Snail
and Slug was observed, respectively, in 19 (50%) and 17
(45%) cases of cervical SCC, 18 (56%) and 15 (47%)
cases of head and neck SCC, and in 11 (44%) and 15
(60%) cases of esophageal SCC. Among these cases, 10
(26%) cervical, 8 (25%) head and neck, and 6 (24%)
esophageal SCC overexpressed both Snail and Slug
transcription factors. In addition, we analyzed p63 expression in all these SCC specimens and observed that
tumors with a strongly positive Snail and/or Slug immunoreactivity were significantly associated with a global
down-regulation of p63, as found by the Student t test
(Figure 4B). This inverse association was also observed in
normal esophageal and exocervical epithelia. Accordingly,
Snail and Slug transcription factors were detected in the
upper epithelial cell layers whereas p63 was only present
in the (para)basal cells of the squamous epithelia (Supplemental Figure 3 at http://ajp.amjpathol.org).
Reactivation of TAp63 and Down-Regulation of
⌬Np63 Occur in Human SCC when Snail
and/or Slug Are Overexpressed
To test whether the expressions of p63 isoforms are
altered during tumorigenesis when Snail and/or Slug transcription factors are up-regulated, we microdissected
frozen biopsies of human SCC (25 cervical, 26 head and
neck, and 14 esophageal SCC), extracted total RNA, and
determined the TA- and ⌬Np63 expression levels by
performing quantitative real-time RT-PCR analysis. Snail
and Slug abundance was determined by immunohistochemistry. After normalizing gene expression levels to
HPRT, we found that ⌬N and TAp63 expressions were,
respectively, significantly down- and up-regulated in cervical and head and neck SCC, which display high Snail
and/or Slug immunoreactivity (Figure 5). Because of the
limited number of specimens, the differences were not
statistically significant in the esophageal SCC group.
⌬Np63 Silencing Alters Cell–Cell Adhesion and
Increases the Migration of Cancer Cells
To investigate the functional contribution of ⌬Np63 downregulation and TAp63 up-regulation in tumor invasion
independently of the well-known epithelial-mesenchymal
transition features induced by Snail and Slug, SCC cell
lines were transiently transfected with a ⌬Np63 siRNA.
Gene silencing efficiency was analyzed by Western blot
(Figure 6A) and real-time RT-PCR (Figure 6B). Results
indicated that ⌬Np63 silencing significantly increases
TAp63 isoform expression, as found by the Student t test
(P ⬍ 0.001). Twenty-four hours after transfection, a cell–
cell adhesion assay was performed and showed that
⌬Np63-silenced SCC cells have a lower ability to aggregate
compared with cells transfected with a control siRNA (Figure 6C). Moreover, we observed that ⌬Np63-silenced SCC
cells exhibited significantly higher migratory properties in
the Boyden Chamber assay (Figure 6D).
Discussion
Events frequently observed in the malignant transformation of epithelial cells include the loss of epithelial differentiation, a decrease in cell– cell contact, and the acqui-
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Figure 5. ⌬N- and TAp63 isoform expressions are, respectively, reduced
and increased in human SCC displaying a high immunoreactivity for Snail
and/or Slug. The expressions of ⌬N- and TAp63 were determined in 25
cervical, 26 head and neck, and 14 esophageal frozen SCC specimens. The
tissue samples were classified in four groups according to Snail and Slug
immunoreactivity (high: ⫹⫹, low: ⫹/⫺). Each real-time RT-PCR experiment
was normalized to the amount of HPRT mRNA from the same sample.
Asterisks indicate statistically significant differences (*P ⬍ 0.05, **P ⬍ 0.01).
sition of invasive and migratory properties. In this regard,
the members of the Snail family of zinc-finger proteins,
Snail and Slug, have been found to play a central role in
this phenomenon referred to as epithelial-to-mesenchy-
mal transition (reviewed by Nieto14). Accordingly, numerous studies have shown that ectopic expression of Snail
and/or Slug in epithelial cells induces dramatic phenotypic changes accompanied by increased cellular motility
and invasiveness.25–28 In addition, these two transcription factors have been reported to correlate negatively in
transformed cell lines with epithelial marker expression
levels such as E-cadherin, desmoplakin, cytokeratin 18,
and MUC-1.26,29 –31
Numerous studies have demonstrated that the p63
proteins play a crucial role both in the initiation of epithelial stratification and in squamous differentiation. Although p63 functions in normal development are well
defined, the role for p63 in tumorigenesis still remains
controversial. Several studies have highlighted the oncogenic potential of ⌬Np63␣.32–34 In contrast, other data
suggest that the p63 gene could act as a tumor suppressor,10,35 although p63 is rarely mutated in human cancer
like classic tumor suppressor genes.
In the present study, we demonstrated that ⌬N and
TAp63 isoform expressions are, respectively, repressed
and increased by both Snail and Slug transcription factors. However, as ⌬Np63 isoforms are more expressed
than TAp63 isoforms, we observed a global reduction in
p63 expression. Our results are in agreement with those
of Higashikawa et al,36 who recently reported that Snailinduced epithelial-to-mesenchymal transition was accompanied by a down-regulation of ⌬Np63␣. Interestingly, we found no up-regulation of TAp63 when Snail
and/or Slug cDNA was transfected in cells lacking ⌬Np63
expression (SiHa cells), suggesting that TAp63 induction
is not directly caused by Snail and Slug transcription
factors but results from the loss of ⌬Np63 isoforms, which
act as dominant-negative inhibitors of TAp63 isoforms.1,7
This hypothesis was strengthened by the increased
TAp63 expression observed after ⌬Np63 silencing.
To evaluate the association between Snail and Slug
transcription factors and p63 isoform expression in hu-
Figure 6. ⌬Np63 silencing reduces cell aggregation and increases migratory abilities of cancer
cells. A: Western blot analysis of ⌬N- and TAp63
isoform expression in the ⌬Np63 siRNA-expressing A431 cells compared with the control cells. A
representative experiment is shown of three independent experiments performed. B: ⌬N- and
TAp63 expression levels were also determined
by real-time RT-PCR. Each experiment was normalized to the amount of HPRT mRNA from the
same sample. Results are the means ⫾ SD of
four independent transfection experiments performed in duplicate. C: Cell– cell adhesion of
⌬Np63 siRNA-expressing cells compared with
control cells. The degree of aggregation was
represented by the aggregation index Nt/N0,
where N0 is the total number of single cells
before incubation and Nt is the total number of
single cells after incubation for t min. D: Analysis
of the migratory abilities of ⌬Np63 siRNA-expressing cells compared with control cells in the
Boyden chamber assay. Data are expressed as
fold induction for ⌬Np63 siRNA-expressing cells
relative to the control cells. Asterisks indicate
statistically significant differences (**P ⬍ 0.01,
***P ⬍ 0.001).
1948
Herfs et al
AJP April 2010, Vol. 176, No. 4
man SCC specimens, we performed immunohistochemical and real-time RT-PCR analysis. Statistical analysis
revealed a significant reduction in p63 immunoreactivity
in SCC when Snail and/or Slug were overexpressed. In
addition, high expression of Snail and/or Slug was also
statistically associated with an up- and down-regulation
of TA- and ⌬Np63, respectively, as already demonstrated
by the in vitro experiments. Previous studies have shown
that global impaired p63 expression is associated with
tumor progression and poor prognosis in bladder cancer.37,38 Furthermore, aberrant overexpression of TAp63
isoforms has been recently reported in head and neck
SCC and associated with tumor development and metastasis.39 Compelling evidence has also accumulated on
the role of ⌬Np63 proteins in squamous differentiation.5,40 – 42 Thus, a reduced expression of ⌬Np63 isoforms could be correlated with epithelial dedifferentiation,
usually related to an aggressive growth pattern. Interestingly, unpublished data have demonstrated a correlation
between p63 and differentiation markers such as keratin
14 in head and neck and esophageal SCC. All these data
suggest a potential prognostic value of p63 and its regulating factors in patient with SCC and may be related to
the effects of these molecules on cell adhesion and migration. Accordingly, we demonstrated that ⌬Np63-silenced SCC cells have a lower ability to aggregate and
exhibit higher migratory properties compared with control
cells in cell– cell adhesion assay and Boyden chamber
assay. In addition, we showed that the silencing of
⌬Np63 in SCC cells results in the acquisition of mesenchymal phenotypic traits associated with a marked upregulation of N-cadherin expression (Supplemental Figure 4 at http://ajp.amjpathol.org). In agreement with our
results, recent studies have reported that the disruption
of p63 in mammary cells and in keratinocytes caused
both a down-regulation of cell-adhesion associated
genes and an up-regulation of genes involved in invasion
and metastasis.43,44 However, in contrast to our results,
siRNAs targeting all p63 transcripts were used in these
studies.
In conclusion, we demonstrated that Snail and Slug
transcription factors affect p63 isoform expression in human SCC, which leads to increased cell migration and
reduced cell– cell adhesion. Although further studies are
needed to identify both the upstream transcriptional regulators and the downstream target genes for each p63
isoform and to understand the complex role of p63 in
tumorigenesis, p63 and its regulating factors could constitute novel prognosis markers in patients with SCC and
attractive targets for the therapeutic modulation of neoplastic cell invasiveness.
Acknowledgments
We thank Patrick Roncarati for his excellent technical
assistance and Dr. Caron de Fromentel (INSERM U590,
Lyon, France) for the generous gift of p63 plasmids.
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